First Report of Bacterial Soft Rot Caused by Pantoea agglomerans on Chinese yam (Dioscorea opposita Thunb.) in China

Chinese yam (Dioscorea opposita Thunb.), which belongs to the family of Dioscorea, is widely naturalized throughout China, due to its high economic and medicinal value. Since 2019, water-soaked lesions were frequently observed in the underground tubers of Chinese yam located in Xinyang City, Henan Province. To identify the causal agent, ten pieces of tissue from the underground tubers with disease symptoms were collected. Those infected tissues (5×5 mm) were crushed in 500 μL sterilized water after surface sterilization and streaked onto Luria-Bertani agar plates. Pale-yellowish, rod-shaped, slimy single bacterial colonies with smooth margin were observed after 24 hours of incubation, and three bacterial colonies (named CY-1, CY-2 and CY-3) were randomly selected for further biochemical and molecular characterization. These bacteria were gram-negative with the cell length of 1.0 to 3.0 μm, width of 0.5 to 1.0 μm, and with peritrichous flagella. Subsequently, the bacteria were biochemically analyzed through BIOLOG (Hayward, CA) and identified as Pantoea agglomerans with 99% probability. Furthermore, the phylogenetic analysis results based on 16S rDNA, DNA gyrase subunit B (gyrB), and RNA polymerase sigma factor (rpoD) showed these three isolates were most closely related to P. agglomerans. The sequence of 16S rDNA, gyrB and rpoD of each strain was submitted to GenBank with the accession numbers MZ541065 MZ541066 and MZ541067 for 16S rDNA; MZ669846, MZ669847 and MZ669848 for gyrB; MZ669849, MZ669850 and MZ669851 for ropD. Pathogenicity test was performed to complete Koch’s postulates. Tubers of Chinese yam were wounded by sterile needle and inoculated with 500 μL 108 CFU/mL bacterial suspension. Sterilized water was used as a control. Five pots were inoculated for each isolate. Water-soaked lesions appeared after five days incubation at 25°C in a biochemical incubator and no lesions were observed on the control. Bacteria re-isolated from the lesions were similar in phenotypic and molecular characteristics to the original isolates. In brief, based on colony morphology, biochemical tests, characteristic sequence analysis, and pathogenicity verification, the pathogen responsible for the soft rot of Chinese yam in Henan Province was identified as P. agglomerans. In China, P. agglomerans has been reported to associate with bacterial soft rot on Chinese cabbage (Guo et al., 2020). To our knowledge, this work is the first report of bacterial rot caused by P. agglomerans on Chinese yam.

First Report of Rhizome Rot Caused by Pectobacterium brasiliense on Ginger in China

