Izbira metode ekstrakcije DNK za spremljanje organizma za biotično zatiranje, Gliocladium catenulatum J1446

Gray mold, caused by the fungus Botrytis cinerea, is one of the most common and serious diseases affecting strawberries. Different fungicides are used to manage this disease but fungi can develop resistance on it. Therefore, much attention is devoted to biological methods of control in recent years. Preparation Prestop® MIX (Verdera Oy, Finland) is available in some European countries. It contains a biocontrol agent (BCA), isolate J1446 of the fungus Gliocladium catenulatum, active against grey mold. In the project Bicopoll, a project of European transnational research cooperation project CORE Organic II, we are monitoring the effectiveness of BCA spreading on strawberry flowers by bees and checking for residues of BSA in bee products (honey, pollen). For this purpose, we developed a new, BCA specific real time PCR, which allows us to detect BCA in different samples and quantify it. In the early stages of development, we focused on the development of DNA extraction methods from product Prestop® MIX

The development of new methods for monitoring biocontrol agent, Gliocladium catenulatum J1446, to control gray mold on strawberries

Gray mold, caused by the fungus Botrytis cinerea, is one of the most common and serious diseases affecting strawberries. Different fungicides are used to manage this disease but can quickly lose their effectiveness and their ability to suppress the disease. Therefore, much attention is devoted to biological methods of control in recent years. Preparation PrestopMIX (Verdera Oy, Finland) is available in some European countries. It contains a biocontrol agent (BCA), isolate J1446 of the fungus Gliocladium catenulatum, active against grey mold. In the project Bicopoll, a project of European transnational research cooperation project CORE Organic II, we are checking for residues of BSA in bee products (honey, pollen) and following BCA distribution to strawberry flowers by bees. For this purpose, we developed a new, BCA specific real time PCR, which allows us to detect BCA in different samples and quantify it. Development of a new method and its application will be presented

Use of honeybees (Apis mellifera) to protect strawberry from grey mould (Botrytis cinerea)

BICOPOLL is a CORE Organic II European project on protecting strawberries from its most important disease, the grey mould (Botrytis cinerea). Protective spores of fungi Gliocladium catenulatum in Prestop® Mix (PM) are delivered to the flowers of strawberry by honeybees. We assessed effectiveness of honeybees as vectors under field conditions. Flower visits of bees, and fruit yield were monitored and departing and returning bees and strawberry flowers were sampled. Bees visited strawberry flowers the whole flowering period, but more abundant were in warm weather and in the afternoon. The quantity of spores on honeybees was determined by plating on media and a new method, qPCR that we have developed specifically for the protective fungi. The highest number of spores on bees was determined directly after administration of PM followed by a steady decline during the day until stabilization at a low number. The spores could also be detected in returning bees at a relatively constant low number. PM increased proportion of healthy berries for approximately 50 %. Results of first field experiment in Slovenia confirmed effectiveness of bees as vector of PM. We suggest some changes in application of PM

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

BACKGROUND: Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. RESULTS: Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. CONCLUSION: The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR

Bakteriozna plamenjača vinove loze - Xylophilus ampelinus

Bacterial blight of grapevine, caused by phytopathogenic bacterium Xylophilus ampelinus, is economically important disease that can significantly affect grapevine production, leading to the overall yield reduction and reduced vitality of infected grapevines. In regions where disease was recorded, losses in production, especially in susceptible varieties, can reach up to 80% of total yield. The bacterium infects only grapevine. In infected plants, the pathogen is located in the vascular tissues (xylem) from where it spreads further, causing a systemic infection of the host plant. During vegetation it is transmitted from plant to plant to short distance and the main source of inoculum for long distance dissemination are infected cuttings used either as rootstocks or grafting material. There are no completely resistant varieties or effective methods for controlling the disease. In Serbia X. ampelinus has a quarantine status. Therefore, it is of utmost importance to prevent introduction of the pathogen by inspecting the health of reproductive plant material, especially of those originating from countries where the pathogen is already present.Bakteriozna plamenjača vinove loze, koju prouzrokuje fitopatogena bakterija Xylophilus ampelinus, je ekonomski značajno oboljenje koje može ugroziti proizvodnju vinove loze umanjujući ukupan prinos i smanjujući dugovečnost zaraženih biljaka. U regionima gde je zabeleženo prisustvo bolesti gubici u proizvodnji, naročito kod osetljivih sorti, mogu dostići i do 80% ukupnog prinosa. Krug domaćina ove bakterije ograničen je samo na vinovu lozu. U zaraženim biljkama patogen se nalazi u sudovnom sistemu (ksilemu) odakle se dalje širi prouzrokujući sistemičnu infekciju biljke domaćina. Tokom vegetacije prenosi se u neposrednoj okolini izvora zaraze sa biljke na biljku, a na veću udaljenost zaraženim sadnim materijalom koji predstavlja primarni izvor inokuluma. Ne postoje potpuno otporne sorte ni dovoljno efikasne metode zaštite od ove bolesti. U Srbiji X. ampelinus ima karantinski status. Stoga je od najvećeg značaja sprečavanje unošenja patogena kontrolom zdravstvene ispravnosti biljnog materijala za reprodukciju, posebno iz zemalja gde je utvrđeno prisustvo patogena

