Publications

4514

Desmodium styracifolium (Osb.) Merr., a member of the Leguminosae family, is an important medicinal plant widely used in traditional Chinese medicine. In September 2024, D. styracifolium plants exhibiting symptoms of little leaf and stunted growth were observed in a field of Zhanjiang, Guangdong province, China. Since the symptoms resembled those associated with phytoplasma infections, total DNA was extracted from the leaves of four symptomatic plants and one healthy plant for molecular identification. Universal primer pairs (P1/P7, R16mF2/mR1) for phytoplasma detection were used to amplify the 16S rDNA fragments (~1.8 kb and ~1.4 kb), while a specific primer pair secY-F/secY-R was employed to amplify a ~1.4 kb segment of the secY gene. Target fragments were successfully amplified from all symptomatic samples but not from the healthy control. These amplicons were cloned and sequenced. The obtained 16S rDNA sequence of D. styracifolium little leaf phytoplasma (DsLFP-GDZJ) showed the highest identity (99.67–100%) with strains of ‘Ca. Phytoplasma australasiaticum’ (subgroup 16SrII-A and 16SrII-D). Phylogenetic analysis also indicated that DsLFP-GDZJ formed a small evolutionary branch with strains of ‘Ca. Phytoplasma australasiaticum’ (subgroup 16SrII-A and 16SrII-D). Virtual RFLP (restriction fragment length polymorphism) analysis of the 16S rDNA sequence demonstrated DsLFP-GDZJ belongs to the 16SrII-A subgroup (GenBank accession number L33765). The secY gene sequence of DsLFP-GDZJ also showed the highest similarity and the closest relationship with those of the 16SrII-A subgroup phytoplasma strains. These results showed that DsLFP-GDZJ is a strain of ‘Ca. Phytoplasma australasiaticum’ (16SrII-A subgroup). To our knowledge, this is the first report of ‘Ca. Phytoplasma australasiaticum’—related phytoplasma associated with D. styracifolium little leaf disease in China, thereby establishing D. styracifolium (Osb.) Merr. as a new host plant of phytoplasma.

The Asian citrus psyllid, Diaphorina citri, transmits the putative causal agent of citrus greening disease, ‘Candidatus Liberibacter asiaticus’. The transmission occurs in a propagative circulative manner. Specific protein-protein interactions are required for the accomplishment of the transmission process. We employed the far-Western technique, also known as protein overlay assay, and mass spectrometry, to identify D. citri proteins that specifically recognize membrane proteins of ‘Ca. L. asiaticus’. Whole-body protein extracts of D. citri were separated by electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were overlayed with sonicated leaf midrib extracts from ‘Ca. L. asiaticus’-infected citrus plants. The protein complexes were detected using different antibodies against ‘Ca. L. asiaticus’ membrane proteins and peptidoglycans associated with lipoproteins. By comparison, spots were extracted from a polyacrylamide gel for LC-MS-MS analysis. Identified proteins included ATP synthase alpha and beta subunits, actin, alpha- and beta-tubulins, transitional endoplasmic reticulum ATPase TER94, 78 kDa glucose-regulated protein, and arginine kinase. These findings emphasized the involvement of energy machinery and muscle proteins in the ‘Ca. L. asiaticus’-D. citri interactions. Understanding the pathogen-vector interactions will lead to better-designed control strategies based on the interference with specific interactions and/or blocking transmission.