In August 2020, ginger (Zingiber officinale) rhizomes (cv. Mianjiang) showing soft rot symptoms were observed in a field in Tayang Village, Fengrun District, Tangshan, Hebei Province (North China). The disease incidence in that field (15 ha in size) was more than 20%. Symptomatic rhizomes (brown and water-soaked) were surface-sterilized in 75% ethanol for 60 sec and then three successive rinses with sterile distilled water. Rhizomes were cut into pieces ca. 0.5 cm in length, and then were soaked in 500 µl 0.9% saline for 20 min. Aliquots (20 μl) of three tenfold dilutions of the tissue specimen soaking solution were plated onto the lysogeny broth (LB) medium. And LB plates were incubated at 28°C for 24 h. Five single colonies were picked from each LB plate and restreaked three times for purity. Endophytic bacteria were also isolated from asymptomatic rhizomes as control. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primer pair 27F/1492R (Weisburg et al. 1991) and sequenced. The results of BLASTN against NCBI nr of the 16S rDNA amplicons suggested that the most isolates (8/10) obtained from the rotten rhizomes belonged to the genus Pectobacterium, and few isolates (2/10) were Enterobacter spp.. Only Enterobacter spp. were isolated from asymptomatic rhizomes. Since all Pectobacterium isolates showed identical 16S rDNA sequence, thus, only two isolates were selected for further analysis. Pectobacterium isolates TS20HJ1 and TS20HJ2 (MZ853520, MZ853521) represent isolates from two plant individuals. To determine the species of the rhizome rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with five housekeeping genes acnA, icdA, mdh, proA and rpoS (MZ994717-MZ994726) (Ma et al. 2007; Waleron et al. 2008), and a phylogenetic tree was reconstructed using RAxML v8.2.12 (github.com/stamatak/standard-RAxML). No sequence variation was observed at any MLSA locus between the two isolates. The result of phylogenetic analysis showed that the ginger rhizome isolates clustered with P. brasiliense type strain IBSBF1692T (Duarte et al. 2004; Nabhan et al. 2012). Ginger seedlings (cv. Mianjiang) were inoculated with the isolate TS20HJ1 by injecting 10 µl of bacterial suspensions (108 CFU·mL-1) into the rhizomes, or injected with 10 µl of 0.9% saline solution as control. The seedlings were grown at 28°C and 50% relative humidity. Ten days after inoculation, only the bacteria-inoculated rhizomes showed diseased symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected rhizomes and were identified with MLSA gene sequencing, fulfilling Koch’s postulates. P. brasiliense causes soft rot of a wide range of economically important crops (Oulghazi et al. 2021). To our knowledge, this is the first report of P. brasiliense causing rhizome rot of ginger in China. The rhizome rot caused 20-25% yield loss on average in Tangshan region in 2020, which poses a significant threat to the local ginger farming. Further research on epidemiology and disease management options is needed.

First Report of Pectobacterium punjabense Causing Blackleg and Soft Rot on Potato in Hebei and Fujian Province, China

Pectobacterium spp. and Dickeya spp. cause blackleg and soft rot on potato worldwide (Charkowski, 2018). Potato plants (cv. Favorita or Jizhang 8#) with blackleg symptoms (vascular browning of crown stems, Fig. S1) were observed in the field in Zhangjiakou, Hebei province in 2018, and in Ningde, Fujian Province in 2019, in China. The disease incidence was around 50% and 10% in Zhangjiakou (5 ha) and Ningde (4 ha), respectively. Diseased plants (3 from each site) were collected to isolate the pathogen. Blackleg symptomatic stems were soaked in 75% ethanol for 2 min, rinsed and ground in sterile distilled water. Serial tenfold dilutions of the above solution were plated onto the crystal violet pectate agar (CVP) plate (Ge et al., 2018). Two to 3 days after incubation at 28°C, 4 bacterial colonies in total which digested pectin from the media and developed pit on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al., 2002). Two colony sequences that showed more than 99% sequence identity to Pectobacterium punjabense type strain SS95 (MH249622) were submitted to the GenBank ( accession numbers: OK510280, MT242589). Additionally, six housekeeping genes proA (OK546205, OK546199), gyrA (OK546206, OK546200), icdA (OK546207, OK546201), mdh (OK546208, OK546202), gapA (OK546209, OK546203), and rpoS (OK546210, OK546204) of these two isolates were amplified and sequenced (Ma et al., 2007, Waleron et al., 2008). All strains show 99% to 100% identity with MH249622T . Phylogenetic trees based on 16S rRNA gene sequences (Fig. S2) and concatenated sequences of the housekeeping genes (Fig. S3) of the 2 isolates were constructed using MEGA 6.0 software (Tamura et al., 2013). Koch’s postulate was performed on potato seedlings and potato tubers (cv. Favorita) by injecting 100 μl bacterial suspension (105 CFU/ml) or sterile phosphate-buffered solution into the crown area of the stems or the tubers and kept at 100% humidity and 21°C for 1 day. Four days after inoculation, the infected area of the inoculated seedlings rotten and turned black, while the controls were symptomless (Fig. S4). Two days after inoculation, the infected tubers rotten and turned black, while the controls were symptomless (Fig. S4). Bacterial colonies were reisolated from these symptomatic tissues and identified using the same methods described above. Blackleg on potato plants or soft rot on potato has been reported to be caused by Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. brasiliense, Pectobacterium parmentieri, Pectobacterium polaris in China (Zhao et al., 2018; Cao et al., 2021; Wang et al., 2021). To our knowledge, this is the first report of blackleg/soft rot of potato caused by Pectobacterium punjabense in China. We believe that this report will draw attention to the management of this pathogen in China.

