The banana Fusarium wilt (BFW) caused by Fusarium oxysporum f. sp. cubense tropical race4 (FocTR4) is difficult to control worldwide, which causes a huge economic losse to banana industry. The purpose of this study was to screen Trichoderma strains with antagonistic activity against FocTR4, to isolate and purify the active compound from the fermentation broth, so as to provide important biocontrol strains and active compound resources. In this work, Trichoderma strains were isolated and screened from the rhizosphere soil of crops, and the strains capable of efficiently inhibiting FocTR4 were screened by plate confrontation, and further confirmed by testing inhibition for the conidial germination and mycelial growth of FocTR4. The phylogenetic tree clarified the taxonomic status of the biocontrol strains. Moreover, the active components in the fermentation broth of the strains were separated and purified by column chromatography, the structure of the most active component was analyzed by nuclear magnetic resonance spectroscopy (NMR), the BFW control effect was tested by pot experiments. We obtained a strain JSHA-CD-1003 with antagonistic activity against FocTR4, and the inhibition rate from plate confrontation was 60.6%. The fermentation broth of JSHA-CD-1003 completely inhibited the germination of FocTR4 conidia within 24 hours. The inhibition rate of FocTR4 hyphae growth was 52.6% within 7 d. A phylogenetic tree was constructed based on the ITS and tef1-α gene tandem sequences, and JSHA-CD-1003 was identified as Trichoderma brevicompactum. Purification and NMR identification showed that the single active compound was trichodermin, and the minimum inhibitory concentration (MIC) was 25 μg/mL. Pot experiments showed that the fermentation broth of strain JSHA-CD-1003 was effective against BFW. The control rate of leaf yellowing was 47.4%, and the rate of bulb browning was 52.0%. Therefore, JSHA-CD-1003 effectively inhibited FocTR4 conidial germination and mycelium growth through producing trichodermin, and showed biocontrol effect on banana wilt caused by FocTR4, thus is a potential biocontrol strain.
Fusarium ; Musa ; Phylogeny ; Trichodermin ; Hypocreales

Zearalenone is one of the most widely polluted Fusarium toxins in the world, seriously endangering livestock and human health. Zearalenone hydrolase (ZHD) derived from Clonostachys rosea can effectively degrade zearalenone. However, the high temperature environment in feed processing hampers the application of this enzyme. Structure-based rational design may provide guidance for engineering the thermal stability of enzymes. In this paper, we used the multiple structure alignment (MSTA) to screen the structural flexibility regions of ZHD. Subsequently, a candidate mutation library was constructed by sequence conservation scoring and conformational free energy calculation, from which 9 single point mutations based on residues 136 and 220 were obtained. The experiments showed that the thermal melting temperature (T_m) of the 9 mutants increased by 0.4-5.6 ℃. The S220R and S220W mutants showed the best thermal stability, the T_m of which increased by 5.6 ℃ and 4.0 ℃ compared to that of the wild type. Moreover, the thermal half-inactivation time at 45 ℃ were 15.4 times and 3.1 times longer, and the relative activities were 70.6% and 57.3% of the wild type. Molecular dynamics simulation analysis showed that the interaction force at and around the mutation site was enhanced, contributing to the improved thermal stability of ZHD. The probability of 220-K130 hydrogen bond of the mutants S220R and S220W increased by 37.1% and 19.3%, and the probability of K130-D223 salt bridge increased by 30.1% and 12.5%, respectively. This work demonstrated the feasibility of thermal stability engineering strategy where the structural and sequence alignment as well as free energy calculation of natural enzymes were integrated, and obtained ZHD variants with enhanced thermal stability, which may facilitate the industrial application of ZHD.
Humans ; Hydrolases ; Zearalenone ; Trichothecenes ; Gene Library ; Hydrogen Bonding

