Isolation of the Novel Phage PHB09 and Its Potential Use against the Plant Pathogen Pseudomonas syringae pv. actinidiae

Bacteriophages are viruses that specifically infect target bacteria. Recently, bacteriophages have been considered potential biological control agents for bacterial pathogens due to their host specificity. Pseudomonas syringae pv. actinidiae (Psa) is a reemerging pathogen that causes bacterial canker of kiwifruit (Actinidia sp.). The economic impact of this pest and the development of resistance to antibiotics and copper sprays in Psa and other pathovars have led to investigation of alternative management strategies. Phage therapy may be a useful alternative to conventional treatments for controlling Psa infections. Although the efficacy of bacteriophage φ6 was evaluated for the control of Psa, the characteristics of other DNA bacteriophages infecting Psa remain unclear. In this study, the PHB09 lytic bacteriophage specific to Psa was isolated from kiwifruit orchard soil. Extensive host range testing using Psa isolated from kiwifruit orchards and other Pseudomonas strains showed PHB09 has a narrow host range. It remained stable over a wide range of temperatures (4–50 °C) and pH values (pH 3–11) and maintained stability for 50 min under ultraviolet irradiation. Complete genome sequence analysis indicated PHB09 might belong to a new myovirus genus in Caudoviricetes. Its genome contains a total of 94,844 bp and 186 predicted genes associated with phage structure, packaging, host lysis, DNA manipulation, transcription, and additional functions. The isolation and identification of PHB09 enrich the research on Pseudomonas phages and provide a promising biocontrol agent against kiwifruit bacterial canker.

Metagenomic Analysis of Marigold: Mixed Infection Including Two New Viruses

Marigold plants with symptoms of mosaic, crinkle, leaf curl and necrosis were observed and small RNA and ribo-depleted total RNA deep sequencing were conducted to identify the associated viruses. Broad bean wilt virus 2, cucumber mosaic virus, turnip mosaic virus, a new potyvirus tentatively named marigold mosaic virus (MMV) and a new partitivirus named as marigold cryptic virus (MCV) were finally identified. Complete genome sequence analysis showed MMV was 9811 nt in length, encoding a large polyprotein with highest aa sequence identity (57%) with the putative potyvirus polygonatumkingianum virus 1. Phylogenetic analysis with the definite potyviruses based on the polyprotein sequence showed MMV clustered closest to plum pox virus. The complete genome of MCV comprised of dsRNA1 (1583 bp) and dsRNA2 (1459 bp), encoding the RNA-dependent RNA polymerase (RdRp), and coat protein (CP), respectively. MCV RdRp shared the highest (75.7%) aa sequence identity with the unclassified partitivirus ambrosia cryptic virus 2, and 59.0%, 57.1%, 56.1%, 54.5% and 33.7% with the corresponding region of the definite delta-partitiviruses, pepper cryptic virus 2, beet cryptic virus 3, beet cryptic virus 2, pepper cryptic virus 1 and fig cryptic virus, respectively. Phylogenetic analysis based on the RdRp aa sequence showed MCV clustered into the delta-partitivirus group. These findings enriched our knowledge of viruses infecting marigold, but the association of the observed symptom and the identified viruses and the biological characterization of the new viruses should be further investigated.

Complete genome sequence analysis of the peanut pathogen Ralstonia solanacearum strain Rs-P.362200

Background Bacterial wilt caused by Ralstonia solanacearum species complex is an important soil-borne disease worldwide that affects more than 450 plant species, including peanut, leading to great yield and quality losses. However, there are no effective measures to control bacterial wilt. The reason is the lack of research on the pathogenic mechanism of bacterial wilt. Results Here, we report the complete genome of a toxic Ralstonia solanacearum species complex strain, Rs-P.362200, a peanut pathogen, with a total genome size of 5.86 Mb, encoding 5056 genes and the average G + C content of 67%. Among the coding genes, 75 type III effector proteins and 12 pseudogenes were predicted. Phylogenetic analysis of 41 strains including Rs-P.362200 shows that genetic distance mainly depended on geographic origins then phylotypes and host species, which associated with the complexity of the strain. The distribution and numbers of effectors and other virulence factors changed among different strains. Comparative genomic analysis showed that 29 families of 113 genes were unique to this strain compared with the other four pathogenic strains. Through the analysis of specific genes, two homologous genes (gene ID: 2_657 and 3_83), encoding virulence protein (such as RipP1) may be associated with the host range of the Rs-P.362200 strain. It was found that the bacteria contained 30 pathogenicity islands and 6 prophages containing 378 genes, 7 effectors and 363 genes, 8 effectors, respectively, which may be related to the mechanism of horizontal gene transfer and pathogenicity evaluation. Although the hosts of HA4–1 and Rs-P.362200 strains are the same, they have specific genes to their own genomes. The number of genomic islands and prophages in HA4–1 genome is more than that in Rs-P.36220, indicating a rapid change of the bacterial wilt pathogens. Conclusion The complete genome sequence analysis of peanut bacterial wilt pathogen enhanced the information of R. solanacearum genome. This research lays a theoretical foundation for future research on the interaction between Ralstonia solanacearum and peanut.

Complete genome sequence analysis of a novel bunyavirus isolated from Paris polyphylla var. yunnanensis

A novel negative-stranded (ns) RNA virus tentatively named “Yunnan manyleaf paris rhizome negative-stranded virus 1” (YMPrNSV1), was isolated from a Paris polyphylla var. yunnanensis plant exhibiting leaf chlorosis and mosaic symptoms in Yunnan. Its complete genome sequence was determined using Illumina and Sanger sequencing. The genomes composed of three RNA segments (L, M and S) with each one containing a single open reading frame. Based on sequence identity and the presence of typical bunya-like domains/motifs, the proteins encoded by YMPrNSV1 were predicted to be: RNA-dependent RNA polymerase (RdRp), putative movement protein (MP), and nucleocapsid protein (NP). Sequence comparison analyses showed that the RdRp, MP and NP of YMPrNSV1 are highly similar to those of watermelon crinkle leaf-associated virus 2 (WCLaV-2), with 69.1%, 50.4% and 60.9% amino acid sequence identities respectively. Phylogenetic analysis based on deduced amino acid sequences of RdRp and NP suggested that YMPrNSV1 clustered with coguviruses in a clade, and that WCLaV-2 is the known closely related species to YMPrNSV1. Base on the above results, YMPrNSV1 should be regarded as a new member of genera Coguvirus, within the family Phenuiviridae.

Human pathogenic Mycobacterium kansasii (former subtype I) with zoonotic potential isolated from a diseased indoor pet cat, Japan

Mycobacterium kansasii is one of the most prevalent and pathogenic nontuberculous mycobacteria in the world. Herein, we report the first case of M. kansasii infection in an indoor domestic cat in Japan. Complete genome sequence analysis of the isolate showed this pathogen is genetically identical to human pathogenic M. kansasii.