Publications

4116

Abstract Psyllids are major vectors of plant diseases, including Candidatus Liberibacter solanacearum (CLso), the bacterial agent associated with “zebra chip” disease in potatoes and “carrot yellows” disease in carrot. Despite their agricultural significance, there is limited knowledge on the genome structure and genetic diversity of psyllids. In this study, we provide chromosome-level genome assemblies for three psyllid species known to transmit CLso: Dyspersa apicalis (carrot psyllid), Dyspersa pallida, and Trioza urticae (nettle psyllid). As D. apicalis is recognized as the primary vector of CLso by carrot growers in Northern Europe, we also resequenced populations of this species from Finland, Norway, and Austria. Genome assemblies were constructed using PacBio HiFi and Hi–C sequencing data, yielding genome sizes of 594.01 Mbp for D. apicalis; 587.80 Mbp for D. pallida; and 655.58 Mbp for T. urticae. Over 90% of sequences anchored into 13 pseudo-chromosomes per species. D. apicalis and D. pallida assemblies exhibited high completeness, capturing over 92% of conserved Hemiptera single-copy orthologs. Furthermore, we identified sequences of the primary psyllid symbiont, Candidatus Carsonella ruddii, in all three species. Gene annotations were produced for each assembly: 17,932 unique protein-coding genes were predicted for D. apicalis; 18,292 for D. pallida; and 16,007 for T. urticae. We observed significant expansions in gene families, particularly those linked to potential insecticide detoxification, within the Dyspersa lineage. Resequencing also revealed the existence of multiple subpopulations of D. apicalis across Europe. These high-quality genome resources will support future research on genome evolution, insect–plant–pest interactions, and disease management strategies.

The ongoing spread of X-disease, which is associated with ‘ Candidatus Phytoplasma pruni’, has resulted in severe economic losses for cherry and stone fruit growers in the U.S. Pacific Northwest in the last decade. Given that this pathogen is transmitted by polyphagous leafhopper species, primarily Colladonus montanus ssp. reductus and C. geminatus in the Pacific Northwest, alternative plant hosts present a significant management concern. Here, we surveyed phytoplasma incidence in non- Prunus plants found in and around stone fruit orchard borders and compared these results to the leafhopper feeding patterns through gut content analysis. We confirmed ‘ Ca. P. pruni’ infection in 21 plant species from 15 families. Of these, 15 species were commonly found in the diets of phytoplasma-carrying leafhoppers. Due to the abundance of common plant species, including Taraxacum, Malva, and Trifolium spp., alternative hosts were found across all growing seasons, although ‘ Ca. P. pruni’ titer was low (<102 cells) in most of these hosts. No geographic patterns were identified; instead, phytoplasma incidence and spread at individual orchards was related to the management of annual and biennial host species, thus removing preferred leafhopper feeding hosts. These data suggest that alternative host removal, along with the removal of infected trees, is an effective means of slowing disease spread.

