Soft Rot Symptoms Research Articles

First Report of Bacterial Soft Rot of Potato Caused by Pectobacterium aroidearum strains in China.

Potato (Solanum tuberosum L.) is a globally important staple crop, and China has contributed more than 20% of the world's production. In Guangdong Province, potato has become one of the most important winter crops, increased the farmer income. However, the potato were seriously affected by soft rot disease caused by Pectobacterium spp. in the past decade. In February 2024, typical symptoms of potato soft rot were observed on the plants of cultivar "Daxiyang" in Enping City, Guangdong Province, where the disease incidences ranged 3%-5% in the investigated fields. The stems adjacent to the tubes showed typical inky black symptoms, and the plants appeared yellow. The interior of the tubes appeared water-soaked and had developed to overall decay, with an evident smell. Eleven symptomatic plants were collected and used to isolate the causal agents. Pieces of tube tissue (about 5×5 mm) were removed between the rot-symptomatic and non-symptomatic margins, and surface sterilized using 75% ethanol for 30 s and 2% NaClO for 1 min. The pieces were rinsed in sterile water for three times, and then plated on Luria-Bertani (LB) medium agar for 36 h at 30°C. The produced colonies were selected for hypersensitive test on tobacco, and the colonies with a positive reaction were subcultured three times. Two representative strains, EP51-2 and EP51-3, were processed for gene sequencing, including house-keeping genes, danX, leuS, and recA (Portier et al. 2019). The gene sequences (GenBank accessions no. PP870934 to PP870939) of these two representative strains were matched with Pectobacterium aroidearum strain NCPPB 929 (CP166097.1) with identities ranging from 97.15 to 100% and the coverage of 100%. The sequences of three genes were concatenated and used for phylogenetic analyses, along with representative strains from 19 other Pectobacterium species. Phylogenetic analyses supported that EP51-2 and EP51-3 were grouped into P. aroidearum with the representative strains. Koch's postulates were applied to determine the pathogenicity of P. aroidearum strain EP51-2. According to the external inoculation method on potato stems (Czajkowski et al. 2010), each of six pots of healthy potato plants was inoculated at the basal stem with 100 µl of bacterial suspension (108 CFU/ml), while six pots inoculated with LB served as controls. The plants were then kept at 30°C and maintained at 95% humidity. After 2 days, all six plants inoculated with the bacteria exhibited typical symptoms on the stems, which progressed to the collapse of the whole plant; the controls remained symptom-free during the observation. Single colonies reisolated from the symptomatic stem of the inoculated plants were confirmed using PCR and sequencing with recA primers as described above, and the causal agent was identified as P. aroidearum strains, fulfilling Koch's postulates. P. aroidearum has been reported on Chinese cabbage (Xie et al. 2018) and leaf mustard (Chu et al. 2024) in China, as well as on crops in the family Araceae, such as konjac (Wei et al. 2021), taro (Zhou et al. 2022), and Pinellia ternata (Du et al. 2024). To our knowledge, this is the first report of P. aroidearum causing potato soft rot in China. This pathogen has been prevalent in the taro fields in Guangdong Province and has caused severe losses. This report highlights an expansion of the host range for this pathogen. Attention should be focused on this newly emerging pathogen affecting potatoes, and immediate measures should be implemented to control its spread.

Beyond the Basics: Taxonomic Classification and Pathogenomics in Recently Discovered Dickeya dadantii Isolates

The genus Dickeya consists of Gram-negative bacteria capable of causing soft rot symptoms in plants, which involves tissue breakdown, particularly in storage organs such as tubers, rhizomes, and bulbs. These bacteria are ranked among the top ten most relevant phytopathogens and seriously threaten economically valuable crops and ornamental plants. This study employs a genomic analysis approach to taxonomically classify and characterize the resistome and virulome of two new strains, CCRMP144 and CCRMP250, identified as Dickeya dadantii. These strains were found to be the causative agents of soft rot symptoms in chili pepper (Capsicum spp.) and lettuce (Lactuca sativa), respectively, in the northeastern region of Brazil. The methodology employed in silico techniques, including tetra correlation search (TCS) and Average Nucleotide Identity (ANI) analysis, in association with a phylogenomic tree inference. TCS and ANI analysis showed that the studied strains belong to the Dickeya dadantii species. The phylogenomic analysis grouped the studied strains in the D. dadantii clade. The genomic characterization demonstrates 68 virulence genes, 54 resistances of biocide and heavy metal genes, and 23 antibiotic resistance genes. As far as we know, this is the first genomic study with Brazilian D. dadantii strains. This study demonstrates the efficacy to taxonomic classification and provides insights into the pathogenesis, host range, and adaptability of these strains which are crucial for the development of more effective management and control strategies for soft rot diseases.

