Glycosyltransferase FvCpsA Regulates Fumonisin Biosynthesis and Virulence in Fusarium verticillioides

Fusarium verticillioides is the major maize pathogen associated with ear rot and stalk rot worldwide. Fumonisin B1 (FB1) produced by F. verticillioides, poses a serious threat to human and animal health. However, our understanding of FB1 synthesis and virulence mechanism in this fungus is still very limited. Glycosylation catalyzed by glycosyltransferases (GTs) has been identified as contributing to fungal infection and secondary metabolism synthesis. In this study, a family 2 glycosyltransferase, FvCpsA, was identified and characterized in F. verticillioides. ΔFvcpsA exhibited significant defects in vegetative growth. Moreover, ΔFvcpsA also increased resistance to osmotic and cell wall stress agents. In addition, expression levels of FUM genes involved in FB1 production were greatly up-regulated in ΔFvcpsA. HPLC (high performance liquid chromatography) analysis revealed that ΔFvcpsA significantly increased FB1 production. Interestingly, we found that the deletion of FvCPSA showed penetration defects on cellophane membrane, and thus led to obvious defects in pathogenicity. Characterization of FvCpsA domain experiments showed that conserved DXD and QXXRW domains were vital for the biological functions of FvCpsA. Taken together, our results indicate that FvCpsA is critical for fungal growth, FB1 biosynthesis and virulence in F. verticillioides.     

Reduction of Mycotoxigenic Fungi Growth and Their Mycotoxin Production by Bacillus subtilis QST 713

The use of chemical pesticides to control the occurrence of mycotoxigenic fungi in crops has led to environmental and human health issues, driving the agriculture sector to a more sustainable system. Biocontrol agents such as Bacillus strains and their antimicrobial metabolites have been proposed as alternatives to chemical pesticides. In the present work, a broth obtained from a commercial product containing Bacillus subtilis QST 713 was tested for its ability to inhibit the growth of mycotoxigenic fungi as well as reduce their mycotoxin production. Mass spectrometry analysis of Bacillus subtilis broth allowed to detect the presence of 14 different lipopeptides, belonging to the iturin, fengycin, and surfactin families, already known for their antifungal properties. Bacillus subtilis broth demonstrated to be a useful tool to inhibit the growth of some of the most important mycotoxigenic fungi such as Aspergillus flavus, Fusarium verticillioides, Fusarium graminearum, Aspergillus carbonarius, and Alternaria alternata. In addition, cell-free Bacillus subtilis broth provided the most promising results against the growth of Fusarium graminearum and Alternaria alternata, where the radial growth was reduced up to 86% with respect to the untreated test. With regard to the mycotoxin reduction, raw Bacillus subtilis broth completely inhibited the production of aflatoxin B1, deoxynivalenol, zearalenone, and tenuazonic acid. Cell-free broth provided promising inhibitory properties toward all of the target mycotoxins, even if the results were less promising than the corresponding raw broth. In conclusion, this work showed that a commercial Bacillus subtilis, characterized by the presence of different lipopeptides, was able to reduce the growth of the main mycotoxigenic fungi and inhibit the production of related mycotoxins.     

Antifungal Activity of Quinofumelin against Fusarium graminearum and Its Inhibitory Effect on DON Biosynthesis

Fusarium graminearum, causal agent of Fusarium head blight (FHB), causes a huge economic loss. No information is available on the activity of quinofumelin, a novel quinoline fungicide, against F. graminearum or other phytopathogens. In this study, we used mycelial growth and spore germination inhibition methods to determine the inhibitory effect of quinofumelin against F. graminearum in vitro. The results indicated that quinofumelin excellently inhibited mycelial growth and spore germination of F. graminearum, with the average EC₅₀ values of 0.019 ± 0.007 μg/mL and 0.087 ± 0.024 μg/mL, respectively. In addition, we found that quinofumelin could significantly decrease deoxynivalenol (DON) production and inhibit the expression of DON-related gene TRI5 in F. graminearum. Furthermore, we found that quinofumelin could disrupt the formation of Fusarium toxisome, a structure for producing DON. Western blot analysis demonstrated that the translation level of TRI1, a marker gene for Fusarium toxisome, was suppressed by quinofumelin. The protective and curative assays indicated that quinofumelin had an excellent control efficiency against F. graminearum on wheat coleoptiles. Taken together, quinofumelin exhibits not only an excellent antifungal activity on mycelial growth and spore germination, but also could inhibit DON biosynthesis in F. graminearum. The findings provide a novel candidate for controlling FHB caused by F. graminearum.     