No abstract available.
Trichothecenes

Twelve healthy rats were divided into the T-2 toxin group receiving gavage of 1 mg/kg T-2 toxin and the control group receiving gavage of normal saline. Total relative concentrations of T-2 toxin and HT-2 toxin in the skeletal system (thighbone, knee joints, and costal cartilage) were significantly higher than those in the heart, liver, and kidneys (P < 0.05). The relative concentrations of T-2 toxin and HT-2 toxin in the skeletal system (thighbone and costal cartilage) were also significantly higher than those in the heart, liver, and kidneys. The rats administered T-2 toxin showed rapid metabolism compared with that in rats administered HT-2 toxin, and the metabolic conversion rates in the different tissues were 68.20%-90.70%.
Animals ; Bone and Bones ; chemistry ; metabolism ; Rats ; Rats, Sprague-Dawley ; T-2 Toxin ; analogs & derivatives ; pharmacokinetics ; toxicity ; Tissue Distribution ; Toxicity Tests, Acute

OBJECTIVETo investigate chondrocyte apoptosis and the expression of biochemical markers associated with apoptosis in Kashin-Beck disease (KBD) and in an established T-2 toxin- and selenium (Se) deficiency-induced rat model.

METHODSCartilages were collected from the hand phalanges of five patients with KBD and five healthy children. Sprague-Dawley rats were administered a selenium-deficient diet for 4 weeks prior to T-2 toxin exposure. The apoptotic chondrocytes were observed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Caspase-3, p53, Bcl-2, and Bax proteins in the cartilages were visualized by immunohistochemistry, their protein levels were determined by Western blotting, and mRNA levels were determined by real-time reverse transcription polymerase chain reaction.

RESULTSIncreased chondrocyte apoptosis was observed in the cartilages of children with KBD. Increased apoptotic and caspase-3-stained cells were observed in the cartilages of rats fed with normal and Se-deficient diets plus T-2 toxin exposure compared to those in rats fed with normal and Se-deficient diets. Caspase-3, p53, and Bax proteins and mRNA levels were higher, whereas Bcl-2 levels were lower in rats fed with normal or Se-deficiency diets supplemented with T-2 toxin than the corresponding levels in rats fed with normal diet.

CONCLUSIONT-2 toxin under a selenium-deficient nutritional status induces chondrocyte death, which emphasizes the role of chondrocyte apoptosis in cartilage damage and progression of KBD.

Adolescent ; Animals ; Apoptosis ; drug effects ; Biomarkers ; Cartilage, Articular ; physiopathology ; Child ; Chondrocytes ; physiology ; Female ; Humans ; Kashin-Beck Disease ; etiology ; physiopathology ; Male ; Matrilin Proteins ; genetics ; metabolism ; Models, Animal ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Selenium ; deficiency ; T-2 Toxin ; pharmacology

OBJECTIVETo elucidate the dietary exposure to deoxynivalenol (DON) and zearalenone (ZEN) from cereal-based products in Chinese populations using the probabilistic assessment approach.

METHODSA total of 292 wheat flours and 347 corn-based products were collected from sampling sites of 107 supermarkets or farmers markets, which were randomly selected from 44 cities of 13 provinces in 2009 by the stratified cluster random sampling method. Then, DON and ZEN contamination levels in these samples above analyzed by UPLC-MS/MS in combination with the food consumption data of 68 959 respondents, who were divided into group 1 aged 3 to 13 years old, and group 2 aged 14 and over 14 years old (≥14 years old), obtained by China National Nutrition and Health Survey in 2002 were investigated. A probabilistic assessment model using Monte Carlo simulation was applied to derive the intake distribution of P(1)-P(99) percentile of dietary exposure to DON and ZEN. Meanwhile, all parameters related to dietary exposure to both toxins were compared with either the provisional maximum tolerable daily intake (PMTDI) of 1 µg·kg(-1)·d(-1) for DON, or the tolerable daily intake (TDI) of 0.25 µg·kg(-1)·d(-1) for ZEN in order to evaluate the risk of dietary intake of two toxins and find the minimum percentile of dietary exposure to these two toxins. The statistical differences of dietary exposure to these two toxins between two groups were achieved by t test.

