Dickeya Fangzhongdai Research Articles

Colonization by orchid mycorrhizal fungi primes induced systemic resistance against necrotrophic pathogen.

Orchids and arbuscular mycorrhiza (AM) plants evolved independently and have different structures and fungal partners, but they both facilitate nutrient uptake. Orchid mycorrhiza (OM) supports orchid seed germination, but unlike AM, its role in disease resistance of mature plants is largely unknown. Here, we examined whether OM induces systemic disease resistance against a necrotrophic pathogen in a similar fashion to AM. We investigated the priming effect of mycorrhizal fungi inoculation on resistance of a terrestrial orchid, Bletilla striata, to soft rot caused by Dickeya fangzhongdai. We found that root colonization by a compatible OM fungus primed B. striata seedlings and induced systemic resistance against the infection. Transcriptome analysis showed that priming was mediated by the downregulation of jasmonate and ethylene pathways and that these pathways are upregulated once infection occurs. Comparison with the reported transcriptome of AM fungus-colonized rice leaves revealed similar mechanisms in B. striata and in rice. These findings highlight a novel aspect of commonality between OM and AM plants in terms of induced systemic resistance.

Dickeya fangzhongdai was prevalent and caused taro soft rot when coexisting with the Pectobacterium complex, with a preference for Araceae plants.

Bacterial soft rot caused by coinfection with Dickeya spp. and Pectobacterium spp. in hosts can cause successive changes in fields, and it is difficult to prevent the spread of and control the infection. Pectobacterium spp. are prevalent in the growing areas of tuberous crops, including taro and potato. Recently, Dickeya fangzhongdai has emerged as a virulent pathogen in taro. To determine the prevalence status of the causal agents and evaluate the potential spreading risks of D. fangzhongdai, screening and taxonomic classification were performed on phytopathogenic bacteria collected from different taro-growing areas in Guangdong Province, China, and biological and genomic characteristics were further compared among typical strains from all defined species. The causative agents were verified to be phytobacterial strains of D. fangzhongdai, Pectobacterium aroidearum and Pectobacterium colocasium. P. aroidearum and P. colocasium were found to form a complex preferring Araceae plants and show intensive genomic differentiation, indicating their ancestor had adapted to taro a long time prior. Compared with Pectobacterium spp., D. fangzhongdai was more virulent to taro corms under conditions of exogenous infection and more adaptable at elevated temperatures. D. fangzhongdai strains isolated from taro possessed genomic components of additional T4SSs, which were accompanied by additional copies of the hcp-vgrG genes of the T6SS, and these contributed to the expansion of their genomes. More gene clusters encoding secondary metabolites were found within the D. fangzhongdai strains than within the Pectobacterium complex; interestingly, distinct gene clusters encoding zeamine and arylpolyene were both most similar to those in D. solani that caused potato soft rot. These comparisons provided genomic evidences for that the newly emerging pathogen was potentially equipped to compete with other pathogens. Diagnostic qPCR verified that D. fangzhongdai was prevalent in most of the taro-growing areas and coexisted with the Pectobacterium complex, while the plants enriching D. fangzhongdai were frequently symptomatic at developing corms and adjacent pseudostems and caused severe symptoms. Thus, the emerging need for intensive monitoring on D. fangzhongdai to prevent it from spreading to other taro-growing areas and to other tuberous crops like potato; the adjustment of control strategies based on different pathopoiesis characteristics is recommended.

Genome Sequence Resource of a Taro Bacterial Soft Rot Pathogen, Dickeya fangzhongdai ZXC1

Dickeya fangzhongdai ZXC1 is a newly identified highly virulent causal agent of taro bacterial soft rot disease isolated from a taro sample showing typical soft rot symptoms in Shaoguan, Guangdong Province, China. The pathogen produces plant cell wall–degrading enzymes that macerate the taro tube tissues. In this study, we report the whole-genome sequencing analysis of strain ZXC1. The results showed that strain ZXC1 has one circular DNA chromosome of 5,129,951 bp with a 56.59% G+C content. In addition to sharing a conserved zms gene cluster that encodes the genes for biosynthesis of phytotoxin zeamines and 11 copies of predicted pectate lyase genes, the strain ZXC1 genome contains more prophage loci and a higher number of type IV secretion system and type IV secretion system gene clusters than the D. fangzhongdai strains isolated from pears or phalaenopsis, which may account for its strong virulence phenotype. The data from this study present a valuable resource for elucidation of the pathogenic mechanisms of D. fangzhongdai and may aid in developing new disease control approaches to safeguard taro production. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Retrospective survey of Dickeya fangzhongdai using a novel validated real-time PCR assay.

