Fungicidal Substances Research Articles

MONITORING OF PESTICIDE RESIDUES IN GRAPEVINE LEAVES UNDER VARIANT FUNGAL DISEASE MANAGEMENT

Food contamination is the result of over-reliance on fungicides in crop production. The presence of pesticide residues in agricultural commodities needs to be strategically solved by applying gentle agrotechnical procedures. The research aimed to evaluate the effect of the treatment of grapevine with fungicides on the content and mobility of 4 active substances in the leaves of the variety Hibernal. The experiment included a control variant treated with fungicides and an untreated variant. We focused on monitoring the active substances of fungicides used in the first half of the vegetation: azoxystrobin, cyflufenamid, difenoconazole, myclobutanil, and another 25 active substances, in terms of multi-residue analysis methodologies. Grape leaves for analysis were collected on two dates: in the phenological phase of berry development BBCH 73 and 14 days after grape harvest – BBCH 91. Samples were prepared according to STN EN 15662. For qualitative analyzes of residues, we used the QuEChERS method in combination with liquid (LC-MS/MS) and gas (GC-MS/MS) chromatography with tandem mass spectrometry. In the untreated variant, the contents of residues from both leaves collection were in undetectable values. In the leaf samples of the treated variant collected in BBCH 73, we established higher values of residues than the MRL determines, namely for azoxystrobin 0.083 mg/kg (MRL – 0.01 mg/kg). Azoxystrobin is the active substance of the Quadris max fungicide, and we applied this preparation 41 days before the first collection of leaves. In the analyzes of leaf sampling from the treated variant after grape harvest in BBCH 91, we found a decrease in the content of all monitored residues below detectable values. We consider the gradual degradation of residues in biological materials to be a very favorable result from the point of view of their impact on consumer health and the environment. Through visual observations of the vegetation, we found out that even the untreated variant had a perfect state of health. Hibernal is a variety suitable for biological production systems, and we assume that even in vintages with high pressure of fungal diseases, it is possible to maintain the health of the grapevine with ecological products. Using the appropriate grape variety combined with the right agrotechnical interventions makes it possible to use safer ecological practices to produce healthy food, maintaining sustainability and a healthy natural environment. The research will continue in the early Table grape varieties, where the vegetation period is shorter, and space for breaking down the residues is more difficult.

RESEARCH OF THE PHYTOTOXIC EFFECT OF TEBUCONAZOLE, РROTHIOCONAZOLE, FLUDIOXONYL AND BASED ON THEM PRODUCTS ON THE GERMINATION POWER AND GROWTH OF SEEDLINGS OF WHEAT AND WHITE MUSTARD

The elaboration of new fungicidal protectants is an urgent issue due to the ability of pathogens to develop resistance. This fact contributes to the constant production of new protectants, so it carries an additional environmental impact. The main problem of development of new fungicides is choice of methods for identifying of optimal combinations and concentrations of fungicides. The optimization of the composition of fungicides is aimed to reduce the amount of active substances due to the synergistic effect. The use of fungicidal preparations with a reduced amount of active substances carries a lower environmental impact and gives a greater economic effect.
 One of the main problems in the application of fungicides is the phytotoxicity of the fungicides in relation to the protected plants. Phytotoxicity in general is the ability of pesticides or other substances to have a depressing (toxic) effect on plants. This problem is solved through researches of the germination of seedlings during the development of protectants.
 Researches of the germination power of seeds of soft winter wheat and white mustard after treatment with tebuconazole, рrothioconazole, and fludioxonyl were considered in the article. The main traits of the influence of the named substances on the germination of wheat grains and white mustard seeds, and morphological features of wheat seedlings, such as the length of their roots and shoots are revealed.
 In accordance to the results of the research, combinations of active substances (a.s.) that provide high effectiveness against the causative agents of diseases, and also do not have a phytotoxic effect on the protected culture have been determined.
 The aim of the work was the identification of traits of the phytotoxic effect of tebuconazole, рrothioconazole, fludioxonyl in the combination of seed protectants which are used to protect seedlings from plant diseases.
 Materials and methods. An assessment of the effect of three active substances of fungicides on wheat of the Grom cultivar and white mustard was given. The analysis was carried out in two replications for 48 possible combinations of tebuconazole, рrothioconazole, and fludioxonyl as well as fot two control variants. The ratio of normal, infected, abnormal and non-germinated seeds was analyzed.
 Results. According to the realized studies, tebuconazole has the greatest suppressive effect on germination and growth of shoots. Prothioconazole had no effect on germination, but it had a stimulating effect on the development and growth of wheat’s roots and shoots. With the combined action of prothioconazole and fludioxonyl, the greatest decrease in germination of wheat was observed.
 Conclusion. The possible phytotoxic effect, which depends on the dose of the protectant, should be taken into account when choosing a protectant and developing combined products. So the results of research demonstrated that the following combinations had no phytotoxic effect on protected plants: tebuconazole with рrothioconazole 5:10 and 5:15, tebuconazole with fludioxonyl 5:10, рrothioconazole with fludioxonyl 5:10.

