Tissue Maceration Research Articles

Parasitism of micromycete Sclerotinia sclerotiorum (lib.) de bary on the dahlia plants (Dahlia cav.) under different weather conditions

The white mold of dahlia is a dangerous disease that remains unexplored in Ukraine. In order to study the symptoms of the disease and the impact of meteorological conditions on its development, a study was conducted in 2015–2017 in Kyiv. In studying the diagnostic signs of the disease, a visual examination, and a biological method of diagnosis were used. The development of the disease was estimated at a six-point scale. As a result of phytopathological monitoring, the stalk form of white mold was found. Substantial damage to plants resulted in tissue maceration and fragmentation of stems and peduncles. The development of the disease of the stem led to plant wilting. During the years of research, white mold on dahlia manifested itself in the autumnal period of plant growth during mass flowering. Meteorological conditions have significantly influenced its distribution and development. In particular, in 2015, Sclerotinia white mold of dahlia manifested itself from the first decade of September for a hydrothermal coefficient (HTC) of 1.0. In 2016, the disease appeared on plants in the first decade of October, when the HTC was 5.7. The first plants affected by white mold in 2017 were marked by the second decade of September for the HTC 1,2. It was that year when the disease has reached the maximum spread and development, which is due to the presence of precipitation from the second decade of September and until the end of October, as well as the absence of low temperatures during this period. Information about the dates of the appearance of the white mold on the dahlia deserves attention when planning a set of protective measures. In prospect in depth analysis of the influence of various meteorological factors on the development of white mold on dahlia should be used in drawing up models of the prediction of disease development in this culture.

Preservation and maceration of amazon açai leaflet tissue to obtain genomic DNA

The objective of this study was to test the efficiency of preservation and maceration methods for Euterpe precatoria leaflet tissue to obtain genomic DNA for molecular studies. The leaflets of E. precatoria were collected in an experimental field at Embrapa Acre, Brazil. The study was conducted in a completely randomized design with 10 replicates, in a 12 × 2 factorial structure, with 12 storage treatments (fresh; lyophiliser 3 days; refrigerator 3, 5, and 7 days; silica gel 7, 10, 20, and 30 days; and transport buffer 3, 5, and 7 days) and two leaf tissue maceration methods (liquid nitrogen and the TissueLyser®). Statistically significant differences in the obtained DNA concentration were found between the maceration and storage treatments. The TissueLyser® macerator produced higher DNA concentrations when compared to liquid nitrogen. For the storage treatments, five groups were formed based on DNA concentration when macerated with the TissueLyser® and two groups when macerated with liquid nitrogen. The DNA concentrations ranged from 285.00 ng/µL (7 days in transport buffer) to 702.00 ng/µL (30 days in silica gel) when the leaflets were macerated with liquid nitrogen, and from 572.73 ng/µL (30 days in silica gel) to 2,850.00 ng/µL (3 days in lyophiliser) using the TissueLyser® macerator. The DNA purity (A260/A280 nm) varied from 1.30 to 1.70 when the leaflets were macerated with liquid nitrogen and from 1.30 to 1.90 with the TissueLyser® macerator. Despite the variations in leaf tissue preservation and DNA concentration, all treatments were effective for DNA isolation and it was possible to amplify genomic regions of microsatellite markers by PCR. It was concluded that leaflets of E. precatoria stored in a lyophiliser and processed with an automatic macerator resulted in satisfactory DNA for molecular studies.

Involvement of abscisic acid in the resistance of citrus fruit to Penicillium digitatum infection

The involvement of hormone abscisic acid (ABA) in citrus fruit resistance to Penicillium digitatum infection was investigated. To this end, the effect of both exogenous ABA and inhibitors of ABA biosynthesis in plants on the resistance of sweet oranges to be infected by this major postharvest pathogen, and also on in vitro fungal growth, was examined. The ability of both P. digitatum to infect wild-type Navelate orange fruit (Citrus sinesis (L.) Osbeck) and its spontaneous ABA-deficient mutant Pinalate was compared. The results showed that the Pinalate orange was more susceptible than its parental to infection, and exogenous ABA reduced the percentage of infected fruits and also the maceration zone of this mutant without affecting in vitro fungal growth. The global results indicated that the ABA of the fruit plays a defensive role in citrus fruit against P. digitatum infection. This work demonstrates, for the first time, the ability of P. digitatum to produce ABA. The hormone decreased in fruit in early stages as a consequence of infection. Thereafter when tissue maceration became evident, ABA increased, and this rise seemed related to the P. digitatum-induced rise in ethylene production. This was suggested by the fact that ABA increased in Navelate orange, but not in its ABA-deficient mutant Pinalate in this infection stage, while exogenous ethylene barely increased ABA in the mutant. Finally, the sharp marked rise in free ABA in later stages was likely related to free ABA being released from conjugated ABA, and also to fungal ABA. However, fungal ABA was not a primary virulence factor in citrus fruit infection.

