Publications

4358

ABSTRACT Phytoplasmas were detected in potato tubers and sugar beet roots in neighbouring fields in Southern Germany using quantitative PCR. Infected potato plants showed phytoplasma‐associated symptoms including yellowing, upward leaf rolling, aerial tubers and early senescence. Sugar beet plants showed similar symptoms to those of syndrome ‘basses richesses’ (SBR) disease, including proliferation, yellowing and necrosis of older leaves. The genetic diversity of the phytoplasmas in the symptomatic potato and sugar beet plants was investigated through sequence analysis of 16S rRNA and stamp , vmp1 , tuf and secY genes. The RFLP profiles and sequences of 16S rRNA were identical among all the sugar beet‐positive samples and aligned with subgroup 16SrXII‐P, whereas the potato phytoplasma strains were identified as belonging to subgroup 16SrXII‐A. Sequence analysis of the non‐ribosomal genes showed that the potato strains belonged to the b1 group of the tuf gene, the V4 and V17 groups of the vmp1 gene and the secY3 and secY4 groups of the secY gene. However, the sugar beet strain was clearly distinct from the potato strains. Together with strain 916/22 from sugar beet in eastern Germany, they formed a new group of tuf , secY , vmp and stamp genes. RFLP assays and multilocus sequence analysis of non‐ribosomal genes of the phytoplasma strains confirmed that the potato phytoplasma strains were molecularly different from the sugar beet phytoplasma strains. In addition, this is the first report on the multilocus sequence analysis of ‘ Candidatus Phytoplasma solani’ strains in potato plants in Germany, suggesting differences in source plants other than sugar beet or interactions with insect vectors specific to each crop.

A Microviridae phage, CLasMV1, was recently identified in ‘ Candidatus Liberibacter asiaticus’ (CLas), the bacterium associated with citrus Huanglongbing (HLB). The CLasMV1 strain GDHZ11D (CP045566) exhibited a circular genome of 8,869 bp containing eight open reading frames (ORFs). Phages of CLas play crucial roles in regulating bacterial population diversity that have potential use for HLB control. However, knowledge of CLasMV1 population diversity is currently limited. In this study, 1,520 CLas samples were collected from 10 citrus-growing provinces in China. PCR screening revealed that 1,042 samples (69%) harbored CLasMV1. Complete genome sequences of 43 CLasMV1 strains were obtained through Illumina HiSeq sequencing with genome sizes ranging from 8,696 to 8,881 bp. Variations were mostly related to single-nucleotide polymorphisms and sequence insertions/deletions. Notably, two hypervariable regions (HVRs) were identified. Two new ORFs were detected, ORF-A (144 bp) in HVR-1 and ORF-B (342 bp) in HVR-2. Whole-genome phylogenetic analysis using 43 strains from this study, 9 previously reported Chinese strains, and 2 Florida strains (CP040636 and MZ382797) delineated five genomic groups. Chinese strains dominated Groups 1 to 4, and Group 5 contained the Florida strains. Further analyses of HVR-1 across all 1,042 CLasMV1 samples detected a 173-bp sequence with suspected mobile activity. Analysis of a PacBio long-read assembly published previously detected a concatemer of the CLasMV1 sequence, suggesting that the phage used the rolling circle mechanism for its replication. These findings significantly enhance our understanding of CLasMV1 genomic diversity and will facilitate future research on CLasMV1 classification and HLB biology.