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

Ginger (Zingiber officinale) is a vital commercial crop in China, valued for its medicinal and nutritional properties. From June to September 2023, soft rot symptoms were observed on ginger rhizomes in fields in Shucheng County, Lu'an City, Anhui Province, China (31°24'N, 116°53'E). Brown, water-soaked lesions emerged at the stem base, accompanied by leaf yellowing and wilting. Approximately 20% of the ginger fields in this region (about 530 hectares) displayed soft rot symptoms. Rhizomes from ten randomly diseased ginger plants from three different locations were surface disinfected (Liu et al. 2020). The disinfected rhizomes were ground in sterile water, and serial dilutions of the supernatant were then placed on Luria-Bertani (LB) agar, resulting in the isolation and purification of 432 colonies. Using the primer set 27F/1492R (Weisburg et al. 1991), the 16S rRNA gene was amplified and sequenced. BLASTn analysis indicated that Pectobacterium species were isolated from all three locations, accounting for one-fifth of the total isolates. Three isolates, PA03, PA05, and PA08, were randomly chosen for further analysis. These colonies appeared milky white, round, with a smooth surface and smooth margins. All three isolates were Gram-negative, tested negative for methyl red and catalase, but positive for urease. The 16S rRNA sequences obtained (GenBank accession No. PP935462, PP935465 and PP935466) were 100% identical to Pectobacterium aroidearum strains KNUB-05-21 (LC777355, 1,375/1,375 bp), 3898C (KY446053, 1,418/1,418 bp), and MY11 (MT834965, 1,373/1,373 bp). The icdA, mdh, and proA genes of the isolates were amplified by PCR using specific primer pairs icdA-F/icdA-R, mdh-F/mdh-R, and proA-F/proA-R, respectively (Ma et al. 2007). BLASTn searches showed that the icdA (PP922142), mdh (PP922148), and proA (PP922145) sequences of PA03 had similarities of 99.25%, 98.26%, and 98.01%, respectively, with those of P. aroidearum strain UNEB3 (MH500095, 563/536 bp; MH507329, 522/394 bp; MH507331, 655/702 bp). Similarly, the icdA (PP922143), mdh (PP922149), and proA (PP922146) sequences of PA05 were 99.05%, 99.09%, and 97.18% identical to those of P. aroidearum strain MTL1911110204 (MT013063, 529/552 bp; MT013079, 498/399 bp; MT013095, 717/714 bp). The icdA (PP922144), mdh (PP922150), and proA (PP922147) sequences of PA08 shared 99.25%, 98.27%, and 97.94% similarity with those of P. aroidearum strain CCRM35 (MH500096, 537/537 bp; MH507330, 513/396 bp; MH507332, 630/705 bp). Phylogenetic trees were generated using MEGA11.0 software based on 16S rRNA and concatenated sequences of icdA-mdh-proA (Tamura et al. 2021). The results of phylogentic analysis revealed that the isolates, PA03, PA05, and PA08, clustered with P. aroidearum strains. To test pathogenicity, 10 µl of bacterial suspensions (1×108 CFU/ml) of isolates PA03, PA05, and PA08 were inoculated into the stem base of ginger plants using a sterile syringe, with sterile water used as a control. Each isolate was inoculated into five pots, with three plants per pot. The plants were maintained in a greenhouse at 28°C with 75% relative humidity. Rhizomes inoculated with bacterial suspensions displayed soft rot symptoms resembling those observed in the field seven days after inoculation, while the control group remained free of symptoms. Bacteria were re-isolated from diseased tissues and identified by PCR with specific primer pairs icdA-F/icdA-R, mdh-F/mdh-R, and proA-F/proA-R. The re-isolated strains had the identical sequences as PA03, PA05, and PA08, confirming their pathogenicity and fulfilling Koch's postulates. P. aroidearum is known to cause soft rot in various crops (Barroso et al. 2019; Morase et al. 2020; Tang et al. 2021). To our knowledge, this is the first report of P. aroidearum causing soft rot disease of ginger rhizomes in China. In 2023, soft rot disease led to a 15 to 20% reduction in ginger yield, posing a substantial risk to ginger cultivation in Lu'an city. Identifying this pathogen is crucial for preventing disease outbreaks and developing effective disease management strategies.

First report of Erwinia papayae associated with papaya mushy canker disease in Guam.

Carica papaya (papaya) in Guam, USA may experience soft rot symptoms, often referred to as mushy canker disease. Disease symptoms first appear as expanding water-soaked dark-green stem lesions or leaf spotting with chlorotic halos. Defoliation at petiole-stem junctions and crown necrosis leads to plant death. Papaya diseases caused by Erwinia spp. are documented in nearby tropic regions such as the Northern Mariana Islands (Trujillo and Schroth 1982), the Philippines (Dela Cueva et al. 2017), Japan (Hanagasaki et al. 2020), and Indonesia (Suharjo et al., 2021). The pathogen was isolated from symptomatic papaya stem sections (cv. Red Lady) from a nursery at the University of Guam Agriculture and Life Sciences building in April 2023. Approximately 20% of seedlings collapsed from stem soft rot, with nearly all plants showing varying degrees of water-soaked lesions on leaves or stems. Stem tissue from lesion margins was excised, surface sterilized with 70% EtOH, and macerated in sterile water. Macerate was plated onto nutrient agar (NA) and incubated at 28°C, yielding colonies that were clear to white in color, smooth, circular and mucoid on NA plates for five suspect isolates (JGD231-235). Strains produced blue diffusible pigment on King's B (KB) media, were Gram-negative rods, and exhibited swimming motility on semi-solid (0.5% agar) NA plates. Crown stab inoculation of ten papaya plants (cv. Red Lady) with isolates resulted in mushy canker symptoms within seven days, while negative control plants stabbed with a sterile probe remained asymptomatic. Koch's postulates were fulfilled by drench-inoculating spontaneous rifampicin-resistant (100µg/ml) mutants, JGD233r and JGD235r, onto ten papaya plants (cv. Solo Sunrise). Nine days post-inoculation, bacterial strains were recovered from symptomatic stem tissue macerate plated on rifampicin (100µg/ml) NA and incubated at 28°C. No symptoms or bacterial cells were recovered from the tissue of negative control plants. Cell morphology, culture phenotypes, and disease symptoms suggested the isolates were Erwinia spp., and blue pigment production on KB further suggested E. papayae (Gardan et al. 2004). Partial 16S rDNA sequences of Guam strains JGD231-235 (sequenced using PCR forward primer 5' - AGAGTTTGATCMTGGCTCAG - 3' and reverse primer 5' - GGTTACCTTGTTACGACTT - 3', GENEWIZ (South Plainfield, NJ)) were deposited into GenBank (OR577627- 631). Highest NCBI BLAST results for all strains showed a 16S rDNA sequence identity of 98.17-98.91% with those of Erwinia sp. I-leaf (LC590218) and E. mallotivora BT-MARDI (HQ456230). A maximum likelihood phylogenetic tree based on concatenated partial atpD, infB, and rpoB sequences of strains JGD232 (PP669340, PP669346, PP669343), JGD233 (PP669341, PP669347, PP669344), and JGD235 (PP669342, PP669348, PP669345) (Brady et al. 2008) constructed using MEGA11 (Tamura et al. 2021) showed all strains formed a monophyletic group with Erwinia sp. I-leaf (Hanagasaki et al. 2020) and E. papayae NCPPB 4294T (Gardan et al. 2004), supported with 98% bootstrap. This note documents the first occurrence of E. papayae as a papaya pathogen in Guam. Papaya cultivation supports sustainable food security for Guam (Bevacqua and Sayama 2023), and Erwinia spp. pathogens threaten papaya on other Pacific islands like Hawaii. These findings convey the need for effective quarantine practices, local disease management, and further research on this pathogen.