PHIALOMYCES MACROSPORUS REDUCES CERCOSPORA COFFEICOLA SURVIVAL ON SYMPTOMATIC COFFEE LEAVES

<p>Saprobe fungi and necrotrophic pathogens share the same niche within crop stubble and the search for fungi non-pathogenic to plants that are able to displace the plant pathogens from its overwintering substrate contributes to the disease management. Brown eye spot (<em>Cercospora coffeicola</em>) is among the most important coffee diseases, it is caused by a necrotrophic pathogen that has decaying leaves as its major source of inoculum. We have screened saprobe fungi for the ability to reduce <em>C. coffeicola</em> sporulation and viability and determined the possible mechanisms involved in the observed biocontrol. A selected saprobe fungus, <em>Phialomyces macrosporus</em>, reduced the pathogen’s viability by 40% both <em>in vitro</em> and <em>in vivo</em>. The fungus acts through antibiosis and competition for nutrients. It produced both volatile and non-volatile compounds that inhibited <em>C. coffeicola</em> growth, sporulation, and viability. It also produced the tissue maceration enzyme (polygalacturonase), which reduces the pathogen both in detached leaves or in planta. The reduction in the fungal viability either by the saprobe fungus or its polygalacturonase-fraction supernatant resulted in the reduction of the disease rate. Therefore, <em>P. macrosporus </em>is a potential microbial agent that can be used in an integrated management of brown eye spot through the reduction of the initial inoculum of the pathogen that survives and builds up in infected leaves.</p><p> </p>

Quantitative Differences in Feeding Behavior of Lygus lineolaris (Hemiptera: Miridae) on Transgenic and Nontransgenic Cotton.

Lygus lineolaris (Palisot de Beauvois) is one of the most important pests on cotton in the United States. Previous research showed that transgenic cotton plants expressing the Bacillus thurigiensis (Bt) crystalline protein Cry51Aa2.834_16 (designated MON 88702) have insecticidal effects on nymphal L. lineolaris. The present study is the first to examine effects of a Bt-expressing cotton on feeding by a heteropteran like L. lineolaris. We compared stylet probing behaviors of third-instar nymphs on pin-head squares (i.e., buds <3 mm wide) of MON 88702 cotton versus nontransgenic (control) DP393 plants using AC-DC electropenetrography. Waveforms were quantified based on appearances previously characterized and correlated with adult L. lineolaris feeding behaviors; nymphal and adult waveforms had the same appearance. Generalized third-instar feeding included maceration of tissues during cell rupturing (waveform CR), tasting/testing during a waveform called transition (T), and ingestion (I); all were similar between MON 88702 and DP393 plants. However, the number of events and duration of each waveform were different between treatments. Relative to nymphs on DP393, those on MON 88702 spent more time overall in stylet probing, due to increased number of maceration events per probe and longer durations of tasting/testing, per waveform event, per probe, and per insect; yet, ingestion events were shorter and more frequent. These findings support that MON 88702 cotton plants were less palatable and/or preorally digestible to L. lineolaris nymphs than DP393, suggesting antixenosis for MON 88702. Transgenic cotton antixenosis could positively affect cotton pest management by reducing feeding of L. lineolaris nymphs and protecting crop yield.

