Publications

4414

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.

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 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.

In the Campania region of southern Italy, a formerly undescribed witches’-broom disease of cultivated strawberry characterized by symptoms similar to those of strawberry witches’-broom and multiplier diseases occurring in North America, has been observed. Strawberry witches’-broom and multiplier diseases are not known to occur in Europe. To elucidate the etiology of the new strawberry disease occurring in southern Italy and to determine the taxonomic position of the presumable causal agent, field observations and PCR assays using universal and group-specific phytoplasma primers followed by multigene sequence analysis were carried out. All of the symptomatic strawberry plants examined tested phytoplasma positive with universal primers and primers specific to the elm yellows (EY) phytoplasma group or 16SrV group. The percentage of diseased plants in the fields was about 30%. Data obtained from sequence and phylogenetic and virtual RFLP analyses of PCR-amplified rDNA (16S rDNA and 16S/23S rDNA spacer region), rpsV (rpl22) and rpsC (rps3), map, imp and groEL gene sequences, showed that the diseased strawberry plants harbored phytoplasma strains which were identical or nearly identical to each other and to strains of the rubus stunt (RuS) agent ‘Ca. Phytoplasma rubi’, a member of the 16SrV group, subgroup 16SrV-E. The 16S rDNA sequence similarity among the strawberry-infecting phytoplasma strains ranged from 99.1 to 99.9%. These strains shared the same range of 16S rDNA sequence similarity with RuS phytoplasma strains including the reference strain RUS of ‘Ca. Phytoplasma rubi’. This is the first report on the occurrence of RuS phytoplasma in naturally affected strawberry plants.

Abstract Citrus greening disease, also known as huanglongbing (HLB), is caused by the gram-negative α-proteobacteria Candidatus Liberibacter species. This disease poses a significant threat to citrus production worldwide, including in Nepal. This study aimed to conduct the diagnosis and phylogenetic analysis of the citrus greening pathogen in Nepal using both conventional PCR and computational methods. A total of 1,026 samples were collected from thirteen districts across six provinces in the country. PCR-based diagnosis was performed using the primer set Las606/LSS, which targets the 16S rRNA gene of Candidatus Liberibacter asiaticus. Additionally, 16S rRNA gene sequencing was performed using sanger sequencing for five samples collected from different geographical regions. The obtained sequences were deposited in GenBank, and a phylogenetic tree was constructed based on these sequences. Among the 1,026 samples tested, 255 were positive, indicating the widespread distribution of HLB across Nepal. All consensus sequences from Nepal showed strong evolutionary relatedness within the C. L. asiaticus cluster, displayed over 99% genetic similarity with reference sequences from various parts of the world. Phylogenetic analysis revealed that the Nepalese sequences were closely related to C. L. asiaticus sequences from India, such as OM522080.1 (Punjab) and MH473394.1 (Meerut). Additionally, sequences obtained from different regions of Nepal clustered closely together. The findings of this research provide valuable insights into the genetic diversity and prevalence of citrus greening disease, which will aid the development of better management strategies to combat this devastating disease. Furthermore, the sequence data deposited in GenBank will serve as a key reference for future molecular studies on citrus greening disease.

A Gram-negative, facultative anaerobic, rod-shaped, motile with peritrichous flagella, fluorescent bacterium, designated ‘Candidatus Pseudomonas auctus’ sp. nov. JDE115, was isolated from soybean root nodules in Virginia and characterized using a comprehensive integrative methodology. Growth of JDE115 occurred with 0–5.0% (w/v) NaCl (optimum 1%), at pH 6.0–10.0 (optimum pH 7.0), and at 10–40°C (optimum 28°C) in LB broth. Phylogenetic analyses based on the 16S rRNA gene placed the isolate as a member of a novel species within the genus Pseudomonas. Phylogenetic analyses based on whole-genome sequences, 16S rRNA, showed JDE115 having the highest similarity to Pseudomonas glycinae MS586. Average Nucleotide Identity (ANI) analysis also revealed the highest similarity of JDE115 to Pseudomonas glycinae MS586 (94.59%), which is below the 95% threshold for species delineation. Genome-to-genome distance analysis (GGDC, Formula 2) showed a maximum value of 57.10% with the same strain, far below the 70% cutoff. The primary isoprenoid quinone detected in JDE115 was ubiquinone-9 (Q-9) and the DNA G + C content was 60.68 mol%. The whole-cell fatty acid profile was dominated by C16:0, C17:0 cyclo, and the summed features 3 (C16:1ω7c and/or C16:1ω6c) and 8 (C18:1ω7c and/or C18:1ω6c). Additional fatty acids detected included 12:0, 14:0, and 18:0. Based on these phenotypic, chemotaxonomic, and phylogenetic data, strain JDE115 is proposed to represent a new species in the genus Pseudomonas, for which the name ‘Candidatus Pseudomonas auctus’ sp. nov. is proposed.

Psyllids are vectors of fastidious plant pathogenic ‘Candidatus Liberibacter’ species that infect both the psyllid vector and plant host. Understanding the molecular and cellular basis of ‘Ca. Liberibacter’ interactions with the psyllid host will aid in identification of effectors involved in invasion and multiplication and facilitate transmission to the host plant. The differential expression of previously identified genes/loci with predicted involvement in tomato host–plant– ‘Ca. L. solanacearum’–prophage–Wolbachia endosymbiont dynamics was quantified by RT-qPCR amplification. Fifteen ‘Ca. Liberibacter solanacearum genes and/or prophage loci and four predicted Wolbachia spp. loci were analyzed in potato psyllids in a 14-day time-course study, post-48-h acquisition-access period by potato psyllids on ‘Ca. L. solanacearum’-infected tomato plants. The ‘Ca. L. solanacearum’-infected tomato host plants were used as an infected host ‘calibrator’ species lacking involvement of psyllid effectors. ‘Ca. L. solanacearum’ genes with predicted functions in adhesion, motility, transport, and virulence that are associated with the prophage lysogenic lifestyle were differentially expressed. In contrast, the prophage-loci expression was synchronous with early or late phase of psyllid-‘Ca. L. solanacearum’ infection, respectively. The observations are consistent with the previously in silico-predicted ‘Ca. L. solanacearum’ gene and prophage/Wolbachia loci functions and time-course global expression patterns. Knockdown of ‘Ca. L. solanacearum’ genes involved in invasion, biofilm formation, and colonization would be expected to impair the vertical and horizontal transmission of ‘Ca. L. solanacearum’ to psyllid offspring and host plants, respectively.