In this study, three Penicillium species, P. expansum, P. crustosum, and P. paneum, were identified in sugar beet roots with blue mold collected at harvest and from overwintering roots, marking a novel finding for Serbia. Notably, our study is the first to document P. crustosum as the causal agent of blue mold in sugar beet in the world. Pathogenicity tests on artificially inoculated sugar beet roots confirmed that all Penicillium isolates can induce rot, with P. expansum demonstrating the highest virulence, followed by P. crustosum and P. paneum. As Penicillium species are important postharvest pathogens linked to decay, economic losses and mycotoxin contamination, further research into their postharvest presence and impact is essential. This study presents the first analysis of Penicillium spp. on sugar beet in Serbia, aimed to characterize isolates both molecularly and morphologically, and to evaluate their pathogenic potential as postharvest pathogens. The results contribute to the current knowledge of Penicillium species capable of colonizing sugar beet roots, and expand our understanding of Penicillium spp. and their diversity and distribution in Serbia.
More frequent application of the biofungicide Bacillus subtilis Ch-13 enhanced its efficacy against natural infestation of Hypomyces odoratus (cobweb disease) in white button mushrooms (Agaricus bisporus), as well as its positive impact on mushroom production. In two different application procedures, strain B. subtilis Ch-13 was used at a final concentration of 60 ml per m2 of casing layer (1 × 108 CFU ml-1). In comparison to the low efficacy of the fungicide prochloraz (53%), optimal efficacy of B. subtilis Ch-13 in the suppression of H. odoratus was recorded. Substantially higher efficacy of this biofungicide in cobweb disease control was achieved when it was applied in three split doses (42%), rather than two (30%), in the large-scale production of white button mushrooms. The greatest improvement in white button mushroom production, in comparison to the untreated control, was achieved when B. subtilis Ch-13 was applied in three split doses (biological efficiency, BE=15%), rather than two (BE=7%). For suppression of the mycopathogen H. odoratus on white button mushrooms, this study supports the application of the biofungicide B. subtilis Ch-13 in three split doses of 30 + 15 + 15 ml m-2 on the second day after casing, two weeks after casing, and after the first fruiting flush, respectively.
The current study aimed to evaluate the efficacy of the biofungicide Bacillus subtilis Ch-13 in the suppression of natural infection of white button mushrooms (Agaricus bisporus) with Hypomyces perniciosus (causal agent of wet bubble disease), as well as its impact on mushroom yield in industrial-scale cultivation. The biofungicide B. subtilis Ch-13 was applied at a total concentration of 60 ml per m2 of casing layer in two different procedures - using either three (30 + 2 × 15 ml m-2) or two split doses (2 × 30 ml m-2) - and then its effects were compared to those of the fungicide prochloraz applied at the standard application rate. The efficacy of the biofungicide was significantly higher when applied in three split doses (29.7%), than in two (15.7%). Though the efficacy of B. subtilis Ch-13 (≈30%) against H. perniciosus was low in comparison to that of prochloraz (≈68%), B. subtilis Ch-13 slightly reduced wet bubble symptoms. Furthermore, the highest increase in mushroom yield was achieved when B. subtilis Ch-13 was applied in three split doses (14%), rather than two (2%), compared to the untreated control. In comparison to prochloraz, three and two split applications of B. subtilis Ch-13 enhanced mushroom yield by up to 17% and 4%, respectively. Regarding its efficacy in wet bubble disease control and augmentation of mushroom yield, B. subtilis Ch-13 was much more effective when applied in three split doses, than in two. Therefore, this study supports the application B. subtilis Ch-13 in three split doses (30 + 2 × 15 ml m-2, on the second day and two weeks after casing, and after the first fruiting flush, respectively) to suppress H. perniciosus and increase mushroom yield.
