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.     

Identification and comprehensive evaluation of a novel biocontrol agent Bacillus atrophaeus JZB120050

Abstract

Bacillus spp. have long been used as biocontrol agents because of their efficient broad-spectrum antimicrobial activity. We identified a novel strain of Bacillus atrophaeus, named JZB120050, from soil. B. atrophaeus JZB120050 had a strong inhibitory effect against Botrytis cinerea and many other phytopathogens. Gas chromatography-mass spectrometry showed that B. atrophaeus JZB120050 produced many secondary metabolites, such as alkanes, alkenes and acids; some of which were related to pathogen inhibition. Enzyme activity analysis showed that B. atrophaeus JZB120050 secreted cell-wall-degrading enzymes, including chitinase, glucanase and protease, which degraded fungal cell walls. Both the novel glucanase gene bglu and chitinase gene chit1 were cloned and heterologously expressed in Escherichia coli and the products showed strong enzyme activity. In addition, B. atrophaeus JZB120050 secreted siderophores and formed a significant biofilm. Future studies should focus on these antimicrobial factors to facilitate widespread application in the field of agricultural biocontrol.     

Bioremediation model for atrazine contaminated agricultural soils using phytoremediation (using Phaseolus vulgaris L.) and a locally adapted microbial consortium

The objective of the present study was to examine a biological model under greenhouse conditions for the bioremediation of atrazine contaminated soils. The model consisted in a combination of phytoremediation (using Phaseolus vulgaris L.) and rhizopheric bio-augmentation using native Trichoderma sp., and Rhizobium sp. microorganisms that showed no inhibitory growth at 10,000 mg L^?1 of herbicide concentration. 33.3 mg of atrazine 50 g^?1 of soil of initial concentration was used and an initial inoculation of 1 × 10⁹ UFC mL^?1 of Rhizobium sp. and 1 × 10⁵ conidia mL^?1 of Trichoderma sp. were set. Four treatments were arranged: Bean + Trichoderma sp. (B+T); Bean + Rhizobium sp. (BR); Bean + Rhizobium sp. + Trichoderma sp. (B+R+T) and Bean (B). 25.51 mg of atrazine 50 g^?1 of soil (76.63%) was removed by the B+T treatment in 40 days (a = 0.050, Tukey). This last indicate that the proposed biological model and methodology developed is useful for atrazine contaminated bioremediation agricultural soils, which can contribute to reduce the effects of agrochemical abuse.     

Inhibitory properties of Enterococcus spp. isolated from faeces of healthy dogs

The aim of the present study was to evaluate the inhibitory effect by the cross-streak method of nine Enterococcus faecium strains isolated from faeces of healthy dogs and their treated and non-treated cell-free supernatant (CFS) by the well-diffusion test on the growth of potentially pathogenic bacteria isolated from clinical cases and aflatoxigenic Aspergillus section Flavi and the consequent aflatoxin B₁ (AFB₁) production. Results obtained from the cross-strake assay showed that E. faecium MF1, GJ18 and GJ40 presented the major inhibitory activity against all pathogenic strains assayed; E. faecium GJ40 produced the larger inhibitory zones (26–27 mm). Well-diffusion test results showed that the majority of the enterococci strains CFS had antimicrobial activity against the pathogenic microorganisms, especially on Gram negative indicators. Cell-free supernatant of E. faecium GJ40 was the one that produced the largest inhibition zones (14 to 21 mm) in the majority of the indicator microorganisms assayed. All supernatants treated with 10 N NaOH (pH6) showed no inhibitory effect on the indicator strain assayed. With respect to fungal inhibition, any of the CFS assayed significantly inhibited the Aspergillus strains growth. But, in general, all CFS reduced AFB₁ production from 8 to 87%. The results demonstrate that enterococci isolated from healthy dog feaces produce substances with the capacity to inhibit some potential pathogenic bacteria growth and the capacity of inhibiting or reducing the AFB₁ production in vitro.     