Biocontrol of Fusarium graminearum,a Causal Agent of Fusarium Head Blight of Wheat,and Deoxynivalenol Accumulation: From In Vitro to In Planta

Crop diseases caused by Fusarium graminearum threaten crop production in both commercial and smallholder farming. F. graminearum produces deoxynivalenol mycotoxin, which is stable during food and feed processing. Therefore, the best way to prevent the sporulation of pathogens is to develop new prevention strategies. Plant-based pesticides, i.e., natural fungicides, have recently gained interest in crop protection as alternatives to synthetic fungicides. Herein we show that treatment with the methanolic extract of medicinal plant Zanthoxylum bungeanum (M20 extract), decreased F. graminearum growth and abrogated DON production. The F. graminearum DNA levels were monitored by a quantitative TaqMan real-time PCR, while DON accumulation was assessed by HPLC quantification. This M20 extract was mainly composed of four flavonoids: quercetin, epicatechin, kaempferol-3-O-rhamnoside, and hyperoside. The in vitro bioassay, which measured the percent inhibition of fungal growth, showed that co-inoculation of four F. graminearum strains with the M20 extract inhibited the fungal growth up to 48.5%. After biocontrol treatments, F. graminearum DNA level was reduced up to 85.5% compared to that of wheat heads, which received F. graminearum mixture only. Moreover, DON production was decreased in wheat heads by 73% after biocontrol treatment; meanwhile in wheat heads inoculated with F. graminearum conidia, an average of 2.263 ± 0.8 mg/kg DON was detected. Overall, this study is a successful case from in vitro research to in planta, giving useful information for wheat protection against F. graminearum responsible for Fusarium Head Blight and DON accumulation in grains. Further studies are needed to study the mechanism by which M20 extract inhibited the DON production and what changes happened to the DON biosynthetic pathway genes.     

H2O2对枯草芽孢杆菌生长代谢以及腺苷发酵的影响

通过摇瓶发酵试验研究H2O2对枯草芽孢杆菌ATCC 21616生长代谢及产苷的影响.结果表明：添加时间、浓度以及两者的交互作用显著影响腺苷的积累,在12h添加20mg/L的H2O2最利于腺苷的合成,产苷量由7.03g/L增至7.79g/L.同时也研究了H2O2添加对代谢过程中糖耗及pH的影响.     

细胞壁缺陷枯草芽胞杆菌DPA合成代谢的研究

目的探讨细胞壁缺陷对枯草芽胞杆菌形成芽胞的能力以及芽胞物质合成代谢的影响及其机制。方法用β-内酰胺抗生素诱导枯草芽胞杆菌(Bacllus subtilis)成为L型并传代培养获得稳定L型纯培养物,采用形态学、生理学及化学分析的方法检测稳定L型的芽胞及吡啶二羧酸(DPA)。结果稳定L型不能产生形态学上的芽胞,但具有吡啶二羧酸合成代谢活性,吡啶二羧酸在菌细胞内大量堆积。结论细胞壁缺陷导致枯草芽胞杆菌丧失了产生芽胞的能力,但激活了吡啶二羧酸的合成代谢机制,从而造成吡啶二羧酸可在L型营养细胞内大量合成与积聚。     

β-甘露聚糖酶产生菌DY-14的发酵优化及分离纯化

利用单因素实验和正交分析方法得到枯草芽孢杆菌DY-14发酵产酶的最佳培养基组分及条件：以魔芋粉为碳源（3%）,蛋白胨为氮源（1.5%）,初始pH 8.0,摇瓶装液量10 mL/100mL,接种量1%,37℃培养24h,最终产生的β-甘露聚糖酶活力可达3216 U/mL。DY-14发酵液经（NH4）2SO4盐析、DEAE-Sepharose阴离子交换层析、Sephadex G-75凝胶过滤,得到了电泳纯的β-甘露聚糖酶。     

Comparison of Ameliorative Effects between Probiotic and Biodegradable Bacillus subtilis on Zearalenone Toxicosis in Gilts

