金针菇单孢菌株菌丝生长速度QTL定位分析

本研究以已经完成基因组测序的单核菌株“6-3”与“6-21”为出发菌株,配对后获得有锁状联合的异核菌株并进行出菇,收集担孢子,单孢分离获得90个菌株构成作图群体,对作图群体的每个菌株进行二代测序并测定菌丝在PDA培养基的生长速度。分析“6-3”与“6-21”两单核菌株的SNP,获得68 914个高质量SNP标记用于遗传连锁群分析,构建了14个遗传连锁群,总长度744.32cM,平均长度为53.17cM,标记间平均遗传距离为1.88cM。QTL分析获得一个控制菌丝生长速度的基因座qMGRP1-LG7,该基因座包含134个基因,富集了与物质代谢有关的通路和基因。     

基于报告基因敲入构建果生刺盘孢细胞核荧光标记菌株

运用基因敲入技术,将GFP、mCherry整合到果生刺盘孢 Colletotrichum fructicola组蛋白histone H1位点,实现融合表达,获得细胞核荧光标记菌株。基于标记菌株可以对分生孢子、营养菌丝、附着胞、侵染菌丝等结构中的细胞核进行实时活体观察。果生刺盘孢存在性亲和的“+”、“-”型菌株分化,GFP“+”型菌株和mCherry“-”型菌株接触形成明显杂交线,杂交线上单子囊内含红绿两种孢子,表明“+”、“-”型菌株间发生杂交;杂交线上子囊壳壁表达mCherry,表明由“-”型菌株发育而来。本研究构建的核荧光标记菌株将是研究果生炭疽菌细胞周期调控和有性繁殖过程的重要材料。     

围小丛壳Glomerella cingulata子囊孢子交配繁殖、生物学特性及致病性

炭疽叶枯病（Glomerella leaf spot）是我国苹果上新发现的一种病害。为了解围小丛壳Glomerella cingulata子囊孢子的交配方式、生物学特性和致病性,从安徽砀山、山东牟平等地采集病害样品,经分离培养和纯化获得单孢菌株。在适宜条件下单孢菌株可产生子囊和子囊孢子,经过毛细管破子囊壁后单孢分离,获得12个子囊,每个子囊有8个子囊孢子。其中10个子囊中有4个“正”孢子（+）和4个“负”孢子（-）,2个子囊中只有“负”孢子。子囊孢子单孢菌株培养72h,“正”菌株菌落白色,以营养生长为主;“负”菌株菌落灰白色,直径略小于正菌株,菌丝稀疏,边缘菌丝白色,中部有大量橙色的分生孢子堆。“正”、“负”菌株异宗配合后,可产生大量可育子囊壳;单独的“正”菌株有性生殖产生稀疏丛簇状的可育子囊壳;单个的“负”菌株只能产生分散且不育的子囊壳。“正”、“负”菌株菌落的生长速度没有差异,对温度、营养、光照和pH值的敏感性也没有差异,但“正”、“负”菌株的致病性存在差异。正菌株的有性生殖没有导致rDNA-ITS、β-tubulin基因碱基序列变异。     

香菇沪香F2菌株重要农艺性状在F2和F3代中的遗传规律

以香菇菌株“沪香F2”及其自交优良F2代菌株“申香1504”为实验材料,收集孢子单核体,对其交配型进行鉴定,然后通过单孢自交的方法,构建F2和F3代群体,并对孢子单核体、F2、F3代群体各阶段培养、出菇情况以及重要农艺性状进行详细统计分析,研究各性状表型分化的情况及遗传规律。结果表明：2个亲本所获得的孢子单核体中A₂B₁交配型比例均为最高,根据孢子单核体交配型数量分别设计了1 028和972个F2和F3代自交配对组合。在2个群体中,配对阶段,分别有15.47%和23.56%的配对组合由于菌丝生长缓慢无法获得后代双核体菌株,且F3代显著高于F2代;生产种培养阶段,出现不良性状的菌株数量分别为7.78%和9.57%;菌棒培养阶段,出现不良性状的菌株数量分别为41.05%和49.28%,且F3代退化菌株比例显著高于F2代,不转色菌株比例显著低于F2代;出菇阶段,分别有3.11%和4.32%的菌株不现蕾,分别有13.04%和4.32%的菌株出畸形菇,分别有19.55%和8.95%的菌株能出正常菇,且F2代出正常菇的菌株比例显著高于F3代。“沪香F2”和“申香1504”分别有26个和8个孢子单核体,多次配对获得的杂交子,出正常菇的概率达50%以上。2个群体的平均单棒产量、平均单棒菇数、平均单菇重表现出明显的分化现象,且两个群体之间均存在极显著性差异。与F2代相比,F3代的产量分布、菇数分布表现出偏分离现象,平均单棒产量低于F2代43.84%,平均单棒菇数低于F2代56.77%。香菇“沪香F2”菌株在F3代中的培养、出菇情况以及农艺性状整体表现劣于F2代,且在F2代中获得表现优于亲本的高产品种,在F3代中获得大朵型品种,对香菇优良菌株选育具有重要的指导作用。     