Isolation of culturable mycobiota from agricultural soils and determination of tolerance to glyphosate of nontoxigenic Aspergillus section Flavi strains

Glyphosate-based herbicides are extensively used in Argentina's agricultural system to control undesirable weeds. This study was conducted to evaluate the culturable mycobiota [colony forming units (CFU) g^?1 and frequency of fungal genera or species] from an agricultural field exposed to pesticides. In addition, we evaluated the tolerance of A. oryzae and nontoxigenic A. flavus strains to high concentrations (100 to 500 mM – 17,000 to 84,500 ppm) of a glyphosate commercial formulation. The analysis of the mycobiota showed that the frequency of the main fungal genera varied according to the analyzed sampling period. Aspergillus spp. or Aspergillus section Flavi strains were isolated from 20 to 100% of the soil samples. Sterilia spp. were also observed throughout the sampling (50 to 100%). Aspergillus section Flavi tolerance assays showed that all of the tested strains were able to develop at the highest glyphosate concentration tested regardless of the water availability conditions. In general, significant reductions in growth rates were observed with increasing concentrations of the herbicide. However, a complete inhibition of fungal growth was not observed with the concentrations assayed. This study contributes to the knowledge of culturable mycobiota from agricultural soils exposed to pesticides and provides evidence on the effective growth ability of A. oryzae and nontoxigenic A. flavus strains exposed to high glyphosate concentrations in vitro.     

The Fate of the Recombinant DNA in Corn During Composting

Abstract

In order to make regulations that safeguard food and the environment, an understanding of the fate of transgenes from genetically modified (GM) plants is of crucial importance. A compost experiment including mature transgenic corn plants and seeds of event Bt 176 (Zea mays L.) was conducted to trace the fate of the transgene cryIA(b) during the period of composting. In bin 1, shredded corn plants including seeds were composted above a layer of cow manure and samples from the corn layer were collected at intervals during a 12-month period. The samples were tested for the transgene persistence and microbial counts and also the compost was monitored for temperature. In bin 2, piles of corn seeds, surrounded by sheep manure and straw, were composted for 12 months. A method combining nested polymerase chain reaction (PCR) and southern hybridization was developed for detection of the transgene in compost. The detection sensitivity was 200 copies of the transgene per gram of dry composted corn material. Composting commenced on day 0, and the transgene was detected in specimens from bin 1 on days 0 and 7 but not on day 14 or thereafter. The transgene in corn seeds was not detectable after 12 months of composting in bin 2. Temperatures in both bins rose to about 50°C within 2 weeks and remained above that temperature for about 3 months, even when the ambient temperature dropped below ?20°C. Extracts from compost were inoculated onto culture plates and then were incubated at 23 to 55°C. Within the first 2 weeks of composting in bin 1, the counts of bacteria incubated at 55°C increased from 3.5 to 7.5 log ₁₀, whereas those incubated at 23°C remained at about 7.5 log ₁₀. The counts of fungi incubated at 45°C increased slightly from 2.5 to 3.1 log₁₀, but those incubated at 23°C decreased from 6.3 to 3.0 log ₁₀. The rapid degradation of the transgene during composting of Bt corn plants suggested that the composting process could be used for safe disposal of transgenic plant wastes.     

不适宜生长条件对混菌降解苯胺的影响

分别从台州和衢州某化工厂的好氧池中分离筛选得到2株苯胺降解菌TZ1和JH1,经16S rDNA测序鉴定为Comamonas sp.TZ1和Pseudomonas sp.JH1,均具有较强的苯胺降解能力,培养24 h后,可使初始浓度为800 mg/L的苯胺去除率达到96.4%~98.4%。在此基础上,按体积比1∶1将2株菌液进行混合构建了混合菌体系,进而对比考察了苯胺初始浓度、pH、盐度和重金属等环境因子对单一菌和混合菌生长量及降解苯胺效果的影响,重点探讨混合菌对不适宜生长环境的适应性及其对苯胺的降解特性。通过单一菌和混合菌对比实验发现,在适宜苯胺初始浓度、pH和盐度条件下,混合菌的生长量略高于单一菌;在不适宜生长的高浓度苯胺、pH和盐度条件下,混合菌也表现出了更强的适应性和苯胺矿化能力。Zn2+和Cr6+耐受实验则表明,对于Cr6+,混合菌表现出了更强的耐受能力,而对于Zn2+并没有表现出更强的耐受能力。     

Antibacterial activity of different natural honeys from Transylvania,Romania

Honey is used in food industry and medicine due to its nutritive, therapeutic and dietetic qualities. The microbiological characteristics of 10 unpasteurized honey samples of known origin, collected from Transylvania beekeepers (Romania) were determined. The antibacterial activity of these types of honey against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Salmonella enteritidis, Salmonella anatum, Salmonella choleraesuis, Bacillus cereus, Bacillus subtilis subsp. spizizenii and Listeria monocytogenes strains was studied. The most sensitive to the antibacterial activity were the two staphylococus strains (the largest diameter of inhibition zone was 18 mm) and B. subtilis strains (13.5 mm). The strains of B. cereus, E. coli, L. monocytogenes and Salmonella spp. were found to present resistance to some of the honey samples. Manna, sunflower and polyfloral honeys presented high antibacterial activity while acacia and linden honeys had a lower activity in terms of the number of sensible strains. Statistical analysis shows that the type of strains and the type of honey have influence on the diameter of inhibition.     