Abstract Huanglongbing (HLB), a devastating citrus disease caused by Candidatus Liberibacter asiaticus (CLas), triggers persistent immune activation marked by excessive callose deposition and reactive oxygen species (ROS) accumulation, which impairs phloem function. This maladaptive response has led to HLB being described as a “pathogen-triggered immune disease”. Overexpression of the Arabidopsis (Arabidopsis thaliana) NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (AtNPR1) gene, a master regulator of systemic acquired resistance (SAR), confers robust HLB tolerance in susceptible citrus varieties, with transgenic lines exhibiting minimal or no disease symptoms following CLas infection. However, the mechanism underlying this tolerance remains unclear. In this study, we demonstrate that AtNPR1 restores immune homeostasis in CLas-infected trees by suppressing callose and ROS hyperaccumulation, thereby alleviating HLB symptom development. Similarly, silencing the Citrus sinensis homolog of NPR3 (CsNPR3), a negative regulator of SAR, mitigates CLas-induced immune overactivation and enhances HLB tolerance. Notably, salicylic acid (SA) levels are lower in AtNPR1-overexpressing citrus plants than wild-type controls after CLas infection, consistent with NPR1's role in negative feedback regulation of pathogen-induced SA accumulation. In Arabidopsis, overexpression of AtNPR1 or disruption of AtNPR3/AtNPR4 also attenuates pathogen-induced callose and ROS responses. Together, these findings reveal conserved roles for NPR1/NPR3/NPR4 in immune regulation across species and suggest that HLB susceptibility in commercial citrus varieties stems from a diminished capacity to maintain immune balance.

ABSTRACT Five draft genome sequences of ‘ Candidatus Liberibacter solanacearum’ haplotype E from five different Bactericera trigonica vector insects sampled in France are reported. Each genome has a size of about 1.20 Mbp, a G + C content of 35% and approximately 1,000 predicted coding DNA sequences.

Huanglongbing (HLB), or citrus greening, remains one of the most destructive diseases affecting citrus production globally. Associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus ( C Las) and vectored by Diaphorina citri , HLB leads to canopy decline, fibrous root loss, and reductions in fruit yield and quality. Recently, the systemic delivery of oxytetracycline (OTC) via trunk injection was approved in Florida as a targeted therapy to reduce C Las titers and improve tree health. In parallel, Individual Protective Covers (IPCs) have been adopted to delay C Las infection in newly planted citrus trees by vector exclusion. This study investigates the combined use of IPCs and trunk injection of OTC for post-IPC therapy. ‘Valencia’ sweet orange trees grafted on US-812 and US-942 rootstocks were planted in December 2020 under HLB-endemic conditions in southwest Florida. IPCs were installed at planting and removed after 18 months. The first OTC injection was performed in May 2023, 10 months after IPC removal. A second injection was performed in May 2024. A 2 × 2 × 2 factorial experimental design evaluated the effects of infection history (early-infected and late-infected), rootstock cultivar (US-812 and US-942), and injection treatment (OTC-injected and non-injected) on tree responses over two consecutive production seasons. In year 1, infection history significantly influenced tree size, fruit yield, total soluble solids (TSS), TSS/titratable acidity ratio, and peel color. Late-infected trees outperformed early-infected trees, regardless of injection treatment and rootstock cultivar. In year 2, OTC-injected trees exhibited significantly higher yields, improved juice quality, and enhanced canopy health regardless of infection history and rootstock cultivar. Fibrous root microbiome analyses based on 16S rRNA sequencing revealed no significant effects of OTC injection on bacterial alpha or beta diversity, with stable community structure observed across treatments and time points. This suggests that targeted vascular delivery of OTC may not cause any major disruption to the root endorhizosphere microbiome. Together, the results from this study demonstrate the efficacy of integrating preventative (use of IPCs) and therapeutic (OTC vascular delivery) strategies for sustainable HLB management while preserving microbial integrity and offering a model for citrus production in parts of the world where HLB is prevalent.

