Incidence and molecular characterization of flavescence dorée and stolbur phytoplasmas in grapevine cultivars from different viticultural areas of Serbia

The presence and distribution of grapevine phytoplasmas was investigated from 2003 to 2005 in some of the most important viticultural areas of Serbia, considering in particular the susceptibility and sensitiveness of both local and imported grapevine cultivars. Both flavescence dorée (FD) and bois noir (BN) phytoplasmas were detected using molecular techniques. The presence of FD phytoplasma at the moment seems limited, while BN phytoplasma appears to be present in the majority of grape growing regions in Serbia. Field surveys demonstrate that grapevine yellows (GY) epidemics in the vineyards inspected in Serbia spread very fast, indeed the incidence of symptomatic plants increased considerably year by year. In particular, the average rate of FD diffusion increased from 45.5 to 93.0 % in the Sićevačko region, while the spread of BN resulted lower. The local cultivar 'Plovdina' appeared to be extremely sensitive to FD phytoplasma showing a percentage of infected plants ranging from 91 to 100 %. PCR-RFLP and phylogenetic analyses based on ribosomal protein (rp) and secY gene sequences performed on Serbian FD grapevine strains demonstrated their close relationship with the Italian FD-C strain present in north-east Italy. Based on both phylogenetic markers, Serbian FD strains represent a new distinct lineage and together with the FD-C strain form a major phylogenetic group within the elm yellows group.

Phenazines producing pseudomonas isolates decrease Alternaria tenuissima growth, pathogenicity and disease incidence on cardoon

Phenazines, secondary metabolites of fluorescent Pseudomonas, represent a group of heterocyclic nitrogen-containing compounds showing a broad spectrum of antibiotic properties. Phenazines producing fluorescent Pseudomonas species are studied extensively for their application in plant disease management. In this study, we examined the antifungal activity of different indigenous Pseudomonas isolates (Q16, B25 and PS2) against the phytopathogenic fungus Alternaria tenuissima, which had infected cardoon (Cynara cardunculus L., Asteraceae). An in vitro experiment demonstrated the antifungal activity of selected indigenous isolates. In addition, an in vivo experiment under gnotobiotic conditions showed suppression of C. cardunculus disease caused by A. tenuissima. The quantification of phenazines revealed significant amounts of phenazine-1-carboxylic acid (PCA) and 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA). PCR analysis confirmed the presence of PCA genes in all examined indigenous Pseudomonas isolates. Based on our results, we assume that these Pseudomonas isolates have potential in controlling plant diseases caused by A. tenuissima. [Projekat Ministarstva nauke Republike Srbije, br. III46007 and br. TR31018

First Report of Stolbur Phytoplasma Affecting Cichorium intybus in Serbia.

Chicory (Cichorium intybus, Asteraceae) is a typical Mediterranean plant indigenous to Europe, western Asia, Egypt, and North America (3). It is commonly consumed as a fresh vegetable in salads. In rural areas of Serbia it grows as a weed in crops, but it is used in folk medicine to treat skin disorders due to its antihepatotoxic activity (3). Methanol extracts of chicory leaves showed moderate antibacterial activity against enteric bacteria (3). A phytoplasma-like disease, expressed as proliferation of chicory shoots and flowers, was observed on wild plants for the first time in Obrenovac vicinity (44°40′ N, 20°20′ E) in July 2012. A flattening of the stem with a large number of filamentous leaves, contortion and abnormal growth of flowers on the stem (typical fasciation symptoms) were observed. Diseased plants did not produce seeds. Total DNA was extracted from the leaf midveins of 15 symptomatic and five symptomless plants (4). PCR amplification of 1.5-kb 16S rDNA fragment was performed using DreamTaq Green master mix (Thermo Scientific, Lithuania) and phytoplasma universal primer pairs P1/16S-Sr (1). Products of nested PCR (1.2 kb) were obtained using primer pair R16F2n/R2 (1). Both amplicons were detected in all diseased samples; however, DNA from symptomless samples yielded no amplicons. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 PCR products was performed in independent reactions using four endonucleases (AluI, TruI1, HhaI and HpaII). RFLP patterns from chicory samples were compared to those of Stolbur (STOL), Aster Yellows (AY), Flavescence Dorée-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas (1). All RFLP profiles from the chicory samples were identical to STOL reference strain, indicating that diseased chicory was affected by a phytoplasma that belongs to ‘Candidatus Phytoplasma solani’ (16SrXII-A group). The 16S rDNA sequence of representative sample from symptomatic plant (Vp4) was deposited under accession number KF661322 in NCBI GenBank. It showed 100% identity to KF263684.1 from Iranian peach, JQ730742.1 from Serbian valerian, and JQ730750 from Serbian corn, all belonging to the ‘Ca. P. solani’ taxon. Puna chicory disease on C. intybus associated with a subgroup 16SrV-B of phytoplasma was detected in China (2). This is the first report of the Stolbur phytoplasma associated with fasciation of C. intybus in Serbia and worldwide

