Pectobacterium araliae sp. nov., a pathogen causing bacterial soft rot of Japanese angelica tree in Japan.

Phytopathogenic bacteria (MAFF 302110T and MAFF 302107) were isolated from lesions on Japanese angelica trees affected by bacterial soft rot in Yamanashi Prefecture, Japan. The strains were Gram-reaction-negative, facultatively anaerobic, motile with peritrichous flagella, rod-shaped, and non-spore-forming. The genomic DNA G+C content was 51.1 mol % and the predominant cellular fatty acids included summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 2 (comprising any combination of C12 : 0 aldehyde, an unknown fatty acid with an equivalent chain length of 10.928, C16 : 1 iso I, and C14 : 0 3OH), and C12 : 0. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences, along with phylogenomic analysis utilizing whole-genome sequences, consistently placed these strains within the genus Pectobacterium. However, their phylogenetic positions did not align with any known species within the genus. Comparative studies involving average nucleotide identity and digital DNA-DNA hybridization with the closely related species indicated values below the thresholds employed for the prokaryotic species delineation (95-96 % and 70 %, respectively), with the highest values observed for Pectobacterium polonicum DPMP315T (92.10 and 47.1 %, respectively). Phenotypic characteristics, cellular fatty acid composition, and a repertoire of secretion systems could differentiate the strains from their closest relatives. The phenotypic, chemotaxonomic, and genotypic data obtained in this study show that MAFF 302110T/MAFF 302107 represent a novel species of the genus Pectobacterium, for which we propose the name Pectobacterium araliae sp. nov., designating MAFF 302110T (=ICMP 25161T) as the type strain.

The endophytic fungal community plays a crucial role in the resistance of host plants to necrotic bacterial pathogens.

Konjac species (Amorphophallus spp.) are the only plant species in the world that are rich in a large amount of konjac glucomannan (KGM). These plants are widely cultivated as cash crops in tropical and subtropical countries in Asia, including China. Pectobacterium carotovorum subsp. carotovorum (Pcc) is one of the most destructive bacterial pathogens of konjac. Here, we analyzed the interactions between Pcc and susceptible and resistant konjac species from multiple perspectives. At the transcriptional and metabolic levels, the susceptible species A. konjac and resistant species A. muelleri exhibit similar molecular responses, activating plant hormone signaling pathways and metabolizing defense compounds such as phenylpropanoids and flavonoids to resist infection. Interestingly, we found that Pcc stress can lead to rapid recombination of endophytic microbial communities within a very short period (96 h). Under conditions of bacterial pathogen infection, the relative abundance of most bacterial communities in konjac tissue decreased sharply compared with that in healthy plants, while the relative abundance of some beneficial fungal communities increased significantly. The relative abundance of Cladosporium increased significantly in both kinds of infected konjac compared to that in healthy plants, and the relative abundance in resistant A. muelleri plants was greater than that in susceptible A. konjac plants. Among the isolated cultivable microorganisms, all three strains of Cladosporium strongly inhibited Pcc growth. Our results further elucidate the potential mechanism underlying konjac resistance to Pcc infection, highlighting the important role of endophytic microbial communities in resisting bacterial pathogen infections, especially the more direct role of fungal communities in inhibiting pathogen growth.

Bacteriophage cocktail for biocontrol of soft rot disease caused by Pectobacterium species in Chinese cabbage.

Pectobacterium spp. are necrotrophic plant pathogens that cause the soft rot disease in Chinese cabbage, resulting in severe yield loss. The use of conventional antimicrobial agents, copper-based bactericides, and antibiotics has encountered several limitations, such as bioaccumulation on plants and microbial resistance. Bacteriophages (phages) are considered promising alternative antimicrobial agents against diverse phytopathogens. In this study, we isolated and characterized two virulent phages (phiPccP-2 and phiPccP-3) to develop a phage cocktail. Morphological and genomic analyses revealed that two phages belonged to the Tevenvirinae and Mccorquodalevirinae subfamilies, respectively. The phiPccP-2 and phiPccP-3 phages, which have a broad host range, were stable at various environmental conditions, such as various pHs and temperatures and exposure to ultraviolet light. The phage cocktail developed using these two lytic phages inhibited the emergence of phage-resistant bacteria compared to single-phage treatments in in vitro challenge assays. The phage cocktail treatment effectively prevented the development of soft rot symptom in matured Chinese cabbage leaves. Additionally, the phage cocktail comprising three phages (phiPccP-1, phiPccP-2, and phiPccP-3) showed superior biocontrol efficacy against the mixture of Pectobacterium strains in Chinese cabbage seedlings. These results suggest that developing phage cocktails is an effective approach for biocontrol of soft rot disease caused by Pectobacterium strains in crops compared to single-phage treatments. KEY POINTS: •Two newly isolated Pectobacterium phages, phiPccP-2 and phiPccP-3, infected diverse Pectobacterium species and effectively inhibited the emergence of phage-resistant bacteria. •Genomic and physiological analyses suggested that both phiPccP-2 and phiPccP-3 are lytic phages and that their lytic activities are stable in the environmental conditions under which Chinese cabbage grows. •Treatment using a phage cocktail comprising phiPccP-2 and phiPccP-3 efficiently suppressed soft rot disease in detached mature leaves and seedlings of Chinese cabbage, indicating the applicability of the phage cocktail as an alternative antimicrobial agent.