药用真菌桑黄液体培养过程中的抗氧化活性研究

桑黄Sanghuangporus sanghuang是一种珍稀的药用真菌,具有较大的应用潜力。本研究主要以菌丝体生物量、多酚含量、黄酮含量、抗坏血酸(Ascorbic acid,AA)含量、总抗氧化能力(Total antioxidant capacity,T-AOC)、抑制羟自由基能力(Restraining ability to hydroxyl free radicals,RAHFR)、超氧阴离子清除能力、1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除能力、2,2’-连氮-双(3-乙基苯并噻唑-6-磺酸)[2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid),ABTS]自由基清除能力、铁离子还原能力(Ferric reducing antioxidant power,FRAP)和亚铁离子螯合能力为测定指标,对桑黄液体培养过程中的抗氧化活性进行了评价。结果显示,菌丝体生物量在2–10d内增长迅速,第10天达到最大值,在此过程中AA含量和T-AOC也出现了峰值,且多酚含量、黄酮含量和FRAP均呈上升趋势,说明菌株的抗氧化活性与其自身的生长状况、次级代谢产物分泌及还原能力等密切相关。此外,该菌株对羟自由基、超氧阴离子、DPPH自由基和ABTS自由基的清除能力也较强,于第12天、第10天、第2天和第14天分别达到73.06U/m L、46.78%、86.47%和94.16%,显示了较强的抗氧化活性。较高的亚铁离子螯合能力也说明桑黄在氧化胁迫下可启动自身的抗氧化系统以阻断自由基链反应。研究结果为更好地研究、开发和利用药用真菌桑黄提供了基础资料。     

Act0988拮抗菌株鉴定及培养条件与菌株生长关系研究

Act0988菌株产对柑橘青霉等多种霉菌具抗菌活性的代谢产物,对菌株进行鉴定、研究培养条件与菌株生长的关系,以为后续开发菌株产抗菌物质的培养基筛选及发酵工艺技术的研究奠定基础。以菌株的形态、培养特征、生理生化特征及16S rDNA序列分析进行菌种鉴定,三角瓶液体振荡培养研究碳氮源、温度、pH及氧与菌株生长的关系。结果表明,Act0988菌株为多产色链霉菌(Streptomyces polychromogenes),菌株生长最佳碳源为可溶性淀粉,发酵液生物量5.8 mg/mL;酵母膏与蛋白胨为最佳氮源,生物量5.7 mg/mL左右。菌株最适温度28℃,生物量6.0 mg/mL。最适初始pH7.0,生物量6.2 mg/mL;最适装液量60 mL/500mL,生物量6.3 mg/mL。Act0988菌株易培养,发酵液抗菌活性强,菌株产抗菌物质具有突出的开发利用潜力。     

野生桑树桑黄和杨树桑黄化学成分及抗氧化活性比较

比较了野生桑树桑黄和杨树桑黄子实体乙醇提取物抗氧化活性和化学成分的差异,探讨其高抗氧化能力的来源。以二苯基三硝基苯肼自由基（DPPH）清除率、超氧阴离子清除率和β-胡萝卜素漂白实验作为抗氧化的指标比较其抗氧化活性差异。结果表明,桑树桑黄和杨树桑黄均具有很强的抗氧化活性,杨树桑黄的抗氧化活性显著强于桑树桑黄;杨树桑黄醇提物的总黄酮和总多酚含量均高于桑树桑黄醇提物。通过超高效液相色谱串联三重四级杆飞行时间质谱法（UPLC-Triple-TOF-MS）比较了桑树桑黄和杨树桑黄乙醇提取物成分差异,桑树桑黄中共鉴定出19种多酚类物质,杨树桑黄中除了与桑树桑黄中相同的19种物质,还另外分析出3种多酚类物质。     