Pulasan (Nephelium mutabile Blume; family Sapindaceae) is a tropical fruit known for its high orilagin, ascorbic acid and thiamine content, known for its potent antioxidant and anti-inflammatory properties. Native to Southeast Asia, pulasan is extensively cultivated in Malaysia, Thailand, Indonesia, Sri Lanka, India and Philippines (Afzaal et al., 2023). In India, pulasan is cultivated in tropics, where it is cherished as a homestead crop. A recent survey conducted in September 2024 at Pangode village (8°45'23.22"N 76°58'48.68"E), Thiruvananthapuram, Kerala, showed little leaf symptoms along with drying of side branches along with stunted growth in pulasan trees (variety P15). Only two pulasan trees were recorded with symptoms out of 20 trees examined in the campus. To investigate the molecular identity of the phytoplasma, leaf samples from two symptomatic and asymptomatic trees were collected and subjected to total DNA extraction by DNA extraction kit (Qiazen). The extracted DNA was amplified using nested PCR assays with universal primers P1/Tint (Smart et al., 1996) followed by 3Fwd/3Rev (Manimekalai et al., 2010) specific for the 16S rRNA gene and tuf gene-specific primer pairs fTufl/rTufl (Schneider et al., 1997). The PCR assays yielded amplicons of ~1.3 kb and ~1.1 kb for the 16S rRNA and tuf genes, respectively, from all the symptomatic leaf samples, whereas no amplification was observed from any asymptomatic samples. One sequence of each gene of pulasan phytoplasma strain (PK-1) were deposited into the GenBank database [(Acc Nos. PQ865399 (16S rRNA gene) and PQ877893 (tuf gene)]. BLASTn analysis of the both gene sequences of the PK strains revealed a 99.62% sequence identity with eggplant little leaf (Acc. No. MW273757; 16S rRNA gene) and 100% sequence similarity with Cucurbita pepo phytoplasma (Acc. No. KX358589; tuf gene) belongs to the clover proliferation phytoplasma subgroup. Phylogenetic analysis using MEGA 11 employing the Maximum likelihood method, demonstrated that the PK phytoplasma strains clustered within the 16SrVI-D subgroup-related strains. The virtual RFLP pattern generated for the 16S rRNA gene of PK phytoplasma strain through pDRAW analysis was found identical to the reference pattern of 16VI-D subgroup (Periwinkle little leaf phytoplasma; GenBank Acc. No. AF228053). Sequence comparison analysis confirmed the presence of a 'Candidatus Phytoplasma trifolii'-related strain associated with pulasan showing little leaf disease and stunting in India. While, phytoplasma associations have been reported in various fruit species globally (Fiore et al., 2018; Mitra et al., 2023), this study represents the first documented case of a phytoplasma disease infecting pulasan worldwide. Notably, 'Ca. P. trifolii' (16SrVI-D subgroup) has previously been identified associated with several economically important agricultural and horticultural crops worldwide (Zhao and Wei, 2020; Rao, 2021). The results of this study revealed that pulasan trees serve as an alternative natural reservoir for the ‘Candidatus Phytoplasma trifolii’ (16SrVI-D) phytoplasma strain, may potentially facilitate its dissemination to other crops through insect vectors. Considering the significant phytosanitary concerns, epidemiological factors and economic impact of pulasan in Southeast Asia for its global export potential, the detection of phytoplasma-associated disease in pulasan is noteworthy as it not only poses a potential threat to cultivation and productivity but may also have broader implications for regional trade and biosecurity protocols.

ABSTRACTHuanglongbing (HLB) is a bacterial disease that affects citrus trees and is considered the most severe citrus disease in the world. The three HLB-associated ‘Ca.Liberibacter’ species harbor prophage regions which have been described to play critical roles in their evolution and biology. In this study, we assembled and characterized the accessory genome of the first circularde novo‘CandidatusLiberibacter asiaticus’ (CLas) assembly (V1R1) from Réunion, one of the sparse areas worldwide hosting CLas and ‘Ca.Liberibacter africanus’ (CLaf). This 1,272 Mb-long whole-genome harbored 1,129 coding sequences and two complete prophages, including a 37,934 bp-long Type 1 prophage, frequently present in CLas genomes, and a 40,501 bp-long undescribed CLas prophage designated as P-V1R1-5. Comparative genomic approaches suggested that P-V1R1-5 have all the genetic elements to produce new viral particles and revealed that it likely belongs to a new CLas Type 5 prophage. While being reported for the first time in a CLas strain, P-V1R1-5-like prophages were previously identified in CLaf genomes, making it the first evidence of shared prophage-like sequence among HLB-associated ‘Ca, Liberibacter’ species. Using PCR amplifications targeting P-V1R1-5, we also showed that 85.7% of the tested CLas strains from Réunion and all tested CLaf strains from Madagascar and Réunion harbored a Type 5-like prophage. The identification of CLas Type 5-like prophages not only expanded our knowledge of CLas genomic diversity in Réunion, but also provided new insights into the role of horizontally transferred elements in the evolution of the sympatric HLB-associated bacteria.IMPORTANCEHuanglongbing (HLB) is the most severe citrus disease worldwide. The disease is associated with three ‘CandidatusLiberibacter’ species harboring prophage regions that have been described to play critical roles in their biology. In this study, we assembled and characterized the accessory genome of the first circularde novo‘Ca.Liberibacter asiaticus’ (CLas) assembly from Réunion, one of the very few areas in the world where both of CLas and ‘Ca.Liberibacter africanus’ (CLaf) coexist. Comparative genomic approaches demonstrated that this genome harbored two complete prophages, including a new CLas Type 5 prophage that was previously identified in CLaf but was reported for the first time in a CLas strain. This first evidence of shared prophage-like sequences among HLB-associated species expands our knowledge of CLas genomic diversity, but also provides new insights into the role of the accessory genome in the evolution of these bacteria.

