Microscopy reveals disease control through novel effects on fungal development: a case study with an early-generation benzophenone fungicide

The benzophenones are a new class of agricultural fungicides that demonstrate protectant, curative and eradicative/antisporulant activity against powdery mildews. The chemistry is represented in the marketplace by the fungicide metrafenone, recently introduced by BASF and discussed in the following paper. The benzophenones show no evidence of acting by previously identified biochemical mechanisms, nor do they show cross-resistance with existing fungicides. The value of microscopy in elucidating fungicide mode of action is demonstrated through identification of the effects of an early benzophenone, eBZO, on mildew development. eBZO caused profound alterations in the morphology of powdery mildews of both monocotyledons and dicotyledons, affecting multiple stages of fungal development, including spore germination, appressorial formation, penetration, surface hyphal morphology and sporogenesis. Identification of analogous effects of eBZO on sporulation in the model organism Aspergillus nidulans (Eidam) Winter provides a unique opportunity to elucidate important morphogenetic regulatory sites in the economically important obligate pathogens, the powdery mildews. Benzophenones provide a further example of the benefits of whole-organism testing in the search for novel fungicide modes of action.     

凡纳滨对虾血蓝蛋白的抗黑曲霉活性

 以凡纳滨对虾(Litopenaeus vannamei)为研究对象, 运用亲和层析、SDS-PAGE、Western-blotting、抗真菌实验等技术探索对虾血蓝蛋白的抗黑曲菌活性。结果发现, 血蓝蛋白在质量浓度为0.1～0.8 mg/mL时对黑曲霉(Aspergillus niger)生长表现出明显的抑制活性。在此基础上, 选取0.2 mg/mL血蓝蛋白作用后的黑曲霉进行显微观察分析。光学显微镜观察结果表明, 血蓝蛋白能抑制黑曲霉孢子生长。扫描电镜分析显示, 在血蓝蛋白作用下, 黑曲霉孢子囊萎缩, 分生孢子集聚成团, 菌丝裂解、扭曲、变短、无分生孢子囊。由此推测, 对虾血蓝蛋白具有抗黑曲菌活性, 其抗菌机制可能与血蓝蛋白抑制黑曲菌孢子生长和促使病态菌丝产生等有关。本研究对揭示血蓝蛋白的非特异性免疫机理和寻找新的对虾疾病免疫学防治途径等具有重要意义。

     

Value added waste of Jatropha curcas residue: Optimization of protease production in solid state fermentation by Taguchi DOE methodology

The oil extraction of Jatropha curcas created the large amount of the by-product from its seeds. An application of solid-state fermentation (SSF) was considered to be of value to these raw materials. This study investigated the potential of a utilization of deoiled J. curcas seed cake as substrate for protease productions by Aspergillus oryzae. While various parameters for SSF was conventionally individually optimized, five parameters were simultaneously examined based on Taguchi method. The effect of three different levels of five factors, including moisture content of substrate, inoculums size, incubation temperature, type of porous substrate and incubation time were examined. The optimum conditions for the protease production by A. oryzae obtained from this experiment were 45% moisture content of substrate, 10% inoculums size, 30 °C incubation temperature, deoiled J. curcas seed cake mixed with cassava bagasse ratio 4:1 as porous substrate at 84 h of incubation time. By adjusting the conditions to these optimum levels, the protease production increased up to 4.6 times as many as the protease yield from the non-optimizing experiment. The use of statistical approach, Taguchi method, provided a satisfactory outcome in defining the optimum conditions for protease production by A. oryzae. Further, the utilization of deoiled J. curcas seed cake as substrate for SSF was proven as the suitable practice for this agricultural waste, in order to develop for an industrial use.     