Chemical constituents of Desmodium triflorum and their antifungal activity against various phytopathogenic fungi.

In the course of finding new antifungal natural compounds against plant pathogens, the methanol extract of Desmodium triflorum was investigated phytochemically. From n-butanol-soluble fraction, seven compounds (1-7) were isolated and structurally elucidated. Of which, six compounds belong to flavone 6- or 8-C-glycoside class (1-6). Three major compounds (1-3) exhibited moderate in vitro antifungal activity against Sclerotium rolfsii, Fusarium oxysporum f. sp. cubense, and Phytophthora palmivora. Compound 1 (IC50 =162.1μg/mL) was most active against S.rolfsii in a dose-dependent manner. At 300μg/mL, compounds 1 and 2 significantly inhibited P.palmivora, whereas compound 3 lacked effectiveness. In addition, the nanoemulsion of the methanol extract with a droplet size of 12.2nm displayed an excellent inhibition against S.rolfsii and P.palmivora compared with the normal extract. The presence of 1 (0.846%) and 2 (0.759%) in the methanol extract may attribute to the antifungal activity of D.triflorum. These results proved the potential of D.triflorum and its C-glycoside flavonoids against phytopathogenic fungi for the first time. Besides, an enhancement in the effectiveness of nanoemulsion containing D.triflorum extract against the fungi was confirmed. The structural characteristics of 1 and 2 could be considered to develop new fungicidal substances in the future.

Antagonism of Bacillus thuringiensis to phytopathogenic micromicetes as pathogens of apple trees

Antagonistic microorganisms, producing bactericidal and fungicidal substances, spread in space and occupy various ecological niches. Antagonistic action of microorganisms can be combined with a number of other properties that can be used by humans for their own purposes. Microorganisms — agents of biological products often have a multifunctional effect on the components of the agroecosystem, which under the influence of various environmental factors could have positive and negative consequences. The aim of the study was to study the effect of new highly active entomopathogenic strains of Bacillus thuringiensis on non-target objects — components of the biocenosis of the apple orchard. When developing technologies for biocontrol of the number of apple trees pests using B. thuringiensis revealed an additional positive protective effect — reducing the number of pathogens of fungal diseases on the leaf surface. Treatment of apple leaves with a liquid culture of strain B. thuringiensis 0376 after 72 hours of application reduced the number of scab of Fusicladium dendriticum by 7 times, and the pathogen of monolisis of Monilia fructigena — 9 times. Strain B. thuringiensis 0371 showed the highest level of antagonism, because in vitro completely inhibited the growth and development of micromycetes on the surface of the nutrient medium. On the leaf surface, the application of culture strain 0371 reduced the number of Fusicladium dendriticum by 92%, and Monilia fructigena — by 86% after 72 hours of treatment. In a field experiment when treating apple trees with a culture of this strain, scab development decreased by 7.1%. Strain B. thuringiensis 787 did not show antagonism against phytopathogenicmicromycetes during co-cultivation on nutrient medium, which may be explained by its inability to produce water-soluble exotoxin, in contrast to strains of B. thuringiensis, which are antagonists of scab and apple monolisis. Strain B. thuringiensis 0371 requires further research and development of its formulations, because it is promising for the creation of a complex biological product with insect-fungicidal properties for use in ecological and agriculture friendly technologies.