First report of bacteria associated with soft rot in yellow pitahaya (Selenicereus megalanthus haw.) in Colombia crops

Colombia is the ninth largest producer of dragon fruit in the world and the department of Boyacá is the largest with 440 hectares planted. Bacterial phytopathogens can cause pitahaya fruit losses and low yields by producing stem soft rot. This research aimed to study and identified possible agents of bacterial diseases of pitahaya in Boyacá, Colombia. Thirteen farms in the region were selected to take 20 samples by means of a targeted and random sampling of stem and fruit tissues with soft rot symptoms in the early stages of the disease. A process of microbiological isolation, biochemical and molecular taxonomic identification of the isolated bacteria. The 16s rRNA gene sequences of the V2-V5 region were edited by removing the primers, assembling and obtaining the consensus sequence of the primers 1100R-337F and 800R -518F. The phylogenetic analysis was performed by BLAST at NCBI, using the “Classifier” and “SeqMatch” tools from the RDP website, and phylogenetic trees were created by multiple alignment using the MUSCLE algorithm and the Tamura Nei distance method was performed. Signs like blisters and mucilaginous fluids, symptoms like yellow and brown chlorotic spots, soft rot, and liquefaction were identified, and possible vectors like flies and ants were report. Twenty-five bacterial morphotypes were identified classifieds in 4 phyla, 9 families and 13 genera. The analysis of the 16S rRNA gene sequences of the bacterial strains showed a 98 to 100% identity with Enterobacter cloacae, Pectobacterium carotovora and Paenibacillus glucanolyticus, reported in other studies as causing the soft rot of the pitahaya stem and fruit. New species were reported as possible pathogenic bacteria of pitahaya: Pantoea cypripedii, Kluyvera intermedia and Klebsiella oxytoca. Infectivity assays did not have positive results. The microbiota identified in the stages of the necrotrophic phase or final stage of soft rot of the stem belong to the genera Achromobacter, Sphingobacterium, Pseudomonas, Klebsiella, Paenibacillus, Bacillus, Stenotrophomonas and Microbacterium. In the fly Leptoglossus zonatus associated with the crop were identified Pseudomonas fulva and Lysinibacillus fusiformis were identified. This is also the first official report of a complex of possible phytopathogen bacteria of the order Enterobacterales (Enterobacteriacea, Erwiniaceae and Pectobacteriaceae family) in symptoms by bacterial disease on pitahaya Colombian crops. Identification of the bacteria in a pathogenic system can guide chemical and biological control practices in order to increase the productive and export potential of exotic and orphan crops from small local farmings.

Pectobacterium punjabense Causing Blackleg and Soft Rot of Potato: The First Report in the Russian Federation.

Phytopathogenic bacteria of the genus Pectobacterium are responsible for several diseases that affect potato (Solanum tuberosum L.) production worldwide, including blackleg and tuber soft rot. These bacteria are highly diverse, with over 17 different species currently identified. However, some of the recently described species, such as Pectobacterium punjabense, are still poorly understood. In this study, we focused on P. punjabense isolates collected from diseased potato tubers in Russia in 2021. Whole-genome sequencing was used to characterise the genomic diversity of the pathogen and determine the biochemical profiles of the isolated bacteria. The ability of these isolates to cause soft rot symptoms was tested. A comparative assessment of the potential pathogenicity of the Pectobacterium isolates was conducted by infecting potato tubers and measuring the accumulation of biomass in a liquid medium during cultivation at different temperatures. A TaqMan qPCR assay was developed for the highly sensitive and specific characterisation of P. punjabense strains, which can be used in diagnostic systems. This is the first report on P. punjabense causing potato disease in the Russian Federation.

First report of Pectobacterium brasiliense causing soft rot on leaf mustard in China.