T6SS and T4SS Redundantly Secrete Effectors to Govern the Virulence and Bacterial Competition in Pectobacterium PccS1.

Previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and virulence in many gram-negative bacteria. However, the role of T6SS in virulence in Pectobacterium atrosepticum remains controversial. We examined a closely related strain, PccS1, and discovered that its T6SS comprises a single-copy cluster of 17 core genes with a higher identity to homologs from P. atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS1, we found that three of the five VgrGs could be classified into group I VgrGs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and were dependent on either the T6SS or type IV secretion system (T4SS). This interaction directly governed virulence and competition. Meanwhile, supernatant proteomic analyses with strains defective in the T6SS and/or T4SS confirmed that effectors, such as FhaB, were secreted redundantly to control the virulence and suppress host callose deposition in the course of infection. Notably, this redundant secretion mechanism between the T6SS and T4SS is believed to be the first of its kind in bacteria.

Inter- and Intra-Specific Variations in Phenotypic Traits of Pectobacterium Strains Isolated from Diverse Eudicots and Monocots in Taiwan.

Pectobacterium spp. are phytopathogenic bacteria whose phylogeny has been continuously revised throughout the years. Previous studies on Pectobacterium's phenotypic diversity often analyzed strains obtained from specific crops or adopted outdated Pectobacterium classification systems. Therefore, a current perspective on trait variations in Pectobacterium species or strains infecting more diverse plant species is limited. This study conducted phylogenetic and phenotypic analyses on strains isolated from eight eudicot and four monocot families in Taiwan. Phylogenetic analysis on 78 strains identified six recognized species, namely, P. brasiliense, P. aroidearum, P. actinidiae, P. colocasium, P. carotovorum, and P. versatile. Among these, the first two were the most predominant species. Patterns suggesting varying host preferences among bacterial species were detected; most P. aroidearum strains were isolated from monocots, whereas P. brasiliense and P. actinidiae tended to exhibit preferences for eudicots. Physiological tests and Biolog analyses conducted on representative strains of each species revealed great within-species phenotypic variations. Despite these strain-level variations, a combination of indole production and phosphatase activity tests was capable of distinguishing all representative strains of P. brasiliense from those of other identified species. Inoculation assays on potato, bok choy, calla lily, and onion showed inter- and intra-specific heterogeneities in the tested strains' maceration potentials. Virulence patterns across Pectobacterium species and strains differed depending on the inoculated host. Altogether, the findings from this work expand the understanding of Pectobacterium's phenotypic diversity and provide implications for pathogen identification and management.

Genome-Wide Identification and Expression Profile Analysis of Sugars Will Eventually Be Exported Transporter (SWEET) Genes in Zantedeschia elliottiana and Their Responsiveness to Pectobacterium carotovora subspecies Carotovora (Pcc) Infection.