Histological Studies of Cassava (Manihot esculenta Crantz) Root Infection Using Aspergillus niger Rhizopus stolonifer, Aspergillus niger, Aspergillus flavus, Botryodiplodia theobromae and Trichoderma viride

Histological studies were carried out to investigate the impact of some pathogenic organisms on infected cassava root after different periods of incubation. Cassava roots inoculated with Aspergillus niger, Rhizopus stolonifer, Aspergillus flavus, and Trichoderma viride showed progressive depletion of the starch grains in the cells and tissue maceration of the cassava root as the period of incubation increased. Cassava root inoculated with A. flavus showed tissue necrosis and slight fragmentation of the starch grains whereas, cassava tissues infected with A. niger showed tissue necrosis and general maceration of the starch grains within the tissue thereby creating empty spaces within the cells with no clear arrangement of the starch grains. Inoculation of the cassava tissues with R. stolonifer resulted in rapid loss of the starch grains and erosion of surrounding cell boundaries. Cassava tissue colonized by T. viride showed tissue necrosis and maceration of the starch grains. Healthy (uninoculated) cassava root tissue showed intact cells packed with large starch grains suggesting that in the host-parasite relationship, the pathogens established themselves inter and intracellularly in the parenchymatous cells of the cortex of the cassava root clearing them of starch grains, and resulting to poor yield and nutritional value of cassava and also economic loss.

Compatible Mixture of Bacterial Antagonists Developed to Protect Potato Tubers from Soft Rot Caused by Pectobacterium spp. and Dickeya spp.

Possibilities to protect potato tubers from rotting caused by Soft Rot Pectobacteriaceae (SRP) under disease favoring conditions were investigated using compatible mixtures of bacterial antagonists and tested with a newly developed stepwise efficacy-based screening protocol. Twenty-two bacterial antagonists were evaluated against a combination of five Pectobacterium and Dickeya strains representing species and subspecies most often associated with potato soft rot in Europe. To enable potential synergistic activity, the antagonists were initially tested against the combination of pathogens in 15 random mixtures containing up to 5 antagonists each. Three mixtures (M2, M4, and M14) out of 15 tested reduced tuber tissue maceration due to soft rot. The individual antagonists derived from M2, M4, and M14 mixtures were tested on potato slices and whole tuber injection assays. These five strains (S. plymuthica strain A294, E. amnigenus strain A167, R. aquatilis strain H145, S. rubidaea strain H440, and S. rubidaea strain H469) were combined to develop a tailored biological control mixture against potato soft rot. The new mixture, designated the Great Five (GF), was tested on seed potato tubers vacuum infiltrated with antagonists and subsequently with the combination of five SRP pathogens. In these experiments, the GF mixture provided stable protection of inoculated potato tubers, reducing soft rot by 46% (P = 0.0016) under high disease pressure conditions. The A294, A167, H145, H440, and H469 antagonists were characterized for features important for viable commercial applications including growth at different temperatures, resistance to antibiotics, and potential toxicity toward Caenorhabditis elegans. The implications for control of soft rot caused by SRP with the use of the GF mixture of antagonists are discussed.

Host-induced gene silencing of Pectobacterium carotovorum quorum sensing gene enhances soft rot disease resistance in potato plants

Quorum sensing is a regulatory mechanism of gene expression in bacteria which regulated many physiological processes such as production of virulence factors in Pectobacterium carotovorum. In this research, the plant expression vector, pBI121, harbouring the anti-quorum sensing gene, aiiA, was transformed into Potato (Solanum tuberosum cv. Agria) explants by Agrobacterium mediated transformation procedure and putative transformants containing pBI121/aiiA were obtained. The results of bioassay test revealed that the expression of aiiA gene in potato plant induces the resistance to the early stage of bacterium pathogenesis. The soft rot symptoms such as wilting, yellowing and tissue maceration were not observed until 48 hr after inoculation of transgenic potatoes with P. carotovorum. Although in non-transgenic plants, the disease symptoms were appeared after 24 hr of plants infection. Hence, this study proves that the heterologous expression of aiiA gene enhances the resistance against potato soft rot disease.

Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications.

Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a variety of tissues, including leaf, cambium and fruit of diverse plant species including annual crops, forest and fruit trees, herbarium leaf material and lyophilized fungal mycelium. We consistently obtained good yields of high purity DNA or RNA in all species tested. The protocol has been validated for thousands of DNA samples by generating high data quality in dense SNP arrays. DNA extracted from Eucalyptus spp. leaf and cambium as well as mycelium from Trichoderma spp. was readily digested with restriction enzymes and performed consistently in AFLP assays. Scaled-up DNA extractions were also suitable for long read sequencing. Successful RNA quality control and good RNA-Seq data for Eucalyptus and cashew confirms the effectiveness of the sorbitol buffer pre-wash for high quality RNA extraction.