Leaf mustard (Brassica juncea [L.] Czern. et Coss.) belongs to Brassicaceae and is an important leaf vegetable widely cultivated in the Yangtze River basin and various southern provinces in China. In August 2023, the rhizome decay symptoms were observed at the stem base of leaf mustard plants (cv. Huarong) in the field of Changde City (29.05 °N; 111.59 °E), Hunan Province, China. The incidence of symptomatic leaf mustard was approximately 30% in several fields (2 ha in total). Brown and water-soaked symptoms appeared at the base of the outer leaves, and hollow rot at the base of the stem, accompanied by a fishy odor. To identify the causal agent, six infected stem samples were collected and surface sterilized by soaking in 75% ethanol for 60 seconds, rinsed three times with sterile distilled water, and finally cut into pieces (5 × 5 mm) in the sterile water. The extract was streaked on nutrient agar medium. After incubation at 28°C for 24 h, 17 strains were obtained and the colonies of all strains were creamy white, roughly circular, and convex elevation. Six single bacterial strains JC23121001-JC23121006, individually isolated from six different diseased stem samples, were selected as representative strains for further study. For preliminary identification, DNA from the six strains was extracted and identified by 16S rDNA sequencing using the universal primer pair 27F/1492R (Weisburg et al. 1991), and the sequences (accession nos. PP784484 to PP784489) showed 99% query coverage and 99.65% identity to Pectobacterium brasiliense type strain IBSBF1692T (Nabhan et al. 2012). In addition, five housekeeping genes acnA, mdh, mltD, pgi, and proA of the six strains were amplified with specially designed primers (Ma et al. 2007), and the resulting sequences from all six strains were 100% identical. The sequences of the representative strain JC23121001 were deposited into GenBank with accession numbers PP108247, PP066857, PP108248, PP066858, and PP066860, respectively. The maximum-likelihood phylogenetic tree clustered JC23121001 with P. brasiliense type strain IBSBF1692T (Nabhan et al. 2012). The pathogenicity test of six strains was carried out on the six-week-old leaf mustard (cv. Huarong) plants grown in the greenhouse by inoculating 10 µl of each bacterial suspension (108 CFU/ml) on needle-like wounds on the stem base of three healthy leaf mustard plants (Singh et al. 2013). Control plants were treated with sterile distilled water. After inoculation, the plants were incubated at 28°C and 90% relative humidity in a growth chamber. This trial was repeated three times. All inoculated mustard stems were slightly water-soaked after 24 hours and eventually developed into soft rot symptoms, consistent with the original symptoms observed. The control plants remained symptom-free. The strains were re-isolated from inoculated plants and re-identified as P. brasiliense by sequencing five housekeeping genes, thus fulfilling Koch's postulates. P. brasiliense has a broad host range and has been reported on other Brassica species, such as Bok choy (Brassica rapa var. chinensis) in China (Li et al. 2023). Soft rot of leaf mustard caused by Pectobacterium aroidearum has also been reported previously (Chu et al. 2023). To our knowledge, this is the first report of P. brasiliense causing soft rot on leaf mustard in China. The soft rot poses a significant threat to the local leaf mustard industry and requires further research into epidemiology and disease management options.

First Report of Fusarium fujikuroi Causing Postharvest Rot of Kiwifruit (Actinidia chinensis) in Jiangxi Province, China.

Kiwifruits (Actinidia chinensis) are among the most widely planted fruit in Jiangxi Province, China. Infected kiwifruits of the cultivars 'Hongyang' and 'Jinyan' were obtained from a commercial orchard in Fengxin county, Jiangxi Province (28°67' N; 115°42' E) from September to November 2022. The 1200 kiwifruits were collected from cold storage (cold stored for 3 months at 2°C), and moved to room temperatures (15 to 20°C), approximately 20% had symptoms of postharvest soft rot 7 days later. The infected fruits had brown or dark gray spots on the peel. Most were round or oval, with a diameter of approximately 1~3 cm. The pulp was milky white, and there was a waterlogged ring at the junction of decay. The pathogen was isolated by removing several small pieces (3×3 mm) of infected tissue from the diseased kiwifruits, which were sterilized with 75% ethanol for 30 s, dipped in 1% NaClO for 1 min, and rinsed three times with sterile distilled water. These pieces were transferred onto potato dextrose agar (PDA) and incubated for 5 days at 28°C, 75% relative humidity (RH), separated, and repurified. Eight unidentified isolates with similar morphology were obtained on PDA (D3-1 to D3-8). These isolates had abundant aerial fluffy mycelia. The colonies were white during the early stage of culture and turned light purple in the later stage. The mycelia grew 5.8 mm day-1 (n=5) on average and produced abundant conidia 10 days later. The microconidia were solitary, transparent, ovoid, with 0 to 1 septa, and 3.6 to 11.2 × 1.6 to 3.5 µm (average 6.5 × 2.9 µm, n = 50). The macroconidia were sickle-shaped, slender and slightly curved, with 3 to 5 septa, and 22.3 to 53.9 × 2.6 to 5.4 µm (average 39.5 × 4.3 µm, n = 50). Chlamydospores were absent. The morphological characteristics enabled the identification of the pathogen as Fusarium spp. (Leslie and Summerell, 2006). Isolate D3-2 was further confirmed, and the primers ITS1/ITS4 (White et al. 1990), 5F2/7CR and EF1/EF2 (O'Donnell et al. 2022) were used to amplify the internal transcribed spacer (ITS) region, RNA polymerase II largest subunit (RPB2) gene and translation elongation factor-1 alpha regions (TEF-1α). The ITS (accession no. PP077075), RPB2 (PP566653) and TEF-1α (PP566654) sequences shared 99.62 to 100% identities with ITS (ON564593.1), RPB2 (ON734380.1) and TEF-1α (ON697186.1) of F. fujikuroi from NCBI, respectively. Thus, the pathogen was identified as F. fujikuroi based on morphological and molecular characteristics. Each of the three isolates was inoculated on surface-disinfected (75% ethanol, 5 min) disease-free kiwifruits of cv. 'Jinyan' and 'Hongyang'. The six kiwifruits were pierced by a sterile inoculation needle and inoculated with 20 μl spore suspension (1×106 spores/ml), and six kiwifruits were treated with spore suspension without any wounds, four control fruits were inoculated with sterile distilled water. All the fruits were sealed in a storage box, kept at an RH of 90%-95%, and incubated at a constant temperature of 28°C for 5 days. After 3 days, the fruit rotted at the inoculation site, and after 5 days, the lesions gradually increased, and the symptoms were the same as those of the original sample. The control fruits remained disease-free. The pathogenicity tests were repeated three times. Koch's postulates were completed by reisolating the fungus from infected kiwifruits, which was identified as F. fujikuroi by sequencing. Although F. solani (Yang et al. 2018) and F. acuminatum (Wang et al. 2015) have been previously reported to rot kiwifruits in China, this is the first report of F. fujikuroi causing postharvest rot on kiwifruits in China. This discovery can alert agronomists to prevent and control this pathogen.

