The functional decline of tomato plants infected by Candidatus Liberbacter solanacearum: an RNA-seq transcriptomic analysis.

Introduction: Candidatus Liberibacter solanacearum (CLso) is a regulated plant pathogen in European and some Asian countries, associated with severe diseases in economically important Apiaceous and Solanaceous crops, including potato, tomato, and carrot. Eleven haplotypes of CLso have been identified based on the difference in rRNA and conserved genes and host and pathogenicity. Although it is pathogenic to a wide range of plants, the mechanisms of plant response and functional decline of host plants are not well defined. This study aims to describe the underlying mechanism of the functional decline of tomato plants infected by CLso by analyzing the transcriptomic response of tomato plants to CLso haplotypes A and B. Methods: Next-generation sequencing (NGS) data were generated from total RNA of tomato plants infected by CLso haplotypes A and B, and uninfected tomato plants, while qPCR analysis was used to validate the in-silico expression analysis. Gene Ontology and KEGG pathways were enriched using differentially expressed genes. Results: Plants infected with CLso haplotype B saw 229 genes upregulated when compared to uninfected plants, while 1,135 were downregulated. Healthy tomato plants and plants infected by haplotype A had similar expression levels, which is consistent with the fact that CLso haplotype A does not show apparent symptoms in tomato plants. Photosynthesis and starch biosynthesis were impaired while starch amylolysis was promoted in plants infected by CLso haplotype B compared with uninfected plants. The changes in pathway gene expression suggest that carbohydrate consumption in infected plants was more extensive than accumulation. In addition, cell-wall-related genes, including steroid biosynthesis pathways, were downregulated in plants infected with CLso haplotype B suggesting a reduction in membrane fluidity, cell signaling, and defense against bacteria. In addition, genes in phenylpropanoid metabolism and DNA replication were generally suppressed by CLso infection, affecting plant growth and defense. Discussion: This study provides insights into plants' defense and functional decline due to pathogenic CLso using whole transcriptome sequencing and qPCR validation. Our results show how tomato plants react in metabolic pathways during the deterioration caused by pathogenic CLso. Understanding the underlying mechanisms can enhance disease control and create opportunities for breeding resistant or tolerant varieties.

Clasnip: a web-based intraspecies classifier and multi-locus sequence typing for pathogenic microorganisms using fragmented sequences.

Bioinformatic approaches for the identification of microorganisms have evolved rapidly, but existing methods are time-consuming, complicated or expensive for massive screening of pathogens and their non-pathogenic relatives. Also, bioinformatic classifiers usually lack automatically generated performance statistics for specific databases. To address this problem, we developed Clasnip (www.clasnip.com), an easy-to-use web-based platform for the classification and similarity evaluation of closely related microorganisms at interspecies and intraspecies levels. Clasnip mainly consists of two modules: database building and sample classification. In database building, labeled nucleotide sequences are mapped to a reference sequence, and then single nucleotide polymorphisms (SNPs) statistics are generated. A probability model of SNPs and classification groups is built using Hidden Markov Models and solved using the maximum likelihood method. Database performance is estimated using three replicates of two-fold cross-validation. Sensitivity (recall), specificity (selectivity), precision, accuracy and other metrics are computed for all samples, training sets, and test sets. In sample classification, Clasnip accepts inputs of genes, short fragments, contigs and even whole genomes. It can report classification probability and a multi-locus sequence typing table for SNPs. The classification performance was tested using short sequences of 16S, 16-23S and 50S rRNA regions for 12 haplotypes of Candidatus Liberibacter solanacearum (CLso), a regulated plant pathogen associated with severe disease in economically important Apiaceous and Solanaceous crops. The program was able to classify CLso samples with even only 1-2 SNPs available, and achieved 97.2%, 98.8% and 100.0% accuracy based on 16S, 16-23S, and 50S rRNA sequences, respectively. In comparison with all existing 12 haplotypes, we proposed that to be classified as a new haplotype, given samples have at least 2 SNPs in the combined region of 16S rRNA (OA2/Lsc2) and 16-23S IGS (Lp Frag 4-1611F/Lp Frag 4-480R) regions, and 2 SNPs in the 50S rplJ/rplL (CL514F/CL514R) regions. Besides, we have included the databases for differentiating Dickeya spp., Pectobacterium spp. and Clavibacter spp. In addition to bacteria, we also tested Clasnip performance on potato virus Y (PVY). 251 PVY genomes were 100% correctly classified into seven groups (PVYC, PVYN, PVYO, PVYNTN, PVYN:O, Poha, and Chile3). In conclusion, Clasnip is a statistically sound and user-friendly bioinformatic application for microorganism classification at the intraspecies level. Clasnip service is freely available at www.clasnip.com.