Polyphosphate-accumulating organisms (PAOs) play a crucial role in enhanced biological phosphorus removal (EBPR) processes. In addition to biosynthesis, they rely on phosphate for energy generation. However, Candidatus Accumulibacter, a model PAO, has been shown to adapt to low phosphate conditions by switching to a glycogen-accumulating metabolism (GAM), with variable success across genus members and experiments. This study aimed to explore the metabolic shift of several Accumulibacter species subjected to low-phosphate concentration in different operating conditions using metatranscriptomics analysis. Furthermore, the study enabled a comparison of the transcriptomic profiles of Accumulibacter with those of Propionivibrio , a glycogen-accumulating organism typically found in EBPR plants. Two sequencing batch reactors were operated with different carbon sources to enrich for different populations of Accumulibacter . After decreasing the influent phosphate concentration, carbon removal performance was maintained while anaerobic phosphate release dropped dramatically, suggesting a shift from a phosphate-accumulating to a glycogen-accumulating metabolism. Analysis of metatranscriptomics data indicated that Accumulibacter regalis (type I) and Propionivibrio aalborgensis remained the most abundant species after the phosphate decrease in the reactor with acetate-propionate and allylthiourea, while Accumulibacter delftensis (type I) and Accumulibacter phosphatis (type II) remained active in the reactor with acetate-glucose and no allylthiourea. Transcription of the genes from the ethylmalonyl-CoA pathway involved in the production of propionyl-CoA and regulation of the anaerobic redox balance was enhanced under low-phosphate conditions, especially for type I Accumulibacter . Conversely, the transcription of the methylmalonyl-CoA pathway was enhanced under low-phosphate conditions in Propionivibrio and type II Accumulibacter .

Abstract Background Phytoplasma research encounters limitations due to the lack of availability of pure cultures of these microorganisms. In this study a culture-independent approach was employed to investigate the genome and pathogenic mechanisms of phytoplasma responsible for witches’ broom disease in Trema levigata (Yunnan province, China). The phytoplasma genome was assembled using Illumina sequencing data and the Phytoassembly pipeline based on mixed samples. Results Nested PCR analysis identified a 16Sr group I, ‘ Candidatus Phytoplasma asteris’ strain in Trema levigate showing witches’ broom disease. Comparative study between infected and healthy plants resulted in an 849.7 kb draft genome with 94.1% coverage, 27.6% GC content, encoding 1356 predicted genes, of which 587 were functionally annotated. Multilocus phylogenetic analysis showed that this phytoplasma is closely related to ‘ Ca. P. asteris’. The genome possesses complete pathways for glycolysis and pyruvate metabolism pathways and harbors specific transporters (spermidine/putrescine, lysine), indicating dependency on host metabolites. Thirty-one putative secreted proteins, including TENGU and SAP05/54-like effectors were identified along with three potential mobile units carrying replication/recombination genes. Conclusions This approach demonstrates the effectiveness of pathogen purification-free analysis for phytoplasma studies in naturally infected host species, thereby enhancing the understanding of phytoplasma-host interactions.

Abstract BACKGROUND Huanglongbing (HLB) is the most devastating citrus disease worldwide, with no resistant commercial citrus varieties identified and no effective chemical treatments currently available on the market. The main challenge in developing effective strategies for controlling HLB is the inability to culture its associated pathogen, the fastidious Candidatus Liberibacter asiaticus ( C Las), in defined media or axenic cultures. RESULTS In this study, we developed an effective method for screening antimicrobials against C Las using periwinkle as a model plant. Compared with citrus, periwinkle offers notable advantages, including reduced time, increased efficiency, and lower costs. This method consists of two main components: screening exogenously applied antimicrobials and evaluating overexpressed endogenous antimicrobial peptides. Using this method, we demonstrated that bromothalonil and cyproconazole effectively inhibit the proliferation of C Las in hydroponically grown periwinkle. Further analyses revealed that bromothalonil and cyproconazole were also highly toxic to other citrus pathogens, including Xanthomonas citri subsp. citri ( Xcc ), Alternaria alternata tangerine pathotype (Z7), and Penicillium digitatum (KH8). In addition, we established an Agrobacterium rhizogenes ‐mediated hairy root genetic transformation system for periwinkle and demonstrated that the proliferation of C Las was significantly inhibited in transgenic roots overexpressing antimicrobial peptide (SAMP). CONCLUSION An efficient antimicrobial screening system for C Las and several other citrus pathogens has been established. By addressing challenges such as high costs, prolonged experimental periods, and heavy workloads, we anticipate that this developed method will serve as a useful tool for the identification of antimicrobials effective against C Las and other citrus pathogens. © 2025 Society of Chemical Industry.