A novel genus of Pectobacterium bacteriophages display broad host range by targeting several species of Danish soft rot isolates

The bacterial diseases black leg and soft rot in potatoes cause heavy losses of potatoes worldwide. Bacteria within the genus Pectobacteriaceae are the causative agents of black leg and soft rot. The use of antibiotics in agriculture is heavily regulated and no other effective treatment currently exists, but bacteriophages (phages) have shown promise as potential biocontrol agents. In this study we isolated soft rot bacteria from potato tubers and plant tissue displaying soft rot or black leg symptoms collected in Danish fields. We then used the isolated bacterial strains as hosts for phage isolation. Using organic waste, we isolated phages targeting different species within Pectobacterium. Here we focus on seven of these phages representing a new genus primarily targeting P. brasiliense; phage Ymer, Amona, Sabo, Abuela, Koroua, Taid and Pappous. TEM image of phage Ymer showed siphovirus morphotype, and the proposed Ymer genus belongs to the class Caudoviricetes, with double-stranded DNA genomes varying from 39 kb to 43 kb. In silico host range prediction using a CRISPR-Cas spacer database suggested both P. brasiliense, P. polaris and P. versatile as natural hosts for phages within the proposed Ymer genus. A following host range experiment, using 47 bacterial isolates from Danish tubers and plants symptomatic with soft rot or black leg disease verified the in silico host range prediction, as the genus as a group were able to infect all three Pectobacterium species. Phages did, however, primarily target P. brasiliense isolates and displayed differences in host range even within the species level. Two of the phages were able to infect two or more Pectobacterium species. Despite no nucleotide similarity with any phages in the NCBI database, the proposed Ymer genus did share some similarity at the protein level, as well as gene synteny, with currently known phages. None of the phages encoded integrases or other genes typically associated with lysogeny. Similarly, no virulence factors nor antimicrobial resistance genes were found, and combined with their ability to infect several soft rot-causing Pectobacterium species from Danish fields, demonstrates their potential as biocontrol agents against soft rot and black leg diseases in potatoes.

Onion (Allium cepa L.) as a new host for 'Candidatus Arsenophonus phytopathogenicus' in Germany.

Onion (Allium cepa L.) is the most produced vegetable after tomato worldwide and is grown on about 15,000 ha in Germany. In Lampertheim, Hesse in southwest Germany (49°40'02.3"N, 8°26'00.0"E) bulbs of the cultivar 'Red Baron F1' were harvested in September 2023 in an apparently healthy state. Four months later some of the onions showed rotting symptoms, which could not be assigned to a known storage disease. At first, the bulbs became glassy, later they showed soft rot. They originated from a field located in a growing region severely affected by "Syndrome Basses Richesses" (SBR). 'Candidatus Arsenophonus phytopathogenicus' as well as 'Candidatus Phytoplasma solani' are associated with this disease in sugar beet (Gatineau et al. 2002). Moreover, 'Ca. A. phytopathogenicus' was recently reported in association of bacterial wilt and yellowing in potato (Behrmann et al. 2023). Both phloem-restricted bacteria are vectored by the polyphagous planthopper Pentastiridius leporinus (Therhaag et al. 2024), which is highly abundant in this region. To examine, if the unknown symptoms in onion might be related to the presence of these pathogens, DNA of 69 bulbs showing a different degree of softening were analyzed. The samples were tested for the presence of 'Ca. Phytoplasma solani' in a TaqMan assay (Behrmann et al. 2022). All showed negative results. To demonstrate the presence of 'Ca. A. phytopathogenicus', universal and genus-specific primers for the amplification of 16S rDNA and a real-time qPCR assay amplifying an hsp20 fragment were employed (Christensen et al. 2004, Zübert and Kube 2021). Two bulbs of the five positive samples were in an apparently healthy state, the other three showed light to moderate softening symptoms. The 16S rDNA fragments from two samples were sequenced on both strands and aligned. Both fragments were homologous. One fragment of 1474 bp fragment showing 100% homology to the 16S rDNA from SBR (accession no. AY057392) was submitted to GenBank (accession no. PP400342). Other taxa of 'Ca. Arsenophonus' showed 16S rDNA homologies of less than 99.3 %. To corroborate the finding onion samples were subjected to PCR reactions employing genus-specific primers for the conserved tufB, secY and manA gene, which had been derived from multiple alignments of 'Ca. A. spp' sequence submissions (Sela et al. 1989, Lee et al. 2010). The tufB, secY and manA primers amplified fragments of about 980 bp, 640 bp and 930 bp, respectively, from all previously positive samples. Samples which had been tested negative for 'Ca. P. phytopathogenicus' remained negative. Fragments from two accessions were sequenced and the sequences from both isolates were 100 % identical. A BLAST search of the partial tufB gene (acc. no. PP950434) showed 98.57 % sequence identity to a yet unnamed Arsenophonus endosymbiont (acc. no. OZ026540) and 91.85 to 91.83 % to 'Ca. A. nasoniae' and 'Ca. A. apicola', respectively. A similar result was obtained for the partial secY sequence (acc. no. PP950433). The manA sequence (acc. no. PP942231) was identical to a partial sequence of 'Ca. A. phytopathogenicus' strain HN (acc. no. OK335757) and 97.42 % to 'Ca. A. nasoniae and about 87 % to related Arsenophonus species. The finding of 'Ca. A. phytopathogenicus' in onion is novel and might indicate an expanding host range of vector and pathogen in the regional crop rotation. As a correlation between the pathogen and the soft rot symptom is unclear at present, further investigations are needed.