Threat of establishment of non-indigenous potato blackleg and tuber soft rot pathogens in Great Britain under climate change.

Potato blackleg and soft rot caused by Pectobacterium and Dickeya species are among the most significant bacterial diseases affecting potato production globally. In this study we estimate the impact of future temperatures on establishment of non-indigenous but confirmed Pectobacterium and Dickeya species in Great Britain (GB). The calculations are based on probabilistic climate change data and a model fitted to disease severity data from a controlled environment tuber assay with the dominant potato blackleg and soft rot-causing species in GB (P. atrosepticum), and three of the main causative agents in Europe (P. carotovorum subsp. brasiliense, P. parmentieri, Dickeya solani). Our aim was to investigate if the European strains could become stronger competitors in the GB potato ecosystem as the climate warms, on the basis of their aggressiveness in tubers at different temperatures. Principally, we found that the tissue macerating capacity of all four pathogens will increase in GB under all emissions scenarios. The predominant Pectobacterium and Dickeya species in Europe are able to cause disease in tubers under field conditions currently seen in GB but are not expected to become widely established in the future, at least on the basis of their aggressiveness in tubers relative to P. atrosepticum under GB conditions. Our key take-home messages are that the GB potato industry is well positioned to continue to thrive via current best management practices and continued reinforcement of existing legislation.

Skeletal malformations of Meox1-deficient zebrafish resemble human Klippel-Feil syndrome.

Klippel-Feil syndrome is a congenital vertebral anomaly, which is characterised by the fusion of at least two cervical vertebrae and a clinically broad set of symptoms, including congenital scoliosis and elevated scapula (Sprengel's deformity). Klippel-Feil syndrome is associated with mutations in MEOX1. The zebrafish mutant choker (cho) carries a mutation in its orthologue, meox1. Although zebrafish is being increasingly employed as fidelitous models of human spinal disease, the vertebral column of Meox1-deficient fish has not been assessed for defects. Here, we describe the skeletal defects of meox1cho mutant zebrafish utilising alizarin red to stain bones and chemical maceration of soft tissue to detect fusions in an unbiased manner. Obtained data reveal that meox1cho mutants feature aspects of a number of described symptoms of patients who suffer from Klippel-Feil syndrome and have mutations in MEOX1. These include vertebral fusion, congenital scoliosis and an asymmetry of the pectoral girdle, which resembles Sprengel's deformity. Thus, the meox1cho mutant zebrafish may serve as a useful tool to study the pathogenesis of the symptoms associated with Klippel-Feil syndrome.

Cell-wall-degrading enzymes produced in vitro and in vivo by Rhizoctonia solani, the causative fungus of peanut sheath blight.

Rhizoctonia solani causes the disease peanut sheath blight, involving symptoms of maceration and necrosis of infected tissue, mainly caused by cell-wall-degrading enzymes (CWDEs). This study investigated the production of CWDEs including polygalacturonase (PG), polymethyl-galacturonase (PMG), cellulase (Cx) and β-glucosidase by R. solani in vitro (in liquid culture) and in vivo (in peanut plants). Significant PG, PMG, Cx and β-glucosidase activities were detected in infected tissues including stalk and leaves of Baisha and Silihong peanut cultivars. Extracts of healthy tissue showed little or no such activities. In shaken liquid cultures of R. solani in medium containing pectin or pectin plus carboxymethyl cellulose (CMC) as the carbon source(s), PG and PMG were notably active. Significant Cx activity was detected in cultures with CMC or pectin plus CMC as the carbon source(s). However, only a very low level of β-glucosidase activity was observed in cultures with any of the tested carbon sources. An increase of pH was recorded in decayed peanut tissues and liquid culture filtrates; the filtrate pH and fungal growth positively correlated. The fungal growth and/or pH were important factors for the production of PG, PMG and Cx in culture with pectin plus CMC as the carbon source. A single active PG isozyme with isoelectric point around 9.2 was detected in culture filtrates and in infected peanut tissues by the method of isoelectric focusing electrophoresis. The crude enzymes extracted from liquid culture of R. solani induced decay of healthy peanut leaves.

