Soft Rot Pathogen Pectobacterium Research Articles

Pseudomonas fluorescens MFE01 delivers a putative type VI secretion amidase that confers biocontrol against the soft-rot pathogen Pectobacterium atrosepticum.

The type VI secretion system (T6SS) is a contractile nanomachine widespread in Gram-negative bacteria. The T6SS injects effectors into target cells including eukaryotic hosts and competitor microbial cells and thus participates in pathogenesis and intermicrobial competition. Pseudomonas fluorescens MFE01 possesses a single T6SS gene cluster that confers biocontrol properties by protecting potato tubers against the phytopathogen Pectobacterium atrosepticum (Pca). Here, we demonstrate that a functional T6SS is essential to protect potato tuber by reducing the pectobacteria population. Fluorescence microscopy experiments showed that MFE01 displays an aggressive behaviour with an offensive T6SS characterized by continuous and intense T6SS firing activity. Interestingly, we observed that T6SS firing is correlated with rounding of Pectobacterium cells, suggesting delivery of a potent cell wall targeting effector. Mutagenesis coupled with functional assays then revealed that a putative T6SS secreted amidase, Tae3Pf , is mainly responsible for MFE01 toxicity towards Pca. Further studies finally demonstrated that Tae3Pf is toxic when produced in the periplasm, and that its toxicity is counteracted by the Tai3Pf inner membrane immunity protein.

PREVALENCE OF CAUSATIVE AGENTS OF RING ROT CLAVIBACTER SEPEDONICUS, BLACKLEG AND WET ROT PECTOBACTERIUM ATROCEPTICUM IN THE 2021 YEAR POTATO HARVEST IN THE TERRITORY OF UKRAINE

Aim. To investigate the prevalence of causative agents of ring rot and soft rot in the potato harvest by the DAS ELISA method. Materials and methods. Samples of the potato harvest from Ternopil, Zhytomyr, Kyiv, Cherkasy, Dnipropetrovsk, Donetsk, Mykolaiv, Odesa, and Kherson regions were used in the study. The detection of bacterial agents was conducted by enzyme-linked immunosorbent assay using commercial test systems LOEWE® Standard Complete Kit (Germany). Results. The highest percentage of affected potatoes was registered in the Donetsk region: 11.1% – by the Clavibacter seedonicus, 31.0% – by the Pectobacterium atrosepticum, and 8.9% – by both bacteria. The causative agent of soft rot was detected twice as often as a causative agent of ring rot in tuber samples from all regions, except the Ternopil region, where the predominance of Clavibacter sepedonicus was revealed. In addition to the Donetsk region, a high prevalence of the causative agent of soft rot was found in the potato harvest from Kyiv (10.2%) and Odesa (10.0%) regions, the highest abundance of the causative agent of ring rot was detected in the Ternopil region. The mixed bacterial infection of potato tubers was diagnosed in 6 areas, and the highest rate was detected in the Donetsk region. Conclusion. The analysis of the distribution of ring rot pathogen Clavibacter sepedonicus and soft rot pathogen Pectobacterium atrocepticum in the potato crop shows the presence of research pathogens in all studied regions. It was discovered the dominance of the representative of pectolytic bacteria in all regions except Ternopil, where the distribution of Clavibacter sepedonicus, the causative agent of ring rot, was dominated. High rates of spread of the studied pathogens were associated with increased local average annual precipitation, as well as with increased annual amplitude of this indicator.

Genome Wide Analysis of the Potato Soft Rot Pathogen Pectobacterium carotovorum Strain ICMP 5702 to Predict Novel Insights into Its Genetic Features.

Pectobacterium carotovorum subsp. carotovorum (Pcc) is a gram-negative, broad host range bacterial pathogen which causes soft rot disease in potatoes as well as other vegetables worldwide. While Pectobacterium infection relies on the production of major cell wall degrading enzymes, other virulence factors and the mechanism of genetic adaptation of this pathogen is not yet clear. In the present study, we have performed an in-depth genome-wide characterization of Pcc strain ICMP5702 isolated from potato and compared it with other pathogenic bacteria from the Pectobacterium genus to identify key virulent determinants. The draft genome of Pcc ICMP5702 contains 4,774,457 bp with a G + C content of 51.90% and 4,520 open reading frames. Genome annotation revealed prominent genes encoding key virulence factors such as plant cell wall degrading enzymes, flagella-based motility, phage proteins, cell membrane structures, and secretion systems. Whereas, a majority of determinants were conserved among the Pectobacterium strains, few notable genes encoding AvrE-family type III secretion system effectors, pectate lyase and metalloprotease in addition to the CRISPR-Cas based adaptive immune system were uniquely represented. Overall, the information generated through this study will contribute to decipher the mechanism of infection and adaptive immunity in Pcc.