Sorption of cyromazine on humic acid: Effects of pH,ionic strength and foreign ions

Cyromazine (CY) is a triazine pesticide used as an insect growth inhibitor for fly control in cattle manure, field crops, vegetables, and fruits. Sorption of CY onto humic acid (HA) may affect its environmental fate. In this study, HA was used to investigate the sorption of CY at different solution chemistry conditions (pH, ionic strength) and in the presence of foreign ions and norfloxacin. All sorption isotherms fitted well with the Freundlich and Langmuir models. The sorption reached a maximum at initial pH 4.0 over the initial pH range of 3.0–7.0, implying that the primary sorption mechanism was cation exchange interaction between CY⁺ species and the negatively charged functional groups of HA. Increasing Ca²⁺ concentration resulted in a considerable reduction in the K _d values of CY, hinting that Ca²⁺ had probably competed with CY⁺ for the cation exchange sites on the surfaces of HA. The sorption of CY on HA in different ionic media followed the order of NH₄Cl ≈ KCl > K₂SO₄ > ZnCl₂ ≈ CaCl₂ at pH 5.0. Spectroscopic evidence demonstrated that the amino groups and triazine ring of CY was responsible for sorption onto HA, while the carboxyl group and the O-alkyl structure of HA participated in adsorbing CY.     

Effects of methanol, ethanol and n-propanol on development of Artemia salina cysts

The current study proposes an ecotoxicological test that makes use of Artemia salina cyst; the results obtained made it possible to calculate the percentage of cyst mortality (no sign of development), and growth inhibition (a delay in normal development), as well as the percentage of larva malformation, and mortality. The dehydrated cysts were directly exposed for forty-eight hours to the action of three alcohols, methanol, ethanol, and n-propanol. No teratogenic or lethal effects on larvae were found at any methanol or ethanol concentrations. Predicted values of effect concentration, EC₁, EC₁₀ and EC₅₀ were calculated from the estimated probit models for larva growth inhibition, GI, and for cyst lethal concentration, LC. At concentrations of 0.21 M methanol and 0.13 M ethanol, there were no signs of alterations in Artemia salina cyst development; these concentrations show a NOEC (No Observed Effect Concentration) value, and properly employed do not interfere with effects due to a given chemical. The n-propanol was only used to verify the extreme resistance of the dried cysts to various environmental factors.     

Biodegradation of s-triazine herbicide atrazine by Enterobacter cloacae and Burkholderia cepacia sp. from long-term treated sugarcane-cultivated soils in Kenya

In this study soils from sugarcane-cultivated fields were screened for bacterial species capable of atrazine (6-chloro-N²-ethyl-N⁴-isopropyl-1,3,5-triazine-2,4-diamine) degradation due to long exposure of the soils to this herbicide. To enrich for atrazine degraders, Minimal Salt Medium containing atrazine as the sole N source and glucose as the C source was inoculated with soils impacted with this herbicide and incubated. Bacterial growth was monitored by measuring optical density. The degradation of atrazine was followed by measuring residual atrazine in liquid cultures over a given time period by high performance liquid chromatography. Bacterial strains isolated from the enrichment cultures were characterized by biochemical tests and identified by 16S rRNA gene sequencing. Two bacterial strains coded ISL 8 and ISL 15 isolated from two different fields were shown to have 94 and 96% 16S rRNA gene sequence similarity to Burkholderia cepacia respectively. Another bacterial sp., ISL 14 was closely related to Enterobacter cloacae with a 96% 16S rRNA gene sequence similarity. There was not much difference between the extents of atrazine degradation by the enrichment cultures with communities (79–82% applied amount) from which pure strains were isolated and the pure strains themselves in liquid cultures that showed a degradation of 53–83% of applied amount. The study showed existence of bacterial strains in different sugarcane-cultivated fields which can use atrazine as a nitrogen source. The bacterial strains isolated can be used to enhance the degradation of atrazine in contaminated soils where atrazine is still considered to be recalcitrant.     

