IDENTIFICATION AND GENETIC DIVERSITY OF PECTOLYTIC PHYTOPATHOGENIC BACTERIA OF MONO- AND DICOTYLEDONOUS ORNAMENTAL PLANTS IN IRAN

Pectobacterium is one of the major destructive causal agent in most crop plants throughout the world. During a survey in spring of 2005 in the rangeland of Kermanshah and Isfahan, provinces of Iran, samples of bulbs and stems of crown imperial with brown spot and soft rot were collected. Eight strains of pectolytic Erwinia were isolated and purified from these samples. Phenotypic tests indicated that the strains were gram-negative, facultative anaerobic, rod shaped, motile with peritrichous flagella. They were oxidase negative, catalase positive and also able to macerate potato slices. Pathogenicity of all the strains were confirmed on corn, philodendron and crown imperial by inoculation of these crops with a bacterial suspension and reisolation of the strain from symptomatic tissues. A pair of specific PCR primers was used to detect these bacterial strains. The primer set (EXPCCF/EXPCCR) amplified a single fragment of the expected size (0.55 kb) from genomic DNA of all strains used in this study. In nested PCR, the primer set (INPCCR/INPCCF) amplified the expected single fragment (0.4 kb) from the PCR product of first PCR amplification. On the basis of the biochemical and phenotypic characteristics and PCR amplification by the specific PCR primers, these strains were identified as Pectobacterium carotovorum subsp. carotovorum. This is the first report of occurrence of crown imperial bacterial soft-rot in Iran.

A severe soft rot disease was observed in some commercial artichoke crops (F1-hybrid cultivars) during 2014 in the Bari, Foggia (Apulia, Southern Italy) and Matera (Basilicata, Southern Italy) provinces. Symptoms were chlorosis and wilting of the older leaves accompanied by dark-green to dark-brown soft rotting of the pith. Several bacterial isolates were obtained from affected plants by culturing on semi-selective crystal violet pectate agar medium. Biochemical, physiological and molecular analyses and pathogenicity tests, performed using five selected isolates, identified three of them as Pectobacterium carotovorum subsp. brasiliense. To the best of our knowledge, this is the first report of a bacterial soft rot caused by P. carotovorum subsp. brasiliense in Italy and the first finding of this bacterial subspecies on artichoke.

During visiting different greenhouses in Mahalat (Markazi province, Iran) in 2012-2013, bacterial soft rot symptoms were observed on Opuntia and Haworthia spp. These consisted of small brown to black water-soaked lesions that increased in size with time, heavily damaging the affected plants. After evaluation of the pectolytic activity on a medium containing polygalacturonic acid using two indicators, 17 bacterial isolates were selected and identified as Pectobacterium carotovorum subsp. carotovorum based on phenotypic features and specific PCR amplification using two sets of primers. All tested isolates were pathogenic and caused the same type of symptoms upon artificial inoculation. A hypersensitive reaction was induced in tobacco leaves infiltrated with bacterial suspensions. In a phylogenetic tree constructed with sequences of the housekeeping genes recA and pmrA based on individual and combined data sets the Iranian isolates clustered together with Pc subsp. carotovorum ATCC15713, in two subgroups with high statistical support. Those from Opuntia sp. were in the same cluster separated from the Haworthia strains by strong bootstrap values, indicating that the two selected genes could identify Pectobacterium at the subspecies level and monitor the diversity of Pcc subspecies.

ABSTRACTAs a result of its domestication, breeding and subsequent commercialization, African violet (Saintpaulia ionantha H. Wendl.) has become the most famous and popular Saintpaulia species. There is interest in producing cultivars that have increased resistance to pests and low temperature, in the introduction of novel horticultural characteristics such as leaf shape, flower colour, size and form, and in improved productivity and enhanced flower duration in planta. In African violet, techniques such as the application of chemical mutagens (ethylmethanesulfonate, N-nitroso-N-methylurea), radiation (gamma (γ)-rays, X-rays, carbon ion beams) and colchicine have been successfully applied to induce mutants. Among these techniques, γ radiation and colchicine have been the most commonly applied mutagens. This review offers a short synthesis of the advances made in African violet breeding, including studies on mutation and somaclonal variation caused by physical and chemical factors, as well as transgenic strategies using Agrobacterium-mediated transformation and particle bombardment. In African violet, Agrobacterium-mediated transformation is affected by the Agrobacterium strain, selection marker, and cutting-induced wounding stress. Somaclonal variation, which arises in tissue cultures, can be problematic in maintaining true-to-type clonal material, but may be a useful tool for obtaining variation in flower colour. The only transgenic African violet plants generated to date with horticulturally useful traits are tolerant to boron (heavy metal) stress, or bear a glucanase-chitinase gene.