AbstractImmunity is generally considered critical for plant health against pathogen infection. Citrus Huanglongbing (HLB) caused by the phloem colonizing bacterial pathogenCandidatusLiberibacter asiaticus (CLas) was suggested to be a pathogen triggered chronic immune disease. However, the genetic evidence and mechanistic understanding for such a disease model is lacking. Here, we show CLas triggers phloem cell death, reactive oxygen species (ROS) production and callose deposition in photosynthesis tissues, but little or none in non-photosynthesis tissues of citrus. We further demonstrate that CLas triggers ROS production in chloroplasts. Overexpression offlavodoxin(Fld), an electron shuttle, in chloroplasts reduced ROS production, cell death and HLB symptoms, but not phloem callose deposition induced by CLas. Knockout, silencing, and overexpression of phloem callose synthase genesCsCalS7a,CsCalS7b, orCsCalS7cdemonstrated that phloem callose deposition also caused phloem cell death. Trunk injection with 2-deoxy-d-glucose, a callose deposition inhibitor, reduced fruit drop and increased fruit yield of HLB symptomatic trees in field trials. Using tomato-Ca. L. psyllaurous (Lpsy) as a model, knockout ofEds1andPad4but notRbohB,Bik1andSobir1abolished ROS production, phloem callose deposition, cell death and disease damages caused by Lpsy. This study provides genetic evidence forCa. Liberibacter-triggered immune disease and reveals that tuning plant immune responses convertsCa. Liberibacter into a benign endophyte, providing a promising strategy for precision breeding to enhance resistance/tolerance against HLB.

ABSTRACTDespite recent genomic studies and increased molecular data, epitheliocystis remains an enigmatic fish disease with no experimental in vitro or in vivo models to aid the advancement of research. In this study, we revert to a classical microscopical approach and screen with the electron microscope the epitheliocystis lesions caused by a Ca. Parilichlamydia sp., infecting mucus cells in Greater amberjack. We report distinct morphological features of this bacterial family, characterised by Intermediate Bodies that closely resemble those of previously described Candidatus similchlamydia, and Elementary Bodies that exhibit morphological similarities to Chlamydia trachomatis. We describe the characteristics of a novel Chlamydial Inclusion Membrane (IM) type, with abundant interdigitations, possibly shaped by fusion of the IM with cytoplasmic vesicles, and moreover discuss the presence of multivesicular bodies in the infected cell. Our observation of immune cells in the infected areas indicates an interaction of macrophages with infected cells, a role for granular cells as pathogens reservoirs and an active phagoptosis process in the nearby areas, overall shedding light on cellular immune processes characterising these infections in fish hosts.