Relationships (synergistic/antagonistic/additive) among three biocontrol agents - the native antagonistic bacterium Bacillus amyloliquefaciens B-241, the yield-stimulating actinobacterium Streptomyces flavovirens A06, and a commercial strain of the entomopathogenic nematode Steinernema feltiae - were investigated for the purpose of evaluating their effects on the suppression of artificially inoculated green mould disease agent Trichoderma aggressivum f. europaeum T77, as well as the suppression of natural infestation of the fungus gnat, Lycoriella ingenua, in an experimental growing chamber of cultivated white button mushroom Agaricus bisporus. Biocontrol agents were applied at standard application rates, or reduced rates of 40% or 20%. The impact of biocontrol agents and their interactions on mushroom productivity was calculated as the ratio of the fresh weight of the total mushroom yield to the weight of the dry spawned substrate. The density of fungus gnat flies was monitored by using yellow sticky traps placed inside each insect-rearing cage with mushroom substrate. The evaluation of disease and pest control efficacy was based on disease and pest incidence in the inoculated control and treatment groups. Simultaneous application of three biocontrol agents revealed mild antagonistic interactions in their efficacy in green mould disease control, an antagonistic relationship in the control of the fungus gnat, while synergy was detected regarding their impact on mushroom yield. The results of this study suggest that each biological agent should be applied three times every seven days to provide efficient pest and disease control: entomopathogenic nematodes used individually at the first day after the casing time (S. feltiae 0.75 × 106 IJ m-2, total amount 2.25 × 106 IJ m-2), and beneficial microorganisms used combined a few days later (B. amyloliquefaciens 1 × 109 CFU ml-1 m-2, total amount 3 × 109 CFU ml-1 m-2, and S. flavovirens 1 × 108 CFU ml-1 m-2, total amount 3 × 108 CFU ml-1 m-2).
Wireworms, the larval stage of click beetles (Elateridae: Agriotes spp.), pose a significant threat to global agriculture, particularly to root vegetables, such as onion. Their subterranean lifestyle, as well as the withdrawal of some traditional synthetic insecticides, make them challenging to control. Therefore, this research aimed to compare the effects of natural, semi-synthetic and synthetic insecticides in controlling wireworm damage in an onion field. A field trial for testing the effects of different insecticidal treatments on plant density, wireworm damage (%) and total onion yield, was conducted at the Institute for Vegetable Crops (Smederevska Palanka, Serbia) in 2024. The experiment consisted of six treatments: an untreated control, three natural insecticides (two formulations of spinosad a.i. - granular and liquid, and Beauveria bassiana ATCC 74040 2.3?107 conidiospores/ml), a semi-synthetic insecticide (a.i. spinetoram) and a synthetic insecticide (a.i. tefluthrin). The insecticides were applied during planting, following their label application rates per hectare. Assessments were conducted 20 and 42 days after treatment (DAT) to determine plant density. Wireworm damage was specifically evaluated 42 DAT, and yield was calculated by weighing the harvested onion bulbs. The results showed that the granular spinosad formulation, applied in furrows at planting, significantly increased plant density 20 DAT, while its liquid formulation, applied as a soil treatment during planting, resulted in the lowest density. Spinetoram showed the highest plant density 42 DAT and the highest percentage of wireworm damage (15%) of all insecticides tested. The control had the highest percentage of damaged plants and the lowest yield. Onion yield was at the maximum after treatment with spinetoram, whereas the lowest yield was achieved after treatment with the granular spinosad formulation. Field trials show that natural and semi-synthetic insecticides can effectively control wireworms, ensuring adequate crop protection and viable yields. This study supports developing and adopting environmentally conscious soil pest management.
In this study, an isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method with diode array detection (DAD) was developed and validated for simultaneous determination of azoxystrobin, trifloxystrobin and prothioconazole in plant-protection products. Chromatographic separation of the active substances was achieved using 0.1% acetic acid and acetonitrile (30:70 v/v) at a flow rate of 0.52 ml/min on a Zorbax Eclipse XDB-C18 (50 mm ? 4.6 mm ? 1.8 ?m) and UV detection at 210 nm. Validation was done by evaluating the linearity and precision of the method, repeatability of injections, accuracy, and limits of detection and quantification (LOD and LOQ). Under the conditions, correlation coefficients of linearity were 0.996-0.997, the precision of method, expressed as relative standard deviation, was lower than the modified Horwitz values, the accuracy of all individual substances was within the range of 94.61-107.35%, while repeatability of the injectons was satisfied with RSD of 0.94-1.35%. LOD and LOQ were 0.0063 mg/ml and 0.019 mg/ml for azoxystrobin, 0.0051 mg/ml and 0.015 mg/ml for prothioconazole and 0.0051 mg/ml and 0.015 mg/ml for trifloxystrobin, respectively. A simple, precise, accurate, and fast analytical method for simultaneous determination of the fungicides azoxystrobin, prothioconazole and trifloxystrobin can be proficiently used for their detection and quantification in formulated products. The developed and validated method was applied to real samples, confirming the method's applicability.
