First Report of Collar and Root Rot of Lettuce Caused by Plectosphaerella cucumerina in Serbia.

In March 2021, unusual plant stuning, collar, and wet root rot of lettuce (Lactuca sativa L.) during the rosette stage was observed in two commercial fields in Serbia (44°58'N, 20°32'E; 44°45'N, 20°43'E). Disease incidence in the fields (≈ 0.9 ha each) was approximately 15 and 20%, respectively. Initial above-ground symptoms were yellowing and wilting of leaves, while below-ground symptoms were collar, wet root rot, and lesions becoming necrotic. Eventually, whole plants wilted, collapsed, and died. A total of 35 symptomatic plants were collected from the fields, and diseased tissues were cut into small pieces, surface sterilized, and plated on potato dextrose agar (PDA). Isolation resulted in 20 morphologically uniform monoconidial isolates. The isolates formed white to creamy colonies, gradually becoming salmon pink, slimy, or moist in appearance, with sparse aerial mycelia. Numerous hyphal coils with conidiophores and hyaline, smooth-surfaced, ellipsoid to ovoid, septate or aseptate conidia were formed (4.5 to 10.1×1.2 to 3.7 µm (n = 100)). To confirm the species identity, the internal transcribed spacer (ITS) region and the D1/D2 region of a selected representative isolate 13-3-c were amplified and sequenced by using primer pairs ITS1/ITS4 (White et al. 1990) and N1/N2 (O'Donnell and Gray 1995), respectively. The sequences were deposited in GenBank (ITS: OR880564 and D1/D2: OR880567). Sequence analysis revealed 100% nucleotide identity with P. cucumerina isolates from different countries deposited in the NCBI GenBank, including isolate MH860704 (Vu et al. 2019) (ITS region) and isolate KY662256 (Su et al. 2017) (D1/D2 region). Neighbor-joining analysis was conducted based on the combined ITS and D1/D2 regions, and the tree was constructed with the substitution models (1,000 bootstrap). The combined phylogeny confirmed that the sequences shared a common clade with P. cucumerina. Hence, morphological, microscopic, and molecular characterization confirmed the pathogen as P. cucumerina (Palm et al., 1995; Carlucci et al., 2012). In a pathogenicity assay, 10 isolates were tested. Five 30-day-old lettuce plants (cv. Majska Kraljica) per isolate were root-dipped in the conidial suspensions (1×105 conidia/ml). The 10 inoculated plants were transplanted into 1 L pots containing sterile substrate (Floragard, Germany). Plants treated with sterile distilled water were used as controls. Plants were maintained in a greenhouse at 25 to 28°C under a 12-hour photoperiod (Cai et al., 2021). Four weeks after inoculation, stunting, chlorosis, and wilting of plants were observed, while collars and roots exhibited typical decaying symptoms. No symptoms were observed on the control plants. The pathogen was reisolated from symptomatic tissue as previously described. Koch's postulates were completed by confirming the identity of reisolates based on morphological features. To our knowledge, this is the first report of P. cucumerina on lettuce or any other crop in Serbia. P. cucumerina is already known as a pathogen of lettuce and other hosts grown in many countries worldwide, as well as in some European countries (Belgium, England, Italy, the Netherlands, and Switzerland) (Zhang et al. 2019). This emerging pathogen may cause significant economic losses in lettuce production in Serbia and in the entire Balkan region. Our results may help to develop effective management strategies based on accurate and timely identification and regular pathogen monitoring.

The value of active follow up of a newly acquired hepatitis B infection - lessons for current approaches.

Abstract: The standard practice of blood borne virus (BBV) follow-up in New South Wales is a passive approach of general-practitioner-led testing. The value of this approach is unknown. We undertook an active contact tracing method with the aims of investigating a potential hepatitis B source, along with accurately measuring the participation rate, to consider the value of this and other follow-up methods for future BBV investigations. Investigation of a newly-acquired hepatitis B infection was undertaken at a dental practice identified as a possible exposure site. To screen for hepatitis B infection among potential source or co-exposed clients, we actively followed up with staff and clients of the practice to request they undertake hepatitis B serology. Eligible staff and clients received up to four phone calls and were provided with a pathology request form by the public health unit (PHU). Access to free serology was offered to people who did not have access to Medicare. Reminder calls were made if serology results were not received by the PHU. As the ordering doctor, the public health physician was responsible for providing results and referring for follow-up care. Of 160 clients, 63 (39%) undertook hepatitis B serology. Of these 63, none were found to have hepatitis B infection. It was estimated the active investigation involved an extra 430 hours of PHU staff time at a cost in Australian dollars of $30,000. Active follow-up allows an accurate participation rate to be documented. Despite intense active follow-up, only 39% of clients undertook testing, bringing into question the yield of the usual approach in which active follow-up of potential mass BBV exposures is not undertaken. While active follow-up is resource intensive, it should be considered where the risks and consequences from the BBV infection are high.