This study was aimed to isolate, identify and characterize Pectobacterium spp. causing soft rot disease of cabbage and Chinese cabbage in Central Poland. Of fifty two plant samples of cabbage and Chinese cabbage showing disease symptoms collected in Central Poland from 2007-2010, 542 bacterial isolates were obtained. Of isolates 117 caused soft rot on cabbage and Chinese cabbage leaves and potato slices and showed pectinolytic activity on crystal violet pectate medium. PCR using Y1/Y2 primers specific for Pectobacterium genus revealed that twenty three of them belonged to this genus. Phenotypic characterization in combination with DNA-based typing methods (rep-PCRs) and sequence analysis of 16S rRNA, and housekeeping genes gyrB , infB , rpoB , atpD , and rpoS (MultiLocus Sequence Typing) done for 12 Polish representatives and reference strains allowed for their identification and revealed genetic diversity. Additional phylogeny analysis of the rpoS gene for Polish and worldwide well defined Pectobacterium spp. strains allowed to classify obtained strains into Pectobacterium spp. phylogenetic clusters (PcI-PcV). The presented studies showed that 8 of 12 isolated Pectobacterium carotovorum strains belong to the Pectobacterium carotovorum subsp. carotovorum ( Pcc ) cluster PcII and other four strains belong to the subspecies Pectobacterium carotovorum subsp. odoriferum ( Pco ) cluster PcIII. To our knowledge, this is the first report that P. carotovorum subsp. odoriferum cause soft rot of cabbage and Chinese cabbage in Central Poland.

A highly efficient genetic transformation system is essential for a successful genetic manipulation of the African violet (Saintpaulia ionantha Wendl.). Developing a particle bombardment-based genetic transformation system for the African violet. A local cultivar of the African violet from Guilan province was used for transformation experiments. The pFF19G and pBin61-Ech42 vectors were used for transient and stable transformation experiments, respectively. The PCR and RT-PCR techniques were used to verify transgene presence and transcript levels in candidate transgenic lines, respectively. Using leaf explants as target tissues, we transferred an endochitinase gene cDNA into African violet. Transgenic plants were regenerated on selection medium at a reasonable frequency (in average, one stable transgenic line per shot). Molecular analysis of transgenic plants by PCR and RT-PCR techniques confirmed successful integration and expression of transgene in several independent transgenic lines. Our results provide an efficient stable transformation system for genetic transformation of African violet.

The main objectives of this study were to evaluate and model the influence of temperature (10, 15 and 20°C), relative humidity (86, 96 and 100%) and initial concentration of bacterial inoculum (105, 107 et 109 CFU ml-1) on the population density of Pectobacterium atrosepticum (Pba) and Pectobacterium carotovorum subsp. carotovorum (Pcc) which are important potato pathogens in temperate climates, and on the development of soft rot symptoms caused by these bacteria at the surface of wounded potatoes tubers under controlled conditions. Experiments were carried out according to a Box-Behnken experimental design, simplifying prediction of the combined effects of three controlled factors. With both bacterial species, statistical analysis showed a significant effect of temperature, relative humidity and initially applied bacterial concentration on population dynamics and soft rot development at the surface of wounded potato tubers. Multiple regression analyses and the contour plots showed that the temperature is the most important factor, followed by the initially applied bacteria concentration and relative humidity. More than 64% of the variability of the soft rot symptoms observed could be explained by the presence of Pba and Pcc at the level of wounded potato tubers under the combined effect of tested factors. The quadratic polynomial models developed in our research should integrate the heterogeneity of tested bacteria belonging to the same species (which was not evaluated in this preliminary investigation) in further research.

Bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is one of the most severe diseases in radish cultivation. To control this plant disease, the most effective method has been known to cultivate resistant cultivars. Previously, we developed an efficient bioassay method for investigating resistance levels with 21 resistant and moderately resistant cultivars of radish against a strain Pcc KACC 10421. In this study, our research expanded to investigate the resistance of radish cultivars against six Pcc strains, KACC 10225, KACC 10421, ATCC 12312, ATCC 15713, LY34, and ECC 301365. To this end, the virulence of the six Pcc strains was determined based on the development of bacterial soft rot in seedlings of four susceptible radish cultivars. The results showed that the Pcc strains exhibited different virulence in the susceptible cultivars. To explore the race differentiation of Pcc strains corresponding to the resistance in radish cultivars, we investigated the occurrence of bacterial soft rot caused by the six Pcc strains on the 21 resistant and moderate resistant cultivars. Our results showed that the average values of the area under the disease progress curve were positively correlated with the virulence of the strains and the number of resistant cultivars decreased as the virulence of Pcc strains increased. Taken together, our results suggest that the resistance to Pcc of the radish cultivars commercialized in Korea is more likely affected by the virulence of Pcc strains rather than by race differentiation of Pcc.