栎生桑黄和忍冬桑黄液体培养过程中发酵液抗氧化能力

桑黄孔菌属Sanghuangporus是药用木生真菌资源中一个重要的属,但是该属仅有少数几个种类被用于人工栽培,且栽培面积较小。此外,桑黄孔菌属中大部分种类的药用功能仍未完全明确。因此,本研究以桑黄孔菌属近期新发表的新种栎生桑黄S. quercicola和关注度较低的忍冬桑黄S. lonicericola为主要研究对象,通过测定它们液体培养过程中第2、4、6、8、10、12和14天的菌丝生物量以及发酵液的粗多糖含量、多酚含量、黄酮含量、抗坏血酸含量、DPPH自由基清除能力、ABTS自由基清除能力、总抗氧化能力、超氧化物歧化酶活性、羟自由基清除能力、超氧阴离子清除能力、铁离子还原能力和亚铁离子螯合能力等12个抗氧化指标,对桑黄的抗氧化能力进行评定。2种桑黄真菌各选取一号菌株,其发酵液均表现出强抗氧化能力。相比之下,栎生桑黄的多糖、抗坏血酸和超氧化物歧化酶含量更高,而忍冬桑黄的多酚和黄酮含量更高。相应的,栎生桑黄和忍冬桑黄在其他一些抗氧化指标上也表现出强弱程度及出现时间的差异。上述研究结果为桑黄孔菌属真菌的药用功能开发提供了新资源,为不同抗氧化代谢产物的分离纯化提供了科学依据。     

Act0988拮抗菌株鉴定及培养条件与菌株生长关系研究北大核心CSCD

Act0988菌株产对柑橘青霉等多种霉菌具抗菌活性的代谢产物,对菌株进行鉴定、研究培养条件与菌株生长的关系,以为后续开发菌株产抗菌物质的培养基筛选及发酵工艺技术的研究奠定基础。以菌株的形态、培养特征、生理生化特征及16S rDNA序列分析进行菌种鉴定,三角瓶液体振荡培养研究碳氮源、温度、pH及氧与菌株生长的关系。结果表明,Act0988菌株为多产色链霉菌(Streptomyces polychromogenes),菌株生长最佳碳源为可溶性淀粉,发酵液生物量5.8 mg/mL;酵母膏与蛋白胨为最佳氮源,生物量5.7 mg/mL左右。菌株最适温度28℃,生物量6.0 mg/mL。最适初始pH7.0,生物量6.2 mg/mL;最适装液量60 mL/500mL,生物量6.3 mg/mL。Act0988菌株易培养,发酵液抗菌活性强,菌株产抗菌物质具有突出的开发利用潜力。     

香菇液体发酵高产多糖菌株筛选

通过评价香菇野生菌株发酵产多糖性能,筛选高产香菇多糖菌株.以采自长白山野生香菇通过组织分离获得的6株菌株和2株人工栽培菌株为出发菌株,对不同发酵培养时间菌丝体生物量、胞内多糖含量、胞外多糖含量等进行测试分析,结果表明,8株菌株随着培养时间的延长,菌丝体生物量均有不同程度的增加,但胞内多糖含量和胞外多糖得率变化趋势不同,...     

Chitinolytic and cellulolytic Pseudomonas sp. antagonistic to fungal pathogens enhances nodulation by Mesorhizobium sp. Cicer in chickpea.

Pseudomonas strains isolated from the rhizosphere of chickpea (Cicer arietinum L.) and green gram (Vigna radiata L.) were screened for the production of chitinases and cellulases. Five Pseudomonas strains were found to produce appreciable amounts of both enzymes in culture-free supernatants and showed growth inhibition of the two fungi Pythium aphanidermatum (Oomycete) and Rhizoctonia solani (Basidiomycete) in plates on potato dextrose agar medium. The fungal growth inhibition was not correlated with cell wall-degrading enzyme activity, which suggested that other antifungal compounds produced by these rhizobacteria were also involved in antagonism. Coinoculation of the Pseudomonas strains with the Mesorhizobium sp. Cicer strain Ca181 resulted in a significant increase in nodule biomass when grown under sterilized chillum jar conditions. The results suggest that hydrolytic enzymes produced by Pseudomonas sp. contribute to suppression of plant diseases by inhibiting growth of phytopathogenic fungi and also promote nodulation of legumes by rhizobia.     