Apple production suffers significant economic losses and fruit quality reduction due to fungal pathogens, particularly ones that cause postharvest fruit rot, such as Botryosphaeriaceae fungi. Isolates used in this study were obtained from symptomatic apples and, based on morphological characteristics and sequence analysis of two genes (EF 1-? and ?-tubulin), they were identified as Diplodia seriata and Botryosphaeria dothidea. Pathogenicity tests on healthy apple fruits revealed that D. seriata was more aggressive than B. dothidea, with significantly higher average values of lesion diameter and depth. Fungicide sensitivity tests showed that D. seriata was more sensitive to the combination fluopyram + tebuconazole (EC50=0.00023 ?g a.i. ml-1), while B. dothidea exhibited higher sensitivity to pyraclostrobin (EC50=0.025 ?g a.i. ml-1). With 98.44% and 97.56% percent growth inhibition (PGI) rate of D. seriata and B. dothidea (respectively) at 10 ?g a.i. ml-1, the tested combination of fungicides surpassed pyraclostrobin in inhibition potential. Four essential oils (thyme, rosemary, lavender and lemongrass) were also tested for antifungal activity using the fumigant macrodilution method. Thyme oil demonstrated the highest antifungal potential, completely inhibiting the mycelial growth of both species at 0.05 ?l ml-1 of air. Strong inhibition potential was also shown by lemongrass oil with 100% inhibition of D. seriata and B. dothidea mycelial growth at 0.07 and 0.09 ?l ml-1 of air, respectively. Rosemary oil showed a moderate inhibition potential, while lavender oil was the least effective. These findings highlight the inhibiting potential of fungicides against D. seriata and B. dothidea, but they also indicate that thyme and lemongrass essential oils could be used as viable alternatives. Further research is needed to determine their effectiveness in in vivo assays and potential impact on fruit quality and the environment.
The protection of quality and safety of stored products requires adequate and regular measures of protection from harmful rodents. Poisonous baits in a variety of formulations, primarily those based on cereals, are the most widely applied method of rodent control. Providing rodenticide baits that are durable over extended periods of time under adverse environmental conditions is an evident challenge for successful rodent control. The present study focused on examining the effects of particle size of maizeand wheat-based rodenticide bait carriers on bait longevity under unfavourable environmental conditions. Maize and wheat grain fractions with particle sizes <0.8 mm, 0.8-1.25 mm, 1.25-2 mm and >2 mm were kept in a climate chamber for 33 days at temperature varying from 30-35 C˚ and humidity from 90-95%. Baits containing different grain fractions, and supplemented with 1% sorbic acid, were tested for mold development. The same grain fractions without the preservative were used as control baits. Test and control baits were exposed to identical environmental conditions in the climate chamber over the same time period. Mold development on preservative-free baits was considerably faster and more intensive, compared to baits that contained sorbic acid. Initial signs of mold development on control baits were observed as early as on the third day of testing. Carrier type and fraction size affected mold development significantly. Regarding maize-based baits, mold development was more intestive on grain fractions >1.25 mm, compared to wheat-based grain which enabled more intensive development of mold on fractions with particle size <1.25 mm.