SWEET, sugars will eventually be exported transporter, is a novel class of sugar transporter proteins that can transport sugars across membranes down a concentration gradient. It plays a key role in plant photosynthetic assimilates, phloem loading, nectar secretion from nectar glands, seed grouting, pollen development, pathogen interactions, and adversity regulation, and has received widespread attention in recent years. To date, systematic analysis of the SWEET family in Zantedeschia has not been documented, although the genome has been reported in Zantedeschia elliottiana. In this study, 19 ZeSWEET genes were genome-wide identified in Z. elliottiana, and unevenly located in 10 chromosomes. They were further clustered into four clades by a phylogenetic tree, and almost every clade has its own unique motifs. Synthetic analysis confirmed two pairs of segmental duplication events of ZeSWEET genes. Heatmaps of tissue-specific and Pectobacterium carotovora subsp. Carotovora (Pcc) infection showed that ZeSWEET genes had different expression patterns, so SWEETs may play widely varying roles in development and stress tolerance in Zantedeschia. Moreover, quantitative reverse transcription-PCR (qRT-PCR) analysis revealed that some of the ZeSWEETs responded to Pcc infection, among which eight genes were significantly upregulated and six genes were significantly downregulated, revealing their potential functions in response to Pcc infection. The promoter sequences of ZeSWEETs contained 51 different types of the 1380 cis-regulatory elements, and each ZeSWEET gene contained at least two phytohormone responsive elements and one stress response element. In addition, a subcellular localization study indicated that ZeSWEET07 and ZeSWEET18 were found to be localized to the plasma membrane. These findings provide insights into the characteristics of SWEET genes and contribute to future studies on the functional characteristics of ZeSWEET genes, and then improve Pcc infection tolerance in Zantedeschia through molecular breeding.

RpoS-Regulated Genes and Phenotypes in the Phytopathogenic Bacterium Pectobacterium atrosepticum.

The alternative sigma factor RpoS is considered to be one of the major regulators providing stress resistance and cross-protection in bacteria. In phytopathogenic bacteria, the effects of RpoS have not been analyzed with regard to cross-protection, and genes whose expression is directly or indirectly controlled by RpoS have not been determined at the whole-transcriptome level. Our study aimed to determine RpoS-regulated genes and phenotypes in the phytopathogenic bacterium Pectobacterium atrosepticum. Knockout of the rpoS gene in P. atrosepticum affected the long-term starvation response, cross-protection, and virulence toward plants with enhanced immune status. The whole-transcriptome profiles of the wild-type P. atrosepticum strain and its ΔrpoS mutant were compared under different experimental conditions, and functional gene groups whose expression was affected by RpoS were determined. The RpoS promoter motif was inferred within the promoter regions of the genes affected by rpoS deletion, and the P. atrosepticum RpoS regulon was predicted. Based on RpoS-controlled phenotypes, transcriptome profiles, and RpoS regulon composition, the regulatory role of RpoS in P. atrosepticum is discussed.

Impact of Homologous Recombination on Core Genome Evolution and Host Adaptation of Pectobacterium parmentieri.

Homologous recombination is a major force mechanism driving bacterial evolution, host adaptability, and acquisition of novel virulence traits. Pectobacterium parmentieri is a plant bacterial pathogen distributed worldwide, primarily affecting potatoes, by causing soft rot and blackleg diseases. The goal of this investigation was to understand the impact of homologous recombination on the genomic evolution of P. parmentieri. Analysis of P. parmentieri genomes using Roary revealed a dynamic pan-genome with 3,742 core genes and over 55% accessory genome variability. Bayesian population structure analysis identified 7 lineages, indicating species heterogeneity. ClonalFrameML analysis displayed 5,125 recombination events, with the lineage 4 exhibiting the highest events. fastGEAR analysis identified 486 ancestral and 941 recent recombination events ranging from 43 bp to 119 kb and 36 bp to 13.96 kb, respectively, suggesting ongoing adaptation. Notably, 11% (412 genes) of the core genome underwent recent recombination, with lineage 1 as the main donor. The prevalence of recent recombination (double compared to ancient) events implies continuous adaptation, possibly driven by global potato trade. Recombination events were found in genes involved in vital cellular processes (DNA replication, DNA repair, RNA processing, homeostasis, and metabolism), pathogenicity determinants (type secretion systems, cell-wall degrading enzymes, iron scavengers, lipopolysaccharides (LPS), flagellum, etc.), antimicrobial compounds (phenazine and colicin) and even CRISPR-Cas genes. Overall, these results emphasize the potential role of homologous recombination in P. parmentieri's evolutionary dynamics, influencing host colonization, pathogenicity, adaptive immunity, and ecological fitness.

Molecular characterization of Pectobacterium atrosepticum infecting potato and its management through chemicals.

