Publications

4364

‘Candidatus Liberibacter asiaticus’ (CLas), the agent associated with the Huanglongbing citrus disease, is commonly detected using quantitative PCR (qPCR) with hydrolysis probes. Internal standards are typically included in the qPCR assays to reduce the risk of false negatives caused by inhibitors. When the internal standard is detected but CLas is not, it is generally assumed that the pathogen is absent from the tested sample. However, our study shows that trace amounts of CLas may go undetected if the internal standard is either overly abundant or too dissimilar to CLas. To overcome these limitations, we developed a synthetic internal standard (IS) that uses the same primer as the CLas target sequence, along with three to four downstream nucleotides, but with a unique internal sequence derived from smooth hammerhead shark (Sphyrna zygaena; IS-SHK). This sequence matches the guanine-cytosine content and melting temperature of the CLas target and was specifically selected to minimize potential interference from other nucleic acid materials in citrus samples. To minimize competition between the IS-SHK standard and the CLas target, an average of only 21 molecules of IS-SHK standard is added to each qPCR reaction. Therefore, when IS-SHK standard is detected at expected levels and CLas is not, the absence of CLas is confirmed rather than inhibition of the detection. Conversely, the absence of both IS-SHK standard and CLas suggests the presence of a qPCR inhibitor, warranting retesting of the sample.

Abstract Citrus Huanglongbing (HLB) is the most destructive disease in citriculture, mainly caused by Candidatus Liberibacter asiaticus (CLas). However, the immune response of citrus to CLas at the cellular level remains to be elucidated. In this study, the first single-cell atlas of rough lemon (Citrus jambhiri Lush.) root apexes were generated using single-nucleus RNA sequencing at 20 weeks post-inoculation with CLas. According to gene expression patterns, the single-cell atlas was partitioned into 20 transcriptionally distinct clusters, and five cell types were identified within these clusters. A significant number of defense-related genes were co-upregulated across the five cell types following CLas infection, whereas genes involved in signal transduction pathways, such as tubulin beta-6 chain (TUBB1) and the phospholipase D alpha 1 (PLD1), were concurrently downregulated. Based on pseudotime trajectory analysis, the key pathways and genes involved in the coordination of cell differentiation and resistance in citrus under CLas infection were characterized. Following CLas infection, the development of phloem cells was significantly delayed, and the differentiation of cambium cells into xylem cells was evident. The expression of genes associated with lignin synthesis was significantly upregulated in these cells. The reduction in phloem cell differentiation and the enhanced differentiation of cambium cells into defense-related xylem cells may represent the primary vascular immune mechanisms exhibited by citrus plants in response to CLas infection. Additionally, DNA-binding one zinc finger transcription factor DOF2.4 was found to potentially serve dual roles in regulating vascular cell development and inducing plant resistance against CLas. In conclusion, this study collectively provides insights into the cellular innate immunity responses of citrus to CLas infection. These findings hold significant implications for the sustainable development of citriculture amidst the ongoing global HLB epidemic, and offer novel insights into vascular immunity and plant defense responses.

Abstract Citrus greening disease, caused by ‘Candidatus Liberibacter asiaticus’, severely impacts citrus production worldwide. The development of sustainable control strategies for this disease is restricted by the unavailability of the bacterium in pure culture. Herein, the metabolic profile of the waxworm larvae, Galleria mellonella, was compared to that of Diaphorina citri, the vector of ‘Ca. L. asiaticus’. Our findings showed that G. mellonella larvae possess the nutritional needs to host ‘Ca. L. asiaticus’, supporting its short-term persistence, and responds to infection with a visible immune reaction by producing melanin upon bacterial invasion. The inoculated larvae exhibit detectable bacterial titers for up to four days when inoculated with infected citrus phloem sap or D. citri haemolymph, after which bacterial titers decline, and infected larvae show reduced survival compared to mock-treated and ‘Ca. L. asiaticus’-free controls. Metabolic profiling of G. mellonella, D. citri, and honeybees (Apis mellifera) reveals distinct chemical compositions in their haemolymph. G. mellonella contains higher levels of amino acids, organic acids, nucleotides, and sugar-nucleotides, providing essential nutrients for ‘Ca. L. asiaticus’, while D. citri is enriched in monosaccharides and sugar-alcohols. Citric acid was detected exclusively in the haemolymph of G. mellonella. These findings suggest G. mellonella as a convenient model that can transiently host ‘Ca. L. asiaticus’ for short-term use, which would facilitate high-throughput screening of antimicrobial compounds against ‘Ca. L. asiaticus’, as well as exploring host-pathogen interactions. This model could accelerate the development of effective treatments against citrus greening and inform broader strategies for managing vector-borne plant diseases.

Abstract Autophagy, a cellular process involved in the degradation and recycling of cellular components, has emerged as a pivotal mechanism for maintaining cellular homeostasis and combating pathogen invasion. Here, we provide evidence that the overexpression of CsATG8c inhibits CLas proliferation in citrus. CsATG8c directly interacts with the CLas effector protein SDE4040, leading to its degradation via the autophagic pathway. The SDE4040 protein acts as a virulence effector, and transgenic citrus plants expressing SDE4040 promote CLas proliferation by suppressing the hypersensitive response (HR) of the host and reducing the levels of jasmonic acid (JA). Collectively, these results demonstrate that ATG8-mediated selective autophagy limits CLas infection by degrading a virulence effector protein, which provides the first evidence for the role of autophagy in combating CLas infection.