RESULTSThe detection frequencies of DON in wheat flours and corn-based products were 100% (292/292) and 97.4% (338/347), respectively. A total of 21 out of 639 samples (wheat flours: 5/292, corn-based products: 16/347) were positive for DON at the levels exceeding the Chinese regulatory limit of 1 000 µg/kg for DON. And the detection frequencies of ZEN in wheat flours and corn-based products were 53.4% (156/292) and 87.6% (304/347), respectively.54 out of 347 corn-based products and no wheat flours were positive for ZEN at the levels exceeding the Chinese regulatory limit of 60 µg/kg for ZEN. Meanwhile, the mean values (95% CI) of the P(50), P(75), P(90), P(95), P(97.5) and P(99) percentile of dietary exposure to DON in populations of 3 to 13 years old were 0.170 (0.170-0.171), 0.762 (0.759-0.765), 2.066 (2.038-2.069), 3.515 (3.501-3.530), 5.342 (5.314-5.372), and 9.220 (9.155-9.279) µg · kg(-1)·d(-1), which were higher than those in populations of ≥14 years old (0.131 (0.130-0.131), 0.500 (0.498-0.501), 1.280 (1.276-1.285), 2.138 (2.128-2.14), 3.510 (3.494-3.527), and 5.512 (5.474-5.546) µg·kg(-1)·d(-1)), with t values of 87.19, 163.87, 164.66, 157.78, 105.47 and 96.31, and all P values less than 0.001. And the mean values (95% CI) of the P(50), P(75), P(90), P(95), P(97.5) and P(99) percentile of dietary exposure to ZEN in populations of 3 to 13 years old were 0.001 (0.001-0.001), 0.006 (0.006-0.006), 0.039 (0.038-0.039), 0.101 (0.100-0.101), 0.195 (0.194-0.197) and 0.378 (0.374-0.381) µg · kg(-1)·d(-1), which were also higher than those in populations of ≥14 years old (0.001 (0.001-0.001), 0.004 (0.004-0.004), 0.026 (0.026-0.026), 0.061 (0.060-0.061), 0.115 (0.115-0.116) and 0.232 (0.231-0.235) µg·kg(-1)·d(-1)) with T-values of 151.11, 73.80, 96.81, 100.81, 91.93 and 76.13, and all P values less than 0.001. Besides, the minimum percentile of dietary exposure to DON in populations of 3 to 13 years old and ≥14 years old exceeded the corresponding PMTDI of 1 µg·kg(-1)·d(-1) was found in the probability distribution of P(76) (99% percentile = 1.03 µg·kg(-1)·d(-1)) and P(84) (95% percentile = 1.01 µg·kg(-1)·d(-1)) percentile, respectively. And the minimum percentile of dietary exposure to ZEN in populations of 3 to 13 years old and ≥14 years old exceeded the corresponding TDI of 0.25 µg·kg(-1)·d(-1) was found in the probability distribution of P(97) (95% percentile = 0.25 µg·kg(-1)·d(-1)) and P(98) (90% percentile = 0.26 µg·kg(-1)·d(-1)) percentile, respectively.

CONCLUSIONThe contamination levels of DON and ZEN in wheat flours and corn-based products and the risk of dietary exposure to both DON and ZEN in populations in Chinese populations were at relatively low levels. The dietary exposure to both DON and ZEN in populations of 3 to 13 years old was higher than those in populations of ≥14 years old . Populations of 3 to 13 years old were the populations at the high risk of dietary exposure to both mycotoxins.

Adolescent ; Adult ; Child ; Child, Preschool ; China ; Diet ; Edible Grain ; Food Contamination ; Humans ; Middle Aged ; Mycotoxins ; Tandem Mass Spectrometry ; Trichothecenes ; Triticum ; Zea mays ; Zearalenone

Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 microg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 microg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.
Animal Feed/analysis ; Animals ; Antibodies, Fungal/analysis ; Antibodies, Monoclonal/analysis ; Chemistry Techniques, Analytical/*methods ; Enzyme-Linked Immunosorbent Assay/*methods/veterinary ; Female ; Food Contamination/*analysis ; Fusarium/immunology ; Imidazoles/chemistry ; Magnetics/methods ; Mice ; Mice, Inbred BALB C ; Mycotoxins/*analysis/chemistry ; Nanoparticles/chemistry ; Ovalbumin/chemistry ; Trichothecenes/*analysis/chemistry

OBJECTIVETo observe the effect of nano-Se-chondroitin sulfate on the growth and apoptosis of chondrocytes from patients with Kashin-Beck disease (KBD) exposed to T-2 toxin in vitro.