Urban ecosystem drives genetic diversity in feral honey bee.

Urbanization can change biodiversity in both directions, positive and negative, and despite the rising global trend of urban beekeeping, little is known about the impact of urbanization on the genetic diversity of honey bees. We investigate how urbanization affects the genetic variability of feral and managed honey bee colonies that are spread throughout the entire city, even in highly urban areas, through genetic analysis of 82 worker bees. We found convincing evidence of high genetic differentiation between these two groups. Additionally, by comparing city samples with 241 samples from 46 apiaries in rural parts of the country, variations in mitochondrial tRNAleu-cox2 intergenic region and microsatellite loci indicated that feral colonies have distinct patterns of genetic diversity. These results, with evidence that feral honey bees find niches within highly modified and human-dominated urban landscapes, lead us to conclude that urbanization is a driver of the genetic diversity of feral honey bees in the city.

Distribution of Y-chromosome haplogroups in Serbian population groups originating from historically and geographically significant distinct parts of the Balkan Peninsula.

Our study enrolled 1200 Serbian males originating from three geographical regions in the Balkan Peninsula inhabited by Serbs: present-day Serbia, regions of Old Herzegovina and Kosovo and Metohija. These samples were genotyped using the combination of 23 Y-chromosomal short tandem repeats (Y-STRs) loci and 17 Ychromosomal single nucleotide polymorphisms (Y-SNPs) loci for the haplotype and haplogroup analysis in order to characterize in detail Y chromosome flow in the recent history. Serbia's borders have changed through history, forcing Serbs constantly to migrate to different regions of Balkan Peninsula. The most significant migration waves in the recent history towards present-day Serbia occurred from the regions of Old- Herzegovina and Kosovo and Metohija that lie in the south-west/south. High haplotype diversity and discrimination capacity were observed in all three datasets, with the highest number of unique haplotypes (381) and discrimination capacity (0.97) detected in the samples originating from the present-day Serbia. Haplogroup composition didn't differ significantly among datasets, with three dominant haplogroups (I-M170, E-P170 and R-M198), and haplogroup I-M170 being the most frequent in all three datasets. Haplogroup E-P170 was the second most dominant in the dataset originating from geographical region of Kosovo and Metohija, whereas haplogroup R-M198 was the second most prevalent in the dataset from historical region of Old Herzegovina. Based on the phylogenetic three for haplogroup I constructed within this study, haplogroup I2a1-P37.2 was the most dominant within all three datasets, especially in the dataset from historical region of Old Herzegovina, where 182 out of 400 samples were derived for SNP P37.2. Genetic distances between three groups of samples, evaluated by the Fst and Rst statistical values, and further visualized through multidimensional scaling plot, showed great genetic similarity between datasets from Old Herzegovina and present-day Serbia. Genetic difference in the haplogroup distribution and frequency between datasets from historical region of Old Herzegovina and from geographical region of Kosovo and Metohija was confirmed with highest Fst and Rst vaules. In this study we have distinguished genetic structure, diversity and haplogroup frequencies within 1200 Serbian males from three datasets, relationships among them as well as with other Balkan and European populations, which is useful for studying recent demographic history.

First Report of Sclerotinia minor on lettuce in Serbia.

