Biljana Todorovic

Possibilities of biological and chemical control of Verticillium wilt in pepper

In pathogen populations in Serbia, the incidence, pathogenic and morphological characters ofVerticillium spp. were studied. Biological and chemical control ofVerticillium was investigated in pepper plants (Capsicum annuum L. cv. ‘Soroksari’) with the biofungicide Polyversum® (Pythium oligandrum) and the conventional fungicides benomyl and propamocarb-hydrochloride. On the basis of macroscopic and microscopic characters of the isolates originating from eight localities in Serbia, it was established that they apparently belong to the speciesVerticillum dahliae. The isolates differed in their pathogenic characters. However, all of them caused marked wilting symptoms on pepper plants 40 days after inoculation, conducted when there were more than nine fully developed leaves on the primary stem. The fungicides were applied either before or after inoculation. Benomyl was the most efficient fungicide in wilt control (88.2% when applied after inoculation and 94.6% when applied before inoculation). Polyversum proved more efficient (66.6%) when applied before rather than after inoculation. Propamocarb-hydrochloride provided sufficient Verticillium wilt control; its efficacy and that of Polyversum were similar, and less efficient than benomyl, but still significantly different from the disease control.

Characterization of Clavibacter michiganensis subsp. michiganensis strains from recent outbreaks of bacterial wilt and canker in Serbia

Sixty-eight Clavibacter michiganensis subsp. michiganensis (Cmm) strains from recent outbreaks of bacterial wilt and canker in Serbia were collected from several tomato growing regions during a three-year period. The pathogen was identified based on bacteriological characteristics and pathogenicity tests and the identity of strains was confirmed by DAS ELISA and PCR amplification using primers CMM5/6 and PSA4/R. The strains showed homogeneity in biochemical and physiological properties. However, pathogenicity tests revealed differences in virulence that are presumably due to a loss of the pat-1 gene. Further strain characterization using DNA-based methods revealed a high diversity of the Serbian Cmm strains. Based on multi-locus sequence typing (MLST) analyses of five genes, Cmm strains were divided into seven groups. The pulsed-field gel electrophoresis (PFGE) pattern of a selection of strains supported the groupings based on trees of the kdpA/sdhA sequences. On the other hand, groupings made according to PFGE and MLST were not correlated to plasmid content in all cases. This study suggested that high genetic variability of the Serbian Cmm strains was detected both in MLST and PFGE analyses, and could have resulted either from new Cmm strains being introduced by seeds from different origins or as a consequence of an intraspecific hybridization process. In addition, this study proposed MLST as an efficient tool in epidemiological studies, population biology investigations and tracking the routes of transmission of pathogens. Four of the five house-keeping genes (kdpA, sdhA, ligA and gyrB) selected to characterize Cmm strains proved to be suitable for the MLST analysis. This is the first study carried out on the characterization of Cmm using MLST.

In vitro toxicity of selected fungicides from the groups of benzimidazoles and demethylation inhibitors to Cladobotryum dendroides and Agaricus bisporus

Twenty microfungal isolates were collected from diseased fruiting bodies of Agaricus bisporus sampled from Serbian mushroom farms during 2003–2007. Based on morphological characteristics and pathogenicity tests, the isolates were identified as Cladobotryum dendroides. The isolates of C. dendroides and A. bisporusF56 and U3 were tested for sensitivity to several selected fungicides in vitro. C. dendroides isolates were found to be more sensitive to prochloraz manganese and flusilazole + carbendazim than to the other fungicides tested (EC50 values were 0.09 and 0.11 mg L− 1, respectively) and weakly resistant to thiophanate-methyl (EC50 values ranged between 6.53 and 12.09 mg L− 1). Selectivity indexes of the tested fungicides on both C. dendroidesand A. bisporusindicated that thiophanate-methyl, cyproconazole + carbendazim and flusilazole + carbendazim had much less selective fungitoxicity than benomyl, carbendazim and prochloraz manganese.

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 EC50 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 EC50 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 (EC50) 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. Gouts scale indicated that 41.7% isolates were moderately sensitive to azoxystrobin and 50% to metalaxyl.

In vitro activity of antimicrobial agents against Pseudomonas tolaasii, pathogen of cultivated button mushroom

In vitro antibacterial activity tests of seven biofungicides (Ekstrasol, Bisolbisan, Bisolbifit, Serenade, Sonata, Timorex, F-Stop) and two disinfectants (colloidal silver alone and in combination with hydrogen peroxide) against the Pseudomonas tolaasii strain (NS3B6) were carried out by the disc-diffusion, broth microdilution and broth macrodilution method. Biofungicides tested in this study did not exhibit any antimicrobial activity in neither one of the methods used. Disc diffusion method revealed high sensitivity of the tested P. tolaasii strain to Ecocute based on colloidal silver and hydrogen peroxide. Both microdilution and macrodilution methods identified the same MICs and MBCs of Ecocute (0.19 mg/L) for P. tolaasii strain. MICs and MBCs values of silver alone were much higher (10 mg/L) compared to silver in combination with hydrogen peroxide.

Toxicity of fungicides with different modes of action to Cladobotryum dendroides and Agaricus bisporus

Isolates of Cladobotryum dendroides from Serbian mushroom farms and Agaricus bisporus F56 were tested for sensitivity to selected fungicides in vitro. Chlorothalonil was the most toxic fungicide to C. dendroides isolates (EC50 values were below 1.68 mg L− 1). Trifloxystrobin and kresoxim-methyl were not effective in growth inhibition of C. dendroides isolates (EC50 values exceeded 300 mg L− 1). Metalaxyl-M+mancozeb was the most toxic fungicide to strain F56 of A. bisporus, and iprodione the least toxic. The fungicide selectivity indexes for both C. dendroides and A. bisporus indicated that iprodione, chlorothalonil, captan and metalaxyl-M+mancozeb had satisfactory selective fungitoxicity. Iprodione had the best selectivity to both the pathogen and the host, although inferior than prochloraz manganese and carbendazim, fungicides officially recommended for mushroom cultivation in European Union (EU) countries.

