Management of bacterial blight of carrots by phenolic compounds treatment.

Bacterial blight is a serious disease of carrot production worldwide. Under favorable conditions, the causal organism Xanthomonas hortorum pv. carotae causes serious loss especially in seed production because of its seed-borne character. Unlike fungal diseases, the treatment of bacterial diseases is limited and methods such as hot water or sodium hypochlorite (bleach) treatment are mainly used by seed companies. Here, we compared the efficacy of hot water treatment, sodium hypochlorite treatment and treatment with three phenolic compounds-carvacrol, thymol and eugenol, to eliminate Xanthomonas growth in vitro and subsequently in vivo on seeds of Xhc low, medium and highly infested carrot seed lots. The complete elimination of Xhc from germinated plants was obtained only for Xhc low infested seed lot with 1% sodium hypochlorite and carvacrol solutions in concentrations of 0.0196%- 0.313%. The significant reduction of Xhc presence in germinated plants of Xhc medium infested seed lot was achieved with 1% sodium hypochlorite treatment and hot water treatment. However, hot water treatment resulted in a significant reduction of seed germination percentage as well. Considering the elimination of Xhc infection from germinated plants and the effect on seed germination and plant vigor, 0.0196% carvacrol solution was suggested as an alternative to 1% sodium hypochlorite treatment regarding additional costs related to the liquidation of used treated water and to hot water treatment that has been proved to be insufficient to obtain disease-free plants.

Combined effect of thyme and clove phenolic compounds on Xanthomonas campestris pv. campestris and biocontrol of black rot disease on cabbage seeds.

The seed-borne bacterium Xanthomonas campestris pv. campestris (Xcc) as a causal organism of black rot disease remains the most serious bacterial problem of agricultural production of cruciferous plants worldwide. The eradication of a primary inoculum originating in seeds is available, but no treatment is totally effective. With the threat of developing chemical resistance and increasing pressure for sustainable disease management, biocontrol methods represent one of the main strategies currently applied in agriculture. Natural antimicrobials, including essential oils, are promising tools in disease management with low risks of environmental pollution and impact on human health. Thyme and clove essential oils were demonstrated to be highly effective in Xanthomonas studies in vitro; therefore, their application in black rot control was evaluated in this study. From five phenolic substances originating from thyme and clove essential oils (carvacrol, eugenol, linalool, p-cymene and thymol), the most promising in vitro results were observed with carvacrol, for which 0.0195% led to the death of all Xcc cells in 30 min. Moreover, a synergistic antibacterial effect of carvacrol and thymol solutions decreased the minimal inhibition concentration to 0.0049% and 0.0195% for carvacrol and thymol, respectively. Using the quadruple bactericidal values, the complete elimination of Xcc from the surface of infested cabbage seeds was obtained for both carvacrol and thymol solutions and their combined mixture at 2 MIC value. The elimination of bacterial infection from germinated cabbage plants was observed for both plate counting and quantitative real-time PCR methods. We also evaluated the effect of the application of phenolic treatment on the seed germination and germinated plants. Our results suggest a high potential of the application of carvacrol and thymol in vegetable seed production, specifically for cabbage, thus representing a suitable alternative to cupric derivatives.

Interaction of the Nanoparticles and Plants in Selective Growth Stages-Usual Effects and Resulting Impact on Usage Perspectives.

Nanotechnologies have received tremendous attention since their discovery. The current studies show a high application potential of nanoparticles for plant treatments, where the general properties of nanoparticles such as their lower concentrations for an appropriate effects, the gradual release of nanoparticle-based nutrients or their antimicrobial effect are especially useful. The presented review, after the general introduction, analyzes the mechanisms that are described so far in the uptake and movement of nanoparticles in plants. The following part evaluates the available literature on the application of nanoparticles in the selective growth stage, namely, it compares the observed effect that they have when they are applied to seeds (nanopriming), to seedlings or adult plants. Based on the research that has been carried out, it is evident that the most common beneficial effects of nanopriming are the improved parameters for seed germination, the reduced contamination by plant pathogens and the higher stress tolerance that they generate. In the case of plant treatments, the most common applications are for the purpose of generating protection against plant pathogens, but better growth and better tolerance to stresses are also frequently observed. Hypotheses explaining these observed effects were also mapped, where, e.g., the influence that they have on photosynthesis parameters is described as a frequent growth-improving factor. From the consortium of the used nanoparticles, those that were most frequently applied included the principal components that were derived from zinc, iron, copper and silver. This observation implies that the beneficial effect that nanoparticles have is not necessarily based on the nutritional supply that comes from the used metal ions, as they can induce these beneficial physiological changes in the treated cells by other means. Finally, a critical evaluation of the strengths and weaknesses of the wider use of nanoparticles in practice is presented.

Drought Influences Fungal Community Dynamics in the Grapevine Rhizosphere and Root Microbiome.

Plant roots support complex microbial communities that can influence nutrition, plant growth, and health. In grapevine, little is known about the impact of abiotic stresses on the belowground microbiome. In this study, we examined the drought-induced shifts in fungal composition in the root endosphere, the rhizosphere and bulk soil by internal transcribed spacer (ITS) high-throughput amplicon sequencing (HTAS). We imposed three irrigation regimes (100%, 50%, and 25% of the field capacity) to one-year old grapevine rootstock plants cv. SO4 when plants had developed 2-3 roots. Root endosphere, rhizosphere, and bulk soil samples were collected 6- and 12-months post-plantation. Drought significantly modified the overall fungal composition of all three compartments, with the root endosphere compartment showing the greatest divergence from well-watered control (100%). The overall response of the fungal microbiota associated with black-foot disease (Dactylonectria and "Cylindrocarpon" genera) and the potential biocontrol agent Trichoderma to drought stress was consistent across compartments, namely that their relative abundances were significantly higher at 50-100% than at 25% irrigation regime. We identified a significant enrichment in several fungal genera such as the arbuscular mycorrhizal fungus Funneliformis during drought at 25% watering regime within the roots. Our results reveal that drought stress, in addition to its well-characterized effects on plant physiology, also results in the restructuring of grapevine root microbial communities, and suggest the possibility that members of the altered grapevine microbiota might contribute to plant survival under extreme environmental conditions.