Lettuce (Lactuca sativa L.) is the world's most popular leafy salad vegetable. One of the major challenges facing lettuce producers are fungal diseases that could, under favorable conditions, devastate the harvest (Raid, 2004). During February 2021, poor growth, plant stunning and blanching of leaves of lettuce plants, cultivated in unheated plastic tunel in Potocanje (Zlatibor region), Serbia, were observed. The crowns were softened with spreading decaying lesions covered with white mycelium, particulary on the leaves near the soil surface. Approximately 2 to 3 weeks before harvest, diseased plants began to wilt and collapse. Estimated disease incidence was 50-55%. In order to identify the causal agent, symptomatic tissues from diseased plants were cut into small pieces, surface sterilized with 70% ethanol for 1 min, rinsed three times in sterile distilled water and placed on potato dextrose agar (PDA). Five isolates with uniform morphology were derived from infected tissue. The colonies had fast-growing, white, cottony aerial mycelium, producing profuse numbers (184 sclerotia/ Petri plate in average) of small, black, irregularly shaped sclerotia, less than 2 mm in diameter. Based on morfological features, the isolates were identified as Sclerotinia minor Jagger (Kohn, 1979). To confirm the species identity, the internal transcribed spacer region of nuclear rDNA of a representative isolate 15-2 was amplified using the primer pair ITS1/ITS4 (White et al. 1990). Sequence analysis of ITS region revealed 100% nucleotide identity between the isolate 15-2 (GenBank Accession No. OL423632.1) and 14 isolates of S. minor from different parts of the world (e.g., accession Nos. MK356551.1, KY707828.1, JF2798801.1). Pathogenicity tests were conducted by artificial inoculation of 55-day-old lettuce plants cv. 'Majska kraljica', grown on commercial growth substrate in l L pots. The obtained isolates were grown on PDA for 7 days and mycelial plugs, 5 mm in diameter, were cut from the margin of the colony and placed mycelium-side down on undamaged ground-level leaves of lettuce plants. Two plugs per isolate were placed onto five plants each for a total of ten replicates per isolate. Negative control plants (5) were inoculated similarly with sterile PDA plugs. Inoculated plants were covered with transparent plastic bags, sprayed with water (under the plastic) twice a day for 3 to 5 days to maintain high humidity, and kept in a growth chamber at 22°C (13 h light). After 7 to 10 days, all pathogen-inoculated plants developed lettuce drop disease symptoms, whereas the control plants remained symptomless. The pathogen was reisolated from symptomatic leaves and Koch's postulates were completed by confirming the identity of the isolates. To our knowledge, this is the first report of S. minor on lettuce in Serbia. More research is needed to better understand this disease, establish control strategies and minimize the spread of the pathogen to other lettuce-producing areas of the country. References: Kohn, L. M. 1979. Delimitation of the economically important plant pathogenic Sclerotinia species. Phytopathology 69: 881-886. White, T. J., et al. 1990. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Raid, R.N., 2004. Lettuce diseases and their management. In Diseases of Fruits and Vegetables: Volume II (pp. 121-147). Springer, Dordrecht. Founding: This work was funded by the Ministry of Education, Science and Technological Development (contract 451-03-9/2021-14/200214).

Further Evidence of Population Admixture in the Serbian Honey Bee Population.

Socioeconomic interests and beekeeper preferences have often taken precedence over the conservation of locally native honey bee subspecies, leading to the predominance of admixture populations in human-dominated areas. To assess the genetic diversity of contemporary managed Serbian honey bee colonies, we used 14 microsatellite loci and analyzed 237 worker bees from 46 apiaries in eight localities of northern and southern Serbia. Furthermore, we compared data for nine microsatellite loci with 338 individuals from Italy, Hungary, Poland, and Spain. The standard parameters of genetic diversity in Serbian honey bee populations were in line with other analyses, although somewhat smaller. STRUCTURE analysis showed the existence of two equally distributed genetic clusters and Analysis of molecular variances could not confirm the presence of a geographically discrete population but showed local differences. Discriminant analysis of principal components showed overlapping of worker bees from different parts of Serbia. Clear genetic differentiation can be observed when comparing all populations between geographical regions and their corresponding subspecies. The absence of the A. m. macedonica subspecies from its historical distribution range in southern Serbia as well as the lack of distinctive geographical groups suggest that selective breeding, queen import, and migratory beekeeping practices strongly influence the genetic structure and diversity of honey bees, leading to the genetic uniformization and creation of the admixture population.

Genetic Diversity Analysis of Mitochondrial Cytb Gene, Phylogeny and Phylogeography of Protected Griffon Vulture (Gyps fulvus) from Serbia.