产纤维素酶真菌的筛选与鉴定

为从秸秆中分离出能够降解纤维的菌株,试验采取秸秆样品接种马铃薯琼脂培养基,在室温环境下培养,取分离菌接种纤维素刚果红琼脂培养基,筛选能形成较大降解圈的细菌,并进行形态学和分子生物学鉴定,测定其纤维素酶性质。结果表明,经分子生物学鉴定,筛选菌为黑曲霉菌,该菌在室温环境能够生长,菌株降解圈直径(H)与菌落直径(C)的比值(H/C)为7.64。筛选菌在20 ℃环境下培养5 d酶活达到最高值,为42.18 U/mL。纤维素酶最适pH为7.0,最适反应温度为20 ℃,在20~40 ℃或pH 6.0~8.0环境下作用1 h,相对酶活力仍保持在90%以上。综上所述,本试验分离的黑曲霉菌株是低温产纤维素酶菌株,产酶能力较强,具有潜在的开发价值。     

土传病害生防菌的分离筛选及鉴定

为了使蛋鸡粪有机肥兼有防治作物土传病害的多功能性。实验从安徽省农科院实验田及蛋鸡养殖场等环境中共分离出170 株真菌,首先将分离菌与辣椒炭疽病(Colletotrichum capsici)进行平板对峙实验,再通过复筛选出7 株菌,同时对该7 株菌进行抑菌谱、耐温实验和蛋鸡粪肥中增殖实验,效果较好的菌株最终进行分子生物学鉴定。结果表明：筛选出的菌株中编号为TCC53 对油菜菌核病和麦纹枯的抑菌率分别可达90.4%、82.3%,TCC157 对辣椒炭疽病和辣椒疫霉的抑菌率分别可达88.6%、71.2%,同时该两株菌均可在40℃以上生长。通过分子生物学鉴定得出TCC53 和TCC157 分别为棘孢曲霉(Aspergillusaculeatus)和皮落青霉(Penicillium crustosum)。皮落青霉和棘孢曲霉均为环境中常见真菌,生长快、不具有致病性,将其添加至蛋鸡粪有机肥中有望提高作物的抗病效果又可改善土壤结构的微生物肥料。     

钾矿物晶体结构对黑曲霉生长代谢及钾与硅的溶出影响

【目的】黑曲霉对钾矿物的分化分解及其中钾（硅）的溶出作用与其产酸及产胞外代谢产物（主要指多糖与蛋白质）的能力密切相关,但研究矿物晶体结构在此过程中的地位与作用的较少。试验采用一株黑曲霉分别与正长石与白云母进行浸出培养以了解不同钾矿物对黑曲霉生长代谢能力的影响,及黑曲霉对含钾硅酸盐矿物的转化作用与其矿物晶体结构的相关性。【方法】在分别含铝土矿、正长石与白云母矿粉的平板固体与液体Czapek’s培养基中培养黑曲霉,并以不含矿粉的纯培养作为对照,观察其在不同矿物环境中的生长特征;采用摇瓶浸出考察黑曲霉对钾矿物的分化分解及其中钾（硅）的溶出效果,通过测定浸出液中的pH值、多糖、蛋白质及K2O与SiO2的浓度,及浸出前后矿物表面的SEM和XRD观察,对比分析正长石与白云母对黑曲霉生长代谢的促进作用及黑曲霉对它们的转化作用的差异。【结果】不同晶体结构的钾矿物对黑曲霉的生长代谢刺激与促进作用不同,与不含矿粉及含钾极少的铝土矿粉培养基相比,黑曲霉在含钾矿粉培养基中的繁殖生长速度明显要快,在固体平板培养基中表现出对钾矿粉的趋化性,在液体培养基中形成的菌体-矿物聚集体更显著,且与正长石相比,黑曲霉在固体平板上的生长过程中对白云母的趋化性要明显,在液体培养环境中形成的菌体-矿物聚集体更显著。黑曲霉在缺钾培养基与分别含正长石与白云母的培养基中培养10 d,可分别代谢产生1.45、1.97、2.45 g•L-1的有机酸,5.11、9.96、12.25 g•L-1的多糖与 6.25、13.78、16.97 g•L-1的蛋白质。浸矿30 d,黑曲霉浸出白云母中K2O与SiO2的量要比浸出正长石的分别高79.10与57.78 mg•L-1。黑曲霉作用前后的白云母与正长石矿粉的SEM与XRD的分析结果表明,白云母较正长石的浸蚀程度更明显,矿物表面沉积了更多的非晶态或晶形不好的细小颗粒,在含少量正长石的白云母矿物或含少量白云母的正长石矿物浸渣的XRD图谱中,反映白云母的主特征峰强度明显下降,且有部分特征峰消失,而反映正长石的特征峰强度略有增强。【结论】钾矿物晶体结构是影响黑曲霉生长所形成的菌体-矿物聚集体、代谢产物、酸碱微环境的差异及钾矿物风化分解与钾（硅）溶出程度不同的重要原因;在缺有效钾环境中,钾矿物对黑曲霉生长代谢具有促进作用,与正长石相比,白云母可以刺激与促进黑曲霉代谢产更多的有机酸、多糖与蛋白质;多种矿物同时存在的情况下,黑曲霉对不同晶体结构的钾矿物的分解有一定的选择性,对具层状结构的白云母较具架状结构的正长石的风化破坏作用要快。     