Natural attenuation along subsurface flow paths based on modeling and monitoring of a pesticide metabolite from three case studies

BackgroundGroundwater—especially for the use as drinking water—is a strictly protected resource in the existing guidelines for pesticide registration and drinking water protection in the EU. One aspect that has hardly played a role in this context so far is the attenuation of pesticide concentrations along the flow path from the regulatory leaching concentration at a depth of 1 m below the applied field to raw water abstraction systems. The soil metabolite N,N-dimethylsulfamide (DMS) is formed from two fungicidal substances: tolylfluanid and dichlofluanid. According to the EU guidance document on relevant metabolites in groundwater, DMS is a “non-relevant” metabolite. However, long-term application of the two active substances on permanent crops has resulted in elevated and quantifiable amounts of DMS in groundwater catchment areas of water supplying plants. Therefore, in the case of DMS, substantial monitoring data is available. This enables in combination with groundwater modeling, a quantitative analysis of the natural attenuation of DMS concentrations over time and distance. To this end, extensive real-world data from three case studies of drinking water catchment areas in Germany were analyzed.ResultsThe environmental fate of DMS in soil and groundwater was evaluated according to the respective data determined at the study sites. Analyses using monitoring data and combined modeling approaches as well, were performed to obtain comparable results. These merged outcomes from monitoring and modeling show total attenuation factors of 12–93 from leachate at 1 m depth down to monitoring wells—close to raw water collection. If concentration attenuation further downwards to collected raw water is considered, the overall attenuation factor is even higher (40–246).ConclusionsThe conditions at the catchment areas of the three case studies are very diverse, thus providing a wide range of attenuating conditions. When following the path of DMS from its formation in soil below a treated field, to its leaching into the aquifer, and within the aquifer down to the raw water collection site, its concentration in water is continuously and consistently decreasing. The results from DMS represent conservative estimations due to non-sorptive, quick transport processes. Extended to other sorptive solutes, it represents the lower end of expected attenuation. Therefore, natural pesticide concentration attenuation processes are suggested for the consideration in regulatory pesticide risk assessments for a more realistic yet still protective evaluation of expected concentrations in raw water.

Investigation on the potential of poplar bark from short-rotation coppices as bio-based fungicidal additives

Bark on trees protects the plant against environmentally adverse conditions as well as fungi and insect attacks. There are different chemical substances, mostly in the outer bark of trees, which can stop fungi from developing. Bark as a by-product of wood plantations is available in high quantity and can serve as an excellent source for the production of eco-fungicides. In the presented article, bark of various poplar clones from two short-rotation coppices (SRC) in Poland and Germany was examined to determine the chemical composition, the possible extraction of potential fungicides (terpenes, aromatic and phenolic substances), and influence of their extracts on slowing the growth of mold fungi. It was proved that the content of the fungicidal substances depends strongly on the clone type. Two methods of extraction—Soxhlet and batch—were compared to obtain fungicidal extracts. Fungicidal substances were found in extracts gained with both approaches. Triterpenes, fatty acids, aldehydes, and alcohols were primarily the active fungicides in the Soxhlet extracts, whereas phenolic substances act as fungicides in the batch extracts.

Sensitivity of micromycetes of the genus Fusarium Link, causative agents of rot of the core of apple fruits, to the active substances of modern fungicides

As a result of the studies, species-specific reactions of strains of the genus Fusarium Link of relative sensitivity to the active substances of chemical fungicides, in vitro, were noted. The drugs showed both very high biological effectiveness (BE) (100%) and very low (0 %). In suppressing the species F. sporotrichioides, the best result was shown by a mixed preparation based on fluopyram and pyrimethanil, as well as single-component - mefentrifluconazole and cyprodinil, for the species F. oxysporum - all three mixed preparations: fluopyram + pyrimethanil; tebuconazole + fluopyram and thiram + difenoconazole. It can be preliminarily concluded that the same active substances and their mixtures exhibit unequal activity against different strains of the same species from the genus Fusarium, the causative agent of apple core rot.