Potato farming is a vital component of food security and the economic stability especially in the under developing countries but it faces many challenges in production, blackleg disease caused by Pectobacterium atrosepticum (Pa) is one of the main reason for damaging crop yield of the potato. Effective management strategies are essential to control these losses and to get sustainable potato crop yield. This study was focused on characterizing the Pa and the investigating new chemical options for its management. The research was involved a systematic survey across the three district of Punjab, Pakistan (Khanewal, Okara, and Multan) to collect samples exhibiting the black leg symptoms. These samples were analyzed in the laboratory where gram-negative bacteria were isolated and identified through biochemical and pathogenicity tests for Pa. DNA sequencing further confirmed these isolates of Pa strains. Six different chemicals were tested to control blackleg problem in both vitro and vivo at different concentrations. In vitro experiment, Cordate demonstrated the highest efficacy with a maximum inhibition zones of 17.139 mm, followed by Air One (13.778 mm), Profiler (10.167 mm), Blue Copper (7.7778 mm), Spot Fix (7.6689 mm), and Strider (7.0667 mm). In vivo, Cordate maintained its effectiveness with the lowest disease incidence of 14.76%, followed by Blue Copper (17.49%), Air One (16.98%), Spot Fix (20.67%), Profiler (21.45%), Strider (24.99%), and the control group (43.00%). The results highlight Cordate's potential as a most effective chemical against Pa, offering promising role for managing blackleg disease in potato and to improve overall productivity.

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.

Multilocus sequence and phenotypic analysis of Pectobacterium and Dickeya type strains for identification of soft rot Pectobacteriaceae from symptomatic potato stems and tubers in Pennsylvania.

Outbreaks of potato blackleg and soft rot caused by Pectobacterium species and more recently Dickeya species across the U.S. mid-Atlantic region have caused yield loss due to poor emergence as well as losses from stem and tuber rot. To develop management strategies for soft rot diseases, we must first identify which members of the soft rot Pectobacteriaceae are present in regional potato plantings. However, the rapidly expanding number of soft rot Pectobacteriaceae species and the lack of readily available comparative data for type strains of Pectobacterium and Dickeya hinder quick identification. This manuscript provides a comparative analysis of soft rot Pectobacteriaceae and a comprehensive comparison of type strains from this group using rep-PCR, MLSA and 16S sequence analysis, as well as phenotypic and physiological analyses using Biolog GEN III plates. These data were used to identify isolates cultured from symptomatic potato stems collected between 2016 and 2018. The isolates were characterized for phenotypic traits and by sequence analysis to identify the bacteria from potatoes with blackleg and soft rot symptoms in Pennsylvania potato fields. In this survey, P. actinidiae, P. brasiliense, P. polonicum, P. polaris, P. punjabense, P. parmentieri, and P. versatile were identified from Pennsylvania for the first time. Importantly, the presence of P. actinidiae in Pennsylvania represents the first report of this organism in the U.S. As expected, P. carotorvorum and D. dianthicola were also isolated. In addition to a resource for future work studying the Dickeya and Pectobacterium associated with potato blackleg and soft rot, we provide recommendations for future surveys to monitor for quarantine or emerging soft rot Pectobacteriace regionally.

Transcriptional Response of Pectobacterium carotovorum to Cinnamaldehyde Treatment.

Cinnamaldehyde is a natural compound extracted from cinnamon bark essential oil, acclaimed for its versatile properties in both pharmaceutical and agricultural fields, including antimicrobial, antioxidant, and anticancer activities. Although potential of cinnamaldehyde against plant pathogenic bacteria like Agrobacterium tumefaciens and Pseudomonas syringae pv. actinidiae causative agents of crown gall and bacterial canker diseases, respectively has been documented, indepth studies into cinnamaldehyde's broader influence on plant pathogenic bacteria are relatively unexplored. Particularly, Pectobacterium spp., gram-negative soil-borne pathogens, notoriously cause soft rot damage across a spectrum of plant families, emphasizing the urgency for effective treatments. Our investigation established that the Minimum Inhibitory Concentrations (MICs) of cinnamaldehyde against strains P. odoriferum JK2, P. carotovorum BP201601, and P. versatile MYP201603 were 250 µg/ml, 125 µg/ml, and 125 µg/ml, respectively. Concurrently, their Minimum Bactericidal Concentrations (MBCs) were found to be 500 µg/ml, 250 µg/ml, and 500 µg/ml, respectively. Using RNA-sequencing analysis, we identified 1,907 differentially expressed genes in P. carotovorum BP201601 treated with 500 µg/ml cinnamaldehyde. Notably, our results indicate that cinnamaldehyde upregulated nitrate reductase pathways while downregulating the citrate cycle, suggesting a potential disruption in the aerobic respiration system of P. carotovorum during cinnamaldehyde exposure. This study serves as a pioneering exploration of the transcriptional response of P. carotovorum to cinnamaldehyde, providing insights into the bactericidal mechanisms employed by cinnamaldehyde against this bacterium.