Once a widespread and common species across the region of southeast Europe, the Griffon vulture is now confined to small and isolated populations across the Balkan Peninsula. The population from Serbia with 290 couples represents its biggest and most viable population that can serve as an important reservoir of genetic diversity from which the birds can be used for the region's reintroduction or recolonization programs. To estimate the level of genetic diversity, the mitochondrial Cytb gene from 58 unrelated birds sampled during the marking in the nests was sequenced and compared to the homologous Griffon vulture sequences available in publicly accessible online databases. Phylogeographic analysis based on Cytb sequences showed that the most frequent haplotype is found in all Griffon vulture populations and that each population possesses private haplotypes. Our data suggest that the Griffon vulture population from Serbia should be used as a source population for restocking and reintroduction programs in the region. The observed genetic differentiation between the populations from the Iberian and Balkan Peninsulas suggest that the introduction of foreign birds from remote populations should be avoided and that birds from indigenous or neighboring populations, if available, should be used instead.

Mutational profile of hereditary breast and ovarian cancer - Establishing genetic testing guidelines in a developing country.

PURPOSE: Because many countries lack the capacity to follow the international guidelines for genetic testing, we suggest the specific approach for establishing local genetic testing guidelines that could be applied in developing countries. We focus on hereditary breast (BC) and ovarian cancer (OC) in Serbia. METHODS: From the cohort of 550 persons who were referred for genetic counseling at the Institute for Oncology and Radiology of Serbia, 392 were selected. Personal and family histories were collected and germline DNA was sequenced with NGS in a panel of 20 genes. RESULTS: Pathogenic (PV) and likely-pathogenic variants (LPV) were detected in 8 genes with the frequency of 23.7%. The most frequent were in BRCA1(7.6%), BRCA2(4.8%), PALB2(4.1%) and CHEK2(3.8%). They were also detected in ATM(1.8%), NBN(0.8%), TP53(0.5%) and RAD51C(0.3%). Whereas high carrier probability (CP), bilateral BC, BC and OC in the same patient and family history (FH) of BC/OC, were the strongest predictors for BRCA1/2 PV/LPV, lower CP values and early age of BC onset without FH were associated with higher frequency of PALB2 and CHEK2 PV/LPV. CONCLUSIONS: Population specific studies to identify specific mutational patterns and predictors of PV/LPV should be conducted in order to make scientifically sound and cost-effective guidelines for genetic testing in developing countries.

Utilization of waste glycerol for the production of biocontrol agents nigericin and niphimycin by Streptomyces hygroscopicus: bioprocess development.

Search for more environment-friendly methods for controlling plant diseases that would contribute to the goal of sustainability in agriculture is in focus. In the present study, the potential of Streptomyces hygroscopicus isolated from soil sample in the production of biocontrol agents, nigericin and niphimycin, effective against Alternaria alternata storage apple pathogen was examined. Also, modelling and optimization of medium composition for biocontrol agent biosynthesis was performed. The results showed that the optimum amount of C3H8O3, (NH4)2SO4 and K2HPO4 in the medium for Streptomyces hygroscopicus biosynthesis is 20, 0.25 and 1.46 g/L, respectively. Scale-up and validation of the obtained results performed in the 3 L laboratory-scale bioreactor showed that on the optimized medium at an aeration rate of 0.7 vvm and an agitation speed of 200 rpm, produced nigericin and niphimycin, showed high activity. Under the same conditions, cultivation of S. hygroscopicus was performed in a 7 L laboratory bioreactor in a medium with waste glycerol instead of pure glycerol. Results showed that the methanol extract of S. hygroscopicus cultivation liquid, containing nigericin and niphimycin, was high effective against two Alternaria isolates. This was confirmed in vitro by obtaining large inhibition zone diameters on A. alternata KA10 (47 mm) and T1Jg3 (44.33 mm) isolates. After successful in vitro analysis, in planta testing was performed. It was found that necrosis diameters that were measured on artificially inoculated apple fruits with A. alternata compared to necrosis diameter measured on untreated, control fruits, were 4.47 and 3.56 times smaller.

Bondi and beyond. Lessons from three waves of COVID-19 from 2020.