黑曲霉固体发酵产酸性蛋白酶条件优化

对经诱变后黑曲霉固体发酵生产酸性蛋白酶的培养基组成及发酵条件进行优化。结果表明,该菌发酵产酶的最适培养基和条件为米糠20 g,碳源玉米粉60 g/kg,氮源硝酸铵5 g/kg,MnCl2 4 g/kg,料液比1.0∶1.5,调初始pH值7,以6mL/100g的接种量接种,在40℃、180 r/min下发酵72 h,摇瓶产酶达到1 120.0 U/mL,比基础培养基提高了近2.9倍。     

黄曲霉野生型及LaeA缺陷型菌株胞外差异蛋白鉴定

【目的】分析黄曲霉野生型产毒株及LaeA缺陷型无毒株在以小麦、玉米为培养基质中生长时的胞外差异蛋白,为进一步明确LaeA因子在黄曲霉产毒调控中的作用提供基础数据。【方法】将小麦及玉米籽粒粉碎后灭菌并加水配制成培养黄曲霉菌的基质,在产毒株(黄曲霉CA14野生型)及无毒株(LaeA缺陷型)培养0、48和72 h时取样,采用SDS-PAGE分别分析黄曲霉毒素B1(AFB1)及胞外差异蛋白,并采用液质联用四极杆飞行时间质谱技术[LC-MS-MS (Q-TOF)]对获得的胞外差异蛋白进行鉴定。【结果】黄曲霉野生型及LaeA缺陷型菌株均能将小麦和玉米基质中的小分子蛋白迅速转化,随着培养时间延长,LaeA缺陷型菌株的胞外蛋白较野生型菌株略少,不产生黄曲霉毒素。产毒株及无毒株胞外差异蛋白条带共有7个,主要为淀粉酶A、碱性蛋白酶、木聚糖酶F3和亮氨酸氨基肽酶A等生理酶类。【结论】黄曲霉产毒株与无毒株的胞外差异蛋白为生理酶类蛋白,该类酶蛋白主要与营养摄入有关,且影响黄曲霉菌丝的生长。     

高效产果胶酶的黑曲霉菌种的诱变及筛选（摘要）

[目的]以原有保藏菌株经诱变并筛选出高产果胶酶的黑曲霉菌种。[方法]涂布平板法选取透明圈和菌落直径较大者作为出发菌株。采用紫外线进行菌种诱变,选择致死率在70%～80%范围内的诱变后菌株进行初筛和复筛。经透明圈检测和摇瓶发酵酶活检测确定其最高酶活和培养时间。[结果]D1-4菌种在适宜培养基中培养96h酶活性最高,达141.13U/ml,比出发菌株酶活提高了1倍。[结论]诱变菌株D1-4有较强分泌果胶酶的能力。     

黑曲霉产木聚糖酶的固态发酵条件优化

[目的]研究培养基组成和培养条件对黑曲霉固态发酵产木聚糖酶的影响。[方法]以木聚糖酶酶活为评价指标,利用单因素试验和L9（34）正交试验考察摇瓶发酵条件下培养基组成、pH值、培养时间、培养温度和接种量对黑曲霉产生木聚糖酶的影响。[结果]最佳培养基组成为：麸皮与玉米芯质量比5∶3,（NH4）2SO4、KH2PO4、CaCl2和MgSO4相对于固体料的质量百分数分别为1.0%、0.2%、0.1%和0.1%）,料液比1∶1.7;最优培养条件为：pH值7.5、培养温度28℃、培养时间60h,其间翻曲2～3次;在此工艺条件下,木聚糖酶的活力可达14698.21IU/g。[结论]该优化条件为木聚糖酶的工业化生产和应用提供了依据。