First Report of Crown Gall of Kiwifruit (Actinidia deliciosa) Caused by Agrobacterium fabacearum in China and the Establishment of Loop-Mediated Isothermal Amplification Technique

Kiwifruit is moderately sweet and sour and quite popular among consumers; it has been widely planted in some areas of the world. In 2019, the crown gall disease of kiwifruit was discovered in the main kiwifruit-producing area of Guizhou Province, China. This disease can weaken and eventually cause the death of the tree. The phylogeny, morphological and biological characteristics of the bacteria were described, and were related to diseases. The pathogenicity of this species follows the Koch hypothesis, confirming that A. fabacearum is the pathogen of crown gall disease of kiwifruit in China. In this study, Loop-mediated isothermal amplification (LAMP) analysis for genome-specific gene sequences was developed for the specific detection of A. fabacearum. The detection limit of the LAMP method is 5 × 10−7 ng/μL, which has high sensitivity. At the same time, the amplified product is stained with SYBR Green I after the reaction is completed, so that the amplification can be detected with the naked eye. LAMP analysis detected the presence of A. fabacearum in the roots and soil samples of the infected kiwifruit plant. The proposed LAMP detection technology in this study offers the advantages of ease of operation, visibility of results, rapidity, accuracy and high sensitivity, making it suitable for the early diagnosis of crown gall disease of kiwifruit.

Genomics Analyses of Rose Crown Gall-Associated Bacteria Revealed Two New Agrobacterium species: Agrobacterium burrii sp. nov. and Agrobacterium shirazense sp. nov.

Agrobacterium tumefaciens species complex contains a set of diverse bacterial strains most of which are well known for their pathogenicity on agricultural plants causing crown gall diseases. Members of A. tumefaciens species complex are classified into several taxonomically distinct lineages called “genomospecies” (13 genomospecies until early 2021). Recently, two genomospecies, i.e., G19 (strains RnrT, Rew and Rnw) and G20 (strains OT33T and R13) infecting Rose sp. plants in Iran were described based on biochemical and molecular-phylogenetic data. Whole genome sequence-based core-genome phylogeny followed by average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) calculations performed in this study suggested that genomospecies G19 and G20 could be described as two novel and standalone species. In the phylogenetic tree, these two new genomospecies were clustered separately from other genomospecies/species of A. tumefaciens species complex. Moreover, both ANI and dDDH indices between the G19/G20 strains and other Rhizobiaceae members are clearly below the accepted thresholds for prokaryotic species description. Hence, Agrobacterium burrii sp. nov. is proposed to encompass the G19 strains, with RnrT = CFBP 8705T = DSM 112541T as type strain. Agrobacterium shirazense sp. nov. is also proposed to include G20 strains, with OT33T = CFBP 8901T = DSM 112540T as type strain.

Origin of Pathogens of Grapevine Crown Gall Disease in Hokkaido in Japan as Characterized by Molecular Epidemiology of Allorhizobium vitis Strains

Crown gall is a globally distributed and economically important disease of grapevine and other important crop plants. The causal agent of grapevine crown gall is tumorigenic Allorhizobium vitis (Ti) strains that harbor a tumor-inducing plasmid (pTi). The epidemic of grapevine crown gall has not been widely elucidated. In this study, we investigated the genetic diversity of 89 strains of Ti and nonpathogenic A. vitis to clarify their molecular epidemiology. Multi-locus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD was performed for molecular typing of A. vitis strains isolated from grapevines with crown gall symptoms grown in 30 different vineyards, five different countries, mainly in Japan, and seven genomic groups A to F were obtained. The results of MLSA and logistic regression indicated that the population of genetic group A was significantly related to a range of prefectures and that the epidemic of group A strains originated mainly in Hokkaido in Japan through soil infection. Moreover, group E strains could have been transported by infected nursery stocks. In conclusion, this study indicates that both soil infection and transporting of infected nursery stocks are working as infection source in Hokkaido.

Complete Genome Sequence Data of the Grapevine Crown Gall inhibiting bacteria Allorhizobium vitis Strain F2/5.