In vitro screening techniques were used to evaluate 46 genotypes of Iranian potato collection for resistance to bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc). One month old in vitro rooted potato plantlets were inoculated by two inoculation techniques under in vitro conditions: 1) sterile toothpicks dipped into bacterial suspension and pressed into the crown of plantlets and 2) the freshly cut crown of plantlets were dipped into bacterial suspension of 108 cfu ∙ ml-1 for 10 min. Typical soft rot disease symptoms, including the percentage of wilted leaves were recorded on inoculated plantlets 3, 6, 9, 12 and 15 days post-inoculation. The potato genotypes which were examined responded differently to Pcc and varying levels of resistance were observed. Potato genotype AG showed the highest level of resistance. Results obtained from in vitro screening were then verified by classical tuber slice assay. The verifications were conducted using five representative cultivars: Milva, Ramus, Picaso, Marfona and Agria which responded similarly to both in vitro and classical evaluation systems. Similar results obtained from these tests indicated that the in vitro screening technique developed in this study could provide a simple and rapid whole plant assay in selecting resistant potato genotypes against bacterial soft rot.

Cabbage is an important vegetable crop worldwide. In Taiwan, during cabbage production, bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum often leads to significant yield losses. Aligning with the Sustainable Development Goals, there is a high demand for sustainable disease control strategies. Silicates are considered to be effective elicitors in activating plant defense responses and are reported to improve resistance to certain plant diseases. Bacillus amyloliquefaciens PMB05 fermentation liquid has been shown to enhance plant immunity and control many bacterial diseases. The supplementation of silicates to the PMB05 fermentation liquid may further improve its efficacy to control bacterial soft rot in cabbage. This study evaluated the effects of sodium metasilicate and potassium silicate on PMB05-mediated plant immune responses and disease control. Initial assays confirmed that treatment with B. amyloliquefaciens PMB05 suspension significantly increased HrpN-triggered reactive oxygen species (ROS) generation and callose deposition; moreover, PMB05 treatment alone reduced bacterial soft rot severity by 39.7%. When combined with B. amyloliquefaceins PMB05 fermentation liquid, sodium metasilicate at 2000 μM further enhanced ROS generation and callose deposition by 100% and 133%, respectively, compared to the treatment of PMB05 alone (p < 0.05). In contrast, potassium silicate exhibited inconsistent effects on ROS production, with both 500 and 1000 µM concentrations significantly reducing ROS generation by 26% and 38%, respectively, while none of the tested concentrations affected callose deposition (p < 0.05). Lastly, disease severity assessments in cabbage inoculated with P. carotovorum subsp. carotovorum PCCSB1 revealed that B. amyloliquefaciens PMB05 fermentation liquid was able to reduce bacterial soft rot symptoms by 60.3%. Supplementation with 1500 and 2000 µM sodium metasilicate further decreased disease severity by 77.9% and 76.4%, respectively (p < 0.05). Although the supplementation of potassium silicate also significantly reduced disease severity compared to P. carotovorum subsp. carotovorum PCCSB1 alone, it was less effective than PMB05 fermentation alone. Overall, these results demonstrate that sodium metasilicate enhances the biocontrol activity of B. amyloliquefaciens PMB05 by further intensifying plant immune responses. This approach may broaden the large-scale use of B. amyloliquefaciens PMB05 fermentation liquid for sustainable soft rot management in cabbage, although the stability and cost-effectiveness of sodium metasilicate under field conditions still require validation.

African violet (Saintpaulia ionantha H. Wendl) is one of the most easily and commonly tissue-cultured ornamental plants. Despite this, there are limited reports on photosynthetic capacity and its impact on the plant quality during acclimatization. Various growth, photosynthetic and biochemical parameters and activities of antioxidant enzymes and dehydrins of micropropagated plants were assessed under three light intensities (35, 70, and 100 µmol m−2 s−1 photosynthetic photon flux density – PPFD). Fresh and dry plant biomass, plant height, and leaf area were optimal with high irradiance (70–100 µmol m−2 s−1 PPFD). Chlorophyll and carotenoid contents and net photosynthesis were optimal in plants grown under 70 µmol m−2 s−1 PPFD. Stomatal resistance, malondialdehyde content, and Fv/Fm values were highest at low light irradiance (35 µmol m−2 s−1 PPFD). The activities of three antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, increased as light irradiance increased, signaling that high light irradiance was an abiotic stress. The accumulation of 55, 33, and 25 kDa dehydrins was observed with all light treatments although the expression levels were highest at 35 µmol m−2 s−1 PPFD. Irradiance at 70 µmol m−2 s−1 PPFD was suitable for the acclimatization of African violet plants. Both low and high irradiance levels (35 and 100 µmol m−2 s−1 PPFD) induced the accumulation of antioxidants and dehydrins in plants which reveals enhanced stress levels and measures to counter it.

