A primeira fase do trabalho objetivou avaliar o efeito do tempo de deslintamento químico, 1:30 e 4:30 min, sobre o nível de infecção de Colletotrichum gossypii inoculado artificialmente em sementes de algodoeiro (Gossypium hirsutum). Sementes de algodoeiro com línter foram inoculadas com C. gossypii, mediante contato das mesmas com colônias do fungo em placas de Petri de 9 cm de diâmetro, por 30 h. Os parâmetros avaliados foram a ocorrência de fungos e o poder germinativo das sementes. Na segunda fase, o objetivo foi avaliar a influência do exsudato de sementes de algodoeiro, em função do tempo da duração do deslintamento com ácido sulfúrico, considerando-se as frações de sedimentação de sementes e diferentes condições de envelhecimento artificial sobre o desenvolvimento de C. gossypii, em condições de laboratório. As sementes foram deslintadas quimicamente com ácido sulfúrico comercial concentrado pelos períodos de 1:30 e 4:30 min, separadas em frações de sedimentação em água e submetidas ao envelhecimento artificial por 0, 72 e 96 h. Os substratos foram obtidos a partir do exsudato resultante da embebição contendo os eletrólitos lixiviados das sementes. O desenvolvimento de C. gossypii na presença do exsudato das sementes foi avaliado em meio agarizado, através da medição do crescimento micelial e da esporulação do fungo. O deslintamento químico, pelo período de 1:30 min, propiciou aumento do percentual de ocorrência de C. gossypii em sementes não-desinfestadas superficialmente, e o crescimento micelial e a esporulação do fungo foram favorecidos pelo substrato proveniente de sementes deslintadas por 4:30 e 1:30 min, respectivamente.

The viability of Clavibacter michiganensis subsp. michiganensis (Cmm) was determined by measuring the intracellular pH (pH in ) as a viability parameter.This was based on the observation that growth of Cmm was inhibited at pH 5Á5 and below.Therefore, viable cells should maintain their pH in above this pH value.The pH in of Cmm was determined using the £uorescent probe 5(and 6 -)-carboxy£uorescein succinimidyl ester (cFSE).The pH in of Cmm cells exposed to acid treatments was determined using £uorescence spectro£uorometry, and for cells exposed to elevated temperatures, the pH in was determined using £uorescence spectro£uorometry and £ow cytometry (FCM). A good correlation was found between the presence of a pH gradient and the number of colony-forming units (cfu) observed in plate counts. However, with the spectro£uorometry technique, the analysis is based on the whole cell population and the detection sensitivity of this technique is rather low, i.e., cell numbers of at least10 7 cfu ml À1 are needed for the analysis. Using FCM, heat-treated and non-treated Cmm cells could be distinguished based on the absence and presence of a pH gradient, respectively. The major advantage of FCM is its high sensitivity, allowing analysis of microbial populations even at low numbers, i.e., 10 2 À103 cfu ml À1

Background: Xanthomonas campestris pv. campestris (Xcc) is a seed-transmitted plant pathogenic bacterium that causes black rot of crucifers. Seed lots and plants are screened for contamination with this pathogen using plating or serological assays. These methods, however, are time consuming and not very sensitive, respectively. Therefore, flow cytometry (FCM) was evaluated as a tool for the rapid detection and quantification of Xcc cells labeled with a mixture of specific fluorescein isothicyanate (FITC)-monoclonal antibodies (mAb) in pure culture, in mixed cultures of Xcc with either the common saprophyte Pseudomonas fluorescens (Psf) or a nonpathogenic X. campestris isolate (Xc), and in crude seed extracts.

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with PI.

 Over the last few years, considerable effort has been spent by Embrapa in the construction of a plant disease database representative enough for the development of effective methods for automatic plant disease detection and recognition. In October of 2016, this database, called PDDB, had 2326 images of 171 diseases and other disorders affecting 21 plant species. PDDB size, although considerable, is not enough to allow the use of powerful techniques such as deep learning. In order to increase its size, each image was subdivided according to certain criteria, increasing the number of images to 46,513. Both the original (PDDB) and subdivided (XDB) databases are now being made freely available for academic research purposes, thus supporting new studies and contributing to speed up the advances in the area. Both collections are expected to grow continuously in order to expand their reach. PDDB and XDB can be accessed in the link https://www.digipathosrep.cnptia.embrapa.br/.