Crown gall disease in grapevine is caused by pathogenic strains of Allorhizobium vitis. A. vitis strain F2/5 is a non-pathogenic biocontrol agent that was previously shown to act as a biological control agent to crown gall disease and first isolated from South Africa. Here, we present the complete assembled genome and is 5.94 Mb in length with 5,414 predicted protein-coding sequences, has two circular chromosomes and five plasmids. The genome sequence has no detectable T-DNA border sequences and is missing key virulence genes which is consistent with the bacteria being non-pathogenic. The F2/5 genome sequence could contribute to understanding the molecular basis underlying the biocontrol activity.

Production of agrocinopine A by Ipomoea batatas agrocinopine synthase in transgenic tobacco and its effect on the rhizosphere microbial community

Agrobacterium tumefaciens is a bacterial pathogen that causes crown gall disease on a wide range of eudicot plants by genetic transformation. Besides T-DNA integrated by natural transformation in vegetative tissues of plants by pathogenic Agrobacterium, previous reports have indicated that T-DNA sequences originating from ancestral Agrobacterium sp. are present in the genomes of all cultivated sweet potato (Ipomoea batatas) analyzed. Expression of Agrobacterium-derived agrocinopine synthase (ACS) gene was detected in leaf and root tissues of sweet potato, suggesting that the plant can produce agrocinopine, a sugar-phosphodiester opine considered to be utilized by Agrobacterium in crown gall. To validate the product synthesized by I. batatas ACS (IbACS), we introduced IbACS into tobacco under a constitutive promoter. High voltage paper electrophoresis followed by alkaline silver nitrate staining detected the production of an agrocinopine-like substance in IbACS1-expressing tobacco, and further MS and NMR analyses of the product confirmed that IbACS can produce agrocinopine A from natural plant substrates. The partially purified compound was biologically active in an agrocinopine A bioassay. 16S rRNA amplicon sequencing and meta-transcriptome analysis revealed that the rhizosphere microbial community of tobacco was affected by the expression of IbACS. A new species of Leifsonia (actinobacteria) was isolated as an enriched bacterium in the rhizosphere of IbACS1-expressing tobacco. This Leifsonia sp. can catabolize agrocinopine A produced in tobacco, indicating that the production of agrocinopine A attracts rhizosphere bacteria which can utilize this sugar-phosphodiester. These results suggest a potential role of IbACS conserved among sweet potato cultivars in manipulating their microbial community.

First report of Agrobacterium tumefaciens causing crown gall disease of roselle (Hibiscus sabdariffa L.) in Taiwan

Roselle (Hibiscus sabdariffa L.) plants, whose calyces are used for production of beverages or jams, are mainly cultivated in Taitung County of eastern Taiwan. Since 2016, large crown galls were observed on the roselle plants in the commercial plantations at Taimali and Jinfong Townships of Taitung County. A follow-up survey in July and August of 2017 revealed spreading of this disease to the neighboring areas including Beinan and Dawu Townships. Disease incidence was estimated to be 0.6-10%. Galls of varying sizes (2-15 cm in diameter) were usually found on the roots and crowns of the roselle plants, starting with small swellings at the infection sites. Galls were light-colored, and smooth and tender in texture at the early stage, but later turned dark-colored, and appeared rough and woody. In some cases, adventitious roots extruding from the larger crown galls could be seen. Isolation of the causal agent was performed by quadrantally streaking bacterial suspension made from surface-sterilized, macerated galls on trypticase soy agar (TSA). After incubating at 28°C for 5 days, single colonies were transferred onto new TSA plates for further cultivation at 28°C. Finally, circular, convex, viscous and milky white colonies with smooth surface similar to colony morphology of Agrobacterium tumefaciens C58 were obtained for further identification. First, all six candidate isolates (TZ-1, TL1-2, TL2-1, TD1-1, TD1-24 and TD2-1) were identified as Agrobacterium spp. using the partial sequences of the 16S rRNA gene (accession numbers MW205820 to MW205825 in the GenBank database). The selected isolates also showed some biochemical and physiological characteristics similar to A. tumefaciens, including oxidase positive, growth at 35°C and in 2% NaCl, and alkalinity from litmus milk. Moreover, they were tested negative for utilization of citrate and acid production on potato dextrose agar (PDA) supplemented with calcium carbonate. Under a transmission electron microscope, the bacterium was rod-shaped and possessed peritrichous flagella. By means of multiplex PCR using primers designed for differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2, a 184 bp product was detected in all six isolates, indicating that they all belong to Agrobacterium biovar 1. Furthermore, the recA allele of each isolate was PCR amplified using primers F2898/F2899, and recA sequence analysis assigned all six isolates to A. tumefaciens genomospecies G7 (GenBank accession numbers MZ570905-MZ570910). Pathogenicity assay was carried out by inoculating the stems of 2-week-old roselle seedlings through wounds made with a sterile needle with bacteria on it. The inoculated seedlings were kept in plastic bags to maintain high humidity. Symptoms similar to those observed in the field developed at the inoculation sites after 7 days, and Koch’s postulates were fulfilled when the bacteria re-isolated from the galls were also identified as A. tumefaciens genomospecies G7 using recA gene sequence analysis. To our knowledge, this is the first report of crown gall disease caused by A. tumefaciens on Hibiscus sabdariffa in Taiwan. This disease may potentially damage the roselle industry if no action is taken to stop its spreading. Identification of the causal agent of roselle crown gall disease could help us further investigate its ecology and develop integrated pest management strategies for prevention of this disease in the future.