The intensity of ethylene release by soybean plants under the influence of fungicides in the early stages of legume-rhizobial symbiosis

The effect of pre-sowing treatment of soybean seeds with fungicides on the intensity of ethylene release, the processes of nodulation and nitrogen fixation in different symbiotic systems in the early stages of ontogenesis were investigated. The objects of the study were selected symbiotic systems formed with the participation of soybean (Glycine max (L.) Merr.) Diamond variety, strains Bradyrhizobium japonicum 634b (active, virulent) and 604k (inactive, highly virulent) and fungicides Maxim XL 035 PS (fludioxonil, 25 g/L, metalaxyl, 10 g/L), and Standak Top (fipronil, 250 g/L, thiophanate methyl, 225 g/L, piraclostrobin, 25 g/L). Before sowing, the seeds of soybean were treated with solutions of fungicides, calculated on the basis of one rate of expenditure of the active substance of each preparation indicated by the producer per ton of seed. One part of the seeds treated with fungicides was inoculated with rhizobium culture for 1 h (the titre of bacteria was 107 cells/mL). To conduct the research we used microbiological, physiological, biochemical methods, gas chromatography and spectrophotometry. It is found that, regardless of the effectiveness of soybean rhizobial symbiosis, the highest level of ethylene release by plants was observed in the stages of primordial leaf and first true leaf. This is due to the initial processes of nodulation – the laying of nodule primordia and the active formation of nodules on the roots of soybeans. The results show that with the participation of fungicides in different symbiotic systems, there are characteristic changes in phytohormone synthesis in the primordial leaf stage, when the nodule primordia are planted on the root system of plants. In particular, in the ineffective symbiotic system, the intensity of phytohormone release decreases, while in the effective symbiotic system it increases. At the same time, a decrease in the number of nodules on soybean roots inoculated with an inactive highly virulent rhizobia 604k strain due to the action of fungicides and an increase in their number in variants with co-treatment of fungicides and active virulent strain 634b into the stage of the second true leaf were revealed. It was shown that despite a decrease in the mass of root nodules, there is an increase in their nitrogen-fixing activity in an effective symbiotic system with the participation of fungicides in the stage of the second true leaf. The highest intensity of ethylene release in both symbiotic systems was recorded in the stage of the first true leaf, which decreased in the stage of the second true leaf and was independent of the nature of the action of the active substances of fungicides. The obtained data prove that the action of fungicides changes the synthesis of ethylene by soybean plants, as well as the processes of nodulation and nitrogen fixation, which depend on the efficiency of the formed soybean-rhizobial systems and their ability to realize their symbiotic potential under appropriate growing conditions.

The effect of combinations of cyproconazole and fludioxonil on infestation of wheat and barley grains

The most popular and affordable means to protect plants from fungal diseases are fungicides. Development of new products is aimed at preparation of the composition of several substances to achieve the effect of mutual enhancement of action. We used the grains of wheat (Triticum sp. variety Julius) and barley (Hordeum sativum variety Gloria), they were significantly infected with phytopathogenic fungi, which allows us to get reliable data on the effectiveness of active substances of fungicides. All wheat grains treated with fungicides were infected less than the control ones. Analysis of the effect of individual fungicides demonstrated that the fungi were more sensitive to fludioxonil, especially at doses of CF 0: 2 (56.7% of infected grains) and CF 0:4 (36.7% of infected grains) 5.0 and 10.0 g/t. After the treatment of cyproconazole, the smallest number of infected grains was obtained when grains were treated at a dose 5.0 g/t (80.0 % of infected grains). The fungistatic effect of cyproconazole (C) and fludioxonil (F) was greatest when ratios of C : F were 2.5:10; 5:10; 10:2.5; 10:5; 10:10 g/t.

CHEMICAL CONSTITUENTS AND ANTIFUNGAL POTENTIAL OF THE Richardia brasiliensis (Gomes) ETHANOLIC EXTRACT

<p>The objective of this work was to evaluate the fungicide potential of the <em>Richardia brasiliensis</em> plant extract or its fractions on <em>Lasiodiplodia theobromae</em> and <em>Colletotrichum gloeosporioides</em>, as well as the isolation and identification of substances present in these fractions. For both the crude and ethanolic extracts, the plants’ aerial part was fractionated and the hexane fractions and ethyl acetate were separately subjected to columns of silica-gel leading to isolation of steroids and triterpene acids respectively. One- and two-dimensional <sup>1</sup>H-NMR and <sup>13</sup>C-NMR spectral analyses were used for structural elucidation. Concentrations of 10; 20; 40; 80; 160 µg 100mL<sup>-1</sup> ethanolic extract, hexane fractions and a pure isolated substance (oleanolic acid) with 5 µL of DMSO were utilized for antifungal activity evaluation. Spectral analysis allowed identification and isolation of <em>β</em>-sitosterol and a mixture of <em>β-</em>sitosterol and stigmasterol. For fungicide action, the hexane and acetate or pure fractions provided reduction of the mycelial growth of <em>C. gloesporioides</em>, at the 160 μg100 mL<sup>-1</sup> concentration. For <em>L. theobromae</em>, the acetate or pure fraction and the ethanolic extract were highlighted in the concentrations of 80 and 160 μg100 mL<sup>-1</sup>. From the results of the evaluation of fungicide potential of the R<em>. brasiliensis</em> extract and its fraction against the fungi <em>C. gloeosporioides</em> and <em>L. Theobromae</em>, as well as the chemical insulation of the substances involved, it can be noted that this weed contains potential fungicidal substances and especially the isolated triterpenes are promising for use in formulations in the control of these pathogens.</p>