Transformation of Inferior Tomato into Preservative: Fermentation by Multi-Bacteriocin Producing Lactobacillus paracasei WX322

Loss and waste of postharvest vegetables are the main challenges facing the world’s vegetable supply. In this study, an innovative method of value-added transformation was provided: production of bacteriocin from vegetable waste, and then its application to preservation of vegetables. Antibacterial activity to soft rot pathogen Pectobacterium cartovorum (Pcb BZA12) indicated that tomato performed best in the nutrition supply for bacteriocin production among 12 tested vegetables. Moreover, the antibacterial activity was from Lactobacillus paracasei WX322, not components of vegetables. During a fermentation period of 10 days in tomato juice, L. paracasei WX322 grew well and antibacterial activity reached the maximum on the tenth day. Thermostability and proteinase sensitivity of the bacteriocin from tomato juice were the same with that from Man-Rogosa-Sharpe broth. Scanning electron microscope images indicated that the bacteriocin from tomato juice caused great damage to Pcb BZA12. At the same time, the bacteriocin from tomato juice significantly reduced the rotten rate of Chinese cabbage from 100% ± 0% to 20% ± 8.16% on the third day during storage. The rotten rate decrease of cucumber, tomato, and green bean was 100% ± 0% to 0% ± 0%, 70% ± 14.14% to 13.33% ± 9.43%, and 76.67% ± 4.71% to 26.67% ± 4.71%, respectively. Bacteriocin treatment did not reduce the rotten rate of balsam pear, but alleviated its symptoms.

Controlling pepper soft rot by Lactobacillus paracasei WX322 and identification of multiple bacteriocins by complete genome sequencing

Bacterial soft rot is a devastating disease that harms vegetables. Lactic acid bacteria (LAB) can be effective antagonist against soft rot pathogens. In this study, Lactobacillus paracasei WX322 and its produced bacteriocin had antibacterial activities against a variety of foodborne pathogens and spoilage bacteria, including both Gram-positive and Gram-negative bacteria, especially the soft rot pathogen Pectobacterium carotovorum subsp. brasiliense BZA12 (Pcb BZA12). The in vivo antibacterial test proved that the bacteriocin produced by the L. paracasei WX322 could control soft rot disease of peppers caused by Pcb BZA12. The L.paracasei WX322 produced bacteriocin after fermentation for 36 h, which had broad antimicrobial activity. In addition, the bacteriocin produced by L. paracasei WX322 had good thermal stability and broad pH adaptation. Then, the complete genome of L. paracasei WX322 was sequenced with one circular chromosome and eight plasmids. Three bacteriocin gene clusters were identified in the genome by antiSMASH. Furthermore, seven genes in the three gene clusters were identified to be bacteriocins. Moreover, three of the seven bacteriocins were novel. The complete genome sequences of the L. paracasei WX322 can provide a better molecular basis of its antibacterial activity and biocontrol effect on bacterial soft rot disease.

Horizontally Acquired Quorum-Sensing Regulators Recruited by the PhoP Regulatory Network Expand the Host Adaptation Repertoire in the Phytopathogen Pectobacterium brasiliense.