Dickeya fangzhongdai, an aggressive plant pathogen, causes symptoms on a variety of crops and ornamental plants including bleeding canker of Asian pear trees. Historical findings stress the need for a specific detection tool for D. fangzhongdai to prevent overlooking the pathogen or assigning it to general Dickeya spp. Therefore, a qualitative real-time PCR for specific detection of D. fangzhongdai has been developed and validated. The developed assay shows selectivity of 100%, diagnostic sensitivity of 76% and limit of detection with 95% confidence interval in plant matrices ranging from 311 to 2,275 cells/mL of plant extracts. The assay was successfully used in a retrospective survey of selected host plants of relevance to Europe and environmental niches relevant to D. fangzhongdai. Samples of potato tubers and plants, plants from the Malinae subtribe (apple, pear, quince, and Asian pear tree) and fresh surface water from Slovenia were analyzed. D. fangzhongdai was not detected in any plant samples, however, 12% of surface water samples were found to be positive.

Metagenomic Analysis of Microbial Communities from Coal Waste

Coal waste contains few macro-and micronutrients, which makes it less likely to become a growth site for microorganisms. One way to screen coal waste resources is to identify the diversity of microbes in them to study the relationship between these microbial communities in contributing to improving environmental pollution using metagenomics to determine microbial diversity. The purpose of this research is to study the diversity of microorganisms in areas contaminated with heavy metals using a metagenomic method. This study was performed using next-generation sequencing techniques, including DNA extraction, 16S rRNA amplification, and gene sequencing analysis. The results of this research found that the 10 most commonly found species were Baekduia soli, Nocardiodes iriomotensis, Nocardioides mesophilus, Nocardioides pakistanensis, Propionibacterium cyclohexanicum, Solirubrobacter ginsenosidimutans, Gemmatirosa kalamazoonensis, Roseisolibacter agri, Kosakonia saccahri, and Dickeya fangzhongdai. Based on this research, it can be concluded that most of the microbial communities from coal waste are dominated by the phylum Actinobaceria. The results of this study can be used as an adaptive microbial germplasm for industrial waste management strategies.

Polyamine signaling communications play a key role in regulating the pathogenicity of Dickeya fangzhongdai.

Dickeya fangzhongdai is a devastating bacterial pathogen infecting a wide range of crops and ornamental plants worldwide. As a newly identified bacterial species in 2016, the regulatory mechanisms that govern its virulence are still a mystery. In this study, we explored the potential roles of polyamine-mediated cell-to-cell communication in regulation of D. fangzhongdai virulence. Null mutation of speA and speC in D. fangzhongdai strain ZXC1, which encodes polyamine biosynthesis through arginine and ornithine pathways, respectively, dramatically reduced bacterial motility, decreased production of plant cell wall degradation (PCWD) enzymes, and attenuated the bacterial virulence on taro and potato. We then tested the effect of various polyamine molecules in the restoration of the mutant phenotypes and showed that putrescine was the most potent signal in the regulation of virulence traits in strain ZXC1. In addition, we found that taro extract contained active signals to rescue putrescine-deficient phenotypes. High-performance liquid chromatography mass spectrometry analysis validated the speA was essential for production of putrescine in D. fangzhongdai ZXC1. We further showed that the putrescine transporters PotF and PlaP are required for putrescine-mediated cell-to-cell communication and virulence against taro and potato tubers. quantitative reverse transcription-PCR analysis demonstrated that putrescine influences the pathogenicity of D. fangzhongdai ZXC1 by regulating the expression of PCWD enzymes, bacterial chemotaxis, and flagellar-related genes. The findings from this study shed a new light for elucidating the pathogenic mechanisms of D. fangzhongdai and present useful clues for developing relevant disease control strategies. IMPORTANCE Dickeya fangzhongdai is a newly identified plant bacterial pathogen with a wide host range. A clear understanding of the cell-to-cell communication systems that modulate the bacterial virulence is of key importance for elucidating its pathogenic mechanisms and for disease control. In this study, we present evidence that putrescine molecules from the pathogen and host plants play an essential role in regulating the bacterial virulence. The significance of this study is in (i) demonstrating that putrescine signaling system regulates D. fangzhongdai virulence mainly through modulating the bacterial motility and production of PCWD enzymes, (ii) outlining the signaling and regulatory mechanisms with which putrescine signaling system modulates the above virulence traits, and (iii) validating that D. fangzhongdai could use both arginine and ornithine pathways to synthesize putrescine signals. To our knowledge, this is the first report to show that putrescine signaling system plays a key role in modulating the pathogenicity of D. fangzhongdai.

