Waltheria indica is a New Host of Phytoplasma Belongs to 16SrI-B Subgroup Associated with Virescence Symptoms in China

Waltheria indica L. is a kind of medicinal plants belonging to the family of Sterculiaceae distributed in China, which extracts with many active compounds used for treatment of rheumatism and sore pains (Hua et al., 2019). During September to November 2020, the plants showing abnormal symptoms including floral virescence, leaf chlorosis and leaflet, as shown in Fig.1, were found in Dingan county of Hainan province, China, with about 70% incidence. The disease symptoms which were suspected to be infected by the phytoplasma, a phloem-limited cell-wall-less prokaryotic pathogen could not be cultured in vitro, severely impacted Waltheria indica growth resulting in financial loss and ecological damage in the location. For identification of the causal pathogen, the total DNA of symptom or symptomless Waltheria indica samples were extracted using 0.10 g fresh plant tissues using CTAB method. PCR reactions were performed using primers R16mF2/R16mR1 (Lee et al., 1993) and AYgroelF/AYgroelR (Mitrović et al., 2011) specific for phytoplasma 16S rRNA and groEL gene fragments. The target productions of the two gene fragments of phytoplasma were detected in the DNA from four symptomatic plant samples whereas not in the DNA from the symptomless plant samples. The PCR productions were sequenced and the data were deposited in GenBank. The two gene fragments of the DNA extracted from the symptom plant samples were all identical, with the length of 1340 bp 16S rRNA (GenBank accession: MW353909) and 1312 bp groEL (MW353709) gene sequence fragments, putatively encoding 437 (groEL) amino acids sequence. The phytoplasma strain was named as Waltheria indica virescence (WiV) phytoplasma, WiV-hnda strain. A Blast search based on the 16S rRNA gene fragment of WiV-hnda phytoplasma strain revealed the highest level of sequence identities (99.85%) with that of 16SrI aster yellows group members (16SrI-B subgroup), such as Onion yellows phytoplasma strain OY-M (AP006628) from Japan (Oshima et al., 2004); Periwinkle virescence phytoplasma strain PeV-hnhk (KP662136), Chinaberry witches’-broom phytoplasma strain CWB-hnsy1 (KP662119) and CWB-hnsy2 (KP662120), all the strains from Hainan island of China (Yu et al., 2017). A Blast search based on the groEL gene sequence fragment of WiV-hnda indicated 99.92% sequence identity with that of 16SrI aster yellows group members (16SrI-B subgroup) such as Onion yellows phytoplasma strain OY-M (AP006628). Homology and phylogenetic analysis by DNAMAN 5.0 and MEGA 7.0 software indicated that the phytoplasma strains of WiV-hnda, OY-M, PeV-hnhk, CWB-hnsy1 and CWB-hnsy2 were clustered into one clade based on the 16S rRNA gene fragments. WiV-hnda, OY-M and Aster yellow witches’-broom (AYWB) (CP000061) phytoplasma strains were clustered into one clade based on the groEL gene fragments. To our knowledge, this was the first time that Waltheria indica virescence disease induced by 16SrI-B subgroup phytoplasma strain was reported in China. Genetic analysis showed that WiV-hnda was closely related to the phytoplasma strains causing Onion yellows in Japan, Periwinkle virescence and Chinaberry witches’-broom disease in China.

Correction to: Detection of Candidatus Neoehrlichia mikurensis in Norway up to the northern limit of Ixodes ricinus distribution using a novel real time PCR test targeting the groEL gene

After publication of our article [1] it came to our notice that the source of the sequence for the control plasmid, pNeo (Materials and methods: Controls) was incorrectly stated as AB094461. The correct accession number is AB074461. The authors apologize for any confusion this may have caused.