Characterization of pectolytic bacteria causing soft rot on sugar beet in the west of Iran

AbstractBacterial soft rots are amongst the most economically important diseases that cause significant yield losses in various crops. A clear identification of the causal agents is required to develop management strategies. In 2022 and 2023, after the widespread occurrence of soft rot disease in sugar beet fields in the Firuzan region, Hamedan Province, Iran, the current study was conducted to identify and characterize the causal agents. A total of 38 bacterial isolates were obtained from sugar beet plants with soft rot symptoms and subjected to biochemical and phenotypical characterization and pathogenicity tests on healthy slices from potato tubers and sugar beet taproots. Twelve isolates from this study along with the type strain Pectobacterium carotovorum ICMP 5702 were selected for subsequent screening by BOX‐PCR. Six isolates were chosen for sequencing the 16SrRNA gene and phylogenetic analysis. In BLAST analyses, the pathogenic isolates S2, S9, S22, S11, S23 and 111 showed the highest similarity values (≥99%) with the reference sequences of Raoultella terrigena, Klebsiella michiganensis, Erwinia (Winslowiella) iniecta, Pantoea agglomerans and P. carotovorum, respectively. These isolates induced soft rot and maceration on plant materials from five species (bell pepper fruits, garlic cloves and slices from sugar beet taproot, potato tubers and carrot roots) 72 h after inoculation. In the greenhouse experiment, they caused soft rot symptoms in the taproots of 2‐month‐old sugar beet plants 3 weeks after inoculation. These soft rot agents had Gram‐negative, catalase‐positive and oxidase‐negative reactions, grew under both aerobic and anaerobic conditions, tolerated and grew at 37°C and 5% NaCl. In general, the results of this research showed that in addition to Pectobacterium, four other genera from the order Enterobacterales including Erwinia, Pantoea, Klebsiella and Raoultella, are amongst the causes of soft rot in sugar beet in Firuzan region of Hamedan.

First Report of Pectobacterium polaris Causing Soft Rot on Broccoli in China.

In April 2023, soft rot symptoms were observed in broccoli (Brassica oleracea L. var. italica) commercial fields in Songming County, Yunnan province, China (103°12'E, 25°31'N). The disease incidence in these fields (6 ha in size) was high, exceeding 50%, and it caused significant yield loss. The affected plants displayed characteristic symptoms, with the roots and stems of broccoli becoming soft, yellowish-brown, rotten, and emitting a foul odor. To identify the causal agent, soft rot symptomatic stems were surface sterilized by dipping them in 75% ethanol for 30 seconds, followed by three successive rinses with sterile distilled water. Tissue specimens were then plated onto nutrient agar (NA) plates and incubated at 28°C for 24 hours. (Wang et al. 2022). Three representative bacterial isolates HYC22041801-HYC22041803 from broccoli were selected for further analysis. The colonies on NA plates appeared as white, small, round, and translucent with smooth edges. Physiological and biochemical tests were performed, along with 96 phenotypic screenings using the BIOLOG GENIII microplate system (Biolog, Hayward, CA, USA). Three isolates were negative for D-arabitol, maltose, and sorbitol, but were positive for cellobiose, α-D-glucose, sucrose, glycerol and gentiobiose tests, which are consistent with the reported type strain P. polaris NIBIO1006T (Chen et al. 2021). Total genomic DNA was extracted from three bacterial isolates using the QIAamp DNA Mini Kit (QIAGEN, USA). The 16S rRNA region and nine housekeeping genes (gapA, icdA, mdh, mtlD, pel, pgi, pmrA, proA and rpoS) were amplified with universal primers 27F/1492R (Monciardini et al., 2006) and designed specific primers (Xie et al., 2018), respectively. All amplicons were sequenced and deposited in GenBank with accession numbers ON723841-ON723843 and ON723846-ON723872. The BLASTn analysis of the 16S rRNA amplicons confirmed that the isolates HYC22041801-HYC22041803 belonged to the genus Pectobacterium. Phylogenetic trees based on 16S rRNA gene sequences and multilocus sequence analysis of other nine housekeeping genes of the three isolates were constructed and the results revealed that three isolates clustered with P. polaris type strain NIBIO1006T, which was previously isolated from potato (Dees et al., 2017). To confirm the pathogenicity, nine broccoli seedlings were stab inoculated with a bacterial suspension (108 CFU·ml-1), while sterile distilled liquid LB medium was used as a negative control. The seedlings were kept at 80% relative humidity and 28°C in a growth chamber. Three trials were conducted per isolate (HYC22041801-HYC22041803). After 3 days, the inoculated petioles showed soft rot symptoms similar to those observed initially in the field, while control plants remained asymptomatic. All three isolates were re-isolated successfully from symptomatic tissues to complete Koch's postulates. P. polaris has been previously reported as the causative agent of blackleg in potato in several countries, including Norway, Poland, Russia, and China (Handique et al. 2022; Wang et al. 2022). Additionally, it was reported to cause soft rot in Chinese cabbage in China (Chen et al. 2021). However, this is the first report of P. polaris causing soft rot disease in broccoli in China. This discovery is of great importance for vegetable growers because this bacterium is well established on Cruciferous vegetables in the local area, and effective measures are needed to manage this disease.

Role of Volatile Organic Compounds Produced by Kosakonia cowanii Cp1 during Competitive Colonization Interaction against Pectobacterium aroidearum SM2.

