Human cadaveric skin allografts are used in the treatment of burns and can be preserved in glycerol at high concentrations. Previously, glycerol has been attributed some antimicrobial effect. In an experimental set-up, we aimed at investigating this effect of prolonged incubation of bacteria in 85% glycerol. Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis were incubated in 85% glycerol. The influence of duration of incubation and temperature on ultrastructure and viability were investigated. Unstressed cultures served as controls. Survival was studied after 24–36 h and 10 days incubation in 85% glycerol at 4°C and 36°C with transmission electron microscopy (TEM) and flow cytometry using viability stains indicating membrane damage (SYTO9, propidium iodide) or esterase activity (carboxyfluorescein diacetate). TEM clearly demonstrated variability in morphological changes of bacteria suggesting different mechanisms of damage. Viability stains supported these findings with faster declining viable cell populations in 85% glycerol at 36°C compared with 4°C. Both methods demonstrated that Gram-negative species were more susceptible than Gram-positive species. In conclusion, 85% glycerol may have some additional antimicrobial effect. Temperature is an important factor herein and Gram-negatives are most susceptible. The latter finding probably reflects the difference in cell wall composition between Gram-positive and Gram-negative bacteria.

Flow cytometric analysis of immuno-stained cells (immuno-FCM) was compared to immunofluorescence microscopy (IF) and dilution plating on a semi-selective medium, for quantitative detection of Xanthomonas axonopodis pv. phaseoli (Xap) in bean seed extracts. Cell concentrations of Xap between 10³-10(7) CFU/mL were added to healthy bean seed extracts. A flow cytometry sorting procedure was developed to separate immuno-stained Xap cells from crude seed extracts and confirming by PCR. FCM was evaluated for direct viable counting (DVC) of Xap using combinations of propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) or PI and SYTO 9 and also the combination of immuno-FCM and PI. Dilution plating and IF allowed detection of Xap in bean seed extracts in a range of 10³-10(6) CFU/mL and immuno-FCM from 10(4)-10(6) CFU/mL. Sorted cells could be detected in crude seed extracts by PCR without further extraction. FCM also allowed quantification of viable cells of Xap after DVC procedures; the red fluorescent dye propidium iodide was used to identify dead cells in combination with the green fluorescent dyes cFDA or SYTO 9, these identifying live cells. The combination of immuno-FCM and PI could be more promising and reliable to detect this pathogen in seeds. A combinação do uso do citômetro de fluxo (FCM) e de anticorpo policlonal (imuno-FCM) foi comparada à microscopia de imunofluorescência (IF) e ao plaqueamento em meio de cultura semi-seletivo, para a detecção de Xanthomonas axonopodis pv. phaseoli (Xap) em sementes de feijão. Concentrações de Xap variando de 10³ a 10(7) CFU/mL foram adicionados aos extratos de sementes. Um método de separação pelo citômetro de fluxo foi desenvolvido para a detecção de Xap em extratos de semente e posterior confirmação por PCR. Para avaliação da viabilidade das células foram usadas sondas fluorescentes, iodeto de propídio (PI)/carboxi diacetato de fluoresceína (cFDA) e PI/SYTO 9 e também, a combinação de imuno-FCM e PI. Em meio semi-seletivo e IF foram detectadas 10³-10(6) UFC/mL e no FCM 10(4)-10(6) UFC/mL, em extratos de sementes artificialmente infestados. Xap somente foi detectada em extratos de sementes por PCR, após o processo de separação pelo FCM. Foi possível pelo FCM a quantificação e identificação de células viáveis (verde fluorescente) e células mortas (vermelho fluorescente) de Xap, pelas sondas cFDA/SYTO 9 e PI, respectivamente. A combinação de immuno-FCM e PI poderá ser uma técnica promissora e segura para a detecção deste patógeno em sementes

This study aimed to detect soft rot caused by Pectobacterium carotovorum subsp. carotovorum in lettuce using images obtained by multispectral sensors mounted on an unmanned aerial vehicle (UAV). A secondary objective was to identify the best sensor and determine the optimal stage after inoculation to detect infected plants. In the field, soft rot lesions and the agronomic traits of lettuce plants inoculated or not with the bacteria were assessed on different days after inoculation (DAI). Classifications were made using the Support Vector Machine (SVM) and Naive Bayes (NB) algorithms to analyze data groups consisting of spectral bands, vegetation indices and a combination of bands and indices obtained from a conventional visible camera and Mapir Survey3W multispectral camera, as well as agronomic parameters. The results confirmed the possibility of pre-symptomatic detection of P. carotovorum subsp. carotovorum in lettuce at the canopy level. With respect to identifying healthy and infected lettuce plants by supervised classification, the best results were obtained at 4 and 8 DAI, especially when using the subsets derived from the Mapir Survey3W camera (RGN sensor), for both classifiers. The subsets obtained with the conventional visible sensor (RGB sensor) produced the best results at 20 and 24 DAI.

Maize white spot, caused by the bacterium Pantoea ananatis, is one of the main leaf diseases of maize in Brazil. However, until now, its presence in corn seeds has not been detected. In this study, P. ananatis was detected in corn seeds using a semi-selective culture medium. A bacterial suspension of P. ananatis was serially diluted ten-fold and plated on 523, YDC or semi-selective PA20 media, with or without the addition of methyl thiophanate. The detection of bacteria in seeds was evaluated in three seed lot samples, collected from field maize plants with white spot symptoms. Seed washings were prepared, ten-fold serially diluted and plated on the same three culture media. Addition of methyl thiophanate to 532, YDC and PA20 culture media did not inhibit bacterial growth. Pantoea ananatis was detected in two corn seed samples on YDC and PA20 media, which was confirmed by biochemical, physiological and molecular characterization as well as by pathogenicity tests. This is the first report of P. ananatis detection in corn seeds.