Publications

4586

Candidatus Liberibacter solanacearum (CLso), a pathogen of concern to the potato industry, which causes zebrachip disease, is vectored by the tomato/potato psyllid [ Bactericera cockerelli (Šulc)]. This study investigated whether Lycium ferocissimum acts as an overwintering green bridge enabling pathogen survival, while potatoes are absent. The potential of L. ferocissimum to act as a green bridge for other pathogens was also investigated. African boxthorn leaf samples were taken in Feb, Mar, and Apr 2023 in Canterbury, New Zealand, and endophytes were isolated and cultured. DNA was extracted from these pure-cultured endophytes as well as from leaves. Molecular methods were used to identify the cultured endophytes and to determine the presence of CLso. This study found several potentially pathogenic endophytes including Alternaria porri, Stemphylium eturmiunum , Stemphylium vesicarium, Pseudomonas spp., Erwinia spp. , Pantoea sp. , and Candidatus Liberibacter solanacearum. The percentage of leaves which tested positive for CLso increased from 2% in Feb 2023 to 39% in Apr 2023. CLso was found in leaves without evidence of psyllid feeding, which may indicate systemic infection of African Boxthorn, suggesting the plant acts as a green bridge plant. It is highly recommended that this plant be eradicated within the vicinity of potatoes and other Solanaceous crops.

Abstract The pear psyllid Cacopsylla pyri is the main vector of ‘ Candidatus Phytoplasma pyri’, the causal agent of pear decline, a disease associated with severe yield losses in pear production. Phytoplasmas are further known to interfere with their vectors metabolism; therefore, we investigated whether an infection with ‘ Ca. P. pyri’ is associated with changes in nutrient storage of the pear psyllids. Additionally, we measured whether the phytoplasma infection is associated with differences in flight performance in C. pyri , as the flight activity of the vector is a key component of phytopathogen spread. Using a flight mill, we quantified flight distance, duration, and speed, and combined qPCR-based infection detection with colorimetric assays to determine protein, carbohydrate, and lipid content from the same individual. Infected psyllids contained significantly higher levels of soluble carbohydrates than uninfected individuals. Lipid content was not affected by the phytoplasma infections but was found to be consistently higher in males than in females, while protein levels did not vary with sex or infection status. Despite infection-related differences in carbohydrate levels, phytoplasma infection did not significantly affect flight duration, distance, or speed. These results indicate that infection-associated shifts in bulk carbohydrate storage do not translate into altered flight motivation or performance, suggesting a functional decoupling between metabolic state and realised dispersal capacity under standardised laboratory conditions.

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 perform the diagnosis and phylogenetic analysis of the citrus greening pathogen in Nepal using both conventional PCR and computational methods. A total of 1,026 citrus leaf 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. The reliability of the PCR was validated by including previously confirmed positive and negative controls in every run. Furthermore, each assay was performed in triplicate to ensure consistency and reproducibility of results. Additionally, 16S rRNA gene sequencing was performed using Sanger sequencing for five samples. 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 Ca. L. asiaticus cluster, with over 99% genetic similarity to reference sequences from various parts of the world. Phylogenetic analysis revealed that the Nepalese sequences were closely related to Ca . L. asiaticus sequences from India (Punjab and Meerut) and sequences obtained from different regions of Nepal clustered closely together. The molecular findings from this study reveal a high prevalence of citrus greening disease across Nepal and underscore the urgent need for integrated management policies, including the use of certified clean planting material and vector (psyllid) control programs. The generated sequence data serves as a vital resource for developing regional diagnostic tools and guiding future surveillance strategies to mitigate HLB’s impact on the world’s citrus industry.