ABSTRACT Insect‐vectored pathogens pose a significant threat to global agriculture. The colonization efficiency of pathogens in vectors plays a central role in these pathosystems, yet studies of the factors that affect this aspect are limited. This study investigates the genetic and microbial symbiont factors influencing the susceptibility of Diaphorina citri to Candidatus Liberibacter asiaticus ( C Las), the pathogen causing citrus greening disease (huanglongbing). Through a microbiome Genome Wide Association Study (mGWAS) based on 16S amplicon sequencing and genomic resequencing of 120 D. citri individuals from six populations, we identified 79 SNPs significantly associated with the relative abundance of C Las within insects. Additionally, some of these SNPs were also associated with the relative abundance of Candidatus Profftella armature, a key endosymbiont of D. citri . SNPs in the regulatory region of gene Dcitr04g11610.1 led to its overexpression in C Las‐susceptible D. citri , and C Las infection further elevated its expression. Conversely, RNAi knockdown of Dcitr04g11610.1 reduced C Las infection rates and abundance, accompanied by increased abundance of Profftella . Phylogenetic analysis revealed Dcitr04g11610.1 ’s high homology to Major Facilitator Superfamily‐type transporter SLC18B1 proteins, suggesting a role in C Las polyamine utilization. These findings highlight the importance and potential interplay of insect genetics and symbiotic microbiota in insect‐vectored plant pathogen systems.

Abstract The African citrus triozid (ACT), Trioza erytreae (Del Guercio, 1918) (Hemiptera: Triozidae), is a significant pest of citrus in southern Africa serving as a vector of African citrus greening disease associated with the phloem-limited gram-negative bacterium “Candidatus Liberibacter africanus” (CLaf). ACT can also vector another Liberibacter species, “Ca. Liberibacter asiaticus” (CLas) associated with Asian citrus greening. Given the confirmed presence of CLas on the African continent, efforts to monitor populations of ACT as well as associated liberibacters in citrus environments have been enhanced since 2021. Trapping and active collections in citrus surroundings indicated the presence of two triozid species with similar morphology to ACT and five other triozids which were morphologically distinct. This study aimed to characterise the diversity of triozid species around citrus and determine their association with liberibacter. Morphological characters and nucleotide sequence phylogeny of the mitochondrial cytochrome c oxidase I (COI) gene region supported the distinction of seven triozid species including ACT. CLaf was only detected in ACT. None of the other triozid species had any liberibacter species associated with them. Our results suggest the presence of other triozid species in other African countries, which probably were misidentified as ACT. Our findings highlight the need for a comprehensive taxonomic revision of triozids associated with citrus. Integrating morphological and molecular characters in a revised classification will be essential for accurate identification of ACT. Molecular confirmation is needed for confidence in distinguishing ACT from closely related non-pest species.

Abstract Segmented filamentous bacteria (SFB) describe morphologically similar gut commensals found in mammals, fish and birds. In mice, SFB intimately colonizes the ileal epithelium at the time of weaning and elicits a strong pleiotropic immune activation that fosters colonization resistance while augmenting disease severity in various disease models. SFB is therefore critical in both health and disease but information regarding SFB in humans remains limited. Here, we first identify and characterize a human SFB species with SFB-specific morphology, including the hook-like tip structure that mediates attachment, and unique genome features, including a starch and glycogen degradation module. This species, which we name Anisomitus miae and establish as the nomenclature type for the SFB genus, is common across Africa. We then bioinformatically identify, based on the 16S rRNA gene V3-V4 variable region sequence, four major, and two minor, human SFB lineages in forty-four countries distributed across all six inhabited continents. We provide evidence towards the co-colonization potential of the SFB lineages and their colonization dynamics, including a potent but short-lived colonization peak in children between one to five years of age. This study establishes the presence of multiple SFB species in the human population and SFB as a minor but wide-spread group of commensals in humans.