In this study, we examine the impact of transcriptional network rearrangements driven by horizontal gene acquisition in PhoP and SlyA regulons using as a case study a phytopathosystem comprised of potato tubers and the soft-rot pathogen Pectobacterium brasiliense 1692 (Pb1692). Genome simulations and statistical analyses uncovered the tendency of PhoP and SlyA networks to mobilize lineage-specific traits predicted as horizontal gene transfer at late infection, highlighting the prominence of regulatory network rearrangements in this stage of infection. The evidence further supports the circumscription of two horizontally acquired quorum-sensing regulators (carR and expR1) by the PhoP network. By recruiting carR and expR1, the PhoP network also impacts certain host adaptation- and bacterial competition-related systems, seemingly in a quorum sensing-dependent manner, such as the type VI secretion system, carbapenem biosynthesis, and plant cell wall-degrading enzymes (PCWDE) like cellulases and pectate lyases. Conversely, polygalacturonases and the type III secretion system (T3SS) exhibit a transcriptional pattern that suggests quorum-sensing-independent regulation by the PhoP network. This includes an uncharacterized novel phage-related gene family within the T3SS gene cluster that has been recently acquired by two Pectobacterium species. The evidence further suggests a PhoP-dependent regulation of carbapenem- and PCWDE-encoding genes based on the synthesized products' optimum pH. The PhoP network also controls slyA expression in planta, which seems to impact carbohydrate metabolism regulation, especially at early infection, when 76.2% of the SlyA-regulated genes from that category also require PhoP to achieve normal expression levels.IMPORTANCE Exchanging genetic material through horizontal transfer is a critical mechanism that drives bacteria to efficiently adapt to host defenses. In this report, we demonstrate that a specific plant-pathogenic species (from the Pectobacterium genus) successfully integrated a population density-based behavior system (quorum sensing) acquired through horizontal transfer into a resident stress-response gene regulatory network controlled by the PhoP protein. Evidence found here underscores that subsets of bacterial weaponry critical for colonization, typically known to respond to quorum sensing, are also controlled by PhoP. Some of these traits include different types of enzymes that can efficiently break down plant cell walls depending on the environmental acidity level. Thus, we hypothesize that PhoP's ability to elicit regulatory responses based on acidity and nutrient availability fluctuations has strongly impacted the fixation of its regulatory connection with quorum sensing. In addition, another global gene regulator, known as SlyA, was found under the PhoP regulatory network. The SlyA regulator controls a series of carbohydrate metabolism-related traits, which also seem to be regulated by PhoP. By centralizing quorum sensing and slyA under PhoP scrutiny, Pectobacterium cells added an advantageous layer of control over those two networks that potentially enhances colonization efficiency.

Efficiency of Vegetable Soft Rot Pathogen Pectobacterium carotovorum subsp. carotovorum(Pcc21) as a Host Cell for Foreign Gene Expression

외래유전자 형질발현을 증폭하기 위해 형질발현 벡터의 개발뿐만 아니라 박테리아, 곰팡이, 효모, 곤충, 식물 및 동물세포와 같은 다양한 숙주세포의 개발이 시도되어 왔다. 특히 DH5α, XL1-blue, Top10 및 BL21과 같은 대장균(Escherichia coli)는 단백질 생산과 형질전환에 가장 널리 사용되고 있는 숙주 중 하나이며, LBA4404, GV3101 및 EHA105와 같은 아그로박테리움(Agrobacterium tumefaciens)은 식물 형질전환에 흔히 사용되고 있다. 그러나 여전히 의학 및 생명공학 분야에서는 단백질 생산을 위한 새로운 숙주세포 개발에 대한 요구가 있다. 최근 감자 무름병균인 펙토박테리아(Pectobacterium carotovorum subsp. carotovorum)는 사용의 편리함 때문에 새로운 숙주세포로써의 가능성에 대한 관심을 얻게 되었다. 이번 연구에서 E. coli(DH5α), A. tumefaciens(LBA4404) 및 P. carotovorum(Pcc21)을 포함하는 다양한 숙주세포에서 GFP 단백질생산 효율을 조사하였다. GFP 전사량은 RT-PCR로 확인하였고 단백질은 Western blot 분석으로 분석하였다. 그 결과 Pcc21은 DH5α와 LBA 4404와 비교했을 때 30% 정도 더 높은 GFP 단백질을 생산하였고 전사량 또한 증가하였다. 따라서 펙토박테리아 Pcc21이 외래 단백질 발현을 위한 새로운 숙주세포로써의 가능성이 높은 것으로 생각된다. 더 나아가 펙토박테리아는 쉽게 식물에 감염되기 때문에 A. tumefaciens 대신 식물의 일시적, 영구적 형질전환을 위한 숙주세포로 사용할 수 있을 것이라 생각된다.

