Response of the mushroom pathogen Cladobotryum mycophilum to prochloraz and metrafenone fungicides and Streptomyces flavovirens actinobacteria.

After an outbreak of cobweb disease of cultivated button mushroom in Serbia in 2003, the isolated fungal pathogen was initially identified as Cladobotryum dendroides (teleomorph Hypomyces rosellus) based on morpho-physiological traits. Molecular analysis indicated re-classification of two strains (isolated in 2004 and 2007) as Cladobotryum mycophilum (teleomorph Hypomyces odoratus). However, subsequent analysis of further five strains (isolated over the period 2003-2010) within the frames of the present study, also confirmed their identification as the exclusive cobweb causal agent C. mycophilum. After artificial inoculation, the symptoms observed on harvested and growing mushrooms were consistent with the appearance of cobweb disease. Pathogen sensitivity to fungicides was estimated by probit analyses. Fungicide susceptibility tests showed that C. mycophilum strains were highly sensitive both to prochloraz (ED50<0.087 µg mL-1) and the newly introduced metrafenone (ED50<0.15 µg mL-1). Furthermore, the growth of all examined strains of C. mycophilum was significantly inhibited by the indigenous actinobacterial strain Streptomyces flavovirens A06. A dual culture assay showed after 72 h that the percentage of radial growth inhibition of the pathogen ranged from 22.38 to 55.73%. Our findings suggest that the antagonistic S. flavovirens A06 might be a potential candidate for controlling the cobweb disease of cultivated button mushroom.

Sensitivity of Trichoderma strains from edible mushrooms to the fungicides prochloraz and metrafenone.

Twenty-two strains of Trichoderma spp. (T. harzianum species complex [THSC], Trichoderma aggressivum f. europaeum, Trichoderma pleuroti, and Trichoderma pleuroticola) causing green mold disease on edible mushrooms (button mushroom, shiitake and oyster mushroom), collected during 2004-2018 from four countries (Serbia, North Macedonia, Croatia, and Hungary) were examined. Based on their ITS (internal transcribed spacer) sequences, strains from shiitake mushroom in Serbia were identified as members of the THSC, while in samples obtained from Serbian and North-Macedonian oyster mushroom farms THSC, T. pleuroti and T. pleuroticola were detected, which represent the first findings in the region. In fungicide susceptibility tests, all examined Trichoderma strains were found to be highly sensitive to prochloraz (ED50<0.4 µg mL-1) and considerably susceptible to metrafenone (ED50 < 4 µg mL-1). The most sensitive taxon to both fungicides was THSC from oyster mushroom. The toxicity of metrafenone was satisfying and strains from oyster mushroom showed the highest sensitivity (ED50 < 1.43 µg mL-1), while strains originating from button mushroom and shiitake displayed similar susceptibilities (ED50 < 3.64 µg mL-1). After additional in vivo trials, metrafenone might also be recommended for the control of green mold disease in mushroom farms.

Bio-herbicidal effects of five essential oils on germination and early seedling growth of velvetleaf (Abutilon theophrasti Medik.).

The evaluation of the inhibition effect exerted by the essential oils of basil (Ocimum basilicum), sage (Salvia officinalis), thyme (Thymus vulgaris), lemon balm (Melissa officinalis) and goldenrod (Solidago virgaurea) on seeds germination and early seedling growth of velvetleaf (Abutilon theophrasti Medik.) weed was examined in a laboratory bioassay. The essential oils were obtained by hydrodistillation and characterized chemically by gas chromatography coupled with both mass spectrometry and flame ionization detector. The working solutions of essential oil emulsified with Tween 20 and dissolved in distilled water were prepared at three concentration levels (0.01%, 0.1% and 1%, vol/vol). The results obtained showed that increase of essential oil concentration leads to decrease of seed germination, shoot and radical length of velvetleaf. The obtained data revealed a highly significant effect (P < 0.05) between control and 1% and 0.1% oil concentrations in all treatments. The essential oils of basil, thyme and lemon balm exhibited more powerful bio-herbicidal effect compared to sage and goldenrod essential oils on the germination and early seedling growth of velvetleaf weed.

Superior cellulolytic activity of Trichoderma guizhouense on raw wheat straw.