Welsh onion (Allium fistulosum) is an important aromatic vegetable crop, particularly in Asia (Kim et al. 2023). In November 2025, two-month-old Welsh onion plants showing leaf proliferation, little leaf, and yellowing symptoms were found at low frequency (< 1%) in Fangyuan Township, Changhua County, Taiwan. The plot was around 880 m 2 in size and about 7,200 clumps were grown. Five symptomatic and three symptomless plants were sampled (bagged separately). The plants’ basal leaf tissues were subjected to DNA extraction and PCR analysis. DNA was extracted using a Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics) using filter tips, and the DNA samples’ quality was verified by successful amplification of a plant 26S rDNA fragment (0.7 kb) using primer pair 28KJ/28C (Cullings 1992). Nested polymerase chain reaction (PCR) assays using phytoplasma 16S rDNA-specific primers were conducted. The nested primer pairs used were P1/P7 and fU5/rU3 (Lorenz et al. 1995), respectively. DNA of periwinkle infected with periwinkle leaf yellowing phytoplasma served as a positive control and no-template controls (nuclease-free water) were included in both PCR rounds. In both tests, only DNA from the five symptomatic plants produced the expected amplicons (1.8 kb and 881 bp for the outer and inner assays). The products from the first and second rounds of PCR were sequenced and assembled. Sequencing chromatograms of the PCR products had single, well-resolved peaks across all positions in all the samples, indicating that only one strain was present. Near-full-length (1,466 bp) 16S rDNA sequences of all five samples were identical to that of a reference ‘Candidatus Phytoplasma australasiaticum’-related strain, NCHU2014 (accession no. CP040925, bp 537,765 to 539,230; 16SrII-A; Rodrigues Jardim et al. 2023), and a representative sequence has been deposited in GenBank (accession no. PX696818). A 1,248 bp fragment (accession no. PX696818, bp 84 to 1,331) of the detected 16S rDNA sequence was excised for analysis using the web tool iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi), and the virtual RFLP patterns obtained were identical to the reference pattern of 16SrII-A (accession no. L33765; similarity coefficient 1.00). Additional PCR tests targeting the protein translocase gene secY and the elongation factor Tu gene (tuf) of 16SrII phytoplasmas were respectively conducted with the infected samples using primer pairs SecYF1(II)/SecYR1(II) and TUF-II-F1/TUF-II-R1 (Al-Subhi et al. 2017; Lee et al. 2010). The secY (accession no. PX737873; 1,263 bp) and tuf (accession no. PX737874; 1,183 bp) sequences were again found identical to those of the reference strain NCHU2014 (16SrII-A phytoplasma; accession no. CP040925, bp 192,846 to 194,108 and bp 139,150 to 140,332). In Taiwan, 16SrII phytoplasma has been reported affecting various plants, such as seseme and purple coneflower (Rodrigues Jardim et al. 2023; Tseng et al. 2014). In A. fistulosum, however, phytoplasma infection has only been associated with group 16SrI (Cho et al. 2019). The present study is the first report of a 16SrII-A phytoplasma affecting Welsh onion and the findings expand the understanding of the width of the subgroup’s host range in the island. Since Welsh onion is an important culinary crop, and the disease significantly affects its morphology and commercial value, ongoing surveillance of its spread in other regions is warranted.

ABSTRACT Crape jasmine is a beautiful evergreen flowering shrub and is native to India. During a survey in 2025, severe yellowing symptoms with stunted growth of the plant was observed on the crap jasmine plant at Bhopal, Central India. For the identification of ‘ Candidatus phytoplasma species’ on crap jasmine, symptomatic and asymptomatic leaf samples were collected and total DNA was extracted. The phytoplasma 16S rRNA gene was successfully amplified by direct and nested PCR using isolated DNA and phytoplasma‐specific primers from three symptomatic plants. The amplified ~1.2 kb nested PCR products from three samples were sequenced, and the resulting 16S rRNA gene nucleotide sequences were submitted to GenBank under the accession numbers PX442245, PX890108 and PX890109. Sequence analysis of all three isolates (PX442245, PX890108 and PX890109) revealed the highest nucleotide sequence identity (99%) and close phylogenetic relationships with reference strains of brinjal little leaf phytoplasma, ‘ Candidatus Phytoplasma trifolii’ (16SrVI group), predominantly belonging to the 16SrVI‐D subgroup. In silico RFLP analysis of the 16S rRNA gene sequences of the phytoplasma isolates under study showed the highest similarity to the reference strain of the 16SrVI‐D subgroup, with similarity coefficients ranging from 0.93 to 0.86. To the best of our knowledge, this is the first report of a ‘ Candidatus Phytoplasma trifolii’‐related strain (16SrVI‐D subgroup) associated with yellows disease of crape jasmine ( Tabernaemontana divaricata ) in India.