Exogenous application of phenylacetic acid promotes root hair growth and induces the systemic resistance of tobacco against bacterial soft-rot pathogen Pectobacterium carotovorum subsp. carotovorum.

Phenylacetic acid (PAA) was evaluated for its capability to promote plant growth and induce systemic resistance in tobacco (Nicotianum tabacum L cv. Xanthi) against the bacterial soft rot pathogen Pectobacterium carotovorum subsp. carotovorum (PCC). Exogenous application of PAA influenced root formation, the activities of defence-related enzymes and the expression of defence and growth-related genes. Increased formation of lateral roots can be observed in tobacco treated with higher PAA concentrations. The highest elicitation of induced systemic resistance (ISR) was found in plants treated with 0.5mM PAA, where the phytotoxic effect was minimal. The activities of the defence enzymes phenylalanine ammonia-lyase (PAL), peroxidase (POD) and polyphnenoloxidase (PPO) were modulated upon treatment with different PAA concentrations. Reverse transcription-PCR analyses showed that 0.5mM PAA modulated the expression of the growth-related genes NtEXP2 and NtEXP6, and the defence-related genes Coi1, NPR1, PR-1a and PR-1b. These results showed that different concentrations of PAA can elicit different responses and effects on tobacco growth and resistance. This study presents the important role of PAA not only on plant growth but also for plant immunity against phytopathogens.

Interference of Quorum Sensing by Delftia sp. VM4 Depends on the Activity of a Novel N-Acylhomoserine Lactone-Acylase.

BackgroundTurf soil bacterial isolate Delftia sp. VM4 can degrade exogenous N-acyl homoserine lactone (AHL), hence it effectively attenuates the virulence of bacterial soft rot pathogen Pectobacterium carotovorum subsp. carotovorum strain BR1 (Pcc BR1) as a consequence of quorum sensing inhibition.Methodology/Principal FindingsIsolated Delftia sp. VM4 can grow in minimal medium supplemented with AHL as a sole source of carbon and energy. It also possesses the ability to degrade various AHL molecules in a short time interval. Delftia sp. VM4 suppresses AHL accumulation and the production of virulence determinant enzymes by Pcc BR1 without interference of the growth during co-culture cultivation. The quorum quenching activity was lost after the treatment with trypsin and proteinase K. The protein with quorum quenching activity was purified by three step process. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) and Mass spectrometry (MS/MS) analysis revealed that the AHL degrading enzyme (82 kDa) demonstrates homology with the NCBI database hypothetical protein (Daci_4366) of D. acidovorans SPH-1. The purified AHL acylase of Delftia sp. VM4 demonstrated optimum activity at 20–40°C and pH 6.2 as well as AHL acylase type mode of action. It possesses similarity with an α/β-hydrolase fold protein, which makes it unique among the known AHL acylases with domains of the N-terminal nucleophile (Ntn)-hydrolase superfamily. In addition, the kinetic and thermodynamic parameters for hydrolysis of the different AHL substrates by purified AHL-acylase were estimated. Here we present the studies that investigate the mode of action and kinetics of AHL-degradation by purified AHL acylase from Delftia sp. VM4.SignificanceWe characterized an AHL-inactivating enzyme from Delftia sp. VM4, identified as AHL acylase showing distinctive similarity with α/β-hydrolase fold protein, described its biochemical and thermodynamic properties for the first time and revealed its potential application as an anti-virulence agent against bacterial soft rot pathogen Pectobacterium carotovorum subsp. carotovorum based on quorum quenching mechanism.

Effect of a biofilm‐degrading enzyme from an oral pathogen in transgenic tobacco on the pathogenicity of Pectobacterium carotovorum subsp. carotovorum

The hypothesis that dispersin B (DspB), an enzyme from the periodontal pathogen Aggregatibacter actinomycetemcomitans that degrades the extracellular matrix polysaccharide PGA, will inhibit biofilm formation of the soft rot pathogen Pectobacterium carotovorum subsp. carotovorum in infected plants was tested by constitutive expression of DspB in tobacco plants. All the transgenic plants expressed properly folded and active DspB enzyme, although at different expression levels. In virulence assays, even the transgenic plant line D10, which produced a low level of DspB compared to other lines, showed significant resistance against P. carotovorum subsp. carotovorum, suggesting that DspB could be a valuable agent for biological control of P. carotovorum subsp. carotovorum infection in crop plants.