Взаємодія процесів асиміляції азоту і вуглецю у рослин сої, оброблених речовинами із фунгіцидною активністю та бульбочковими бактеріями, інкубованими з лектином

Взаємодія процесів асиміляції азоту і вуглецю у рослин сої, оброблених речовинами із фунгіцидною активністю та бульбочковими бактеріями, інкубованими з лектином

Biotic aspects involved in the control of damping-off producing agents: The role of the thermotolerant microbiota isolated from composting of plant waste

Along with the high temperatures generated during the composting process, the microbial community of a compost pile collaborates actively in the control of damping-off producing agents. Such collaboration could be justified on the basis of the ability of the microbiota to produce fungicide substances that are involved in the control of damping-off or other bioactive compounds that affect the growth promotion of plants. Thanks to ex situ experiments, a remarkable suppressive effect was corroborated against different agents producing damping-off. The microbial strains involved in this effect were detected mainly in the bio-oxidative and maturation phases of the composting process. However, only 3% of the total collection of strains proved to have a multipotential character with respect to its spectrum of action against the damping-off producing agents, as well as in relation to its capacity to produce substances of agronomic interest. It is worth mentioning the presence of two thermophilic isolates identified as Geobacillus thermodenitrificans and Bacillus aerius that showed the ability to inhibit the symptoms caused by Phytophthora capsici in vivo. Both strains were able to produce siderophores, salicylic acid and chitinase enzymes in vitro, but only G. thermodenitrificans was able to stimulate the development of the root in pepper seedlings. In this sense, it is suspected that the production of cyanide by this thermophilic bacteria could be related to this latter effect.

Black and white teas as potential agents to combine with amphotericin B and protect red blood cells from amphotericin B-mediated toxicity.

Amphotericin B is a fungicidal substance that is treatment of choice for most systemic fungal infections affecting immunocompromised patients. However, severe side effects have limited the utility of this drug. The aim of this study was to evaluate the antifungal effect of the combination of amphotericin B with black tea or white tea and protective of citotoxic effect. The present study shows that white and black teas have additive effects with amphotericin B against some species Candida. In addition, the combination of white and black tea with amphotericin B may reduce the toxicity of amphotericin B to red blood cells. Our results suggest that white and black tea is a potential agent to combine with amphotericin for antifungal efficacy and to reduce the amphotericin dose to lessen side effects.

Antifungal activity of essential oils on two Venturia inaequalis strains with different sensitivities to tebuconazole.

The antifungal activity of seven essential oils (eucalyptus, clove, mint, oregano, savory, tea tree, and thyme) was studied on Venturia inaequalis, the fungus responsible for apple scab. The composition of the essential oils was checked by gas chromatography-mass spectrometry. Each essential oil had its main compound. Liquid tests were performed to calculate the IC50 of essential oils as well as their majority compounds. The tests were made on two strains with different sensitivities to tebuconazole: S755, the sensitive strain, and rs552, the strain with reduced sensitivity. Copper sulfate was selected as the reference mineral fungicidal substance. IC50 with confidence intervals were calculated after three independent experiments. The results showed that all essential oils and all major compounds had in vitro antifungal activities. Moreover, it was highlighted that the effectiveness of four essential oils (clove, eucalyptus, mint, and savory) was higher than copper sulfate on both strains. For each strain, the best activity was obtained using clove and eucalyptus essential oils. For clove, the IC50 obtained on the sensitive strain (5.2mg/L [4.0-6.7mg/L]) was statistically lower than the IC50 of reduced sensitivity strain (14mg/L [11.1-17.5mg/L]). In contrast, for eucalyptus essential oil, the IC50 were not different with respectively 9.4-13.0 and 12.2-17.9mg/L for S755 and rs552 strains. For mint, origano, savory, tea tree, and thyme, IC50 were always the best on rs552 strain. The majority compounds were not necessarily more efficient than their corresponding oils; only eugenol (for clove) and carvacrol (for oregano and savory) seemed to be more effective on S755 strain. On the other hand, rs552 strain seemed to be more sensitive to essential oils than S755 strain. In overall, it was shown that essential oils have different antifungal activities but do not have the same antifungal activities depending on the fungus strain used.