Soft rot disease of Belamcanda chinensis caused by Dickeya fangzhongdai in China

The rhizome of Belamcanda chinensis (belamcanda sinensis/Shegan) is an important traditional Chinese medicine, which is often used to treat lung diseases. However, soft rot disease seriously threatens the production of B. chinensis, causing its rhizomes to rot and the entire plant to die. Based on morphological characteristic and multigenic phylogenetic analyses of 16S rDNA, pel (pectate lyases encoding genes) and mdh (malic dehydrogenase encoding genes), the pathogenic isolates were identified as Dickeya fangzhongdai. One representative isolate SG-17 was selected for biological characteristics and pathogenicity tests analysis. The results showed that the optimum temperature and pH for SG-17 growth were 28 °C and pH 6, respectively. SG-17 caused typical soft rot symptoms on healthy B. chinensis plants, and produced symptoms of similar severity on Iris japonica (fringed iris) and Iris tectorum (iris), the other two species in Iridaceae family of economic and ornamental importance. Moreover, we also evaluated the impact of SG-17 on the contents of bioactive ingredients in B. chinensis rhizomes. The results show that the contents of tectorigenin and irigenin of B. chinensis rhizomes were increased significantly after infected with SG-17, while the contents of irisflorentin was decreased. As revealed by bactericides screen experiments, Ethylicin and Carbendazim · tetramethylthiuram disulfide exhibited good antibacterial activities, which could be used as the preferred agents for the control of soft rot of B. chinensis. As far as we know, this is the first report of Dickeya fangzhongdai causing soft rot on B. chinensis in the world.

First Report of Canker Caused by Pectobacterium actinidiae on Asian Pear Trees (Pyrus pyrifolia) in South Korea.

In July 2020, pear trees (Pyrus pyrifolia cv. Niitaka) with cankers displaying dark-red bacterial ooze on the trunk and branches were found in two pear orchards located in Naju, Jeollanam-do, South Korea (34°57'50″ N, 126°43'52″ E and 34°56'14″ N, 126°33'42″ E). The incidence was 1.5% (3 out of 200 trees) and 0.83% (1 out of 120 trees), respectively. The symptoms were similar to those of the bleeding canker caused by Dickeya fangzhongdai (Choi et al. 2021), which is typically observed in October. The bacterial ooze was suspended in sterile water and streaked in Luria-Bertani (LB) medium to isolate single bacterial colonies. Two isolates (PRI-B16 and PRI-B17) from representative diseased trees were selected for investigation. Physiological and biochemical characteristics of the isolates analyzed using the BIOLOG GEN III MicroPlate™ system (Biolog, Hayward, CA, USA) were similar to the characteristics of Pectobacterium actinidiae (Portier et al. 2019). These isolates were positively utilized stachyose, L-galactonic acid-g-lactone, guanidine hydrochloride and weakly utilized (-)-D-arabitol (Portier et al. 2019). Bacterial genomic DNA was extracted from cell cultured in 5 ml LB at 28C for 2 days using G-spin DNA extraction kit (iNtRON Biotechnology, Korea) according to the manufacturer's protocol. PCR amplification was amplified as Portier et al. (2019). The generated their sequences of the small subunit ribosomal RNA (16S rRNA) using primers 27f and 1492r (Heuer et al. 1997) (Genebank accession numbers: ON951863 and ON951864) were 99.86% and 99.76% identical, respectively, to that of P. actinidiae isolate SCPJ-1 (KY307837.1) by a BLAST search against gene bank databases. The dnaX (Genebank accession nos: ON960281 and ON960282), leuS (Genebank accession nos: ON960283 and ON960284), and recA (Genebank accession nos: ON960285 and ON960286) genes of these isolates were also amplified and sequenced by previously described Stawiak et al. (2009) for dnaX and leuS, and Waleron et al. (2002) for recA. A neighbor-joining phylogenetic analysis based on the concatenated dnaX, leuS, and recA sequences placed the two isolates in a clade containing previously identified P. actinidiae isolates. A pathogenicity test was conducted using two-year-old pear (P. pyrifolia cv. Nittaka) trees grown in a greenhouse. Wounded and unwounded pear tree branches were inoculated with 10 µL of the bacterial suspension (108 CFU/ml) or sterile water as a control. The inoculated plants were maintained at 30°C without light for 2 days under 85-90% humidity. At 7 days post-inoculation, bacterial ooze was observed on the branches inoculated with a bacterial suspension, whereas branches subjected to unwounded inoculation and water inoculation exhibited no symptoms. This assay was performed three times. We reisolated two colonies from each sample showing typical bleeding symptoms and confirmed their identity by sequencing the dnaX locus. Pectobacterium actinidiae has been reported to cause canker in pear trees in Brazil (Araujo et al. 2021) as well as kiwifruit in South Korea (Koh et al. 2012). This is the first report of P. actinidiae causing canker on pear trees in South Korea and is, therefore, pathologically significant.

Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18–20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

First Report of Dickeya fangzhongdai Causing Peduncle Soft Rot of Banana in China.

Banana (Musa spp.) is a popular fruit all over the world, and it's also an important cash crop with a planting area of 358,924 ha in southern China. In July 2020, a peduncle soft rot disease occurred on dwarf banana (Musa sp. cv. Guangfen) in Guigang city (N22°50'29″, E109° 43'34″), Guangxi province, China. More than 20% plants were infected in the banana plantation. The first external sign of the disease appeared on the incisional wound after the flower bud was cut off from the peduncle. The symptom initially appeared as a black lesion on the wound, then extended into the internal tissue of the whole peduncle. In the later stages, the internal tissue became soft and rot, occasionally formed a necrotic cavity, and eventually led to the black rot of the whole peduncle with a foul smell. To isolate the pathogen, the internal lesion tissues of 5 mm × 5 mm were collected between the border of symptomatic and healthy tissue, treated with 75% ethanol for 10 s, and 0.1% HgCl2 for 3 min, then rinsed with sterile water for three times. Sterilized tissue fragments were cut to pieces with sterilized surgical shears and soaked in 5 mL sterile water, then shaken for 10 min in a vortex oscillator. The suspension was diluted 1000 times with sterilized water，then plated on nutrient-agar medium and incubated at 28℃ in darkness for 24 h. Among the 32 isolates, 23 pure bacterial cultures with similar morphology were predominantly obtained from the samples. These bacteria were gram-negative, and their colonies were initially yellowish white with irregular edges and smooth surfaces, then turned to grayish blue after 72 h incubated at 28℃. The representative isolates GZF2-2 and GZF1-8 were selected for further identification. Genomic DNA was isolated from the bacteria and the 16S rDNA was amplified with primers 27F/1492R (Weisburg et al. 1991) and sequenced. The obtained sequences (GenBank Accession No. MZ768922 and OK668082) showed >99% identities to several records of Dickeya fangzhongdai deposited in NCBI GenBank (1400/1404 bps for GZF2-2 to KT992690, 1409/1417 bps for GZF1-8 to MT613398) based on BLAST analysis. In addition, the recA, fusA, gapA, purA, rplB, dnaX genes and the 16S-23S intergenic spacer (IGS) regions of the two isolates were also amplified and sequenced (GenBank Accession Nos. OK634381-OK634382, OK634369- OK634370, OK634373-OK634374, OK634377-OK634378, OK634385-OK634386, OK634365- OK634366 and OK631722-OK631723) as described by Tian et al. (2016). All the DNA sequences matched that of D. fangzhongdai strains JS5T (percent identities>99.06%), PA1 and ECM-1 in GenBank. Neighbor-joining phylogenetic analysis by software MegaX (Kumar et al. 2018) based on the 16S rDNA sequences revealed that the two isolates were in the same clade with reported D. fangzhongdai strains. Multilocus sequence analysis of the other seven regions also showed the two representative isolates were belong to D. fangzhongdai. Therefore, the isolates were identified as D. fangzhongdai. Pathogenicity of isolate GZF2-2 was investigated to demonstrate Koch's postulate. The end of the banana peduncles of 6 healthy plants were cut off, and 10 mL bacterial suspension (108 CFU/mL) was inoculated to the fresh wound on the plants using sterile brushes. Six control plants were inoculated with sterilized water. All the inoculated peduncles were covered with plastic bags to maintain high humidity. After 28 days, all the peduncles inoculated with strain GZF2-2 showed soft rot symptoms similar to those observed in the field, while the controls remained symptomless. The same bacteria were re-isolated from the symptomatic peduncles and confirmed by sequencing the 16S rDNA. D. fangzhongdai has been reported to cause soft rot on onion (Ma et al. 2020) and bleeding cankers on pear trees (Chen et al. 2020). To the best of our knowledge, this is the first report of D. fangzhongdai causing peduncle soft rot on banana in China.