OBJECTIVES: To describe local operational aspects of the coronavirus disease 2019 (COVID-19) response during the first three waves of outbreaks in New South Wales (NSW), Australia, which began in January, July and December 2020. Type of program or service: Public health outbreak response. METHODS: Narrative with epidemiological linking and genomic testing. RESULTS: Epidemiological linking and genomic testing found that during the first wave of COVID-19 in NSW, a large number of community transmissions went undetected because of limited testing for the virus and limited contact tracing of cases. The second wave of COVID-19 in NSW emerged following reintroduction from the second wave in Victoria, Australia in July 2020, and the third wave followed undetected introduction from overseas. By the second and third waves, cases could be more effectively detected and isolated through an increased ability to test and contact trace, and to rapidly genomic sequence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolates, allowing most cases to be identified and epidemiologically linked. This greater certainty in understanding chains of transmission resulted in control of the outbreaks despite less stringent restrictions on the community, by using a refined strategy of targeted shutdown, restrictions on cases, their close contacts, identified hotspots and venues of concern rather than a whole of community lockdown. Risk assessments of potential transmission sites were constantly updated through our evolving experience with transmission events. However, this refined strategy did leave the potential for large point source outbreaks should any cases go undetected. [Addendum] A fourth wave that began in Sydney in June 2021 challenged this strategy due to the more transmissible nature of the Delta variant of SARS-CoV-2. LESSONS LEARNT: A wave of COVID-19 infections can develop quickly from one infected person. The community needs to remain vigilant, adhering to physical distancing measures, signing in to venues they visit, and getting tested if they have any symptoms. Signing out of venues on exit allows public health resources to be used more efficiently to respond to outbreaks.

MtDNA Analysis Indicates Human-Induced Temporal Changes of Serbian Honey Bees Diversity.

Local populations of Apis mellifera are rapidly changing by modern beekeeping through the introduction of nonnative queens, selection and migratory beekeeping. To assess the genetic diversity of contemporary managed honey bees in Serbia, we sequenced mitochondrial tRNAleu-cox2 intergenic region of 241 worker bees from 46 apiaries at eight localities. Nine haplotypes were observed in our samples, with C2d being the most common and widespread. To evaluate genetic diversity patterns, we compared our data with 1696 sequences from the NCBI GenBank from neighbouring countries and Serbia. All 32 detected haplotypes belonged to the Southeast Europe lineage C, with two newly described haplotypes from our sample. The most frequent haplotype was C2d, followed by C2c and C1a. To distinguish A. m. carnica from A. m. macedonica, both previously reported in Serbia, PCR-RFLP analysis on the COI gene segment of mtDNA was used, and the result showed only the presence of A.m. carnica subspecies. An MDS plot constructed on pairwise FST values showed significant geographical stratification. Our samples are grouped together, but distant from the Serbian dataset from the GenBank. This, with the absence of A. m. macedonica subspecies from its historic range of distribution in southern Serbia, indicates that honey bee populations are changing rapidly due to the anthropogenic influence.

Exploring the real-world effect of the SARS-CoV-2 pandemic on the molecular diagnostics for cancer patients and high-risk individuals.

Background: The SARS-CoV-2 pandemic introduced a global distraction effect in cancer patients' care. The aim of this study was to explore the effect of the pandemic on the largest molecular diagnostics center for cancer patients and high-risk individuals in Serbia.Research design and methods: EGFR, KRAS/NRAS, BRAF, and BRCA1/2 mutation testing were performed by qPCR and NGS. NGS was used for panel testing of hereditary breast/ovarian cancer and cancers associated with Lynch syndrome. The analytical output during the state of emergency (SoE) was compared to the period before and after the outbreak using one-way ANOVA. Statistical significance was set at p < 0.05.Results: A 38% reduction in the number of analysis was detected during the SoE. After the SoE, a 19% reduction was noted compared to SoE and 50% compared to the period before the SoE (p = 0.038). Three of the 48 scheduled appointments for pretest genetic counseling were carried out during the SoE, but the number of NGS tests increased by 50%.Conclusions: The SARS-CoV-2 pandemic had a profound negative effect on the diagnostic output of our centralized molecular diagnostics center. The only positive effect was shortening of waiting lists for hereditary cancer patients and high-risk individuals.