Fungicide sensitivity of Trichoderma spp. from Agaricus bisporus farms in Serbia

Trichoderma species, the causal agents of green mould disease, induce great losses in Agaricus bisporus farms. Fungicides are widely used to control mushroom diseases although green mould control is encumbered with difficulties. The aims of this study were, therefore, to research in vitro toxicity of several commercial fungicides to Trichoderma isolates originating from Serbian and Bosnia-Herzegovina farms, and to evaluate the effects of pH and light on their growth. The majority of isolates demonstrated optimal growth at pH 5.0, and the rest at pH 6.0. A few isolates also grew well at pH 7. The weakest mycelial growth was noted at pH 8.0–9.0. Generally, light had an inhibitory effect on the growth of tested isolates. The isolates showed the highest susceptibility to chlorothalonil and carbendazim (ED50 less than 1 mg L−1), and were less sensitive to iprodione (ED50 ranged 0.84–6.72 mg L−1), weakly resistant to thiophanate-methyl (ED50 = 3.75–24.13 mg L−1), and resistant to trifloxystrobin (ED50 = 10.25–178.23 mg L−1). Considering the toxicity of fungicides to A. bisporus, carbendazim showed the best selective toxicity (0.02), iprodione and chlorothalonil moderate (0.16), and thiophanate-methyl the lowest (1.24), while trifloxystrobin toxicity to A. bisporus was not tested because of its inefficiency against Trichoderma isolates.

First report of Pseudomonas tolaasii on Agaricus bisporus in Serbia

Symptoms of brown blotch were observed on cultivated Agaricus bisporus on several mushroom farms in Serbia. Subsequently, samples were collected from nine localities during 2006–2010 and fluorescent Gram-negative bacterial strains were isolated. All strains caused sunken brown lesions on A. bisporus tissue blocks after artificial inoculation. Isolated strains showed the LOPAT characteristics of the group Va being negative for levan production; the ability to produce a hypersensitivity reaction in tobacco; were oxidase and arginine dihydrolase positive; and lacked pectolytic activity. The results from other tests were as follows: oxidative metabolism of glucose; catalase activity positive; aesculin hydrolisis negative; Tween 80 hydrolysis positive; casein hydrolysis positive; gelatine hydrolysis and nitrate reduction negative; mannitol, erythritol, sorbitol, inositol and trehalose were used as a carbon source but not sucrose, arabinose and D(-) tartrate. Based on these features bacterial strains were identified as Pseudomonas tolaasii. The identity of isolated strains was confirmed by polymerase chain reaction. This is the first report of P. tolaasii in Serbia.

Toxicity of twenty-two plant essential oils against pathogenic bacteria of vegetables and mushrooms

ASBTRACT Toxicity of twenty-two essential oils to three bacterial pathogens in different horticultural systems: Xanthomonas campestris pv. phaseoli (causing blight of bean), Clavibacter michiganensis subsp. michiganensis (bacterial wilt and canker of tomato), and Pseudomonas tolaasii (causal agent of bacterial brown blotch on cultivated mushrooms) was tested. Control of bacterial diseases is very difficult due to antibiotic resistance and ineffectiveness of chemical products, to that essential oils offer a promising alternative. Minimal inhibitory and bactericidal concentrations are determined by applying a single drop of oil onto the inner side of each plate cover in macrodilution assays. Among all tested substances, the strongest and broadest activity was shown by the oils of wintergreen (Gaultheria procumbens), oregano (Origanum vulgare), and lemongrass (Cymbopogon flexuosus. Carvacrol (64.0–75.8%) was the dominant component of oregano oils, while geranial (40.7%) and neral (26.7%) were the major constituents of lemongrass oil. Xanthomonas campestris pv. phaseoli was the most sensitive to plant essential oils, being susceptible to 19 oils, while 11 oils were bactericidal to the pathogen. Sixteen oils inhibited the growth of Clavibacter michiganensis subsp. michiganensis and seven oils showed bactericidal effects to the pathogen. The least sensitive species was Pseudomonas tolaasii as five oils inhibited bacterial growth and two oils were bactericidal. Wintergreen, oregano, and lemongrass oils should be formulated as potential biochemical bactericides against different horticultural pathogens.

Efficacy of seaweed concentrate from Ecklonia maxima (Osbeck) and conventional fungicides in the control of Verticillium wilt of pepper

In order to control the causal agent of Verticillium wilt of pepper (Verticillium dahliae), the efficacy of two conventional fungicides, thiophanate-methyl and carbendazim, and seaweed concentrate (SWC) from Ecklonia maxima was evaluated in greenhouse conditions. Pepper plants were inoculated with selected V. dahliae isolate in the stage of more than nine fully developed leaves on primary stem. The tested fungicides and SWC were applied three days before inoculation of pepper plants. Carbendazim was the most efficient fungicide among tested substances (69.64%). SWC proved to be more effective when applied at 1.0% concentarion (41.96%). The use of thiophanate-methyl provided good Verticillium wilt control in pepper (60.71%). SWC was less efficient than thiophanate-methyl and carbendazim, but still significantly better compared to the disease control plot.