Five Plant Natural Products Are Potential Type III Secretion System Inhibitors to Effectively Control Soft-Rot Disease Caused by Dickeya.

Dickeya zeae, a plant soft-rot pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors, infecting a wide variety of monocotyledonous and dicotyledonous plants and causing serious losses to the production of economic crops. In order to alleviate the problem of pesticide resistance during bacterial disease treatment, compounds targeting at T3SS have been screened using a hrpA-gfp bioreporter. After screening by Multifunctional Microplate Reader and determining by flow cytometer, five compounds including salicylic acid (SA), p-hydroxybenzoic acid (PHBA), cinnamyl alcohol (CA), p-coumaric acid (PCA), and hydrocinnamic acid (HA) significantly inhibiting hrpA promoter activity without affecting bacterial growth have been screened out. All the five compounds reduced hypersensitive response (HR) on non-host tobacco leaves and downregulated the expression of T3SS, especially the master regulator encoding gene hrpL. Inhibition efficacy of the five compounds against soft rot were also evaluated and results confirmed that the above compounds significantly lessened the soft-rot symptoms caused by Dickeya dadantii 3937 on potato, Dickeya fangzhongdai CL3 on taro, Dickeya oryzae EC1 on rice, and D. zeae MS2 on banana seedlings. Findings in this study provide potential biocontrol agents for prevention of soft-rot disease caused by Dickeya spp.

Identification, biological characteristics, and control of pathogen causing Pinellia ternata soft rot in Hubei province

This study was designed to identify the pathogen causing soft rot of Pinellia ternata in Qianjiang of Hubei province and screen out the effective bactericides, so as to provide a theoretical basis for the control of soft rot of P. ternata. In this study, the pathogen was identified based on molecular biology and physiological biochemistry, followed by the detection of pathogenicity and pathogenicity spectrum via plant tissue inoculation in vitro and the indoor toxicity determination using the inhibition zone method to screen out bactericide with good antibacterial effects. The control effect of the bactericide against P. ternata soft rot was verified by the leave and tuber inoculation in vitro. The phylogenetic tree was constructed based on the 16 S rDNA, dnaX gene, and recA gene sequences, respectively, and the result showed that the pathogen belonged to the same branch as the type strain Dickeya fangzhongdai JS5. The physiological and biochemical tests showed that the pathogen was identical to D. fangzhongdai, which proved that the pathogen was D. fangzhongdai. The pathogenicity test indicated that the pathogen could obviously infect leaves at 24 h and tubers in 3 d. As revealed by the indoor toxicity test, 0.3% tetramycin, 5% allicin, and 80% ethylicin had good antibacterial activities, with EC_(50) values all less than 50 mg·L~(-1). Tests in tissues in vitro showed that 5% allicin exhibited the best control effect, followed by 0.3% tetramycin and 10% zhongshengmycin oligosaccharide, and their preventive effects were better than curative effects. Therefore, 5% allicin can be used as the preferred agent for the control of P. ternata soft rot, and 0.3% tetramycin and 10% zhongshengmycin oligosaccharide as the alternatives. This study has provided a certain theoretical basis for the control of P. ternata soft rot.

Dickeya fangzhongdai

This datasheet on Dickeya fangzhongdai covers Identity, Distribution, Hosts/Species Affected, Further Information.

Soft rot disease caused by Dickeya fangzhongdai in epiphytic orchids in Vietnam

Soft rot diseases are becoming problematic in the cultivation of epiphytic orchids in Vietnam, but the causal agents are unknown. We investigated soft rot in Dendrobium anosmum, Phalaenopsis amabilis and Paphiopedilum concolor. Leaves of diseased plants with soft rot symptoms were sampled and cultures of Dickeya were obtained. The pathogen was identified as Dickeya fangzhongdai based on a phylogenetic analysis of 16s rRNA, dnaX, gapA and rplB. The pathogenicity of nine D. fangzhongdai isolates was confirmed in D. anosmum, Ph. amabilis and Pa. concolor in a nursery trial. The efficacy of a range of chemical agents was evaluated on the growth of D. fangzhongdai in vitro, and on D. fangzhongdai soft rot disease management in 1-year-old D. anosmum, Ph. amabilis and Pa. concolor plants. Cuprous oxide and Cu-bismerthiazol strongly inhibited the in vitro growth of D. fangzhongdai, and they reduced the soft rot lesion area of infected plants in vivo. These findings provide an interim option for producers to improve management of D. fangzhongdai soft rot disease in production nurseries in Vietnam.