First report of Lasiodiplodia theobromae causing decline of blueberry (Vaccinium corymbosum L.) in the Czech Republic.

Popularity of blueberries (Vaccinium corymbosum L.) has been rising worldwide, due to their nutritional bioactive compounds such as flavonoids, anthocyanins and polyphenols (Sinelli et al. 2008). These factors led to an increase of the demand for fresh blueberries in the Czech market, which resulted in increased blueberry planted in the Czech Republic. In spring 2018, about 200,000 1-year-old blueberry plants originally from the Netherlands were planted on 47 ha in the South Moravian region. In September 2019, wilting of leaves and shoots, bark necrosis and dieback of the plant were observed. The occurrence of blueberry decline symptoms in the field was near 2% of 5,000 observed plants which resulted in significant yield losses at the beginning of the production. Twenty shoots of symptomatic plants were collected (one shoot from each plant, twenty plants in total) and surface-disinfected using 75% ethanol. Necrotic tissues of xylem were aseptically placed on potato dextrose agar (PDA) (Himedia, India) amended with streptomycin and incubated at 25°C in the dark for one week. Nine isolates of Botryosphaeriaceae-like fungi were obtained from twenty plants and were transferred on PDA. Aerial mycelium grew rapidly, initially white, and then became grey to black. To enhance the conidia production, the isolates were transferred on water agar (Himedia, India) with autoclaved poplar twigs, placed near day light and incubated at room temperature. Three weeks later, black pycnidia were observed on the poplar twigs. Mature pycnidia exuded white/grey coloured masses of conidiospores. Conidia were granular, subcylindrical to elliptical. Immature conidia were single-celled and hyaline measuring 9.73 ± 1.39 × 16.86 ± 1.81 µm (n=60). Mature conidia were thick-walled with a septum from 11.30 ± 1.17 × 16 ± 1.58 µm (n=60). Based on conidiogenous cells, paraphyses and longitudinal striations on mature conidia characteristics, the isolates were morphologically assigned to the Lasiodiplodia genus (Phillips et al. 2013). Isolates were single-spored and total DNA was extracted with NucleoSpin Tissue (Macherey-Nagel, Germany) following manufacturer's protocol. ITS region, including the 5.8S, and part of the translation elongation factor 1 alpha (TEF-1α) were amplified with ITS1/ITS4 and EF1-728F/EF1-986R primers, respectively (Carbone and Kohn 1999; White et al. 1990). Consensus sequences were blasted using NCBI nucleotide BLASTn search. The sequences of ITS region were in 468/469 nts (MEND-F-00167) and 486/490 nts (MEND-F-00168) similar to Lasiodiplodia theobromae isolate CBS 287.47 and 286/289 nts (MEND-F-00167), 287/290 nts (MEND-F-00168) in case of TEF-1α. Sequences of two representative isolates (MEND-F-00167 and MEND-F-00168) were deposited in GenBank under accession numbers MN983133 and MN983134 for ITS and MN989914 and MN989915 for TEF-1α. For pathogenicity tests, 6 mm agar plugs with mycelium obtained from ten day old mycelia of both fungal isolates were inoculated on ten 1-year-old blueberry shoots cv. Top Shelf collected from one plant, using a cork borer. Five shoots and pure agar plugs were used as a control. Plugs were covered with wet cotton wool to keep moisture and fixed with Parafilm. After three weeks, characteristic discoloration and lesions appeared on the inoculated shoots. Control shoots remained symptomless. Necrotic tissue was aseptically cut and L. theobromae was re-isolated on PDA. Identity of fungus was confirmed by morphological characterization and sequencing, fulfilling Koch´s postulates. Blueberry decline caused by Botryosphaeriaceae spp. has already been described around the world (Hilário et al. 2020; Tennakoon et al. 2018; Wiseman et al. 2017; Xu et al. 2015). To our knowledge, this is the first report of L. theobromae on blueberry in the Czech Republic.

High-Throughput Sequencing Analysis of the Bacterial Community in Stone Fruit Phloem Tissues Infected by "Candidatus Phytoplasma prunorum".

"Candidatus Phytoplasma prunorum" (CPp) is a highly destructive phytopathogenic agent in many stone fruit-growing regions in Europe and the surrounding countries. In this work, we focused on documenting entire bacterial community in the phloem tissues of 60 stone fruit trees. Nested PCR and two real-time PCR assays were used to select CPp-positive (group A) and CPp-negative samples (group B). Afterwards, high-throughput amplicon sequencing was performed to assess bacterial community compositions in phloem tissues. The bacterial composition in phloem tissue consisted of 118 distinct genera, represented mainly by Pseudomonas, Acinetobacter, Methylobacterium, Sphingomonas, and Rhizobium. Statistics showed that CPp influenced the bacterial composition of infected plants (group A) and that the bacterial community depended on the geographical origin of the sample. This is the first work focusing on an analysis of the influence of CPp on the bacteria coexisting in the phloem tissues of stone fruit trees.