Occurrence of Crown Gall Disease on Japanese Spindle (Euonymus japonicas var. Green Rocket) Caused by Agrobacterium rosae in Iran

Crown gall disease caused by diverse Agrobacterium species is one of the main biotic constraints in the ornamental plants industry in Iran (Mafakheri et al. 2017). In August 2019, Japanese spindle (Euonymus japonicus var. Green Rocket) plants showing crown gall symptoms were observed in a commercial greenhouse in Tehran, Iran. Infected plants were characterized by a visible overgrowth on their stems and crown. Bacterial isolation from the gall tissues was performed on nutrient agar (NA) and 1A media as described by Moore et al. (2001). The six resulted bacterial strains (A.E1 to A.E6) were evaluated using PCR primer pair F8360/F8361 amplifying a 453 bp DNA fragment in recA gene and confirmed as Agrobacterium sp. (Shams et al. 2013). Pathogenicity of the strains was evaluated in two independent assays on Japanese spindle plantlets as well as 10-15 day old tomato (Solanum lycopersicum cv. Sunseed 6189) and sunflower (Helianthus annuus cv. Armavirski) plants in greenhouse conditions using the needle prick method as described previously (Mafakheri et al. 2019). The reference strain A. radiobacter ICMP 5856 and sterile distilled water were used as positive and negative controls, respectively. Crown gall symptoms appeared 20-25 days post inoculation on the Japanese spindle plantlets as well as tomato and sunflower plants inoculated with the strains isolated in this study, while the negative control plants remained asymptomatic. Koch’s postulates were accomplished by re-isolating on NA medium and PCR-based identification of the inoculated strains from the symptomatic plants. The representative strain A.E1 was subjected to multilocus sequence analysis (MLSA) using the sequences of four housekeeping genes (i.e. atpD, gyrB, recA, and rpoB) as described previously (Mafakheri et al. 2019). MLSA results revealed that the strain A.E1 is phylogenetically closely related to A. rosae. The sequences were deposited into GenBank under the accession numbers MT007962 to MT007965 for atpD, gyrB, recA, and rpoB, respectively. Further, the strain A.E1 was subjected to whole genome sequencing using Illumina HiSeq X platform. DNA extraction was performed using NucleoSpin Microbial DNA kit (Macherey-Nagel, Germany), DNA libraries were obtained with Nextera XT DNA Library Prep Kit (Illumina, USA), and de novo sequence assembly was performed using SPAdes genome assembler. The resulting whole genome sequence was deposited into the GenBank database under the accession number JAFJZW000000000. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were calculated among all the type strains of Agrobacterium species/genomospecies using standard criteria as detailed previously (Osdaghi et al. 2020; Chen et al. 2021). The strain A.E1 had 97% ANI and 72% dDDH values with A. rosae strain NCPPB 1650, suggesting that the bacterial strains isolated from Japanese spindle in Iran belong to A. rosae. This is the first report of A. rosae causing crown gall disease on Japanese spindle in Iran. The new crown gall disease could negatively affect the ornamental shrub production industry in central Iran unless strict sanitary measures are taken into the account in the nurseries in these areas. Further nationwide surveys and samplings are warranted to elucidate the economic impact of the pathogen on ornamental plant industry in the country.