Genetic diversity of the Griffon vulture population in Serbia and its importance for conservation efforts in the Balkans.

The Griffon vulture was once a widespread species across the region of Southeast Europe, but it is now endangered and in some parts is completely extinct. In the Balkan Peninsula the largest Griffon vulture inland population inhabits the territory of Serbia. We present, for the first time, the genetic data of this valuable population that could be a source for future reintroduction programs planned in South-eastern Europe. To characterize the genetic structure of this population we used microsatellite markers from ten loci. Blood samples were collected from 57 chicks directly in the nests during the ongoing monitoring program. We performed a comparative analysis of the obtained data with the existing data from three native populations from French Pyrenees, Croatia, and Israel. We have assessed the genetic differentiation between different native populations and determined the existence of two genetic clusters that differentiate the populations from the Balkan and Iberian Peninsulas. Furthermore, we analysed whether the recent bottleneck events influenced the genetic structure of the populations studied, and we found that all native populations experienced a recent bottleneck event, and that the population of Israel was the least affected. Nevertheless, the parameters of genetic diversity suggest that all analysed populations have retained a similar level of genetic diversity and that the Griffon vulture population from Serbia exhibits the highest value for private alleles. The results of this study suggest that the Griffon vulture populations of the Balkan Peninsula are genetically differentiated from the populations of the Iberian Peninsula, which is an important information for future reintroduction strategies.

EGFR mutation testing from liquid biopsy of non-small cell lung cancer at the Institute for Oncology and Radiology of Serbia.

PURPOSE: Resistance to tyrosine kinase inhibitors (TKIs) in lung cancer often occurs, so mutation testing from liquid biopsy is the method of choice as a minimally invasive approach that quickly provides information for additional therapeutic options. The purpose of this study was to assess the success rate and usefulness of EGFR testing from liquid biopsy at the Institute for Oncology and Radiology of Serbia (IORS). METHODS: EGFR mutation testing was performed by real-time qPCR in 4750 tumor samples using the Cobas® EGFR Mutation Test v2. EGFR testing from 104 liquid biopsy samples was used to track the resistance on first-line EGFR-TKIs as well as for initial testing of 124 patients without tissue biopsies. RESULTS: Liquid biopsy samples were tested in cases with inadequate material for DNA isolation or without tissue biopsy at diagnosis. Nine mutated samples were detected (7.3 %) with a 99.2 % testing success rate. Testing liquid biopsy samples of patients who progressed on EGFR-TKIs showed an accordance rate of 67% with driver mutations, and 49% of mutated patients had the T790M mutation which rendered them eligible for third-generation EGFR-TKIs. An additional 5 patients tested EGFR wild type from plasma after progression were rebiopsied and 3 of them had the T790M mutation. CONCLUSIONS: EGFR mutation testing from liquid biopsy has been successfully implemented in Serbia and has proven invaluable for detecting molecular resistance mechanisms to EGFR-TKIs and as an alternative sample source for patients with scarce biopsy material or without any at all.

Identification of a broad spectrum of mammalian and avian species using the short fragment of the mitochondrially encoded cytochrome b gene.

Mitochondrial DNA (mtDNA), especially the gene for cytochrome b (MT-CYB), has been found to be highly informative for species identification. In this study, we present the results of the analysis of a 127 bp long fragment of MT-CYB, amplified using universal primers, variable enough to be used for species identification and discrimination, even in highly degraded animal samples. The total number of analyzed species in this study was 30, including 17 mammalian and 13 bird species. Using a newly created primer pair, we successfully amplified and sequenced the target sequence in almost all tested species. The amplification was incomplete in just two species, and as a result, partial, but still variable sequences, were obtained. Using the target fragment we successfully identified all tested samples. Initial results suggested that the intraspecies genetic diversity of the target region, in all tested species, was low - from 0 to 4.72%. The interspecies genetic diversity of the target region, crucial for successful discrimination, showed relatively high diversity, ranging from 8.36% to 42.52%. Given its short length, the target region should be used for species determination, particularly in samples that are degraded or are low in DNA quantity.

Toxicity of mancozeb, chlorothalonil, captan, fluopyram, boscalid, and difenoconazole to Didymella applanata isolates from Serbia.