Lignocellulosic plant biomass is the world's most abundant carbon source and has consequently attracted attention as a renewable resource for production of biofuels and commodity chemicals that could replace fossil resources. Due to its recalcitrant nature, it must be pretreated by chemical, physical or biological means prior to hydrolysis, introducing additional costs. In this paper, we tested the hypothesis that fungi which thrive on lignocellulosic material (straw, bark or soil) would be efficient in degrading untreated lignocellulose. Wheat straw was used as a model. We developed a fast and simple screening method for cellulase producers and tested one hundred Trichoderma strains isolated from wheat straw. The most potent strain-UB483FTG2/ TUCIM 4455, was isolated from substrate used for mushroom cultivation and was identified as T. guizhouense. After optimization of growth medium, high cellulase activity was already achieved after 72 h of fermentation on raw wheat straw, while the model cellulase overproducing strain T. reesei QM 9414 took 170 h and reached only 45% of the cellulase activity secreted by T. guizhouense. Maximum production levels were 1.1 U/mL (measured with CMC as cellulase substrate) and 0.7 U/mL (ß-glucosidase assay). The T. guizhouense cellulase cocktail hydrolyzed raw wheat straw within 35 h. Our study shows that screening for fungi that successfully compete for special substrates in nature will lead to the isolation of strains with qualitatively and quantitatively superior enzymes needed for their digestion which could be used for industrial purposes.

Impact of a native Streptomyces flavovirens from mushroom compost on green mold control and yield of Agaricus bisporus.

Thirty-five actinobacterial isolates, obtained from button mushroom (Agaricus bisporus) substrates (i.e., compost in different phases of composting, black peat or casing layer) in Serbia in 2014-2016 were tested in vitro against the causal agents of green mold in cultivated mushroom. Out of six most promising isolates, A06 induced 42.4% in vitro growth inhibition of Trichoderma harzianum T54, and 27.6% inhibition of T. aggressivum f. europaeum T77. The novel strain A06 was identified as Streptomyces flavovirens based on macroscopic and cultural characteristics and 16S rDNA sequence and used in mushroom growing room experiments. Actinobacteria had no negative influence on mycelial growth of the cultivated mushroom in compost in situ. Isolate S. flavovirens A06 enhanced mushroom yield significantly, up to 31.5%. The A06 isolate was more efficient in enhancing yield after inoculation with the compost mold T. aggressivum (26.1%), compared to casing mold T. harzianum (8%). Considering disease incidence, actinobacteria significantly prevented green mold in compost caused by T. aggressivum (6.8%). However, fungicide prochloraz-Mn had a more significant role in reducing symptoms of casing mold, T. harzianum, in comparison with actinobacteria (24.2 and 11.8%, respectively). No significant differences between efficacies of S. flavovirens A06 and the fungicide prochloraz-Mn against T. aggressivum were revealed. These results imply that S. flavovirens A06 can be used to increase mushroom yield and contribute to disease control against the aggressive compost green mold disease caused by Trichoderma aggressivum.

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.

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.

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.

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.

Training History, Cardiac Autonomic Recovery from Submaximal Exercise and Associated Performance in Recreational Runners.

This study investigated the effect of prolonged exertion on cardiac parasympathetic (cPS) reorganization and associated aerobic performance in response to repeated short-lasting submaximal exercise bouts (SSE) performed for 7 days following prolonged exertion. In 19 recreational runners, heart rate (HR) and HR variability (HRV) indices (lnRMSSD, lnHF, and lnLF/HF) were monitored pre- and post-submaximal graded cycling performed on consecutive days following a half-marathon (HM) and compared with the baseline, pre-HM values. Additionally, HR recovery (HRR), aerobic performance, and rate of perceived exertion (RPE) were determined. HR, HRV indices, and HRR were tested for correlation with exercise performance. A significant time effect was found in HR, HRR, and HRV indices as well as in aerobic performance and RPE during the study period. Most of the measured parameters differed from their baseline values only on the same day following HM. However, HRR and HR measured in recovery after SSE were additionally affected one day following the half-marathon yet in opposite directions to those recorded on the same day as the HM. Thus, postSSE HR and HRR exhibited a bivariate time response (postSSE HR: 102 ± 14 bpm; p < 0.001; 82 ± 11 bpm; p = 0.007 vs. 88 ± 11 bpm; HRR in 30 s after SSE cessation: 14.9 ± 4.9 bpm; p < 0.001; 30.1 ± 13.3 bpm; p = 0.006 vs. 24.4 ± 10.8 bpm), potentially indicating a cPS dysfunction phase on the same day and cPS rebound phase one day following HM reflected also in consecutive changes in aerobic power. Correlations were found between the changes in measured cardiac indices with respect to baseline and the changes in aerobic performance indices throughout the study period. The effect of exercise history on cPS reorganization is more pronounced in response to SSE than at rest. Accordingly, we conclude that SSE performed repeatedly on a daily basis following prolonged exertion offers a noninvasive tool to evaluate the impact of training history on cPS recovery and associated aerobic power output in recreational athletes.

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 (EC(50) values were 0.09 and 0.11 mg L(- 1), respectively) and weakly resistant to thiophanate-methyl (EC(50) 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 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 (EC(50) values were below 1.68 mg L(-1)). Trifloxystrobin and kresoxim-methyl were not effective in growth inhibition of C. dendroides isolates (EC(50) 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.