The effect of the N-acyl-homoserine lactonase on the properties of Pectobacterium betavasculorum

Bacteria use quorum sensing (QS) as a mechanism of cell to cell communication that enables them to coordinate essential behaviours. To study the role of QS regulation in directing the cellular processes and virulence of Pectobacterium betavasculorum, the effect of the aiiA gene on this bacterium was investigated. In this study, plasmid pVLT33/aiiA was introduced into bacterial cells to gain Pb/pVLT33/aiiA. The pVLT33/aiiA plasmid contains a heterologous aiiA gene (encoding AiiA enzyme), which catalyzes the degradation of QS signalling molecules of the of N-acyl-homoserine lactones (AHLs). An absence or reduction of AHLs in enclosed medium was assessed with the biosensor Chromobacterium violaceum CV026 in a co-culture assay. To evaluate the effects of AHL on virulence behaviour of the mutated P. betavasculorum, motility, biofilm formation and pathogenicity assays on potato tubers were performed. The results revealed that swimming motility and biofilm formation in the Pb/pVLT33/aiiA strain were significantly influenced, while the introduction of the aiiA gene had no effect on the hypersensitive response. The enzymatic bioassay revealed that the aiiA gene completely abolished secretion of pectate lyase (Pel) and polygalacturonase (Peh) enzymes, while the breakdown of cellulose was slightly reduced. Furthermore, aiiA expression in P. betavasculorum caused reduction of tissue maceration and pathogenicity on potato tubers and cactus. It can be concluded that the main virulence behaviours of P. betavasculorum are controlled by the QS system. Hence, application of anti-QS mechanisms is a promising strategy for the prevention of diseases in which virulence is regulated by QS.

Reduction of Tissue Maceration in Potatoes by Rose Essential Oil

Erwinia carotovora is a phytopathogen which causes soft-rot disease in a wide variety of economically important plants. E. carotovora is known to produce a range of exoenzymes that enhance its ability to damage the host tissue and cause disease. A cell to cell communication mechanism called quorum sensing which is mediated by small signalling molecules regulates exoenzymes (cellulase, pectinase and protease) and carbapenem production in E. carotovora. Thus the exploration of new strategies to manipulate this communication pathway for the prevention of E. carotovora infections is valuable. In this study, the inhibitory effects of the rose, orange, lavender, clove, cinnamon, black pepper and cumin oils on the production of the exoenzymes (cellulase, pectinase and protease) and carbapenem in the Erwinia carotovora subsp. carotovora ATCC 39048 were investigated. And potato tissue maceration was also tested in the presence of oils. Rose and lavender essential oils markedly inhibited the production of pectinases by 38.7 and 9.7%, cellulases by 36.6 and 31.7% and proteases by 29 and 16.1%, carbapenem by 61.9 and 54%, and maceration of potatoes by 61.4 and 30.7% in the E. carotovora respectively without affecting the growth of cells. Although several studies have reported antibacterial effects of rose and lavender essential oils, there is no report describing their antivirulence potential. To the best of our knowledge, this is the first report on the rose and lavender essential oils with potential antivirulence components against soft rot caused by E. carotovora.