The Periplasmic Oxidoreductase DsbA Is Required for Virulence of the Phytopathogen Dickeya solani

In bacteria, the DsbA oxidoreductase is a crucial factor responsible for the introduction of disulfide bonds to extracytoplasmic proteins, which include important virulence factors. A lack of proper disulfide bonds frequently leads to instability and/or loss of protein function; therefore, improper disulfide bonding may lead to avirulent phenotypes. The importance of the DsbA function in phytopathogens has not been extensively studied yet. Dickeya solani is a bacterium from the Soft Rot Pectobacteriaceae family which is responsible for very high economic losses mainly in potato. In this work, we constructed a D. solani dsbA mutant and demonstrated that a lack of DsbA caused a loss of virulence. The mutant bacteria showed lower activities of secreted virulence determinants and were unable to develop disease symptoms in a potato plant. The SWATH-MS-based proteomic analysis revealed that the dsbA mutation led to multifaceted effects in the D. solani cells, including not only lower levels of secreted virulence factors, but also the induction of stress responses. Finally, the outer membrane barrier seemed to be disturbed by the mutation. Our results clearly demonstrate that the function played by the DsbA oxidoreductase is crucial for D. solani virulence, and a lack of DsbA significantly disturbs cellular physiology.

Relationship of Resistance-Related Enzyme activity and Salicylic Acid content in Phalaenopsis Species with different levels of resistance to DIckeya Dadantii

ABSTRACT Orchids (Phalaenopsis) are ornamental plants that are cultivated commercially and in great demand in the market. Soft-rot disease (SRD) caused by the necrotrophic pathogen Dickeya dadantii is a cause of considerable economic loss to cultivators of many orchid species. Our previous experiment identified a limited number of species that were resistant to D. dadantii. This study aimed to validate the resistance level of four Phalaenopsis species in a detached leaf inoculation protocol to identify the resistance mechanism(s) involved. Soft-rot symptom diameter was measured from 6 to 18 hours post-inoculation (HPI) with D. dadantii. Disease assessment confirmed that P. amboinensis is a resistant species, P. pantherina is a susceptible species, and P. amabilis and P. schilleriana are very susceptible species. There was no difference in the lignin content between the resistant and very susceptible species. Detailed observation of resistant and very susceptible species, P. amboinensis vs. P. amabilis, revealed higher phenylalanine ammonia-lyase (PAL) and peroxidase (POD) in P. amabilis than in P. amboinensis. In contrast, there was higher salicylic acid (SA) content in P. amboinensis than in P. amabilis. These results suggest that POD and PAL activities may not be effective in defense against soft-rot disease, while SA plays an important role in the resistance of P. amboinensis to D. dadantii. Low PAL activity in P. amboinensis implies that the SA contents from the isochorismate pathway may be involved in the mechanism of P. amboinensis resistance to D. dadantii. Therefore, endogenous SA content may be a good indicator for screening resistant species in Phalaenopsis.

Salicylic acid effect on the mechanism of Lelliottia amnigena causing potato soft rot