The competitive colonization of bacteria on similar ecological niches has a significant impact during their establishment. The synthesis speeds of different chemical classes of molecules during early competitive colonization can reduce the number of competitors through metabolic effects. In this work, we demonstrate for the first time that Kosakonia cowanii Cp1 previously isolated from the seeds of Capsicum pubescens R. P. produced volatile organic compounds (VOCs) during competitive colonization against Pectobacterium aroidearum SM2, affecting soft rot symptoms in serrano chili (Capsicum annuum L.). The pathogen P. aroidearum SM2 was isolated from the fruits of C. annuum var. Serrano with soft rot symptoms. The genome of the SM2 strain carries a 5,037,920 bp chromosome with 51.46% G + C content and 4925 predicted protein-coding genes. It presents 12 genes encoding plant-cell-wall-degrading enzymes (PCDEWs), 139 genes involved in five types of secretion systems, and 16 genes related to invasion motility. Pathogenic essays showed soft rot symptoms in the fruits of C. annuum L., Solanum lycopersicum, and Physalis philadelphica and the tubers of Solanum tuberosum. During the growth phases of K. cowanii Cp1, a mix of VOCs was identified by means of HS-SPME-GC-MS. Of these compounds, 2,5-dimethyl-pyrazine showed bactericidal effects and synergy with acetoin during the competitive colonization of K. cowanii Cp1 to completely reduce soft rot symptoms. This work provides novel evidence grounding a better understanding of bacterial interactions during competitive colonization on plant tissue, where VOC synthesis is essential and has a high potential capacity to control pathogenic microorganisms in agricultural systems.

Surviving the Potato Stems: Differences in Genes Required for Fitness by Dickeya dadantii and Dickeya dianthicola.

Bacteria belonging to the genus Dickeya cause blackleg and soft rot symptoms on many plant hosts, including potato. Although there is considerable knowledge about the genetic determinants that allow Dickeya to colonize host plants, as well as the genes that contribute to virulence, much is still unknown. To identify the genes important for fitness in potato stems, we constructed and evaluated randomly barcoded transposon mutant (RB-TnSeq) libraries of Dickeya dadantii and Dickeya dianthicola. We identified 169 and 157 genes important for growth in D. dadantii and D. dianthicola in stems, respectively. This included genes related to metabolic pathways, chemotaxis and motility, transcriptional regulation, transport across membranes, membrane biogenesis, detoxification mechanisms, and virulence-related genes, including a potential virulence cluster srfABC, c-di-GMP modulating genes, and pectin degradation genes. When we compared the results of the stem assay with other datasets, we identified genes important for growth in stems versus tubers and in vitro conditions. Additionally, our data showed differences in fitness determinants for D. dadantii and D. dianthicola. These data provide important insights into the mechanisms used by Dickeya when interacting with and colonizing plants and thus might provide targets for management.

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 .

First report of Pseudomonas marginalis causing tuber soft rot of potato in Iran

In 2016, potato tuber soft rot symptoms were observed in major potato production fields in Jiroft county (southern Kerman province, Iran). Bacteria, which appeared as white to creamy colonies, were isolated from diseased tissues and shown to be pathogenic on potato tubers after inoculation and re-isolation of the isolates. Based on the assessment of colony morphology, biochemical characteristics, and analysis of 16S rRNA and gyrB gene sequences, the pathogen was identified as Pseudomonas marginalis. To our knowledge, this is the first report of P. marginalis causing potato tuber soft rot disease in Iran.

First Report of Pectobacterium aroidearum Causing Soft Rot on Ficus carica in Korea

In July 2021, symptoms of soft rot were observed on the stems of <i>Ficus carica</i> in Yeongam, Jeollanamdo, Korea. To accurately diagnose the cause, infected stem was collected and bacterial strain was isolated. Among these, the pathogenic strain KNUB-08-21 was identified as <i>Pectobacterium aroidearum</i> through 16S rRNA gene sequencing and phylogenetic analysis based on the concatenated sequences of the <i>dnaX</i>, <i>leuS</i>, and <i>recA</i> genes. The affiliation of the isolate with this bacterial species was also confirmed by its biochemical characteristics obtained using API ID 32 GN system. Artificial inoculation confirmed the strain’s pathogenicity in figs, causing significant damage to both stems and fruits. To our knowledge, this is the first report of <i>P. aroidearum</i> causing soft rot disease in <i>F. carica</i> in Korea.

First report of Lasiodiplodia pseudotheobromae causing soft rot of plum in China.