First Report of Stem Rot on Potato Caused by Dickeya chrysanthemi in Minnesota

Bacterial soft rot and black leg diseases of potato occur sporadically in the Upper Midwestern United States. Multiple members of the soft rot Enterobacteriaceae (SRE) cause diseases in potato. The taxonomy of SREs has undergone extensive revision, resulting in the establishment of new genera and species to better reflect the phylogeny and diversity of strains formerly classified as Erwinia carotovora or E. chrysanthemi (Marrero et al. 2013). One such species, Dickeya dianthicola, has been associated with recent severe black leg outbreaks in the eastern United States. To identify the SREs in the Upper Midwest, a 2016 survey of commercial and seed potato fields in Minnesota and North Dakota was conducted. SREs (123) were isolated from 78 tissue samples exhibiting symptoms of black leg, stem rot, and soft rot. Small tissue samples from the margin of symptomatic and healthy tissues were suspended in 0.02 M potassium phosphate buffer, pH 7.2, for 30 min. Cell suspensions were serially diluted and spread onto crystal violet pectate (CVP) agar. Bacterial colonies producing pitting on CVP were subsequently reisolated on CVP and purified on nutrient broth yeast extract medium. Three isolates from one sample were obtained from a stem sample collected in Sherburne County, Minnesota. One isolate, SR64, was retained for phenotypic characterization and genetic identification. Symptoms associated with SR64 included dry, black-brown necrotic stem lesions with cracking and exposed vasculature. SR64 was determined to be gram negative based on reaction in 3% KOH. Growth on King’s B agar medium failed to produce fluorescence characteristic of Pseudomonas. SR64 also exhibited facultative anaerobic respiration in Hugh-Leifson medium and the ability to grow at 37°C on nutrient agar. Reinoculation of potato tuber slices with SR64 and incubation at 25°C for 2 days produced tissue maceration and dark ooze typical of SRE. DNA extracted from a pure culture of SR64 using DNeasy blood and tissue kits (Qiagen, Valencia, CA, U.S.A.) was used as template for genetic analysis. PCR was conducted using two primer sets: ADE1/ADE2 (Nassar et al. 1996) and Df/Dr (Laurila et al. 2010). SR64 template DNA produced 420 and 119 bp amplicons via ADE1/ADE2 and Df/Dr primers, respectively, as expected for Dickeya. The 16S rDNA was amplified using primer set F27/R1492 (Monciardini et al. 2002), and the ∼1.2 kb amplicon was sequenced and submitted for GenBank nucleotide BLAST analysis. SR64 shared 98% identity with that of the 16S rDNA sequence of type strain Dickeya chrysanthemi LMG 2804 (NZ_CM001974) (Samson et al. 2005). To confirm the species identification, additional housekeeping genes, dnaJ (DCH402_RS02710), dnaX (DCH402_RS15110), and gyrB (DCH402_RS00180) were amplified using primer sets reported by Marrero et al. (2013). Housekeeping gene sequences were concatenated creating a 1,955 bp fragment for analysis with CLC Main Workbench software (Qiagen). The pairwise percent identity between the concatenated sequences of SR64 and D. chrysanthemi LMG 2804ᵀ, D. zeae LMG 2505ᵀ, D. dianthicola LMG 2485ᵀ, D. dadantii subsp. dadantii LMG 25991ᵀ, and D. solani LMG 25993ᵀ was 99.13, 89.42, 90.85, 89.88, and 85.89%, respectively. The low incidence of Dickeya observed in this survey could be owing to limitations of the protocols used to isolate SREs. Alternatively, it could indicate that Dickeya is not currently prevalent in the region. This is the first report of D. chrysanthemi on potato in Minnesota.

土壌分離糸状菌Scopulariopsis brevicaulis MIB301によるバレイショ組織のマセレーション

土壌分離糸状菌Scopulariopsis brevicaulis MIB301によるバレイショ組織のマセレーション

Population Structure and Biodiversity of Pectobacterium parmentieri Isolated from Potato Fields in Temperate Climate.

Pectobacterium parmentieri (formerly Pectobacterium wasabiae) is a newly established species of pectinolytic plant-pathogenic bacteria responsible for the symptoms of soft rot and blackleg on potato. In this work, we describe biodiversity and the population structure of P. parmentieri strains isolated during two consecutive growing seasons from the seed potato fields in Poland. About 450 samples of diseased potato tubers, potato plants, or accompanying weeds were collected throughout the country and tested for the presence of P. parmentieri by molecular identification methods. We found that P. parmentieri strains commonly occur in almost all regions of Poland. Furthermore, these isolates constituted significant fraction of pectinolytic bacteria from seed potato fields because 16% (2013) and 13% (2014) of the analyzed plant samples were infected with P. parmentieri. Subsequently, a detailed characterization of the obtained strains was conducted basing on repetitive sequences profiling, recA-gene-based phylogeny, and phenotypic features. By applying repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR), we revealed the presence of five distinct genomic profiles among P. parmentieri strains, with profile I being the most abundant (approximately 44%). The performed recA gene-based phylogenetic analysis divided P. parmentieri isolates into two distinct clades, although the strains originating from different years did not group separately. Evaluation of the phenotypic traits playing crucial roles for the virulence of pectinolytic bacteria (namely, pectinase, cellulase and protease activities, and siderophore production, in addition to potato tissue maceration, swimming, and swarming motility) indicated some differences among the characterized strains. To the best of our knowledge, this is the first study that describes biodiversity and the population structure of P. parmentieri isolated in two growing seasons under temperate climate conditions and, hence, illustrates high heterogeneity within this species.