Highly scalable combinatorial mixing of samples with target-specific primers for rapid pathogen detection on a centrifugal platform

In application areas such as crop genotyping, plant diagnostics, pharmaceuticals and forensics, screening a large number of M samples for specific responses to a library of N active agents in a time- and cost-efficient manner is of critical importance. Parameters of interest include response of cells to a specific drug compound, identification of specific genes or plant pathogens in crops using DNA markers and DNA traceability for food safety. The cost of reagents as well as the liquid handling ro-botics required to perform the enormous number of pipetting steps severely hamper the proliferation of such key technologies into smaller laboratories

Retrospective survey of Dickeya fangzhongdai using a novel validated real-time PCR assay

Dickeya fangzhongdai, an aggressive plant pathogen, causes symptoms on a variety of crops and ornamental plants including bleeding canker of Asian pear trees. Historical findings stress the need for a specific detection tool for D. fangzhongdai to prevent overlooking the pathogen or assigning it to general Dickeya spp. Therefore, a qualitative real-time PCR for specific detection of D. fangzhongdai has been developed and validated. The developed assay shows selectivity of 100%, diagnostic sensitivity of 76% and limit of detection with 95% confidence interval in plant matrices ranging from 311 to 2,275 cells/mL of plant extracts. The assay was successfully used in a retrospective survey of selected host plants of relevance to Europe and environmental niches relevant to D. fangzhongdai. Samples of potato tubers and plants, plants from the Malinae subtribe (apple, pear, quince, and Asian pear tree) and fresh surface water from Slovenia were analyzed. D. fangzhongdai was not detected in any plant samples, however, 12% of surface water samples were found to be positive

Newly Isolated Bacteriophages from the Podoviridae, Siphoviridae, and Myoviridae Families Have Variable Effects on Putative Novel Dickeya spp.

Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This study reports the isolation of three bacteriophages active against putative novel Dickeya spp. isolates from commercially produced infected orchids that show variable host-range profiles. Bacteriophages were isolated through enrichment from Dickeya-infected orchid tissue. Convective interaction media monolith chromatography was used to isolate bacteriophages from wastewaters, demonstrating its suitability for the isolation of infective bacteriophages from natural sources. Based on bacteriophage morphology, all isolated bacteriophages were classified as being in the order Caudovirales, belonging to three different families, Podoviridae, Myoviridae, and Siphoviridae. The presence of three different groups of bacteriophages was confirmed by analyzing the bacteriophage specificity of bacterial hosts, restriction fragment length polymorphism and plaque morphology. Bacteriophage BF25/12, the first reported Podoviridae bacteriophage effective against Dickeya spp., was selected for further characterization. Its genome sequence determined by next-generation sequencing showed limited similarity to other characterized Podoviridae bacteriophages. Interactions among the bacteriophages and Dickeya spp. were examined using transmission electron microscopy, which revealed degradation of electron-dense granules in response to bacteriophage infection in some Dickeya strains. The temperature stability of the chosen Podoviridae bacteriophage monitored over 1 year showed a substantial decrease in the survival of bacteriophages stored at -20°C over longer periods. It showed susceptibility to low pH and UV radiation but was stable in neutral and alkaline pH. Furthermore, the stability of the tested bacteriophage was also connected to the incubation medium and bacteriophage concentration at certain pH values. Finally, the emergence of bacteriophage-resistant bacterial colonies is highly connected to the concentration of bacteriophages in the bacterial environment. This is the first report on bacteriophages against Dickeya from the Podoviridae family to expand on potential bacteriophages to include in bacteriophage cocktails as biocontrol agents. Some of these bacteriophage isolates also showed activity against Dickeya solani, an aggressive strain that causes the soft rot of potatoes, which indicates their broad potential as biocontrol agents

Draft Genome Sequences of Dickeya sp. Isolates B16 (NIB Z 2098) and S1 (NIB Z 2099) Causing Soft Rot of Phalaenopsis Orchids

The genus Dickeya contains bacteria causing soft rot of economically important crops and ornamental plants. Here, we report the draft genome sequences of two Dickeya sp. isolates from rotted leaves of Phalaenopsis orchid

Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites

Summary Erwinia amylovora causes a major disease of pome fruit trees worldwide, and is regulated as a quarantine organism in many countries. While some diversity of isolates has been observed, molecular epidemiology of this bacterium is hindered by a lack of simple molecular typing techniques with sufficiently high resolution. We report a molecular typing system of E. amylovora based on variable number of tandem repeats (VNTR) analysis. Repeats in the E. amylovora genome were identified with comparative genomic tools, and VNTR markers were developed and validated. A Multiple-Locus VNTR Analysis (MLVA) was applied to E. amylovora isolates from bacterial collections representing global and regional distribution of the pathogen. Based on six repeats, MLVA allowed the distinction of 227 haplotypes among a collection of 833 isolates of worldwide origin. Three geographically separated groups were recognized among global isolates using Bayesian clustering methods. Analysis of regional outbreaks confirmed presence of diverse haplotypes but also high representation of certain haplotypes during outbreaks. MLVA analysis is a practical method for epidemiological studies of E. amylovora, identifying previously unresolved population structure within outbreaks. Knowledge of such structure can increase our understanding on how plant diseases emerge and spread over a given geographical region