Control of brown rats (Rattus norvegicus) is an indispensable measure of protection of human population and domestic animals, aimed at preventing the spread of infectious diseases, and a measure of protection of goods and commodities that the brown rat can damage by its presence and activities. Rodent control procedures in environments with unfavourable conditions, such as high temperature and humidity, may fail due to reduced bait palatability caused by its degradation. Baits were prepared based on OEPP/ EPPO recommendations, while ECHA recommendation was followed for bait exposure to unfavourable environmental conditions. Placebo baits were prepared by mixing ground maize grain and paraffin. Test baits, made by mixing the placebo bait with sorbic acid, were exposed to unfavourable environmental conditions (temperature 30-35 ⁰C and humidity 90-95%). Brown rats previously captured from a wild population were used as the test organism. After an acclimation period, the animals were subjected to a four-day choice feeding test in which they were simultaneously offered placebo and test baits. The average bait palatability was unchanged, and the palatability of bait containing the preservative was 48.85 %. Furthermore, the preservative had no influence on bait consumption, compared to baits in the pre-test period, as there was no statistical difference in total amounts of bait consumed in the tests. The results indicate that 1% sorbic acid can be used as a potentially good additive to baits that are planned to be used in sewage systems and other areas where unfavorable environmental conditions prevail and there is a risk of rapid mold development and bait decay.
The effects of biopesticide products based on oxymatrine, azadirachtin and Beauveria bassiana (strain ATCC 74040) on demographic parameters of the two-spotted spider mite, Tetranychus urticaeKoch (Acari: Tetranychidae), were evaluated in laboratory bioassays. The biopesticides were applied by spraying 24 h old eggs laid on bean leaves, using the following concentrations: 50 pl/l (oxymatrine-based product), 0.75 g/l (azadirachtin-based product) and 3 ml/l (B. bassiana-based product). These concentrations were within 95% confidence limits of the LC50s estimated in acute toxicity bioassays. The mites that hatched from treated eggs completed their juvenile development on the same leaf discs, i.e. the toxic effect was caused by topical treatment and residual exposure. When the surviving females entered their preovipositional period, cohorts of 40 control and 40 treated females were transferred to untreated leaf discs (1 female/disc). Females were transferred daily to new discs until the death of the last one. Using the data on their age-specific survival and age-specific fertility (production of fem ale offspring), the following dem ographic parameters were estimated: gross reproductive rate (GRR), net reproductive rate (R0), intrinsic rate of increase (rm), finite rate of increase (A), and mean generation time (T). The biopesticides significantly affected these dem ographic parameters in fem ales that survived treatm ents. All three biopesticides significantly reduced the GRR and R0values. In the bioassays with oxymatrine-and azadirachtin-based products, the rm and A values were reduced by 22% and 5%, and 16% and 4%, respectively, due to reduced survival and fertility, as well as extended juvenile developmental time of females that survived treatment, compared to control females. In the bioassay with the B. bassiana-based product, the rm and A values were reduced by 7% and 2%, respectively, mostly due to the reduced survival of treated females. The oxymatrine-and azadirachtin-based products significantly extended, while B. bassiana-based product reduced the T values.
In 2016, the species Neopulvinaria innumerabilis (Rathvon, 1854), belonging to the scale insect family Coccidae, was for the first time found in Serbia, in the location Radmilovac on Cornus sanguinea L. It reproduces by gamogenesis, develops one generation annually, and overwinters as a fertilized female on the trunk or branches of its host plant. During this research, the species was detected on five host plants at six locations in Serbia. It feeds by sucking sap from leaves, young shoots and branches of infested plants, causing desiccation of leaves and individual twigs. Four species of its natural enemies were collected and reared from colonies of N. innumerabilis. Two species of parasitoid wasps, Coccophagus lycimnia (Walker) (Aphelinidae) and Metaphycus hageni Daane and Caltagirone (Encyrtidae), were found, while the ladybird Exochomus quadripustulatus L. (Coccinellidae) and the fly Leucopomyia silesiaca (Egger) (Chamaemyiidae) were identified as predators. The most abundant entomophagous species was L. silesiaca.
Preservation of the appearance, structure and most importantly the attractiveness of baits targeting harmful rodents over extended periods of their exposure is one of the main tasks in the process of bait formulation. The impact of several preservatives, different methods of homogenization of main bait components, and base-paraffin ratios on the extension of longevity of bait base was examined under controlled laboratory conditions. Baits based on ground maize grain were exposed to unfavourable environmental conditions, such as high temperature (30 °C-35 °C) and air humidity (90%-95%). The exposure period was 9 days, and potential mold development was monitored over the same period of time. Sodium benzoate was found deficient in providing a satisfactory effect on bait longevity (<5 days), in contrast to potassium sorbate (>7 days) and sorbic acid, which provided the longest extension in bait sustainability (>9 days). Preservative application can significantly extend bait longevity under unfavourable environmental conditions. Extended bait functionality is important for rodent pest control procedures in habitats where unfavourable environmental conditions prevail, such as sewers and water supply grids (collectors and pumping stations), public areas, housing facilities (boiler rooms, moist sellers, etc.).