Formulation and Characterization of Antibacterial Hydrogels Based on Polyurethane Microstructures and 1,2,4-Triazole Derivatives

Antibacterial gels offer an efficient hygiene in the absence of soap and water. The most part of these products are based on mixtures of different antibacterial and fungicide substances dissolved in an alcohol. Unfortunately, all cosmetic products containing alcohols wash the sebum and dry the skin. In the present study, alcohol-free antibacterial hydrogels were obtained through the synthesis of polyurethane (PU) microstructures with an increased amount of surfactant; 5-mercapto-1,2,4-triazole derivatives were used as active compounds inside the PU microstructures as antibacterial agents. There were studied: the pH of samples, the size and stability of PU microstructures, the skin irritation, sebum level and the hydration of stratum corneum using the mouse model and their efficiency against Escherichia coli. The results indicate that were obtained hydrogels with a slightly acid pH, PU microstructures with sizes between 185 and 265 nm and with a medium stability. It was found that these new antibacterial hydrogels do not produce an important modification of skin parameters (erythema, melanin, sebum and hydration of stratum corneum) and are efficient against E. coli.

Evaluation of pesticide residue dynamics in Chinese cabbage, head cabbage and cauliflower

ABSTRACTPesticide residues from the time of application until harvest were analysed for 20, 17 and 18 active insecticidal and fungicidal substances in Chinese cabbage, head cabbage and cauliflower, respectively. In total, 40 mathematical models of residue degradation were developed using a first-order kinetic equation, and from these models it was possible to forecast the action pre-harvest interval for a given action threshold for low-residue production in Brassica vegetables as a percentage of the maximum residue level. Additionally, it was possible to establish an action pre-harvest interval based on an action threshold of 0.01 mg kg‒1 for the production of Brassica vegetables for baby food. Among the evaluated commodities, the speed of residue degradation was highest in head cabbage, medium in Chinese cabbage and lowest in cauliflower. The half-lives of pesticide in various vegetables were also determined: they ranged from 1.55 to 5.25 days in Chinese cabbage, from 0.47 to 6.54 days in head cabbage and from 1.88 to 7.22 days in cauliflower.

Exploring polyamine metabolism of Alternaria alternata to target new substances to control the fungal infection

Polyamines are essential for all living organisms as they are involved in several vital cell functions. The biosynthetic pathway of polyamines and its regulation is well established and, in this sense, the ornithine descarboxylase (ODC) enzyme acts as one of the controlling factors of the entire pathway. In this work we assessed the inhibition of the ODC with D, l-α-difluoromethylornithine (DFMO) on Alternaria alternata and we observed that fungal growth and mycotoxin production were reduced. This inhibition was not completely restored by the addition of exogenous putrescine. Actually, increasing concentrations of putrescine on the growth media negatively affected mycotoxin production, which was corroborated by the downregulation of pksJ and altR, both genes involved in mycotoxin biosynthesis. We also studied the polyamine metabolism of A. alternata with the goal of finding new targets that compromise its growth and its mycotoxin production capacity. In this sense, we tested two different polyamine analogs, AMXT-2455 and AMXT-3016, and we observed that they partially controlled A. alternata’s viability in vitro and in vivo using tomato plants. Finding strategies to design new fungicide substances is becoming a matter of interest as resistance problems are emerging.

Bacillus thuringiensis and Bacillus weihenstephanensis Inhibit the Growth of Phytopathogenic Verticillium Species.