Metagenomic Insights into the Effects of Fructooligosaccharides (FOS) on the Composition of Luminal and Mucosal Microbiota in C57BL/6J Mice, Especially the Bifidobacterium Composition

Fructooligosaccharides (FOS) are considered prebiotics and have been proven to selectively promote the growth of Bifidobacterium in the gut. This study aimed to clarify the effects of FOS intake on the composition of luminal and mucosal microbiota in mice. Briefly, mice were fed a 0% or 25% FOS (w/w)-supplemented diet for four weeks, and the composition of luminal and mucosal microbiota, especially the Bifidobacterium, was analyzed by sequencing the V3–V4 region of 16S rRNA and groEL gene, respectively. After FOS intervention, there were significant increases in the total and wall weights of the cecum and the amount of total short-chain fatty acids (SCFAs) in the cecal contents of the mice. At the phylum level, the results showed a significant increase in the relative abundance of Actinobacteria in the contents and mucosa from the cecum to the distal colon in the FOS group. Besides Bifidobacterium, a significant increase was observed in the relative abundance of Coprococcus in all samples at the genus level, which may be partially related to the increase in butyric acid levels in the luminal contents. Furthermore, groEL sequencing revealed that Bifidobacterium pseudolongum was almost the sole bifidobacterial species in the luminal contents (>98%) and mucosa (>89%). These results indicated that FOS can selectively promote B. pseudolongum proliferation in the intestine, either in the lumen or the mucosa from the cecum to the distal colon. Further studies are required to reveal the competitive advantage of B. pseudolongum over other FOS-metabolizing bacteria and the response mechanisms of B. pseudolongum to FOS.

groEL Gene-Based Phylogenetic Analysis of Lactobacillus Species by High-Throughput Sequencing

Lactobacillus is a fairly diverse genus of bacteria with more than 260 species and subspecies. Many profiling methods have been developed to carry out phylogenetic analysis of this complex and diverse genus, but limitations remain since there is still a lack of comprehensive and accurate analytical method to profile this genus at species level. To overcome these limitations, a Lactobacillus-specific primer set was developed targeting a hypervariable region in the groEL gene—a single-copy gene that has undergone rapid mutation and evolution. The results showed that this methodology could accurately perform taxonomic identification of Lactobacillus down to the species level. Its detection limit was as low as 104 colony-forming units (cfu)/mL for Lactobacillus species. The assessment of detection specificity using the Lactobacillus groEL profiling method found that Lactobacillus, Pediococcus, Weissella, and Leuconostoc genus could be distinguished, but non-Lactobacillus Genus Complex could not be detected. The groEL gene sequencing and Miseq high-throughput approach were adopted to estimate the richness and diversity of Lactobacillus species in different ecosystems. The method was tested using kurut (fermented yak milk) samples and fecal samples of human, rat, and mouse. The results indicated that Lactobacillus mucosae was the predominant gut Lactobacillus species among Chinese, and L. johnsonii accounted for the majority of lactobacilli in rat and mouse gut. Meanwhile, L. delbrueckii subsp. bulgaricus had the highest relative abundance of Lactobacillus in kurut. Thus, this groEL gene profiling method is expected to promote the application of Lactobacillus for industrial production and therapeutic purpose.

Diagnosis and molecular characterization of Anaplasma platys in dog patients in Yogyakarta area, Indonesia

Anaplasma platys is a tick-borne, Gram-negative bacterium that causes anaplasmosis, a companion vector-borne disease impacting dogs. Information on this disease remains limited in Indonesia. Its symptoms are not specific, so molecular analysis is required for a rapid and accurate diagnosis. GroEL is an essential gene commonly used for classification and species identification of many groups of bacteria, including Anaplasma spp. In this study, a molecular diagnosis of anaplasmosis based on the groEL gene sequence was conducted using PCR. In addition, the genetic diversity of Anaplasma platys in infected dogs was determined. Blood samples were collected from 51 dogs suspected of anaplasmosis from Prof. Dr. Soeparwi Animal Hospital, animal clinics, and pet shops in the Yogyakarta area, Indonesia, based on anamnesis, histories of tick infestations, and clinical symptom examinations. DNA extraction and PCR targeting the groEL gene were performed, followed by sequencing. Phylogenetic tree analysis and construction were carried out using the BLAST and MEGA programs. Positive PCR sample results (amplicon length of 624 bp) were found in 6 of 51 dogs. Samples A1 (KHJ/C2), A2 (KHJ/A2), A3 (KSK/L), A4 (KHJ/L), and A5 (KNP/M2) had close ties to Anaplasma platys (AF478129.1) from GenBank. Phylogenetic analysis showed a very high homology value (100%) and bootstrap value of 100%. It can be concluded that there was no genetic diversity in the Anaplasma platys found in infected dogs in the Yogyakarta area.