Differences in the constituents of bacterial microbiota of soils collected from two fields of diverse potato blackleg and soft rot diseases incidences, a case study.

The presence of bacteria from the Dickeya spp. and Pectobacterium spp. in farmlands leads to global crop losses of over $420 million annually. Since 1982, the scientists have started to suspect that the development of disease symptoms in crops might be inhibited by bacteria present in the soil. Here, we characterized in terms of physicochemical properties and the composition of bacterial soil microbiota two fields differing, on the basis of long-term studies, in the occurrence of Dickeya spp.- and Pectobacterium spp.-triggered infections. Majority, i.e. 17 of the investigated physicochemical features of the soils collected from two fields of either low or high potato blackleg and soft rot diseases incidences turned out to be similar, in contrast to the observed 4 deviations in relation to Mg, Mn, organic C and organic substance contents. By performing microbial cultures and molecular diagnostics-based identification, 20 Pectobacterium spp. strains were acquired from the field showing high blackleg and soft rot incidences. In addition, 16S rRNA gene amplicon sequencing followed by bioinformatic analysis revealed differences at various taxonomic levels in the soil bacterial microbiota of the studied fields. We observed that bacteria from the genera Bacillus, Rumeliibacillus, Acidobacterium and Gaiella turned out to be more abundant in the soil samples originating from the field of low comparing to high frequency of pectinolytic bacterial infections. In the herein presented case study, it is shown for the first time that the composition of bacterial soil microbiota varies between two fields differing in the incidences of soft rot and blackleg infections.

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

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

Diversity of pectobacterium strains by biochemical, physiological, and molecular characterization / Diversidade de cepas de Pectobacterium por caracterização bioquímica, fisiológica e molecular

Pectobacterium is a complex taxon of strains with diverse characteristics. It comprises several genera, including Erwinia, Brenneria, Pectobacterium, Dickeya, and Pantoea. Pectobacterium and Dickeya cause diseases in a wide range of plants, including potatoes, where they are causative agents of soft rot in tubers and blackleg in field-grown plants.Characterizing Pectobacterium species allows for the analysis of the diversity of pectinolytic bacteria, which may support control strategies for plant bacterial diseases. The aim of this study was to perform biochemical, physiological, and molecular characterizations of Pectobacteriumspp. from different sites and host plants. The isolated strains were characterized by the glucose fermentation test, Gram staining, catalase activity, oxidase activity, growth at 37 ºC, reducing substances from sucrose, phosphatase activity, indole production, acid production from different sources (sorbitol, melibiose, citrate, and lactose), pathogenicity in potato, and hypersensitivity reactions. Molecular characterization was performed with species-specific primers ECA1f/ECA2r and EXPCCF/EXPCCR, which identify P.atrosepticum and P.carotovorum subsp. carotovorum (Pcc), respectively, and with primers 1491f/L1RA/L1RG and Br1f/L1RA/L1RG that differentiate Pcc from Dickeya chrysanthemi and from P. carotovorum subsp. brasiliensis. The strains were identified as belonging to the genus Pectobacterium, though they did not fit the biochemical nor the molecular classification standards for subspecies differentiation, indicating significant diversity among the strains.

Pectobacterium é um táxon complexo de isolados bacterianos com características diversas. Compreende vários gêneros como Erwinia, Brenneria, Pectobacterium, Dickeya e Pantoea. Pectobacterium e Dickeya causam doenças em ampla variedade de plantas, incluindo a batateira, na qual são os agentes etiológicos da podridão mole dos tubérculos e da canela-preta de plantas cultivadas em campo.A caracterização de espécies de Pectobacterium permite a análise da diversidade de bactérias pectolíticas, podendo auxiliar estratégias de controle de doenças bacterianas em plantas. O objetivo deste trabalho foi caracterizar bioquímica, fisiológica e molecularmente isolados de Pectobacterium sp. provenientes de diferentes locais e hospedeiros. Os isolados foram caracterizados pelos testes de fermentação de glicose, Gram, catalase, oxidase, crescimento à 37 ºC, redução de substâncias a partir de sacarose, atividade da fosfatase, produção de indol, produção de ácido a partir de sorbitol, melibiose, citrato e lactose, patogenicidade em batata e reação de hipersensibilidade. Para a caracterização molecular, foram utilizados os pares de primersECA1f/ECA2r e EXPCCF/EXPCCR [específicos para P. atrosepticum e P.carotovorum subsp. carotovorum(Pcc), respectivamente] e as tríades de primers 1491f/L1RA/L1RG e Br1f/L1RA/L1RG, para diferenciar Pcc de Dickeya chrysanthemi e de P. carotovorum subsp. brasiliensis. Os isolados foram identificados como pertencentes ao gênero Pectobacterium, no entanto, não se enquadraram na classificação bioquímica e tampouco molecular para diferenciação das subespécies, demonstrando a grande diversidade dos mesmos.