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 (Alič 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.

First Report of Bacterial Soft Rot Disease on Taro Caused by Dickeya fangzhongdai in China.

Taro [Colocasia esculenta (L.) Schott.] is an important root crop in the world with great economic value. In recent years, outbreaks of soft rot were observed on taro plants in several plantation areas located in Shaoguan, Guangdong Province, China (25°7'57" N, 113°19'5" E). Root tubers of taro (Paodan variety) infected by soft rot had water-soaked lesions with a dark brown-black margin including a rotten smell, they also had internal rot that was also found in root tubers with no external symptoms. In some areas, the incidence of soft rot can reach up to 30%. To isolate the causal agent, ten pieces of taro root tubers with typical symptoms were surface-sterilized with 75% ethanol and 0.1% HgCl2 solution and then washed thrice with sterile water. The tuber slices were soaked in 50 ml sterile water and shaken at 28°C, 200 rpm for 2 h, and 100 µl was streaked onto the modified Yeast Extract Beef (YEB) agar medium (1% peptone, 0.5% yeast extract, 0.5% sucrose, 0.5% NaCl, 1 Mmol/L MgSO4•7H2O, 1.5% agar, pH 7.0) plates (Zhou et al. 2011) and incubated at 28°C for 24 h. Single colonies grown on YEB were selected for preliminary inoculation onto healthy taro (Paodan variety) slices. Two of the Gram-negative bacteria, named as ZXC1 and MPC2, developed symptoms consistent in rotted decay inside the root tubers after incubation for 24h at 30°C. ZXC1 and MPC2 were biochemically profiled using a Biolog Gen III MicroPlate (Microlog 3, 5.2) (Shen et al. 2019) and resulted Dickeya sp. (SIM 0.856 and 0.704). To determine the species of the Dickeya isolates, 16S rRNA sequences were amplified by primers 27F and 1492R (Hauben et al. 1998). Housekeeping genes including gyrB, atpD, rpoB, and infB were also amplified using degenerate primers (Brady et al. 2008). Results from the BLASTn analysis of the 16S rRNA (GenBank accession numbers MN853405, MN853406), gyrB (GenBank accession numbers MN866299, MN866303), atpD (GenBank accession numbers MN866298, MN866302), rpoB (GenBank accession numbers MN866301, MN866305), and infB (GenBank accession numbers MN866300, MN866304) genes in the isolates ZXC1 and MPC2 showed 99% identities to those of the previously reported D. fangzhongdai isolates from Phalaenopsis (Zhang et al. 2018). Multilocus sequence analysis (MLSA) by MEGA 7.0 performed with four housekeeping genes (gyrB, atpD, rpoB, infB) showed that they clustered with D. fangzhongdai isolates. Analyses using scanning and transmission electron microscopy showed that ZXC1 and MPC2 bacteria were rod-shaped, 0.5-1.0 μm × 1.0-3.0 µm, with peritrichous flagella. Pathogenicity tests were performed thrice using surface-sterilized 2-month-old taro seedlings (Paodan variety). Six individual seedlings were inoculated using a sterile syringe with ten microliters of bacterial suspension (108 CFU/ml) in Tris buffer (0.1 mol/L Tris and 0.1 mol/L HCl, pH 7.4). Taro seedlings injected with sterile Tris buffer were used as the negative control. These taro seedlings were grown in the greenhouse (30 ± 2°C, 90 ± 5% relative humidity). At the 25th day post inoculation, soft rot symptoms were observed in inoculated taro, while all control taro plants remained symptom-free. Small and pale yellow with irregular margins colonies consistent with morphological characteristics of those of D. fangzhongdai were re-isolated from symptomatic taro tubers and the housekeeping genes presence was verified by sequencing as described above, fulfilling Koch's postulates. D. fangzhongdai is a newly emerging bacterial pathogen, which causes bleeding cankers in pear trees (Tian et al. 2016), and soft rot of Phalaenopsis (Zhang et al. 2018). This is the first report of D. fangzhongdai causing soft rot disease in taro. Considering the high incidence of soft rot, this pathogen might pose a significant threat to taro and other economically important crops. Therefore, further researches are needed to investigate host range of the pathogen and develop appropriate integrated management to contain this disease spreading.

Occurrence of Dickeya fangzhongdai Causing Soft Rot of Banxia (Pinellia ternata) in China.