Plum (Prunus salicina) is one of the most important fruit tree species worldwide (Valderrama-Soto et al. 2021). In June 2023, the postharvest soft rot symptoms were observed on plum fruits in several fruit markets of Guiyang city, Guizhou province, China. The disease incidence in these markets ranged from 20 to 25% with 70% disease severity. Plum fruits showed rotting, which was characterized by water soaked fruit tissue, softening and presence of whitish mycelia four days post inoculation. In severe conditions, whole fruits become rotted and were covered with white fungal mycelia. Small sections (5 × 3 mm) from 6 diseased plum fruits were surface sterilized by using 75% ethanol for 30 s followed by 0.1% mercuric chloride solution for 5 min, rinsed three times with ddH2O, and then transferred onto potato dextrose agar (PDA) and incubated at 25 ± 2°C for three days. Three pure cultures (GUCC23-0001 to GUCC23-0003) were obtained by transferring a single hyphal tip to new PDA plates. Colonies of these isolates were grayish-white initially, gradually turning to whitish brown with fluffy aerial mycelia and uneven edges and finally turned to a dark gray colony after five days of inoculation. The pseudoparaphyses were hyaline, cylindrical, aseptate, and rounded at apex. Conidia were ellipsoidal, hyaline, unicellular, and 24.2 to 28.6 × 12.3 to 15.5 µm in size (n = 30) (Fig. S1), which were similar to the morphology of Lasiodiplodia pseudotheobromae (Alves et al. 2008). Furthermore, fungal DNA was extracted from fresh mycelia of PDA after seven days by using fungus genomic DNA extraction kit (Biomiga, USA). Partial DNA sequences from four loci including internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1), beta-tubulin (tub2), and polymerase II second largest subunit (rpb2) were amplified with ITS1 and ITS4 (White et al. 1990), EF1-688F and EF1-1251R (Alves et al. 2008), Bt2a and Bt2b (Glass and Donaldson 1995), and RPB2-LasF and RPB2-LasR, respectively (Cruywagen et al. 2017). GenBank accession numbers are OR361680, OR361681, OR361682 for ITS, OR423394, OR423395, OR423396 for tef1, OR423397, OR423398, OR423399 for tub2, and OR423391, OR423392, OR423393 for rpb2, and gene sequencing showed 99.6 to 100% identity with ex-type strain of L. pseudotheobromae (CBS 116459). Phylogenetic analysis also placed our isolates in a highly supported clade with the reference isolate of L. pseudotheobromae (Fig. S2). Another experiment was designed to confirm the pathogenicity test for additional confirmation. Five mm mycelial plugs of L. pseudotheobromae from a three day old culture on PDA were placed on five surface-sterilized and non-wounded plum fruits for 12 hours and incubated at 25°C ± 2°C for four days. Sterilized fungus free PDA plugs were used as a negative control. Mycelial plugs were removed after 12 hours following which whole fruits were incubated in plastic boxes at 25°C ± 2°C. The experiment was repeated twice. The pathogenicity was evaluated under control conditions in laboratory (relative humidity, 70 ± 5% and temperature 25 ± 5˚C). Plum fruits showed rotting, which was characterized by water soaked fruit tissue, softening and presence of whitish mycelia four days post inoculation. These symptoms and signs were similar to the initially observed symptoms on plums in the markets. No disease symptoms were observed on the control fruits. The re-isolated fungus obtained from inoculated plum fruits was very similar to those isolated from diseased samples in morphology, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of L. pseudotheobromae causing postharvest fruit rot of plum in China. In 2022, the total planting area of plum was 1946.5 thousand hectares, which produces approximately 6626300 tons of plum (Food and Agriculture Organization of the United Nations, 2022). Based on the disease incidence and severity reported in the current study, soft rot of plum may be responsible for nearly 35% of yield losses under severe. Therefore, our study laid a theoretical foundation for the prevention and control of this post-harvest disease of plum.

Identification of non-canonical antagonistic bacteria via interspecies contact-dependent killing.

Canonical biocontrol bacteria were considered to inhibit pathogenic bacteria mainly by secreting antibiotic metabolites or enzymes. Recent studies revealed that some biocontrol bacteria can inhibit pathogenic bacteria through contact-dependent killing (CDK) mediated by contact-dependent secretion systems. The CDK was independent of antibiotic metabolites and often ignored in normal biocontrol activity assay. In this study, we aimed to use a pathogen enrichment strategy to isolate non-canonical bacteria with CDK ability. Rhizosphere soil samples from Chinese cabbage showing soft rot symptom were collected and Pectobacterium carotovorum subsp. carotovorum (Pcc), the pathogen of cabbage soft rot, were added into these samples to enrich bacteria which attached on Pcc cells. By co-culture with Pcc, four bacteria strains (named as PcE1, PcE8, PcE12 and PcE13) showing antibacterial activity were isolated from Chinese cabbage rhizosphere. These four bacteria strains showed CDK abilities to different pathogenic bacteria of horticultural plants. Among them, PcE1 was identified as Chryseobacterium cucumeris. Genome sequencing showed that PcE1 genome encoded a type VI secretion system (T6SS) gene cluster. By heterologous expression, four predicted T6SS effectors of PcE1 showed antibacterial activity to Escherichia coli. Overall, this study isolated four bacteria strains with CDK activity to various horticultural plant pathogens, and revealed possible involvement of T6SS of Chryseobacterium cucumeris in antibacterial activity. These results provide valuable insight for potential application of CDK activity in biocontrol bacteria. © 2024 Society of Chemical Industry.

Inoculation Methods to Determine Resistance of Phalaenopsis amabilis (L.) Regeneratded from Irradiated Protocorms to Dickeya dadantii

Soft-rot disease (SRD) in Phalaenopsis, caused Dickeya dadantii, has resulted in significant losses in the orchid sector in Indonesia. This study aimed to evaluate the inoculation method of Dickeya dadantii and identify the resistance response of individual regenerated plantlets of Phal. amabilis from irradiated protocorm. A detached leaf assay was used to evaluate the inoculation method and resistance response of SRD. Based on the results of this study, Dickeya dadantii bacteria could only infect the leaves through wounding tissue. The density of bacteria that could infect leaf tissue was OD600 = 0.2. All dilution factors tested caused soft rot symptoms in P. amabilis. On the other hand, Vanilla planifolia only showed symptoms at a dilution factor of 10-0. Four accessions of regenerated plantlets from irradiated protocorms were resistant to SRD. They were from irradiation 5 Gy (IP 05 Gy-23, IP 05 Gy-31, and IP 05 Gy-33) and one accession from the control treatment or without irradiation (IP 0 Gy -1). These results showed that 5 Gy irradiation increased plant resistance to SRD in Phalaenopsis. A dose of 5 Gy can potentially produce mutant lines resistant to SRD in Phalaenopsis or other plants, too.