Sugar beet cultivation in Europe is threatened by two vector-borne diseases: syndrome “basses richesses”, caused by the phloem-limited pathogen ‘Candidatus Arsenophonus phytopathogenicus’, and phytoplasmoses associated with ‘Ca. Phytoplasma solani’ subgroup 16SrXII-A and the related subgroup 16SrXII-P. Infections lead to reduced sugar yield, biomass and growth abnormalities. In Germany, Pentastiridius leporinus represents the main vector. Despite their importance, genetic diversity remains poorly understood. During a two-year survey, barcoded amplicons were generated from infected sugar beet samples from Germany and neighbouring countries using the phytoplasma markers 16S rRNA-ITS-23S rRNA, tuf, and groEL-stamp-nadE, as well as rplO-secY-rpmJ and groEL for ‘Ca. A. phytopathogenicus’. Amplicon pools underwent single-molecule real-time sequencing and amplicon-sequence-variant inference. Additionally, planthopper samples from sugar beet in Germany were analysed and compared to sugar beet data for ‘Ca. A. phytopathogenicus’. No genetic diversity of ‘Ca. A. phytopathogenicus’ was detected, whereas 16SrXII-A and -P showed variation below the subgroup level. 16SrXII-A exhibited higher diversity than 16SrXII-P. In Germany, 16SrXII-A formed a single cluster, while 16SrXII-P comprised two clusters based on 16S rRNA-ITS-23S rRNA. In neighbouring countries, only 16SrXII-A showed diversity, resolving up to four clusters by groEL-stamp-nadE. These results provide a basis for the identification of dominant strains supporting comparative variety evaluation for tolerance.

Citrus huanglongbing (HLB) is recognized as the most destructive bacterial disease affecting citrus. Among the causal agents, Candidatus Liberibacter asiaticus (Las) is the predominant species causing HLB epidemics worldwide. Detecting this phloem limited bacterium at early or low titer stages and assessing its cellular activity remain major challenges for HLB research and management. To address these limitations, we developed a one step reverse transcription quantitative PCR (RT qPCR) assay for highly sensitive Las detection using total nucleic acids and the established Li 16S rRNA/rDNA primer–probe set. This method simultaneously amplifies both Las 16S rRNA and rDNA in a single reaction, increasing sensitivity by up to 500 fold compared with qPCR, which targets only 16S rDNA. The improved sensitivity reduced false negatives, expanded sampling capacity, and enabled detection of low titer infections previously undetectable. In addition, the higher abundance of 16S rRNA generated a consistent Ct gap between RT qPCR and qPCR, which we used to estimate relative Las cell activity in vivo in host insects and plants and to identify the most effective inoculum sources. This comparative detection strategy was further applied to evaluate inoculum potential, monitor changes in relative cell activity during in vitro cultures, and assess antimicrobial treatments targeting Las. The persistent detection of long lasting, low titer Las infections underscores the complex biology of the HLB causal agent and highlights ongoing challenges for effective disease management.

Abstract Mauritius is one of the two countries where two huanglongbing (HLB)-associated bacteria, ‘ Candidatus Liberibacter asiaticus’ (CLas) and ‘ Ca . Liberibacter africanus’ (CLaf), have coexisted since at least the 1990s, offering insights into their interactions, particularly given their divergent thermal tolerances. The objective of the study was to revisit the unique HLB epidemiological situation in Mauritius using modern diagnostic and genotyping techniques in the broader context of global warming. Using multiple-locus variable number tandem repeat analysis (MLVA), multilocus sequence analysis (MLSA), a real-time PCR assay, and a loop-mediated isothermal amplification (LAMP) assay, we detected both species. While CLas was commonly detected from sea level up to 580 m above sea level (masl), CLaf was commonly detected between 300 and 600 masl, where temperatures appear higher than in African regions where CLaf has been reported, suggesting putative adaptation to warmer temperatures of Mauritian CLaf. LAMP data also identified some limitations in inclusivity in Keremane’s system. Based on tandem-repeat (TR) data, a single population of CLas was identified whose level of diversity did not differ statistically from that of Réunion, a geographically proximate island with a similar epidemiological situation. These two populations were only weakly differentiated, consistent with the detection of very few private alleles in Mauritius. Prophage- and miniature inverted-repeat transposable element (MITE)-based genotyping confirmed the high genetic relatedness between samples from these islands. Altogether, our results suggest a putative identical source for CLas and/or epidemiological links between these islands. We discuss the implications of our results for surveillance and management of HLB.