Banxia [Pinellia ternata (Thunb.) Breit., Araceae] is a perennial herbaceous plant, where the tuber is commonly used in traditional Chinese herbal medicine. In the summer of 2020, an outbreak of soft rot of Banxia was observed in Zhugentan Town (30°50'N, 112°91'E), Qianjiang City, Hubei Province, with about 56% percentage of infected plants. Symptomatic plants initially appeared with small water-soaked spots on leaves that progressed into extensive translucent spots when facing a light source. The bacteria further spread to the stems and tubers. Infected tubers appeared normal, but inner macerated inclusions exuded when touched. The whole plant was macerated and collapsed within a few days. Ten leaves with typical symptoms were obtained from a diseased field, by surface sterilizing in 75% ethanol for 30 s and 0.3% NaClO for 5 min, washing the tissue sections three times in sterile water. Small pieces of tissue (5 × 5 mm) were removed from lesion borders, plated on nutrient ager medium, and cultivated at 37 ℃ for 48 h. Five representative isolates were selected for further identification. Colonies were all smooth and transparent. In addition, these strains were Gram-negative, and had the ability to reduce D-arabinose, melibiose, galactose, raffinose, rhamnose, inositol, and mannitol, but not reduce 5-keto-D-gluconate, L-xylose, amygdalin, and sorbitol. Genomic DNA was extracted from isolate stain ZG5. The 16S rDNA gene, recombinase A (recA) gene, and DNA polymerase III subunits gamma and tau (dnaX) were amplified by PCR with the primers 27f/1492r (Weisburg et al. 1991), recF/recR (Waleron et al. 2002), and dnaXf/dnaXr (Sławiak et al. 2009), respectively. The PCR products were sequenced, then submitted to GenBank (GenBank MW332472, MW349833, MW349834, respectively). BLAST search showed that the sequences of 16S rDNA, recA, and dnaX respectively matched ≥99% with D. fangzhongdai strains DSM 101947 (CP025003), QZH3 (CP031507), and PA1 (CP020872). Pathogenicity tests were performed on 10 healthy, 3-month-old P. ternate plants. Five plants were injected with 20 μl of bacterial suspension (108 CFU/ml) of isolate ZG5, and other plants were injected with sterile water as a negative control. All tested plants were incubated at 28 ℃ and individually covered with a plastic bag. After 24 h, soft rot symptoms all appeared on the pathogen-inoculated leaves, whereas no symptoms on the control leaves. The pathogenicity test was repeated three times and obtained same results. Koch's postulates were fulfilled by reisolating D. fangzhongdai from inoculated plants. Meanwhile, PCR were performed on the reisolated bacteria as above described, and the pathogen was identified and confirmed as D. fangzhongdai. Here we report that D. fangzhongdai causes soft rot of P. ternata in China. The disease progressed very rapidly, and reduced the yield and quality of tubers. Thus, more research is needed to implement effective strategies to manage this disease.

First Report of Bleeding Canker of Pear Tree Trunks Caused by Dickeya fangzhongdai in Korea.