Selection of indigenous fluorescent pseudomonad isolates from maize rhizospheric soil in Vojvodina as possible PGPR

Among 268 bacterial isolates obtained from maize rhizospheric soil from Novi Sad, Vojvodina. Serbia. 59 were fluorescent pseudomonads. According to intrinsic antibiotic resistance (JAR) and heavy metal tolerance (HMT) patterns, the six representative isolates were selected and their diversity was assessed. Lytic enzyme activity (protease, chitinase, lipase, phospolipase, cellulase, gellatinase, pectinase, urease), plant-growth promoting treats (P-solubilization, siderophores, HCN and IAA production) and plant pathogenicity estimation (on lilac leaf apple and bean pods) resulted in selection of three isolates (PS2, P54, PS6) with the outlook for agriculture application. Antifungal activity was estimated on dual culture with eight phytopathogenic fungi (Curvularia lunata, Fusarium semitectum, Fusarium equiseti from Salvia officinalis L., F.equiseti from Matricaria chamomilla L.. Myrothecium verrucaria, Verticillium sp., Diaporte eres complex and Sclerotinia sclerotiorum) isolated from medicinal plants in Serbia. Isolate PS2 showed hyphal deformation of all investigated fungi and effective inhibition of mycelial growth of 7 out of 8 phytopatogenic fungi, partly due to production of chitinases, siderpohores and lytic enzymes. Abundant production of IAA (14 to 37 mM) and siderophores, phosphate solubilization and especially fungal growth inhibition make it suitable for further investigation, field trials and possible application in maize cultivation as biocontrol agent

First Report of Blueberry Reddening Disease in Serbia Associated with 16SrXII-A (Stolbur) Phytoplasma

Blueberries (Vaccinium corymbosum) are among the healthiest fruits due to their high antioxidant content. The total growing area of blueberries in Serbia ranges from 80 to 90 ha. A phytoplasma-like disease was observed for the first time during July 2009 in three blueberry cultivars (Bluecrop, Duke, and Spartan) grown in central Serbia, locality Kopljare (44°20′10.9″ N, 20°38′39.3″ E). Symptoms of yellowing and reddening were observed on the upper leaves and proliferating shoots, similar to those already described on blueberries (4). There was uneven ripening of the fruits on affected plants. Incidence of affected plants within a single field was estimated to be greater than 20% in 2009 and 50% in 2010. Blueberry leaves, together with petioles, were collected during two seasons, 2009 and 2010, and six samples from diseased plants and one from symptomless plants from each cultivar, resulting in 42 samples in total. For phytoplasma detection, total DNA was extracted from the veins of symptomatic and asymptomatic leaves of V. corymbosum using the protocol of Angelini et al. (1). Universal oligonucleotide primers P1/P7 were used to amplify a 1.8-kb DNA fragment containing the 16S rRNA gene, the 16S-23S spacer region, and the 5′ end of the 23S rRNA gene. Subsequently, a 1.2-kb fragment of the 16S rRNA gene was amplified by nested PCR with the R16F2n/R16R2 primers. Reactions were performed in a volume of 50 μl using Dream Taq Green master mix (Thermo Scientific, Lithuania). PCR reaction conditions were as reported (3), except for R16F2n/R2 primers set (annealing for 30 s at 58°C). PCR products were obtained only from the DNA of symptomatic plants. Fragments of 1.2 kb were further characterized by the PCR-RFLP analysis, using AluI, HpaII, HhaI, and Tru1I restriction enzymes (Thermo Scientific, Lithuania), as recommended by the manufacturer. The products of restriction enzyme digestion were separated by electrophoresis on 2.5% agarose gel. All R16F2n/R2 amplicons showed identical RFLP patterns corresponding to the profile of the Stolbur phytoplasma (subgroup 16SrXII-A). The results were confirmed by sequencing the nested PCR product from the representative strain Br1. The sequence was deposited in NCBI GenBank database under accession number KC960486. Phylogenetic analysis showed maximal similarities with SH1 isolate from Vitis vinifera, Jordan (KC835139.1), Bushehr (Iran) eggplant big bud phytoplasma (JX483703.1), BA strain isolated from insect in Italy (JQ868436.1), and also with several plants from Serbia: Arnica montana L. (JX891383.1), corn (JQ730750.1), Hypericum perforatum (JQ033928.1), tobacco (JQ730740.1), etc. In conclusion, our results demonstrate that leaf discoloration of V. corymbosum was associated with a phytoplasma belonging to the 16SrXII-A subgroup. The wild European blueberry (Vaccinium myrtillus L.) is already detected as a host plant of 16SrIII-F phytoplasma in Germany, North America, and Lithuania (4). The main vector of the Stolbur phytoplasma, Hyalesthes obsoletus Signoret, was already detected in Serbia (2). The first report of Stolbur phytoplasma occurrence on blueberry in Serbia is significant for the management of the pathogen spreading in blueberry fields. Since the cultivation of blueberry has a great economic potential in the region, it is important to identify emerging disease concerns in order to ensure sustainable production