Pectobacterium carotovorum subsp. carotovorum causing soft rot of calla (Zantedeschia aethiopica) plants is reported in central Italy. Bacterial isolates were identified by biochemical, physiological and pathogenicity tests and characterized by the API 50 CHE Kit and rep-PCR.

Chinese cabbage (Brassica rapa L. ssp. pekinensis), an important vegetable crop, can succumb to diseases such as bacterial soft rot, resulting in significant loss of crop productivity and quality. Pectobacterium carotovorum ssp. carotovorum (Pcc) causes soft rot disease in various plants, including Chinese cabbage. To overcome crop loss caused by bacterial soft rot, a gene from Chinese cabbage was isolated and characterised in this study. We isolated the BrWRKY12 gene from Chinese cabbage, which is a group II member of the WRKY transcription factor superfamily. The 645-bp coding sequence of BrWRKY12 translates to a protein with a molecular mass of approximately 24.4 kDa, and BrWRKY12 was exclusively localised in the nucleus. Transcripts of BrWRKY12 were induced by Pcc infection in Brassica. Heterologous expression of BrWRKY12 resulted in reduced susceptibility to Pcc but not to Pseudomonas syringae pv. tomato in Arabidopsis. Defence-associated genes, such as AtPDF1.2 and AtPGIP2, were constitutively expressed in transgenic lines overexpressing BrWRKY12. The expression of AtWKRY12, which is the closest orthologue of BrWRKY12, was down-regulated by Pcc in Arabidopsis. However, the Atwrky12-2 mutants did not show any difference in response to Pcc, pointing to a difference in function of WRKY12 in Brassica and Arabidopsis. Furthermore, BrWRKY12 in Chinese cabbage also exhibited enhanced resistance to bacterial soft rot and increased the expression of defence-associated genes. In summary, BrWRKY12 confers enhanced resistance to Pcc through transcriptional activation of defence-related genes.

Bacterial soft rots of banana and plantains involve two different diseases caused by related but distinct pathogens: rhizome soft rot or head rot by Pectobacterium carotovorum. subsp. carotovorum and pseudostem wet rot and rhizome rot - in some parts of the world called rhizome necrosis and corm rot-, caused by Dickeya paradisiaca. Although considered minor diseases, they cause losses of individual plants, yield reduction and raise the cost of production. Bacterial soft rots are found in wet humid soils with poor drainage, and high pH, during prolonged wet periods. Affected plants show poor shoot emergence, dwarfing, scorching of the lower leaves, and wilting and rot of the youngest emerging leaves. Rhizome infection causes a pseudostem wet rot progressing upward from infected rhizome and from petioles downward and a spongy necrotic collar at the union of rhizome with the pseudostem. Bacterial soft rot pathogens can be diagnosed by microbiological and molecular techniques. Disease management relies on use of healthy planting material, plant to plant tools disinfection and improving soil drainage and cropping practices.

The integral defense responses of plants triggered by the small molecules of plant pathogens are regarded as plant immunity. The pathogen-associated molecular pattern-triggered immunity (PTI) occurs on the recognition of a pathogen by receptors on plant cell surfaces as an infection begins. During the activation of PTI, the effectiveness of a plant's photosynthetic system may be altered. In this study, chlorophyll fluorescence was used to assay the dynamic changes of PTI. When we used flg22Pst as an elicitor, we found that the photosynthetic electron transport rate (ETR) of Arabidopsis thaliana Col-0 was significantly decreased at 2, 4, and 24 h on treatment with a PTI-intensifying protein, plant ferredoxin-like protein (PFLP). In addition, this reduction in the photosynthetic ETR was also carried out with a PTI-intensifying Bacillus amyloliquefaciens strain, PMB05, on the induction of flg22Pst. The disease resistance against bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) was still enhanced by PMB05. Interestingly, among the eight tested Bacillus species strains, the PTI triggered by HrpNPcc from P. carotovorum subsp. carotovorum exhibited an ETR that was significantly decreased by PMB05. Furthermore, this decrease was consistent with rapid H2O2 generation and callose deposition triggered by HrpNPcc and the disease resistance against bacterial soft rot. Taken together, such results led us to conclude that the assay based on the ETR established in this study can be used as a model for evaluating the effectiveness of plant immunity-intensifying microbes for controlling plant diseases.