Identificación, clonación y actividad lactonasa de la proteína recombinante de N-ácil homoserina lactonasa (AiiA) de Bacillus thuringiensis cepa 147-115-16 / Identification, cloning and lactonase activity of recombinant protein of N-acyl homoserine lactonase (AiiA) from Bacillus thuringiensis 147-115-16 strain

The quorum-quenching N-acyl homoserine lactonases are a family of bacterial metalloenzymes that participate in degradation of N-acyl homoserine lactones (AHLs), disrupting the quorum sensing system of gram negative bacterial species. From a collection of Bacillus thuringiensis strains isolated in Colombia from plants and exhibiting toxic activity against lepidopteran insects, 310 bacterial isolates were tested to determine lactonase activity by using biosensor systems in presence of synthetic N-hexanoyl-L-homoserine lactone (C6-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). From them, 251 strains showed degrading activity to both C6-HSL and C8-HSL, 57% exhibited degrading activity to C6-HSL and 43% to C8-HSL. One B. thuringiensis strain, denoted as 147-115-16, that exhibit high degrading activity to C6-HSL and C8-HSL, was able to attenuate soft rot symptoms in infected potato slices with Pectobacterium carotovorum. This strain contains an homologous of the aiiA gene that was cloned, sequenced and expressed in Esherichia coli DE3. The recombinant protein AiiA147-11516 display activity to C6-HSL, C8-HSL, N-(β-ketocaproyl) (3-O-C6-HSL) and N-3-oxo-dodecanoyl (3-O-C12-HSL). The recombinant strain in the presence of P. caratovorum cultures was able to attenuate the infection, suggesting that it interferes either on the accumulation or response to the AHLs signals. Acording to this data and based on previous report from recombinant AiiA147-11516, this enzyme exhibit activity to wide range of catalytic substrates suggesting its industrial application in the disease control programs by plants transformation.

Las N-acíl homoserina lactonasas son una familia de metaloenzimas bacterianas que participan en la degradación de N-acil homoserina lactonas (AHLs) interrumpiendo el sistema de detección de quórum sensing de bacterias Gram negativas. A partir de una colección de cepas de Bacillus thuringiensis aisladas del filoplano de plantas colombianas que presentaron actividad tóxina contra insectos lepidópteros, 310 fueron probadas para determinar actividad lactonasa mediante el uso de sistemas de biosensores en presencia de N-hexanoilo-L-homoserina lactona (C6-HSL) y la N-octanoilo-L-homoserina lactona (C8-HSL) sintéticas. De estas cepas, el 251 mostraron actividad para ambas lactonas y de estas, el 57% mostró actividad a C6-HSL y el 43% a C8-HSL. Una cepa de B. thuringiensis- denominada 147-115-16- que mostró alta actividad para C6-HSL y C8-HSL, fue capaz de atenuar los síntomas de la pudrición blanda en rodajas de papa infectadas con Pectobacterium carotovorum. Esta cepa contiene un gen homólogo a aiiA, el cual este fue clonado, secuenciado y expresado en Escherichia coli DE3. La proteína recombinante AiiA147-11516 exhibe actividad para C6-HSL y C8-HSL, así como para N-(β-cetocaproil) (3-O-C6-HSL) y N-3-oxo-dodecanoil (3-O-C12-HSL). La cepa recombinante en presencia de P. caratovorum fue capaz de atenuar la infección, sugiriendo que interfiere con la acumulación o respuesta de las señales AHLs. Según estos datos y basados en el reporte previo sobre la actividad hidrolítica de la proteína recombinante AiiA147-11516, esta enzima posee un actividad contra un amplio número de sustratos lo cual sugiere su aplicación en la industria en el control de enfermedades, mediante la transformación de plantas.

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.