Field isolates of Didymella applanata, the causal agent of spur blight of raspberry, were evaluated in vitro for their sensitivity to mancozeb, chlorothalonil, captan, fluopyram, boscalid and difenoconazole. A total of 10 isolates, collected during 2013 at five localities in the major raspberry growing region in Serbia, and characterized as copper hydroxide, dithianon, and tebuconazole (sensitive), pyraclostrobin (sensitive or highly resistant) and fluazinam (sensitive or moderately resistant), were used in this study. The EC50 values for the isolates ranged from 1.33 to 2.88 mg L(-1) for mancozeb, from 3.18 to 6.65 mg L(-1) for chlorothalonil, from 15.75 to 24.69 mg L(-1) for captan and from 1.80 to 8.20 mg L(-1) for fluopyram. The narrowest range of EC50 values was recorded for difenoconazole (0.23-0.49 mg L(-1)), whereas the widest range was obtained for boscalid (4.49-49.25 mg L(-1)). The calculated resistance factors showed that all D. applanata isolates were sensitive to mancozeb, chlorothalonil, captan, and difenoconazole. Four isolates were moderately resistant to boscalid, while three of them were also moderately resistant to fluopyram. This finding of moderately resistant isolates to these SDHI fungicides indicates a possible cross-resistance which should be clarified in further investigations.

Toxicity of copper hydroxide, dithianon, fluazinam, tebuconazole and pyraclostrobin to Didymella applanata isolates from Serbia.

A study of the in vitro sensitivity of 10 isolates of Didymella applanata to copper hydroxide, dithianon, fluazinam, tebuconazole and pyraclostrobin, was conducted. The isolates were derived from diseased raspberry canes sampled during 2013 at five localities in western part of Serbia, known as the main raspberry growing region of the country. Prior to sensitivity testing experimental conditions for radial growth assay were optimized. The results showed that the temperature of 22 °C, oatmeal agar medium and 12/12 hrs light/ darkness light regimen provided the best conditions for sensitivity tests. Most of D. applanata isolates were sensitive to the tested fungicides. The narrowest range of EC50 values was recorded for tebuconazole (1.42-2.66 mg L(-1)). The widest range of EC50 values was obtained for pyraclostrobin, ranging from 0.17 mg L(-1) to 55.33 mg L(-1). The EC50 values for the studied isolates were 39.48-51.19 mg L(-1) for copper hydroxide, 12.12-18.73 mg L(-1) for dithianon and 5.72-42.56 mg L(-1) for fluazinam. According to resistance factor values, all D. applanata isolates were sensitive to copper hydroxide, dithianon and tebuconazole. Among tested isolates, six were highly resistant to pyraclostrobin (RFs in the range of 207.1-325.5) and two moderately resistant to fluazinam (RFs were 3 and 7.4), respectively.

Toxicity of metalaxyl, azoxystrobin, dimethomorph, cymoxanil, zoxamide and mancozeb to Phytophthora infestans isolates from Serbia.

A study of the in vitro sensitivity of 12 isolates of Phytophthora infestans to metalaxyl, azoxystrobin, dimethomorph, cymoxanil, zoxamide and mancozeb, was conducted. The isolates derived from infected potato leaves collected at eight different localities in Serbia during 2005-2007. The widest range of EC(50) values for mycelial growth of the isolates was recorded for metalaxyl. They varied from 0.3 to 3.9 µg mL(-1) and were higher than those expected in a susceptible population of P. infestans. The EC(50) values of the isolates were 0.16-0.30 µg mL(-1) for dimethomorph, 0.27-0.57 µg mL(-1) for cymoxanil, 0.0026-0.0049 µg mL(-1) for zoxamide and 2.9-5.0 µg mL(-1) for mancozeb. The results indicated that according to effective concentration (EC(50)) the 12 isolates of P. infestans were sensitive to azoxystrobin (0.019-0.074 µg mL(-1)), and intermediate resistant to metalaxyl, dimethomorph and cymoxanil. According to resistance factor, all P. infestans isolates were sensitive to dimethomorph, cymoxanil, mancozeb and zoxamide, 58.3% of isolates were sensitive to azoxystrobin and 50% to metalaxyl. Gout's scale indicated that 41.7% isolates were moderately sensitive to azoxystrobin and 50% to metalaxyl.