METHODSSamples of the articular cartilage were obtained from 6 patients with grade II/III KBD diagnosed in line with the National Clinical Diagnostic Criteria of KBD (WS/T 207-2010) for chondrocyte separation and culture in vitro. The separated chondrocytes were treated with synthesized nano-Se-chondroitin sulfate particles and T-2 toxin, alone or in combination, and the cell growth and apoptosis were observed using MTT assay, HE staining and flow cytometry.

RESULTSThe synthesized nano-Se-chondroitin sulfate, with a selenium entrapment ratio of 10.1%, spontaneously formed nanoparticles in distilled water with sizes ranging from 30 to 200 nm. Fourier-transform infrared spectroscopy suggested a possible covalent bond that bound Nano-Se and chondroitin sulfate. Within the concentration range of 50-200 ng/ml, nano-Se-chondroitin sulfate significantly inhibited T-2 toxin-induced apoptosis of the cultured chondrocytes and reduced the early apoptosis rate to (8.64∓1.57)% (P<0.05).

CONCLUSIONNano-Se-chondroitin sulfate can inhibit T-2 toxin-induced apoptosis of cultured chondrocytes from KBD patients in vitro, and serves as a promising candidate therapeutic agent for KBD.

Apoptosis ; drug effects ; Cells, Cultured ; Chondrocytes ; drug effects ; pathology ; Chondroitin Sulfates ; administration & dosage ; pharmacology ; Humans ; Kashin-Beck Disease ; pathology ; Middle Aged ; Nanostructures ; T-2 Toxin ; toxicity

Podostroma cornu-damae is a rare species of fungus belonging to the Hyocreaceae family. Its fruit body is highly toxic, as it contains trichothecene mycotoxins. Unfortunately, it highly resembles Ganoderma lucidum and Cordyceps, well-known health foods; this can lead to poisoning. We experienced such a case of a 42-year old man who received mushroom poisoning by injesting Podostroma cornu-damae. The patient was presented with severe pancytopenia and infection. The patient recovered without any complications after conservative care, antibiotics therapy, and granulocyte colony stimulating factor administration. The most common complications of podostroma cornu-damae intoxication were reported pancytopenia, infection, disseminated intravascular coagulation, acute renal failure, etc. It is important to provide enough fluid therapy, use of antibiotics to infection and granulocyte colony stimulating factor administration.
Acute Kidney Injury ; Agaricales ; Anti-Bacterial Agents ; Colony-Stimulating Factors ; Cordyceps ; Disseminated Intravascular Coagulation ; Fluid Therapy ; Fruit ; Fungi ; Granulocytes ; Humans ; Mushroom Poisoning ; Mycotoxins ; Pancytopenia ; Reishi ; Trichothecenes

The present study was carried out to investigate the effect of barley and barley bran contaminated with Fusarium spp on growth performance and feed efficiency of fattening and growing pigs. In experiment 1, total 48 fattening Landrace pigs were used in a fattening trial for 71 days. Pigs weighing around 75 kg were allocated into different substitution groups containing 0, 10, 20 and 30% of barley contaminated Fusarium spp. In experiment 2, total 16 growing Landrace pigs were used in a growing trial for 45 days. Pigs weighing around 29.4 kg were allocated into different substitution groups containing 0, 5, 10 and 20% of barley bran contaminated Fusarium spp. Mycotoxin concentrations of barley and barley bran contaminated with 30% Fusarium spp were 0.452 and 1.049 ppm for deoxynivalenol, 8.125 and 17.646 ppm for nivalenol and 0.023 and 0.029 ppm for zearalenone, respectively. In experiment 1, no differences were found in weight gain and feed intake between control group (0%) and 10 or 20% substitution groups, but in 30% substitution group, weight gain and feed intake were significantly lower (p < 0.05) than those in control group. After slaughtering, the extended haemorrhage of the fundus region in stomach was observed in 20 or 30% substitution groups. In experiment 2, weight gain and feed intake were not significantly different among treatment groups. After slaughtering of experimental pigs, the extended haemorrhage of the fundus region in stomach was observed in pigs fed diet with 20% substitution group. These results suggest that the feeding of diet with contaminated highly levels of Fusarium spp was negative effect on growth and feed efficiency in growing and fattening pig.
Diet ; Fusarium ; Hordeum ; Stomach ; Swine ; Trichothecenes ; Weight Gain ; Zearalenone