Pectobacterium carotovorumElicits Plant Cell Death with DspE/F but theP. carotovorumDspE Does Not Suppress Callose or Induce Expression of Plant Genes Early in Plant–Microbe Interactions

The broad-host-range bacterial soft rot pathogen Pectobacterium carotovorum causes a DspE/F-dependent plant cell death on Nicotiana benthamiana within 24 h postinoculation (hpi) followed by leaf maceration within 48 hpi. P. carotovorum strains with mutations in type III secretion system (T3SS) regulatory and structural genes, including the dspE/F operon, did not cause hypersensitive response (HR)-like cell death and or leaf maceration. A strain with a mutation in the type II secretion system caused HR-like plant cell death but no maceration. P. carotovorum was unable to impede callose deposition in N. benthamiana leaves, suggesting that P. carotovorum does not suppress this basal immunity function. Within 24 hpi, there was callose deposition along leaf veins and examination showed that the pathogen cells were localized along the veins. To further examine HR-like plant cell death induced by P. carotovorum, gene expression profiles in N. benthamiana leaves inoculated with wild-type and mutant P. carotovorum and Pseudomonas syringae strains were compared. The N. benthamiana gene expression profile of leaves infiltrated with Pectobacterium carotovorum was similar to leaves infiltrated with a Pseudomonas syringae T3SS mutant. These data support a model where Pectobacterium carotovorum uses the T3SS to induce plant cell death in order to promote leaf maceration rather than to suppress plant immunity.

Combining flow cytometry andgfpreporter gene for quantitative evaluation ofPectpbacterium carotovorumssp.carotovoruminOrnithogalum dubiumplantlets

Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta. Several calibration steps were required to distinctly gate the GFP-labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads. The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta. The combination of time- and cost-saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.

Identification of an ISR‐related metabolite produced by rhizobacterium Klebsiella oxytoca C1036 active against soft‐rot disease pathogen in tobacco

Klebsiella oxytoca C1036 (C1036) causes induced systemic resistance (ISR) activity against the soft-rot pathogen Pectobacterium carotovorum subsp. carotovorum SCC1 (SCC1). However, microbial metabolites from C1036 involved in ISR activity remain unknown. The present study was performed to identify an ISR-related metabolite produced by C1036. The supernatants of C1036 cultures grown on Luria-Bertani medium were subjected to solvent extraction, repeated column chromatography and preparative liquid chromatography for isolation of an ISR-related metabolite. High-resolution mass spectrometer analysis of the isolated metabolite indicated a C9H15O3N compound with a mass of 185.11. Low-resolution mass spectrometer analysis of the metabolite showed a molecular ion peak at 185 and its fragment ions at 84 and 56. Nuclear magnetic resonance spectrometer analyses characterised all protons and carbons of the isolated metabolite. Based on the data, the isolated metabolite was determined to be butyl 2-pyrrolidone-5-carboxylate (BPC). BPC at 12 mM significantly suppressed the disease symptoms in ISR bioassays against SCC1. This is the first report identifying BPC as an ISR-related metabolite produced by C1036. C1036 may play a role in promoting plant growth because it produces ISR-related metabolites against the plant pathogen SCC1.

Overexpression of the pineapple fruit bromelain gene (BAA) in transgenic Chinese cabbage (Brassica rapa) results in enhanced resistance to bacterial soft rot

Bromelain is a crude protein extract obtained from pineapple stems, which comprises a variety of proteolytic enzymes. It exhibits potential therapeutic activities against trauma, inflammation, autoimmune diseases and malignant disorders. In this study, we cloned BAA1 (the gene encoding fruit bromelain) into a plant expression vector that was then used to transform Brassica rapa and overexpress BAA1 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. We demonstrate that constitutive overexpression of BAA1 in B. rapa confers enhanced resistance to the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum. These results suggest that it could be utilized for protecting plants from attack by bacterial pathogens.