Comparative genomics analysis and virulence-related factors in novel Aliarcobacter faecis and Aliarcobacter lanthieri species identified as potential opportunistic pathogens.

BACKGROUND: Emerging pathogenic bacteria are an increasing threat to public health. Two recently described species of the genus Aliarcobacter, A. faecis and A. lanthieri, isolated from human or livestock feces, are closely related to Aliarcobacter zoonotic pathogens (A. cryaerophilus, A. skirrowii, and A. butzleri). In this study, comparative genomics analysis was carried out to examine the virulence-related, including virulence, antibiotic, and toxin (VAT) factors in the reference strains of A. faecis and A. lanthieri that may enable them to become potentially opportunistic zoonotic pathogens. RESULTS: Our results showed that the genomes of the reference strains of both species have flagella genes (flaA, flaB, flgG, flhA, flhB, fliI, fliP, motA and cheY1) as motility and export apparatus, as well as genes encoding the Twin-arginine translocation (Tat) (tatA, tatB and tatC), type II (pulE and pulF) and III (fliF, fliN and ylqH) secretory pathways, allowing them to secrete proteins into the periplasm and host cells. Invasion and immune evasion genes (ciaB, iamA, mviN, pldA, irgA and fur2) are found in both species, while adherence genes (cadF and cj1349) are only found in A. lanthieri. Acid (clpB), heat (clpA and clpB), osmotic (mviN), and low-iron (irgA and fur2) stress resistance genes were observed in both species, although urease genes were not found in them. In addition, arcB, gyrA and gyrB were found in both species, mutations of which may mediate the resistance to quaternary ammonium compounds (QACs). Furthermore, 11 VAT genes including six virulence (cadF, ciaB, irgA, mviN, pldA, and tlyA), two antibiotic resistance [tet(O) and tet(W)] and three cytolethal distending toxin (cdtA, cdtB, and cdtC) genes were validated with the PCR assays. A. lanthieri tested positive for all 11 VAT genes. By contrast, A. faecis showed positive for ten genes except for cdtB because no PCR assay for this gene was available for this species. CONCLUSIONS: The identification of the virulence, antibiotic-resistance, and toxin genes in the genomes of A. faecis and A. lanthieri reference strains through comparative genomics analysis and PCR assays highlighted the potential zoonotic pathogenicity of these two species. However, it is necessary to extend this study to include more clinical and environmental strains to explore inter-species and strain-level genetic variations in virulence-related genes and assess their potential to be opportunistic pathogens for animals and humans.

First Report of Dickeya fangzhongdai causing soft rot in Orchids in Canada.