This study was conducted to compare the potential ameliorative effects between probiotic Bacillus subtilis and biodegradable Bacillus subtilis on zearalenone (ZEN) toxicosis in gilts. Thirty-six Landrace×Yorkshire gilts (average BW = 64 kg) were randomly divided into four groups: (1) Normal control diet group (NC) fed the basal diet containing few ZEN (17.5 μg/kg); (2) ZEN contaminated group (ZC) fed the contaminated diet containing an exceeded limit dose of ZEN (about 300 μg/kg); (3) Probiotic agent group (PB) fed the ZC diet with added 5 × 10⁹ CFU/kg of probiotic Bacillus subtilis ANSB010; (4) Biodegradable agent group (DA) fed the ZC diet with added 5 × 10⁹ CFU/kg of biodegradable Bacillus subtilis ANSB01G. Results showed that Bacillus subtilis ANSB010 and ANSB01G isolated from broiler intestinal chyme had similar inhibitory activities against common pathogenic bacteria. In addition, the feed conversion ratio and the vulva size in DA group were significantly lower than ZC group (p < 0.05). The levels of IgG, IgM, IL-2 and TNFα in the ZC group were significantly higher than PB and DA groups (p < 0.05). The levels of estradiol and prolactin in the ZC group was significantly higher than those of the NC and DA groups (p < 0.05). Additionally, the residual ZEN in the feces of the ZC and PB groups were higher than those of the NC and DA groups (p < 0.05). In summary, the ZEN-contaminated diet had a damaging impact on growth performance, plasma immune function and hormone secretion of gilts. Although probiotic and biodegradable Bacillus subtilis have similar antimicrobial capacities, only biodegradable Bacillus subtilis could eliminate these negative effects through its biodegradable property to ZEN.     

In Vitro Effects of Lemon Balm Extracts in Reducing the Growth and Mycotoxins Biosynthesis of Fusarium culmorum and F. proliferatum

The objectives of this research were to obtain the extracts of lemon balm (Melissa officinalis) using supercritical CO₂ (SC-CO₂) and methanol as co-solvent and evaluate the antifungal activity of those extracts against two selected strains of Fusarium species (Fusarium culmorum and Fusarium proliferatum). The extraction conditions were set at 40 and 60 °C and 250 bar. The obtained extracts were characterized in terms of antifungal activity on potato dextrose agar media (PDA). The results showed that the extraction parameters had different effects on mycelium growth and mycotoxins biosynthesis reduction. All studied lemon balm extracts (1, 2.5, 5, 7.5, and 10%) inhibited the growth of F. proliferatum and F. culmorum mycelia compared to the control. The lemon balm extracts significantly reduced ergosterol content and synthesized mycotoxins in both tested strains. These findings support the antifungal activity of lemon balm extracts against F. proliferatum and F. culmorum. However, more research on other Fusarium species is needed, as well as in vivo applications, before considering lemon balm extracts as a natural alternative to synthetic fungicides.     

Population Structure and Genetic Diversity of the Fusarium graminearum Species Complex

The Fusarium graminearum species complex (Fg complex) consists of phylogenetically distinct species some of which cannot be discriminated based on their morphology. Their chemotypes and geographic distributions are dramatically different, and these highlight the challenges that Fusarium head blight (FHB) poses to plant disease specialists and plant breeders, thereby requiring that quarantine officials employ molecular diagnostic tools in their active surveillance programs. Molecular marker technologies play essential roles in species identification of the Fg complex, and they are being used widely to assess the genetic diversity of the clade. The utility, applicability and limitations of molecular methods for assessing the population structure and genetic diversity within the Fg complex are discussed with suitable examples. Knowledge gained from these studies will provide a baseline for monitoring changes in FHB pathogen diversity and mycotoxin potential over time, both of which are critical to the ultimate control and elimination of this economically devastating disease.     

Population Genetic Structure and Chemotype Diversity of Fusarium graminearum Populations from Wheat in Canada and North Eastern United States

Fusarium head blight (FHB) is a major disease in wheat causing severe economic losses globally by reducing yield and contaminating grain with mycotoxins. In Canada, Fusarium graminearum is the principal etiological agent of FHB in wheat, producing mainly the trichothecene mycotoxin, deoxynivalenol (DON) and its acetyl derivatives (15-acetyl deoxynivalenol (15ADON) and 3-acetyl deoxynivalenol (3ADON)). Understanding the population biology of F. graminearum such as the genetic variability, as well as mycotoxin chemotype diversity among isolates is important in developing sustainable disease management tools. In this study, 570 F. graminearum isolates collected from commercial wheat crops in five geographic regions in three provinces in Canada in 2018 and 2019 were analyzed for population diversity and structure using 10 variable number of tandem repeats (VNTR) markers. A subset of isolates collected from the north-eastern United States was also included for comparative analysis. About 75% of the isolates collected in the Canadian provinces of Saskatchewan and Manitoba were 3ADON indicating a 6-fold increase in Saskatchewan and a 2.5-fold increase in Manitoba within the past 15 years. All isolates from Ontario and those collected from the United States were 15ADON and isolates had a similar population structure. There was high gene diversity (H = 0.803–0.893) in the F. graminearum populations in all regions. Gene flow was high between Saskatchewan and Manitoba (Nm = 4.971–21.750), indicating no genetic differentiation between these regions. In contrast, less gene flow was observed among the western provinces and Ontario (Nm = 3.829–9.756) and USA isolates ((Nm = 2.803–6.150). However, Bayesian clustering model analyses of trichothecene chemotype subpopulations divided the populations into two clusters, which was correlated with trichothecene types. Additionally, population cluster analysis revealed there was more admixture of isolates among isolates of the 3ADON chemotypes than among the 15ADON chemotype, an observation that could play a role in the increased virulence of F. graminearum. Understanding the population genetic structure and mycotoxin chemotype variations of the pathogen will assist in developing FHB resistant wheat cultivars and in mycotoxin risk assessment in Canada.     