Use of fungi for the bioconversion of rice straw into cellulase enzyme

Cellulase production was carried out by solid state bioconversion (SSB) method using rice straw, a lignocellulosic material and agricultural waste, as the substrate of three Trichoderma spp. and Phanerochaete chrysosporium in lab-scale experiments. The results were compared to select the best fungi among them for the production of cellulase. Phanerochaete chrysosporium was found to be the best among these species of fungi, which produced the highest cellulase enzyme of 1.43 IU/mL of filter paper activity (FPase) and 2.40 IU/mL of carboxymethylcellulose activity (CMCase). The “glucosamine” and “reducing sugar” parameters were observed to evaluate the growth and substrate utilization in the experiments. In the case of Phanerochaete Chrysosporium, the highest glucosamine concentration was 1.60 g/L and a high concentration of the release of reducing sugar was measured as 2.58 g/L obtained on the 4th day of fermentation. The pH values were also recorded. The range of the pH was about 5.15 to 5.56 in the case of Phanerochaete Chrysosporium.     

钻井废水的生物强化处理简

从受钻井废水污染的土壤样品中筛选菌株进行生物处理实验，确定7株菌进行菌剂配伍。通过正交实验剔除可能有抑制作用的菌株，并确定菌剂各组成菌株的最佳配比，制成复合微生物菌剂。考察5种添加物对菌剂的影响，结果显示，当硫酸铵的添加量为20 mg/L时降解率为60%，高于其他添加物。生物强化实验结果显示，投加菌剂的反应器对钻井废水的平均降解率为42%，比未投加菌剂的对照实验的平均降解率（16%）高，而且耐冲击负荷性和降解性能稳定性优于对照实验。     

Analysis of metabolic changes in Trichoderma asperellum TJ01 at different fermentation time-points by LC-QQQ-MS

Trichoderma spp. are among the most widely recognized biocontrol fungi used to inhibit pathogens and promote plant growth. These functions are related to primary and secondary metabolites. This study investigated the different metabolites in Trichoderma asperellum TJ01 cultured for 24 and 72?h using liquid chromatography with triple-quadrupole mass spectrometry. Compared to the 24?h culture of T. asperellum TJ01, the 72?h culture with amino acid metabolism tended to decrease while sugar and lipid metabolisms tended to increase. Furthermore, the 72?h culture had a higher proportion of upregulated flavonoids, in combination with a higher proportion of downregulated alkaloids, and equal proportions of upregulated and downregulated polyphenols and hormones. This study also identified a few valuable medicinal substances such as trigonelline and 5-hydroxytryptophan in T. asperellum TJ01 fermentation cultures.     

Influence of herbicide glyphosate on growth and aflatoxin B1 production by Aspergillus section Flavi strains isolated from soil on in vitro assay

The effect of six glyphosate concentrations on growth rate and aflatoxin B₁ (AFB₁) production by Aspergillus section Flavi strains under different water activity (a_W) on maize-based medium was investigated. In general, the lag phase decreased as glyphosate concentration increased and all the strains showed the same behavior at the different conditions tested. The glyphosate increased significantly the growth of all Aspergillus section Flavi strains in different percentages with respect to control depending on pesticide concentration. At 5.0 and 10 mM this fact was more evident; however significant differences between both concentrations were not observed in most strains. Aflatoxin B₁ production did not show noticeable differences among different pesticide concentrations assayed at all a_W in both strains. This study has shown that these Aspergillus flavus and A. parasiticus strains are able to grow effectively and produce aflatoxins in high nutrient status media over a range of glyphosate concentrations under different water activity conditions.     

Selective effects of natural and synthetic insecticides on mortality of Spodoptera frugiperda (Lepidoptera: Noctuidae) and its predator Eriopis connexa (Coleoptera: Coccinellidae)

Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) is a serious pest of corn in several American countries. It is mainly controlled with synthetic insecticides. The objectives of this study were to evaluate the effects of the natural products, neem oil and pyroligneous extract, and the synthetic insecticide, lufenuron, at 2.50mL water (0.25 %) on the mortality of 2-, 4- and 6-day-old caterpillars of S. frugiperda, and their selectivities against fourth instar larvae of Eriopis connnexa Germar (Coleoptera: Coccinellidae). Four- and 6-day-old S. frugiperda caterpillars showed higher mortality after exposure to neem oil (83.33 ± 0.83 and 89.58 ± 0.90 %, respectively) and lufenuron (95.83 ± 0.96 and 85.41 ± 0.83 %), compared to pyroligneous extract (68.75 ± 0.69 and 31.25 ± 0.31 %). The deleterious effect of pyroligneous extract was higher in 2- (83.33 ± 0.83 % mortality) and 4-day-old (68.75 ± 0.69 %) S. frugiperda caterpillars than in 6-day-old caterpillars (31.25 ± 0.31 %). Larval mortality of the predator E. connexa was lower with neem oil and pyroligneous extract (25.00 ± 0.33 %) than with lufenuron (91.66 ± 1.22 %). Neem oil is thus recommended for control of S. frugiperda because of its high toxicity, combined with its relatively low toxicity to larvae of the natural enemy E. connexa.     