Persistent seed banks are equally important in agriculture and invasion biology. While seed vitality persistence exemplifies an eternal uphill battle for weed control in agriculture, it signals a potential invasiveness of species in invasion biology. Considering yield losses caused by Amaranthus retroflexus, Abutilon theophrasti, Chenopodium album and Datura stramonium in agriculture, and the importance of Ambrosia trifida as an emerging invader in Europe, the objective of this study was to test the viability and longevity of the aged seeds of these economically important weeds. Three seed viability/longevity tests were conducted: the crush test, germination test in Petri dishes, and 3,5-triphenyltetrazolium chloride (TTC) test. The results revealed a significant variation in germination potential among the tested species. The highest vitality was observed in 7-year-old seeds of A. retroflexus (41.67 %), followed by 16-year-old A. theophrasti seeds (17.78 %), 13-year-old C. album seeds (15.00 %) and 17-year-old D. stramonium seeds (7.5 %). Furthermore, a remarkable seed longevity was documented in the tested species (with the exception of A. trifida), preserving their germination potential for over half a century. Seed germination was recorded in 49-year-old seeds of D. stramonium, 53-year-old seeds of A. retroflexus, 58-year-old seeds of A. theophrasti and 59-year-old seeds of C. album, in strong evidence of the persistence of these weed species' seeds in the environment.
Hyalesthes obsoletus (Hemiptera: Fulgoromorpha: Cixiidae) is a pan-European polyphagous planthopper known as a significant vector of the plant pathogenic bacterium 'Candidatus Phytoplasma solani' (stolbur phytoplasma), which poses threats to various agricultural crops. A population of H. obsoletus associated with Convolvulus arvensis in Serbia was studied to investigate the presence and genetic diversity of Wolbachia, an endosymbiotic bacterium known for its promising biological control applications. Both insect-associated microorganisms, 'Ca. P. solani' and Wolbachia, were found in the assessed H. obsoletus population. The analyzed vector population had a 'Ca. P. solani' infection rate of 50%, while Wolbachia showed a high infection rate of 80%. Wolbachia presence displayed minimal variation across genders and was independent of individuals' phytoplasma-infection status. Genotyping of the identified 'Ca. P. solani' strains revealed four previously described stamp genotypes (Rqg50/St1, Rqg31/St2, STOL/St4 and M5/St28). Notably, a single novel Wolbachia ftsZ genotype, designated WHo1, was found in the assessed H. obsoletus population, providing a valuable insight into the genetic diversity of Wolbachia endosymbionts within the Cixiidae family. Phylogenetic analysis demonstrated intricate relationships between WHo1 and other Wolbachia strains infecting hosts from diverse hemipteran suborders. Although Wolbachia-based strategies show promise for phytoplasma vector control, further research is needed to elucidate its potential interactions with 'Ca. P. solani' and its effects on vector reproduction and fitness.
While pesticides undeniably contribute to enhancing agricultural productivity, the escalating trend in their usage has given rise to a myriad of environmental and public health challenges over time. Tetrachlorvinphos, an organophosphate pesticide deemed potentially carcinogenic by the International Agency for Research on Cancer, is commonly employed to combat flies, mites, and larvae in animals, safeguarding public health in open spaces, and managing pest issues in domestic animals. We aimed to investigate the genotoxic and cytostatic effects of tetrachlorvinphos on human lymphocytes in the G0 phase of the cell cycle using the sister chromatid exchange (SCE) assay. We found that tetrachlorvinphos increased SCE values at 3 concentrations (5, 25, 50 ?M). On the other hand, the increase in SCE values was found to be statistically significant only at the highest concentration (50 ?M, p<0.05). We also found that the SCE value showed a linear dose-dependent increase (p=0.005). We concluded that exposure to tetrachlorvinphos had genotoxic potential on human lymphocytes in the G0 phase of the cell cycle. Additionally, exposure of cells in the G0 phase of cell cycle to tetrachlorvinphos was found to have no discernible impact on cell cycle kinetics.