Pears (Pyrus pylifolia L.) are cultivated nationwide as one of the most economically important fruit trees in Korea. At the end of October 2019, bleeding canker was observed in a pear orchard located in Naju, Jeonnam Province (34°53'50.54″ N, 126°39'00.32″ E). The canker was observed on trunks and branches of two 25-year-old trees, and the diseased trunks and branches displayed partial die-back or complete death. When the bark was peeled off from the diseased trunks or branches, brown spots or red streaks were found in the trees. Bacterial ooze showed a rusty color and the lesion was sap-filled with a yeasty smell. Trunks displaying bleeding symptoms were collected from two trees. Infected bark tissues (3 × 3 mm) from the samples were immersed in 70% ethanol for 1 minute, rinsed three times in sterilized water, ground to fine powder using a mortar and pestle, and suspended in sterilized water. After streaking each suspension on Luria-Bertani (LB) agar, the plates were incubated at 25°C without light for 2 days. Small yellow-white bacterial colonies with irregular margins were predominantly obtained from all the samples. Three representative isolates (ECM-1, ECM-2 and ECM-3) were subjected to further characterization. These isolates were cultivated at 39 C, and utilized (-)-D-arabinose, (+) melibiose, (+)raffinose, mannitol and myo-inositol but not 5-keto-D-gluconate, -gentiobiose, or casein. These isolates were identified as Dickeya sp. based on the sequence of 16S rRNA (MT820458-820460) gene amplified using primers 27f and 1492r (Heuer et al. 2000). The 16S rRNA sequences matched with D. fangzhongdai strain ND14b (99.93%; CP009460.1) and D. fangzhongdai strain PA1(99.86%; CP020872.1). The recA, fusA, gapA, purA, rplB, and dnaX genes and the intergenic spacer (IGS) regions were also sequenced as described in Van der wolf et al. (2014). The recA (MT820437-820439), fusA (MT820440-820442), gapA (MT820443-820445), purA (MT820446-820448), rplB (MT820449-820451), dnaX (MT820452-820454) and IGS (MT820455-820457) sequences matched with D. fangzhongdai strains JS5, LN1 and QZH3 (KT992693-992695, KT992697-992699, KT992701-992703, KT992705-992707, KT992709-992711, KT992713-992715, and KT992717-992719, respectively). A neighbor-joining phylogenetic analysis based on the concatenated recA, fusA, gapA, purA, rplB, dnaX and IGS sequences placed the representative isolates within a clade comprising D. fangzhongdai. ECM-1 to 3 were grouped into a clade with one strain isolated from waterfall, D. fangzhongdai ND14b from Malaysia. Pathogenicity test was performed using isolate ECM-1. Three two-year-old branches and flower buds on 10-year-old pear tree (cv. Nittaka), grown at the National Institute of Horticultural and Herbal Science Pear Research Institute (Naju, Jeonnam Province in Korea), were inoculated with 10 μl and 2 μl of a bacterial suspension (108 cfu/ml), respectively, after wounding inoculation site with a sterile scalpel (for branch) or injecting with syringe (for flower bud). Control plants were inoculated with water. Inoculated branches and buds in a plastic bag were placed in a 30℃ incubator without light for 2 days (Chen et al. 2020). Both colorless and transparent bacterial ooze and typical bleeding canker were observed on both branches and buds at 3 and 2 weeks post inoculation, respectively. No symptoms were observed on control branches and buds. This pathogenicity assay was conducted three times. We reisolated three colonies from samples displaying the typical symptoms and checked the identity of one by sequencing the dnaX locus. Dickeya fangzhongdai has been reported to cause bleeding canker on pears in China (Tian et al. 2016; Chen et al. 2020). This study will contribute to facilitate identification and control strategies of this disease in Korea. This is the first report of D. fangzhongdai causing bleeding canker on pears in Korea.

Characterization of Dickeya fangzhongdai causing bacterial soft rot disease on Dendrobium nobile in India

Soft-rot disease on Dendrobium nobile observed in a commercial farm in the Nilgiris district of Tamil Nadu, India was investigated. Disease etiology is reported along with phenotypic, molecular identification of the pathogen. Infected leaves showed rot symptoms characterized by irregular water-soaked lesions on the leaf margin that rapidly spread over the entire leaf. Milky exudation on the cut end of infected leaves indicated the bacterial etiology of the disease. The bacterial colonies isolated were irregular, whitish-yellow, spindle-shaped with a characteristic raised center. All isolates showed identical phenotypic traits and induced a hypersensitive reaction on Nicotiana tabacum leaves. Pathogenicity assay performed on D. nobile confirmed their pathogenic nature as all three bacterial isolates incited typical soft-rot within 12 h post-inoculation (hpi). In vitro host range studies revealed that all the isolates could cause rot on cut slices of other vegetable crops including potato, carrot, and onion. Additionally, the bacterium incited soft rot on Aloe vera, but not on chrysanthemum and banana transplants. The identity of the bacterial isolates was confirmed as Dickeya fangzhongdai using 16S rRNA gene sequences as well as by their pathogenic behavior. Complete understanding of the etiology of the disease and of the pathogen would be useful to devise suitable management practices for the containment of disease spread in the future.

Bleeding canker of pears caused by Dickeya fangzhongdai: Symptoms, etiology and biology

Bleeding canker, a devastating disease of pear trees (Pyrus pyrifolia L.), was first reported in the 1970s in Jiangsu, China and more recently in other provinces in China. Trees infected with bleeding canker pathogen, Dickeya fangzhongdai, develop cankers on the trunks and branches, and a rust-colored mixture of bacterial ooze and tree sap could be seen all over the trunks and branches. In this study, we provided detail descriptions of the symptoms and epidemiology of bleeding canker disease. Based on pathogenic and phenotypic characterizations, we identified the causal agent of bleeding canker of pear as D. fangzhongdai. Dickeya fangzhongdai strains isolated from pear were also pathogenic on Solanum tuberosum, Brassica pekinensis, Lycopersicon esculentum, and Phalaenopsis aphrodite based on artificial inoculation, and the pathogen were more virulent on potato than that of D. solani strain. This study provides new information about this disease and bleeding canker disease of pear.