豆豉中纳豆杆菌的筛选和纳豆激酶的初步分离

利用改良的培养基 纤维蛋白平板法 ,从我国传统食品豆豉中筛选出具有高纤溶活性的纳豆杆菌菌株 ;菌株液体发酵法制得粗酶液 ,采用沉淀法及柱层析等方法从发酵液中初步分离纳豆激酶 ;并应用体外溶栓实验考察分析了纳豆激酶的纤溶活性 ,为开发新型溶栓药物及保健食品提供了实验参考依据     

Influence of H2O2-Induced Oxidative Stress on In Vitro Growth and Moniliformin and Fumonisins Accumulation by Fusarium proliferatum and Fusarium subglutinans

Fusarium proliferatum and Fusarium subglutinans are common pathogens of maize which are known to produce mycotoxins, including moniliformin (MON) and fumonisins (FBs). Fungal secondary metabolism and response to oxidative stress are interlaced, where hydrogen peroxide (H₂O₂) plays a pivotal role in the modulation of mycotoxin production. The objective of this study is to examine the effect of H₂O₂-induced oxidative stress on fungal growth, as well as MON and FBs production, in different isolates of these fungi. When these isolates were cultured in the presence of 1, 2, 5, and 10 mM H₂O₂, the fungal biomass of F. subglutinans isolates showed a strong sensitivity to increasing oxidative conditions (27–58% reduction), whereas F. proliferatum isolates were not affected or even slightly improved (45% increase). H₂O₂ treatment at the lower concentration of 1 mM caused an almost total disappearance of MON and a strong reduction of FBs content in the two fungal species and isolates tested. The catalase activity, surveyed due to its crucial role as an H₂O₂ scavenger, showed no significant changes at 1 mM H₂O₂ treatment, thus indicating a lack of correlation with MON and FB changes. H₂O₂ treatment was also able to reduce MON and FB content in certified maize material, and the same behavior was observed in the presence and absence of these fungi, highlighting a direct effect of H₂O₂ on the stability of these mycotoxins. Taken together, these data provide insights into the role of H₂O₂ which, when increased under stress conditions, could affect the vegetative response and mycotoxin production (and degradation) of these fungi.     

Detoxification and Excretion of Trichothecenes in Transgenic Arabidopsis thaliana Expressing Fusarium graminearum Trichothecene 3-O-acetyltransferase

Fusarium graminearum, the causal agent of Fusarium head blight (FHB), produces trichothecenes including deoxynivalenol (DON), nivalenol (NIV), and 3,7,15-trihydroxy-12,13-epoxytrichothec-9-ene (NX-3). These toxins contaminate grains and cause profound health problems in humans and animals. To explore exploiting a fungal self-protection mechanism in plants, we examined the ability of F. graminearum trichothecene 3-O-acetyltransferase (FgTri101) to detoxify several key trichothecenes produced by F. graminearum: DON, 15-ADON, NX-3, and NIV. FgTri101 was cloned from F. graminearum and expressed in Arabidopsis plants. We compared the phytotoxic effects of purified DON, NIV, and NX-3 on the root growth of transgenic Arabidopsis expressing FgTri101. Compared to wild type and GUS controls, FgTri101 transgenic Arabidopsis plants displayed significantly longer root length on media containing DON and NX-3. Furthermore, we confirmed that the FgTri101 transgenic plants acetylated DON to 3-ADON, 15-ADON to 3,15-diADON, and NX-3 to NX-2, but did not acetylate NIV. Approximately 90% of the converted toxins were excreted into the media. Our study indicates that transgenic Arabidopsis expressing FgTri101 can provide plant protection by detoxifying trichothecenes and excreting the acetylated toxins out of plant cells. Characterization of plant transporters involved in trichothecene efflux will provide novel targets to reduce FHB and mycotoxin contamination in economically important plant crops.     