First Report of 16SrXII-A Subgroup Phytoplasma (Stolbur) Associated with Reddening of Oenothera biennis in Serbia

Evening primrose (Oenothera biennis L.) is a biennial medicinal, edible, and ornamental plant species. It has attracted great interest for its seed oil that contains gamma linolenic acid, thus distinguishing this plant as a main commercial source of this essential fatty acid (4). This species has been grown as a permanent member of a medicinal plant collection established near Backi Petrovac (northern Serbia) for 22 years. The first disease symptoms were recognized as red spots on leaf rosette in July 2011, spreading gradually during vegetative growth and covering 1/3 to 1/2 of the leaf surface. Symptoms, observed on 16% of the plants (32 of 200) in the second half of May 2012 and on 23% (69 of 300) at the beginning of May 2013, appeared as reddening of lower leaves of flower-bearing stems. Affected plants exhibited stunted growth, while reddening spread over other leaves of flower-bearing stems. In severely affected plants, the flower-bearing stems were poorly developed, frequently forming witches' brooms. For that reason, 30 reddened and 20 symptomless leaves (2 leaves per plant) were sampled in both July 2012 and 2013 and total nucleic acids were extracted. Direct PCR assays were performed using phytoplasma universal primer pair P1/P7 (2) to amplify 1,800-bp fragments (the 16S rRNA gene, the 16S-23S intergenic spacer region, and a part of the 5′ region of the 23S rRNA gene). PCR products were used in nested PCR with primers R16F2n/R2 (2) to amplify 1,200-bp fragments. The identification of phytoplasmas was done using RFLP (restriction fragments length polymorphisms) analyses of R16F2n/R2 amplicons digested with AluI, Kpn I, HpaII, TruI1, or HhaI endonucleases (Thermo Scientific, Lithuania) (2). RFLP patterns were identical to that of STOL reference strain of the 16SrXII-A subgroup, indicating that symptomatic plants were infected with phytoplasma (2). The 16S rDNA nucleotide sequence of representative strain E7 was deposited in GenBank under accession number KF850526. The BLASTn search showed 100% homology to an Iranian strain (KF263684.1) from peach and Serbian strains JQ730742.1 and JQ730750 from valerian and corn, respectively, all belonging to ‘Candidatus Phytoplasma solani’ (Stolbur). Sequencing data confirmed the association of Stolbur phytoplasma with affected O. biennis plants. It has already been reported that phytoplasma infection caused yellows disease of O. biennis (1). Also, the virescence of O. hookeri was associated with phytoplasma strain OAY from aster yellows (AY) group (subgroups 16SrI-B), and selected as the reference strain for the novel taxon ‘Ca. P. asteris’ (3). Here we provide the first report of naturally occurring Stolbur phytoplasma disease of O. biennis in Serbia

Phenolic composition and biological activities of geographically different type of propolis and black cottonwood resins against oral streptococci, vaginal microbiota and phytopathogenic Fusarium species

Aims: A multidisciplinary approach was used to compare phenolic composition, radical scavenging and antimicrobial activity of propolis samples from different geographical localities, and plant resin against various microorganisms. Methods and Results: Using UHPLC-qqqMS quantitative analysis, 28 phenolic compounds were determined. Caffeic and p-coumaric acids were identified as main phenolic acids in poplar propolis samples, except samples from Russia (P6) and China (P7). Radical scavenging activity (applying DPPH spectrophotometric assay) showed the highest activity of Serbian (40·51%) and Chinese (53·21%) propolis samples. Broth microdilution method was used for the oral cavity, fungal phytopathogenic and human vaginal isolates which have been identified at a molecular level. The most sensitive bacterial isolates were Lactobacillus acidophilus (MIC of 0·03–0·13 mg ml−1) and the oral streptococci isolates (MIC values of 0·19–0·13 mg ml−1). The most sensitive fungal phytopathogenic isolate was Fusarium oxysporum (MIC 0·003 mg ml−1). All samples, except propolis from Serbia (P4) and Turkey (P5), showed a strong antifungal activity against Fusarium sporotrichioides, Fusarium subglutinans and Fusarium proliferatum. Conclusion: The results of various tests indicate good radical scavenging and antimicrobial activity against important human and plant pathogens. Significance and Impact of the Study: A detailed propolis analysis is important when proposing a preparation of new biological antimicrobial products which have a positive impact on human health and reduce antibacterial resistance