AbstractPurple sulfur bacteria (PSB) of the family Chromatiaceae (Gammaproteobacteria) can perform chemo‐ and photo‐lithoautotrophy (through anoxygenic photosynthesis) in anoxic layers of freshwater stratified (including meromictic) lakes. This group has been extensively studied via physiological and ecological approaches, albeit their genomics has lagged behind. Here, we monitored a small, shallow, karstic lake, Lagunillo de Cardenillas, that developed a pink coloration throughout the whole water column and prevailed for ca. 2 years across seasons of the limnological cycle. Combining the study of physical/chemical parameters, amplicon sequencing, metagenomics, genomics, and microscopy, we observed this phenomenon was caused by blooms of a novel Thiocapsa species, which represented ca. 40% of the total microbial biomass of the lake's water column during the autumn/winter mixing period, and ca. 36% in the anoxic layers during spring/summer stratification. The dominance of this microbe was attributed to the high sulfur concentrations and biogeochemical features of the lake combined with various genomic footprints/abilities of this microbe to utilize different nutrient sources under anoxic and oxic/microaerophilic conditions. The latter included nitrogen (cyanate and ethanolamine hydrolysis, N fixation, dissimilatory nitrate reduction, ammonia assimilation, denitrification), carbon (anoxygenic photosynthesis and the presence of α‐carboxysomes and type IA RuBisCOs) and sulfur (dimethylsulfide [DMS] and thiosulfate oxidation, dimethylsulfoxide [DMSO] reduction). In addition, this novel species possessed genes for gas vesicle formation, anoxic/oxic respiration pathways, hydrogenases, oxic stress response, and a CRISPR‐Cas array. Thus, its extensive genomic repertoire helped explain its versatility and success in colonizing both the anoxic layers and the oxic/anoxic interphase in this lake.

‘Candidatus Phytoplasma mali’ is associated with apple proliferation, a devastating disease in fruit production. Using genome analysis, a gene encoding a hemolysin-like protein was identified. It was postulated that this protein could be an effector. However, the function of this protein is unknown. It is shown that the hemolysin-like protein binds to a GTP binding protein, Toc33, of Arabidopsis thaliana in yeast two-hybrid analysis and that the Toc33-binding domain is located in the C-terminus of the domain of unknown function (DUF21) of the protein. The biochemical studies reveal that the protein can hydrolyze phosphate of purine and pyrimidine nucleotides. Transgenic Nicotiana benthamiana plants expressing the protein show no discernible change in phenotype. Phytoplasma have a much-reduced genome, lacking important genes for catabolic pathways or nucleotide production; therefore, the hemolysin-like protein plays a role in the uptake of plant nucleotides from their host and hydrolyzes these nucleotides for energy and their own biosynthesis.

Huanglongbing (citrus greening disease) is caused by the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) (Alphaproteobacteria) and is one of the most destructive bacterial-vector diseases affecting the citrus industry. The bacterium is transmitted by the Asian citrus psyllid (ACP; Diaphorina citri). Early detection in citrus trees is challenging due to uneven distribution of CLas throughout the tree and a long pre-symptomatic phase of the disease. Due to these limitations, ACP sampling has been suggested as a more reliable early detection strategy. The objective of this study was to develop and optimize approaches for detecting CLas in ACP adults and nymphs collected in citrus groves in California using real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). The goal was to establish the optimal number of ACP adults and nymphal instar life stages (stages 1–2, 3, or 4–5) that yielded the most reliable detection of CLas (Cq values ≤ 38). Results indicated that CLas detection correlated with psyllid developmental stage, with the 4th–5th instar nymphs (sample size of five to ten per tube) or adult ACP (sample size of three to ten per tube) providing the most consistent qPCR detection. While CLas detection rates increased with adult ACP age, nymphs were preferred for field sampling as adult ACP might have dispersed from non-infected trees, potentially misrepresenting the grove’s CLas status. Detection by droplet digital PCR confirmed the presence and genome copies of CLas in a subset of ACP across life stages. In field populations, detection rates in nymphs were consistent or stable throughout the year, whereas CLas detection in adults exhibited seasonal variation, with CLas detection and genome load peaking in January. These targeted ACP sampling strategies and optimized laboratory processing methods will facilitate CLas detection in psyllids for streamlining citrus greening disease management.