Salicylic acid (SA) plays an important role in protecting plants from biotic stresses. Lelliottia amnigena is a newly identified potato soft rot pathogen and there are no adequate studies on this soft rot pathogen. Therefore, this paper focussed on the effect of SA on the mechanism under which L. amnigena causes potato soft rot. L. amnigena was examined and detected to secrete pectinase, proteases, pectin lyase and cellulase, which are the most important pathogenic enzymes involved in the production of plant diseases. Sterilised healthy potato tubers were inoculated with 0.2 mL of L. amnigena suspension (3.69 CFU · mL−1 × 107 CFU · mL−1). After 24 h, 200 μL of four different SA concentrations (0.5 mM, 1.0 mM, 1.5 mM and 2.0 mM) were used to treat the tubers. Co-culture of L. amnigena and SA significantly reduced the activity of pectinase, protease, pectin lyase and cellulase by an average of 33.8%, 43.4%, 67.7% and 46.9%, across the four concentrations (0.5 mM, 1.0 mM, 1.5 mM and 2.0 mM), respectively, compared to the control. The average disease index was reduced by 54.7% across the four SA concentrations. Treatment with SA induced transcriptional levels of the superoxide dismutase, peroxide, catalase and glutathione S-transferase across the four levels by an average of 3.87, 3.25, 3.97 and 3.94-fold, respectively, compared to control. Based on our results, we could state that SA could reduce the activities of these extracellular enzymes produced by L. amnigena by modulating both enzymatic and non-enzymatic antioxidant activities and gene expression that induce natural resistance in plants against bacterial infections.

Efficacy of essential oils in the management of soft rot caused by Pectobacterium aroidearum in lettuce

Lettuce is susceptible to several diseases, especially soft rot caused by bacteria of the genus Pectobacterium. Due to the adaptability of this pathogen and the lack of disease control registered for the crop, the objective of this work was to evaluate the effects of essential oils in the management of soft rot caused by P. aroidearum in lettuce. The study was developed at the Universidade do Estado da Bahia, Juazeiro, BA, Brazil, and the essential oils (EOs) of orange, bergamot, lemongrass, palmarosa, citronella, cloves, tea tree, rosemary, sage, and ginger were used in concentrations of 0.25; 0.5; 0.75 and 1.0% to assess the in vitro growth inhibition of the bacterium. Subsequently, the curative effects of the disease were evaluated by applying the EOs that obtained the best results in vitro in lettuce plants of the susceptible variety “Mônica”. The treatments were applied, via spraying, 12 hours after inoculation using the bite method with bacterial suspension. The best in vivo treatment was selected to assess its preventive and curative activity, as well as to find the ideal concentration for reducing epidemiological variables and chromatographic characterization. The EOs of palmarosa, sage, citronella, lemongrass, and cloves (0.25%), and that of sage (0.75%), inhibited bacterial growth in vitro. The EO of salvia showed the best results in vivo, inhibiting the growth of the disease in concentrations of 0.50 and 0.75%, so it was selected for the preventive and curative control tests alone. The preventive treatment was not efficient for the management of soft rot in lettuce, however, from the regression analysis, a concentration of 0.64% of the salvia EO was found as a potential for curative control of this bacteriosis, as it reduces the incidence and severity of the disease. Linalyl acetate and linalool were found in higher concentrations in the chromatographic analysis. These components, probably, conferred the bactericidal capacity of the EO of sage, being potential for the use in the control of P. aroidearum in lettuce.

Biological Control of soft rot bacteria of onion in Bangladesh

An investigation was conducted to search antagonistic bacteria as biological control agents of soft rotting bacterial pathogen of onion (Allium cepa L.) in vitro and in storage. Antibacterial activity of previously isolated 91 bacterial isolates was tested in vitro against onion soft rot bacteria Burkholderia cepacia O-15. Two isolates namely, R-15 and E-37 were found antagonistic against onion soft rot bacteria. Isolate R-15 was identified as the genus Bacillus and the isolate E-37 to Lactobacillus sp. Isolate R-15 proved to be a strong antagonist against onion soft rot bacteria was selected for bio-control of onion in storage. That was also effectively reduces the soft rot disease of onion in storage condition. Percentage of disease reduction (PDR) due to treatment with antagonistic bacteria was 72.4% compared to untreated control. It is therefore suggested that this isolate could be exploited as biocontrol agent for onion soft rot in Bangladesh. Bangladesh J. Sci. Ind. Res.56(4), 231-240, 2021

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.