Dickeya fangzhongdai was originally described as the causal agent of bleeding canker of pear tree in China. Recently, D. fangzhongdai was isolated and identified as the causal agent of soft rot in an orchid plant purchased in a local supermarket in Prince Edward Island, Canada. A water-soaked dark green spot on the leaf surface was observed and later became larger soft rot symptom. The origin of the orchid plants was traced back to a producer in Ontario, Canada who propagated them from with cuttings originally imported from the Netherlands and Taiwan. Bacterial isolations were made from a soft rot lesion on an orchid leaf by surface sterilization of small pieces of marginal tissue of the diseased leaf in 70% alcohol. The small pieces of leaf tissue were then washed three time using sterile water, and immersed in drops of sterile water. Bacterial streaming was observed under the microscope and non-fluorescing bacterial colonies were isolated on King's B and casamino acid-peptone-glucose agar plates and purified as isolates 908, 909, 910 and 911. The DNA samples were extracted from the four isolates, as well as the diseased leaf tissue, and tested by using a qPCR assay with the specific primer/probe set (DfF/DfR/DfP) for D. fangzhongdai (Tian et al. 2020). The assay yielded PCR amplicons of 135 bp with a melting temperature of 86.5±0.6 °C as did two control reactions using genomic DNA from D. fangzhongdai strains JS5T and QZH3 originally isolated in China, providing presumptive identification of the orchid isolates as D. fangzhongdai. To fulfill Koch's postulates, freshly purchased healthy orchid plants (n=4) were inoculated by leaf injection with the bacterial isolates obtained in this study and strains JS5 T and QZH3 at ~107 CFU/ml. Three leaves of the same side of the plants were inoculated with the same strains as triplicates. Sterile water was used as the negative control. Inoculated plants were incubated in a growth chamber with a 16 h photoperiod at 23 °C. Water soaked lesions developed in 3-5 days after inoculation followed by soft rotting in leaves inoculated with the new bacterial strains from orchid plants while strain QZH3 caused soft rot in 10 days after inoculation (Fig. S1). The non-fluorescing bacteria on King's B plates with colony morphology similar to those inoculated were re-isolated from the inoculated leaves and confirmed to be D. fangzhongdai by qPCR. Phylogenetic analysis of the assembled 16S rRNA sequence of isolate 908 (GenBank accession number: MT984340), together with GenBank data of all Dickeya spp. and some Pectobacterium spp, using neighbor-joining (NJ) method inferred with MEGA X software (Kumar et al. 2018) showed that isolate 908 clustered with strains JS5T and QZH3 at a phylogenetic distance of 0.0007. This clearly indicated that isolate 908 and JS5T and QZH3 belong to the same genus. Species-level identification of isolate 908 was achieved by genome sequencing and analysis based on average nucleotide identity (ANI). Genomic DNA of isolate 908 was sequenced with Illumina MiSeq to provide approximately 180X genome coverage. After quality checking using FastQC (Andrews 2010), de-novo assembly was performed with VelvetOptimiser v2.2.6 (Zerbino and Birney 2008). The draft genome size of strain 908 was 4,938,027 bp consisting of 76 contigs with 56.8% G+C content and 63,801 bp as N50. The draft genome was checked for misassembled fragments using QUAST v5.0.2 (Gurevich et al. 2013) and found to be of good quality. The draft genome sequence is deposited in GenBank under the accession number of JADCNJ000000000. The draft genome sequence of strain 908 was compared to that of D. fangzhongdai JS5T type strain genome using FastANI v1.2 (Jain et al. 2018) resulting in an ANI value of 98.9%, which is above the 95% cut-off for the same species. Previously, it was reported that D. fangzhongdai caused soft rot in orchid in Europe (Alic et al. 2018) and in onions in New York (Ma et al. 2020). The difference in virulence among D. fangzhongdai strains warrants further investigation and their pathogenicity on potato is being investigated to evaluate any threat to the potato industry. To our knowledge, this is the first report of D. fangzhongdai causing soft rot disease on orchids in Canada and North America.

Description of Clavibacter zhangzhiyongii sp. nov., a phytopathogenic actinobacterium isolated from barley seeds, causing leaf brown spot and decline.