Phytoremediation potential of Arabidopsis thaliana, expressing ectopically a vacuolar proton pump, for the industrial waste phosphogypsum

Phosphogypsum (PG) is a by-product of the phosphorus–fertiliser industry and represents an environmental concern since it contains pollutants such as cadmium (Cd). We have recently shown that the overexpression of a proton pump gene (TaVP1) in transgenic tobacco (Nicotiana tabacum) led to an enhanced Cd tolerance and accumulation. The aim of this study was to evaluate the potential of transgenic Arabidopsis thaliana plants harbouring the TaVP1 gene to phytoremediate phosphogypsum. A pot experiment was carried out under greenhouse conditions. Transgenic A. thaliana plants harbouring the TaVP1 gene were grown on various substrates containing phosphogypsum (0, 25, 50 and 100 %) for 40 days. At the end of the growth period, we examined the growth (germination, root length, fresh weight) and physiological parameters (chlorophyll and protein contents, catalase activity and proteolysis) as well as the cadmium, Mg, Ca, and P contents of the A. thaliana plants. In order to evaluate Cd tolerance of the A. thaliana lines harbouring the TaVP1 gene, an in vitro experiment was also carried out. One week-old seedlings were transferred to Murashige and Skoog agar plates containing various concentrations of cadmium; the germination, total leaf area and root length were determined. The growth and physiological parameters of all A. thaliana plants were significantly altered by PG. The germination capacity, root growth and biomass production of wild-type (WT) plants were more severely inhibited by PG compared with the TaVP1 transgenic A. thaliana lines. In addition, TaVP1 transgenic A. thaliana plants maintained a higher antioxidant capacity than the WT. Interestingly, elemental analysis of leaf material derived from plants grown on PG revealed that the transgenic A. thaliana line accumulated up to ten times more Cd than WT. Despite its higher Cd content, the transgenic A. thaliana line performed better than the WT counterpart. In vitro evaluation of Cd tolerance showed that TaVP1 transgenic A. thaliana lines were more Cd-tolerant than the WT plants. These results suggested that ectopic expression of a vacuolar proton pump in A. thaliana plants can lead to various biotechnological applications including the phytoremediation of industrial wastes.     

Acute toxicity of the insect larvicide abate® (temephos) on the fish tilapia melanopleura and the dragonfly larvae neurocordelia virginiensis.

Abstract

Acute bioassay tests on the toxic effects of the insect larvicide Abate® (temephos) on the mouth brooder cichlid fish Tilapia melanopleum and the dragonfly larvae (Odonata) Neurocordulia virginiensis were conducted in static non renewal toxicity test set ups. The 96h‐LC50 (95% confidence intervals) was 30.2 (20.5‐ 44.20) mg/L for the fish and 2.0 (1.16–2.0) mg/L for the dragonfly larvae. The dragonfly larvae were 15 times more susceptible to the larvicide than the tilapia. The calculated NOEC (No Observable Effect Concentration) was 14.1 mg/L for the fishand less than 1.0 mg/L for the insect larvae. The estimated ‘safe’ concentration of the pesticide to the fish was 3.0 mg/L and 0.2 mg/L for insect larvae. These figures are far above the concentrations approved for use in the control of mosquito larvae(0.0004–0.01 mg/L). It appears that the application of toxic levels of the insecticide for the elimination of some aquatic invertebrates may be ‘safe’ for normal survival, growth and reproduction offish and some aquatic insect larvae.     

Influence of air permeation of pheromone on response of male lesser peachtree borers1 and peachtree borers2

Abstract

Air permeation trials were conducted with Z,Z and E,Z isomers of 3,13‐octadecadien‐1‐ol acetate (ODDA) to determine response difference between males of the peachtree borer (PTB), Synanthedon exitiosa (Say), and lesser peachtree borer (LPTB), S. pictipes (Grote & Robinson), within and adjacent to permeated plots of a peach orchard. The pheromone was released from hollow fiber dispensers attached to the trees in the orchard.

In the pheromone‐treated plots, both E,Z and Z,Z‐ODDA significantly reduced trap captures of PTB relative to the untreated plot. The E,Z isomer also significantly reduced captures of LPTB males but captures in the Z,Z treatment were higher than in the untreated plots. The pheromone treatments also influenced trap capture of both PTB and LPTB male moths outside the permeated plots.