Verticillium wilt causes severe yield losses in a broad range of economically important crops worldwide. As many soil fumigants have a severe environmental impact, new biocontrol strategies are needed. Members of the genus Bacillus are known as plant growth-promoting bacteria (PGPB) as well as biocontrol agents of pests and diseases. In this study, we isolated 267 Bacillus strains from root-associated soil of field-grown tomato plants. We evaluated the antifungal potential of 20 phenotypically diverse strains according to their antagonistic activity against the two phytopathogenic fungi Verticillium dahliae and Verticillium longisporum. In addition, the 20 strains were sequenced and phylogenetically characterized by multi-locus sequence typing (MLST) resulting in 7 different Bacillus thuringiensis and 13 Bacillus weihenstephanensis strains. All B. thuringiensis isolates inhibited in vitro the tomato pathogen V. dahliae JR2, but had only low efficacy against the tomato-foreign pathogen V. longisporum 43. All B. weihenstephanensis isolates exhibited no fungicidal activity whereas three B. weihenstephanensis isolates showed antagonistic effects on both phytopathogens. These strains had a rhizoid colony morphology, which has not been described for B. weihenstephanensis strains previously. Genome analysis of all isolates revealed putative genes encoding fungicidal substances and resulted in identification of 304 secondary metabolite gene clusters including 101 non-ribosomal polypeptide synthetases and 203 ribosomal-synthesized and post-translationally modified peptides. All genomes encoded genes for the synthesis of the antifungal siderophore bacillibactin. In the genome of one B. thuringiensis strain, a gene cluster for zwittermicin A was detected. Isolates which either exhibited an inhibitory or an interfering effect on the growth of the phytopathogens carried one or two genes encoding putative mycolitic chitinases, which might contribute to antifungal activities. This indicates that chitinases contribute to antifungal activities. The present study identified B. thuringiensis isolates from tomato roots which exhibited in vitro antifungal activity against Verticillium species.

Chemical Basis of the Fungicidal Activity of Tobacco Extracts against Valsa mali

Under pressure from social criticism and an unclear future, tobacco researchers have begun to seek alternative uses for the product. Here, we present our study on isolating tobacco compounds with fungicidal activity, which could be used as plant-derived pesticides. Using Valsa mali as the target fungus, agar plate tests were conducted to evaluate the fungicidal activity of various tobacco extracts, including tobacco leaves extracts prepared with different solvents, extracts of different tobacco cultivars, and samples from different tobacco organs. Fungal growth morphology was used as the criterion to evaluate the fungicidal activity of tobacco extracts. Correlation analyses between the fungicidal activities and the chemical components of tobacco extracts indicated the major chemical constituents with fungicidal activity. Then, the active compounds were isolated and their effects on the ultra-microstructures of V. mali was analyzed using scanning- and transmission-electron microscopy. The results suggested that tobacco extracts prepared with solvents of weaker polarity had higher fungicidal activity, and the inhibitory activity of tobacco extracts against V. mali was also cultivar dependent. Furthermore, the fungicidal effects of tobacco flower extracts were higher than those of the leaf extracts. Chemical analysis indicated that cembranoids were the main fungicidal substances, which act by destroying the endometrial structure of the fungus. Tobacco cembranoids at 80 μg/mL could completely inhibit the growth of V. mali, with an EC50 value of 13.18 μg/mL. Our study therefore suggests that tobacco leaves and inflorescences are excellent plant resources for the biological control of V. mali.

Quercetin and rutin as potential agents antifungal against Cryptococcus spp.

Amphotericin B is a fungicidal substance that is treatment of choice for most systemic fungal infections affecting as cryptococcosis the immunocompromised patients. However, severe side effects have limited the utility of this drug. The aim of this study was to evaluate the antifungal effect of the combination of amphotericin B with quercetin or rutin and as a protective of citotoxic effect. The antifungal activity to amphotericin B, quercetin and rutin alone and in combination was determined in Candida sp and Cryptococcus neoformans strains. Cytotoxicity test on erythrocytes was performed by spectrophotometric absorbance of hemoglobin. The amphotericin B MIC was reduced when used in combination with quercetin or rutin to C. neoformans ATCC strain and reduced when combined with rutin to a clinical isolate of C. neoformans. In addition, the combination of quercetin with amphotericin B may reduce the toxicity of amphotericin B to red blood cells. Our results suggest that quercetin and rutin are potential agents to combine with amphotericin B in order to reduce the amphotericin dose to lessen side effects and improve antifungal efficacy.