枯草杆菌溶栓酶的分离纯化研究

目的 :以枯草杆菌HL 986发酵液为原料 ,对枯草杆菌溶栓酶的分离纯化技术进行研究。方法 :采用磷酸钙吸附沉淀除去部分色素和杂蛋白 ,经超滤脱盐、CM 5 2离子交换分离、丙酮沉淀和SephadexG 75凝胶过滤处理 ,得到具有溶栓活性的酶。结果 :得到的溶栓酶在SDS PAGE上为单一谱带 ,其分子量约为 2 7kD ,比活为980 0U/mg,活性得率为 6 3.8%。结论 :以较高的得率获得了高纯度的枯草杆菌溶栓酶。     

枯草芽孢杆菌纤溶酶的生物分析方法及其在猕猴体内的药动学研究

目的：研究猕猴单次静脉滴注注射用枯草芽孢杆菌纤溶酶（BSFE）的药动学。方法：6只猕猴静脉滴注BSFE2.5mg/kg,采用ELISA法测定动物的血药浓度,实验数据用DAS2.0药动程序拟合并计算药动参数。结果：6只猕猴静脉滴注2.5mg/kg的BSFE,浓度-时间数据经药代动力学参数计算,表明药物消除符合二房室模型。6只猕猴的达峰时间（tmax）均为0.5h,峰浓度（Cmax）均值为（592.3±150.9）μg/L;平均消除t1/2β为（5.78±1.19）h;平均清除率（CL）为（4.42±1.19）L.h^-1.kg^-1;药时AUC0-24.5h均值为（557.1±211.1）μg.L^-1.h。结论：本试验所建立的双抗体夹心ELISA法,灵敏度高、特异性强、操作简便,适于BFSE的药代动力学研究。     

枯草杆菌屎肠球菌二联活菌胶囊治疗急慢性腹泻的疗效及安全性

目的:临床验证枯草杆菌、屎肠球菌(美常安)治疗急慢性腹泻的疗效及安全性.方法:多中心、随机、盲法、平行对照研究.其中急性腹泻95例,慢性腹泻48例;急性腹泻疗程5d,慢性腹泻疗程14d,观察腹泻缓解时间及腹泻伴随症状(腹疼痛、腹胀、里急后重等)变化.结果:急性腹泻于疗程结束时,试验组腹泻缓解率为91.67%(44/48);大便成形率为78.05%(32/41);腹泻伴随症状积分明显下降,腹泻综合疗效评估显效78.72%(37/47),有效率为10.64%(5/47),与对照组比较差异无显著性(P>0.05).慢性腹泻组3d时腹泻缓解率为37.50%(9/24),起效比对照组迅速,2组比较P=0.039 5;疗程结束时腹泻缓解率为91.67%(22/24),大便成形率为62.50%(15/24),腹泻伴随症状明显下降;综合疗效评估显效66.67%(16/24),有效25.00%(6/24).安全性研究未发现不良反应.结论:枯草杆菌屎肠球菌二联活菌胶囊治疗成人急慢性腹泻安全有效.     

枯草杆菌、肠球菌二联活菌肠溶胶囊治疗溃疡性结肠炎的疗效分析

目的 探讨枯草杆菌、肠球菌二联活菌肠溶胶囊治疗溃疡性结肠炎的效果.方法 收集本院2014年1月至2015年6月收治的100例溃疡性结肠炎患者,随机分为对照组与实验组.对照组给予美沙拉嗪治疗,实验组加服枯草杆菌、肠球菌二联活菌肠溶胶囊,比较两组整体治疗效果、复发率、缓解与维持时间、ESR、CRP与不良反应.结果 实验组治疗总有效率(94％)与维持时间[(0.69±0.24)年]显著高于对照组[74％, (0.51±0.16)年],缓解时间[(50.14±9.15)d]显著低于对照组[(57.47±9.84)d],差异有统计学意义(x2=7.440,t=3.922、3.857,P＜0.01);实验组治疗后ESR[(14.71± 7.04)mm/h]与CRP水平[(10.15± 3.53)mg/L]显著低于对照组[(20.15± 8.25)mm/h, (19.53±4.39)mg/L],差异有统计学意义(t=3.547、11.774,P＜0.01),复发率(14％)明显低于对照组(32％),差异有统计学意义(x2=4.534,P＜0.05);两组总体不良反应发生率差异无统计学意义(16％ vs.10％,x2=0.796,P＞ 0.05).结论 枯草杆菌、肠球菌二联活菌肠溶胶囊治疗溃疡性结肠炎的疗效显著,且安全性较高,具有借鉴性.