Spectrophotometric Determinations of Chloroplastidic Pigments in Physalis angulata L. Leaves Using Different Methodologies

The aim of this study was to compare different protocols for extraction and quantification of photosynthetic pigments, considering different solvents, extraction time and temperature, to determine the best methodology for Physalis angulata L. leaves. The efficiency of immersion of leaf discs in 95% ethanol, 80% acetone and dimethyl sulfoxide (DMSO) was evaluated for 24, 48 and 72 hours. The effect of temperature on extraction with DMSO was also evaluated, with the samples incubated at room temperature and in a water bath at 65 °C. These protocols were compared with the classical methods of plant tissue maceration in 80% acetone followed by filtration, which proved to be the most efficient for Physalis angulata plants and indicated for use in the laboratory. The use of high temperatures (65 °C) is not necessary for the chlorophyll extraction process with DMSO, the room temperature is most efficient in the extraction of carotenoids. Considering the total chlorophyll content, the leaf discs immersion technique in a solvent is acceptable, with 95% ethanol and 24 hours incubation and the most economically viable alternative.

Activated carbon-plasticised agarose composite films for the adsorption of thiol as a model of wound malodour.

Conditions such as diabetes, cardiovascular disease and long-term immobilisation can precipitate the development of chronic dermal ulcers. Such wounds are associated with inflammation and bacterial contamination which in turn can lead to the liberation of offensive odours that cause patient embarrassment and, in some instances, social isolation. Activated carbon-containing dressings have been used to manage the odours from such wounds. However, these can be bulky and can become fouled by wound exudate. Agarose is a natural polysaccharide derived from seaweed that forms brittle free-standing films that can be made pliable by addition of a plasticiser. In this study, activated carbon-containing plasticised agarose films were evaluated for their ability to sequester thiol-containing molecules from solution and the gaseous phase. The water vapour transmission rate was also evaluated to determine the potential breathability of these films should they be considered for application to the skin. It was found that the adsorption of thiols was directly proportional to the activated carbon content of the films. Water vapour was found to pass relatively freely through the films indicating that sweat-induced tissue maceration would be unlikely to occur if applied clinically. In conclusion, activated carbon-containing plasticised agarose films have some potential in the sequestration of malodourous molecules such as those liberated from chronic dermal wounds.

Isolation and phenotypic and morphological characterization of the first Podoviridae lytic bacteriophages \u03d5A38 and \u03d5A41 infecting Pectobacterium parmentieri (former Pectobacterium wasabiae)

Two bacteriophages, ϕA38 and ϕA41, infecting Pectobacterium parmentieri strain SCC 3193 (former Pectobacterium wasabiae strain SCC 3193) were isolated from arable soil samples collected in different regions of Poland. ϕA38 and ϕA41 have a typical morphology of the members of the family Podoviride and order Caudovirales, with a head diameter of ca. 60 nm and tail length of ca. 20 nm. Phages ϕA38 and ϕA41 exhibited a similar RFLP pattern with Csp6I restriction endonuclease. They were stable in a range of pHs, temperatures and osmolarities but were rapidly inactivated by UV light. During the first 20 min., 74 and 69% of ϕA38 and ϕA41 phages, respectively, were adsorbed to SCC 3193 cells. In one-step growth experiments, ϕA38 and ϕA41 showed latent period of ca. 20–30 min and burst size of 102 and 141 phages, respectively. The optimal multiplicity of infection (MOI) was calculated to be 0.01 for both bacteriophages. In the host range experiments, both phages were able to infect six from 21 of the tested P. parmentieri isolates but the phages were unable to infect other members of the Pectobacterium spp. or Dickeya spp. In the proof-of-concept experiments, ϕA38 and ϕA41 were able to inhibit the growth of P. parmentieri strain SCC 3193 and to protect potato tuber tissue maceration caused by the bacterium. The potential use of ϕA38 and ϕA41 bacteriophages for the biocontrol of P. parmentieri in potato is discussed.