Publications

4481

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.

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.

A novel bacterial strain UR115T belonging to the class Alphaproteobacteria was isolated from tomato rhizosphere soil. Strain UR115T was Gram-stain-negative, motile, facultative anaerobic, catalase positive, oxidase positive and rod shaped. Strain UR115T showed growth at 15–37 °C, pH 5.5–9.5 and up to 4.0% NaCl. Strain UR115T exhibited 16S rRNA gene sequence similarities of <90.40% with members of the class Alphaproteobacteria. Based on the phylogenetic analysis inferred from both 16S rRNA gene and whole-genome sequences, strain UR115T formed a separate and distinct clade, leading to its classification as a member of a novel order within the class Alphaproteobacteria. The genome size and G+C content of strain UR115T were 5,059,753 bp and 63.9%, respectively. Average amino acid identity values between strain UR115ᵀ and reference genomes ranged from 47.85% to 59.96%, and pairwise Percentage of Conserved Protein values ranged from 4.12% to 46.63%. The major cellular fatty acids of strain UR115T were C19 : 0 cyclo ω8c, C16 : 0 and summed feature 8 (C18 : 1  ω7c and/or C18 : 1  ω6c). The polar lipids of strain UR115T were phosphatidylethanolamine, phosphatidylglycerol, unidentified aminophospholipid, two unidentified aminolipids and two unidentified lipids. The predominant quinone was ubiquinone-10. Based on its distinct phylogenetic position, unique genomic characteristics and chemotaxonomic profiles, we proposed the novel species Radicibacter daui gen. nov., sp. nov., the novel family Radicibacteraceae fam. nov. and the novel order Radicibacterales ord. nov. The type strain is strain UR115T (=KACC 23674T=JCM 36738T).

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.

‘Candidatus Phytoplasma solani’ is the causal agent of the Bois noir (BN), affecting grapevine worldwide. The complex epidemiology of BN, which involves multiple ‘Ca. P. solani’ host plants and insect vectors, as well as the occurrence of recovery (loss of symptoms on grapevine canopy), makes disease investigations and containment in vineyards difficult. To achieve early detection of ‘Ca. P. solani’, a droplet digital PCR (ddPCR)-based approach and quantitative (q)PCR assay were compared, testing specific primers based on the elongation factor Tu (tuf) gene using SYBR Green chemistry. The regression curve analysis of the ddPCR assay showed good linearity. Compared with the qPCR method, the sensitivity of ddPCR improved about 10-fold. The analysis of grapevine roots spiked with serial dilutions of ‘Ca P. solani’. PCR tuf fragments showed that qPCR was inhibited, while ddPCR was not affected. Testing 66 grapevine samples from 50 grapevine plants, the ddPCR provided superior diagnostic performance compared to qPCR in roots of symptomatic plants (75% detected by ddPCR, 41.6% by qPCR), roots of recovered plants (58.8% detected by ddPCR, 25% by qPCR), and asymptomatic leaf tissues from recovered plants (75% detected by ddPCR, 25% by qPCR). The ddPCR analysis allowed us to detect ‘Ca. P. solani’ on 40% of leaf samples from recovered plants and 20% of roots from asymptomatic plants. No differences among ddPCR and qPCR were found in detecting phytoplasma on symptomatic leaf samples. The ddPCR assay allowed the absolute quantification of ‘Ca. P. solani’ in complex matrices, such as roots, and when low titer of phytoplasma is present.

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.

This is a general protocol for the vegetative propagation of citrus material infected with the bacterium Candidatus Liberibacter asiaticus (CLas), the presumed causal agent of citrus greening disease (HLB), using an aeroponic cloning apparatus. The following procedure details a workflow using an EZ-Clone Pro Low Cloning System from Hydrobuilder.com. This system comes in varying cell-sizes (number of clones). We have found the most versatile to be the 16- or 32-cell size based on ease of preparation, keeping algae growth to a minimum, and maintenance. Cloning versus seed sowing has been useful in producing infected material for experimental assays requiring cloned, mature citrus trees as opposed to genetically variable plants from seeds. Where seed germination can take up to 12+ months to achieve plants with stem diameters ≥ 10 mm and 5+ years for flowering, infected branches from flowering trees can be directly propagated using this protocol within a few months and retain their maturity. Clone viability rate differs by citrus cultivar and general health of the cutting used, with citron and CLas negative material having the highest success rate. Infected citron cuttings have had a 95% survival rate, regardless of the diameter of the cutting. The cutting diameter ranged from 2mm-12mm and was limited to 12mm maximum, which is the size of the collar on the EZ-Clone Pro Low cloner.