Clavibacter michiganensis is a Gram-stain-positive bacterium with eight subspecies, five of which have been redefined as different species on the basis of their genome sequence data. On the basis of the results of phylogenetic analysis of dnaA gene sequences, strains of members of the genus Clavibacter isolated from barley have been grouped in a separate clade from other species and subspecies of the genus Clavibacter. In this study, the biochemical, physiological, fatty acids and genetic characteristics of strains DM1T and DM3, which represented the barley isolates, were examined. On the basis of results from multi-locus sequence typing and other biochemical and physiological features, including colony colour, carbon source utilisation and enzyme activities, DM1T and DM3 are categorically differentiated from the aforementioned eight species and subspecies of the genus Clavibacter. Moreover, the results of genomic analysis reveal that the DNA G+C contents of DM1T and DM3 are 73.7 and 73.5 %, respectively, and the average nucleotide identity (ANI) values between DM1T and DM3 and other species and subspecies range from 90.4 to 92.0 %. The ANI value between DM1T and DM3 is 98.0 %. These results indicate that DM1T and DM3 are distinct from other known species and subspecies of the genus Clavibacter. Therefore, we propose a novel species, C. zhangzhiyongii, with DM1T (=CFCC 16553 T=LMG 31970T) as the type strain.

Interaction of environmental eukaryotic microorganisms and fungi in the pond-cultured carps: new insights into the potential pathogenic fungi in the freshwater aquaculture.

The quality and safety of the aquatic products have gradually become the focus of global attention. In this study, the environmental eukaryotic and fungi communities in pond-cultured grass carp (Ctenopharyngodon idellus) and the koi carp (Cyprinus carpio L.) were investigated. For comparative analysis, the alpha diversity shows that the environmental microbial abundance in the koi carp groups were higher than that in the grass carp groups, while beta diversity reveals that the differences of the microbial community composition and structures in the grass carp groups were significantly higher than those in the koi carp groups. Meanwhile, the environmental microbial diversity of grass carp groups was higher than that of koi carp groups at phylum level, but showed no significant difference at genus level. Additionally, the dominant total phyla were Opisthokonta, Stramenopiles plusAlveolates plusRhizaria, Archaeplastida, Cryptophyceae, and Centrohelida for the 18S rRNA gene and Ciliophora, Chlorophyta, and Ascomycota for the ITS2 rRNA gene in both of the two carp groups. Additionally, annotation analysis showed that the biomarkers in the grass carp groups are significantly higher than those of the koi carp groups. Furthermore, the functional prediction of Funguild showed significant difference in outputs, while similarity in trophic modes and guild types between the two carp groups. Meanwhile, the total relative abundances of animal pathogen, fungal parasite, and plant pathogen were extremely similar between the two carp groups. Surprisingly, one pathogenic fungus of genus Fusarium was identified in both the environments of two carp groups based on filtered operational taxonomic unit tables. Overall, this is the first robust report to understand the characteristics of environmental eukaryotic microorganisms and fungi in the edible and ornamental carps. Our results also provide the basic data for the prevention of fungal diseases and the healthy culture of the carps.

Atria: an ultra-fast and accurate trimmer for adapter and quality trimming.

With advances in next-generation sequencing, adapters attached to reads and low-quality bases directly and implicitly hinder downstream analysis. For example, they can produce false-positive single nucleotide polymorphisms (SNP), and generate fragmented assemblies. There is a need for a fast trimming algorithm to remove adapters precisely, especially in read tails with relatively low quality. Here, we present Atria, a trimming program that matches the adapters in paired reads and finds possible overlapped regions using a fast and carefully designed byte-based matching algorithm (O (n) time with O (1) space). Atria also implements multi-threading in both sequence processing and file compression and supports single-end reads. Compared with other trimmers, Atria performs favorably in various trimming and runtime benchmarks of both simulated and real data. We also provide a fast and lightweight byte-based matching algorithm, which can be used in various short-sequence matching applications, such as primer search and seed scanning before alignment.