Phytoplasmas (‘Candidatus Phytoplasma’) are intracellular pleomorphic plant pathogens belonging to the class Mollicutes. They colonize both plant hosts and insect vectors in their life cycle. Apple proliferation (AP) is one of the most important phytoplasmoses present in Europe, causing significant economic losses in apple production. The causal agent, ‘Ca. P. mali’, was identified in apple and Cacopsylla picta samples using both real-time PCR and nested PCR based on the amplification of 16S rDNA. The objective of this study was to gain deeper insights into the epidemiology of apple proliferation in Croatia. Variability of genetic markers other than 16S rRNA was used for characterization of strains. Four molecular markers differing in level of conservation, aceF, pnp, imp, and secY, were selected in line with previously typed fruit tree phytoplasmas. New genotypes were discerned for each genetic marker, and 20 different sequence types were revealed in the Croatian strains of ‘Ca. P. mali’. On the basis of this comprehensive analysis, the founder sequence type ST1 (A13–P10–S12–I21) can be proposed. This is the first extensive research and multigene typing performed on Croatian ‘Ca. P. mali’ strains. Obtained results reveal considerable genetic diversity of epidemiological relevance limited to only two locations in north-western Croatia. Additionally, novel primers were constructed to amplify fragments larger than the entire coding region for all four genes in order to further expand the phytoplasma multi-locus sequence typing scheme.

ABSTRACT Polyphosphate-accumulating organisms (PAOs) are the main bacteria responsible for phosphorus removal and recovery in full-scale wastewater treatment plants (WWTPs). They encompass members of the genera Candidatus Accumulibacter, Azonexus (formerly Dechloromonas ), and Candidatus Phosphoribacter (formerly Tetrasphaera ), with most studies focusing on Ca . Accumulibacter, primarily using lab-scale enrichment cultures. Although members from the three genera often co-exist in full-scale WWTPs, the metabolic capabilities and traits that determine the niche differentiation of the specific species are still unknown. We retrieved 214 high-quality metagenome-assembled genomes from a full-scale plant with phosphorus removal and examined the polyphosphate-related metabolic pathways using genome-resolved metatranscriptomics in the different process tanks in situ and by using short-term incubations ex situ . We observed the co-existence of nine uncultured PAO species from the three genera with clear niche differentiation in the utilization of different carbon sources and involvement in the denitrification process. Additionally, we observed several physiological differences among species of the same genus, indicating variations in niche specialization. This suggests that biological P removal and other processes in full-scale WWTPs are carried out by a complex and diverse PAO community that together ensures stable plant performance. IMPORTANCE The current understanding of the ecology and physiology of polyphosphate-accumulating organisms (PAOs) is mostly based on Candidatus Accumulibacter, primarily studied in enriched lab-scale studies. Recent taxonomic reclassification revealed that the most studied Ca . Accumulibacter species are either not present or present in low abundance in full-scale wastewater treatment plants (WWTPs). This raises concerns that knowledge from lab-scale studies may not apply to species in full-scale plants. Additionally, the indication of a distinct PAO physiology in Candidatus Phosphoribacter compared to Ca . Accumulibacter and the other abundant PAO Ca . Azonexus poses further questions about the accuracy of the current PAO model. Here, we show that in full-scale plant species from Ca . Accumulibacter, Ca . Azonexus, and Ca . Phosphoribacter always co-exist, and they have distinct niche separations in terms of carbon source utilization and the use of electron acceptors. This co-existence and metabolic diversity indicate that a complex microbial community is crucial for efficient phosphorus removal in full-scale WWTPs.

Abstract Bactericera maculipennis (Crawford) and Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) share hosts within the Solanaceae and Convolvulaceae (Solanales), and both are associated with “Candidatus Liberibacter solanacearum” (Lso). Lso, transmitted by B. cockerelli, causes diseases in solanaceous crops including zebra chip disease of potato. Up to 50% of B. maculipennis adults also harbor Lso, but transmission of Lso to plants by this psyllid has not been confirmed yet. The only documented field host of B. maculipennis in the Pacific Northwest is Convolvulus arvensis L. (Convolvulaceae) but diagnostic methods fail to detect Lso in leaves of this plant. It is therefore unclear how Lso persists within B. maculipennis populations. We surveyed species of Convolvulaceae and Solanaceae for B. maculipennis and report a widespread association between B. maculipennis and Lso throughout the western United States. Diagnostic polymerase chain reaction failed to detect Lso from leaves of C. arvensis yet readily detected Lso from stems where B. maculipennis nymphs tend to feed. Bactericera maculipennis transmitted Lso to species of Convolvulaceae in greenhouse experiments, confirming vector competency. We report high rates of Lso infection in populations of both B. maculipennis and B. cockerelli occurring on C. arvensis, but occurrence of B. cockerelli on C. arvensis was limited to autumn months only and with very low populations. Results suggest C. arvensis is a non-crop reservoir of Lso but do not suggest that B. maculipennis is a direct threat to solanaceous crops or that C. arvensis is a major source of Lso-infected B. cockerelli colonizing potato fields.