Ginger is one of the most valuable cash crops for farmers in different parts of Ethiopia. Bacterial wilt disease, caused by Ralstonia solanacearum, creates major limitation to production of ginger in Ethiopia. Heavy losses due to the disease occur regularly, causing an additional impediment for production in infected areas. Field tests were conducted at Tepi, South- Western Ethiopia, during the 2019 and 2020 main cropping seasons to assess the effects of soil solarization and botanical mulch on epidemics of bacterial wilt of ginger. Four soil solarization periods, lasting two, four, six or eight weeks before planting were integrated with four different botanical mulch treatments after planting: vetivar grass, lemon grass, Chinese chive and Lantana camara. Unsolarized and unmulched plots were used as the control for comparison. Treatments were arranged in factorial arrangements with randomized complete block design with three replications. The outcomes indicated that soil solarization integrated with lemon grass mulch treatments significantly reduced bacterial wilt mean incidence by 22.1% up to 42.2%, compared to control plots. These treatments also dramatically reduced AUDPC and disease progress rates. Soil solarization for eight weeks integrated with lemon grass mulch resulted in the lowest (42.2%) final mean disease severity and AUDPC (33.8%) in comparison to the control. Typical results of this study indicated that soil solarization integrated with botanical mulch treatments were effective in slowing dawn the epidemics of bacterial wilt and in recovering ginger production and productivity, and they are consequently recommended for application in the study areas along with other crop management schemes.
The original published version of the article contained two errors: (1) In Table 1B: tomato was not indicated as a host plant for T. evansi (2) In Figure 1: After carefully looking at the previous drawings of T. ludeni body parts, we concluded that it is necessary to provide new and more precise drawings of the male aedeagus and female tibia/tarsus I
The study was focused on improvement of the integrated management strategy against green mould disease agent Trichoderma aggressivum Samuels & Gams and mushroom fly Lycoriella ingenua (Dufour) as pests of the white button mushroom Agaricus bisporus (Lange) Imbach. The impact of neem cake amendment in casing soil on regulation of the abundance of mushroom sciarid fly adults, efficacy in controlling the green mould disease agent, and mushroom yield was evaluated. Casing soil was supplemented with different concentrations of neem cake: 1, 2.5, 5, 10 and 15%. Neem cake added as a supplement to casing soil at a rate of 2.5% reduced the number of mushroom fly adults by 83.93% and green mould disease incidence by 59.6% in comparison to the control. No adverse effect on mushroom formation, yield and quality of fruiting bodies was observed at that concentration. Amendment of 2.5% neem cake in the casing soil could be recommended for application in mushroom production to control L. ingenua and symptoms of green mould disease without negative impact on mushroom yield.
The original published version of the article contained an error: (1) Figure 2 contains research data repeated from Figure 1. Corrigendum presents the corrected version of Figure 2.
Sclerotinia species are economically important, necrotrophic and aggressive plant pathogens with a broad host range and worldwide distribution. They act as airborne or soilborne pathogens, and can be transmitted by seed. These pathogens can affect crops both during the growing season and after harvest. Yield losses due to Sclerotinia diseases in susceptible crops vary and may be as high as 100%. The most common pathogen from the genus Sclerotinia in Serbia is S. sclerotiorum. It occurs regularly on sunflower and its incidence may exceed 50% in some years, thus causing economically important crop losses in Vojvodina. Recently, two new species were detected in Serbia: S. trifolium in alfalfa and S. minor in lettuce plants. Diseases caused by Sclerotinia spp. are difficult to control due to the long-term survival of sclerotia in the soil and development of airborne ascospores. As with many other diseases, there is no single treatment that can completely control these pathogens. Implementation of multiple strategies, such as cultural practices (sanitation, crop rotation and tillage), physical, chemical and biological protection, as well as deployment of resistant cultivars, is necessary for effective disease management.