Criteria for the selection of strains of entomopathogenic fungi Verticillium lecanii for solid state cultivation

The objective of this study was the selection of strains of Verticillium lecanii for solid-state fermentation (SSF) containing cuticle of Sphenarium purpurascens as an inducer of proteases and chitinases. The selection criteria were: growth at low water activity (a_W), enzymatic activities (proteases and chitinases) and CO₂ production rate. Three strains of V. lecanii were studied ATCC 26854, ATCC 46578 and a wild strain (WS). The strains ATCC 26854 and WS presented the best biomass production at low a_W (0.957). Highest rates of clearing zones of casein and chitin were obtained for strains ATCC 26854 and WS. Best results of CO₂ production in SSF were obtained by using V. lecanii ATCC 26854 which showed a maximal value (2.3 mg CO₂ g IDM⁻¹ h⁻¹) at 36 h of cultivation. Although clearing zones of casein and chitin were partial criteria for strain selection. It was concluded that growth a low water activities and CO₂ production rate, were more reliable criteria for selecting strains of V. lecanii for solid state culture using cuticle of insect as the main C and N source.     

共生菌促进斜生栅藻生长和油脂合成

从斜生栅藻藻际分离共生菌群并进行16S rDNA序列鉴定,构建藻菌共培养体系,并检测斜生栅藻生长、生理生化及产油特性。共分离出7个菌群/株,包括微球菌(菌株1-1、1-2和1-3)、假单胞菌(菌株2-1和2-2)、微小杆菌(菌株-3)和葡萄球菌(菌株-4)。微球菌(菌株1-2)和假单胞菌(菌株2-1)为优势促生菌株,能显著促进微藻生长及色素和油脂积累。斜生栅藻与微球菌(菌株1-2)1∶10共培养体系培养8 d后,栅藻生物量高达4.27 g·L^-1,比对照增加了46.0%;叶绿素a、叶绿素b和类胡萝卜素分别比对照提高12.1%、16.7%和25.0%;含油量为25.7%,比对照增加了14.0%,单不饱和油酸含量(16.4%)也显著高于对照。另一个优异共培养体系是斜生栅藻与假单胞菌(菌株2-1)1∶5共培养体系,在培养8 d后,微藻生物量、叶绿素a、叶绿素b和类胡萝卜素分别比对照提高47.9%、16.0%、17.5%和19.9%;总油脂(27.1%)和单不饱和油酸含量(18.2%)分别比对照增加了20.4%和64.0%。表明藻际共生菌假单胞菌和微球菌可分别与斜生栅藻互作,能够显著促进斜生栅藻生物量和优质油脂的富集,可应用于栅藻商业生产。     

改变培养基碳源复壮草菇退化菌种

碳源是草菇重要的营养源之一,草菇菌种在添加葡萄糖的传统PDA上长期继代会导致菌种退化。本研究用蔗糖、果糖、甘露醇、海藻糖代替PDA中的葡萄糖,对草菇原种（D0）和退化菌株（D1-D3）进行复壮。结果表明：改变碳源对D0影响不显著。蔗糖、果糖、甘露醇、海藻糖均可提高D1-D3的气生菌丝密度、菌丝生长速度和生物量,诱导厚垣孢子产生,并增加菌丝多糖和蛋白质含量;有效抑制了活性氧O₂ ^-·、H₂O₂的积累;提升了超氧化物歧化酶（SOD）和过氧化物酶（POD）活性,但过氧化氢酶（CAT）活性变化不显著;且菌种退化程度越高,复壮效果越好,海藻糖的效果最佳。与对照组相比,海藻糖处理组使退化最严重的D3的菌丝生长速度和生物量分别提高了75.00%和66.67%,多糖和蛋白质含量分别增加了22.49%和16.58%;O₂ ^-·和H₂O₂分别降低了12.50%、12.83%;POD和SOD分别提高了33.33%和255.56%。本研究通过改变培养基的碳源,有效复壮了草菇退化菌种的菌丝特性;也为延缓食用菌菌种退化研究提供参考。     

外源茉莉酸甲酯诱导对香菇多糖代谢及关键酶基因表达的影响

香菇多糖是重要的生物活性成分,其药用价值高,茉莉酸甲酯有助于提高食用菌多糖含量,但关于其对香菇多糖合成的影响尚不清楚。本研究以香菇新808为实验材料,对不同浓度茉莉酸甲酯（MeJA）诱导下香菇菌丝生物量和生长速率、香菇多糖代谢关键酶活性及基因相对表达量、香菇多糖含量进行比较分析。结果表明：10μmol/L的茉莉酸甲酯可显著促进香菇菌丝生长并提高菌丝体生物量,分别是对照的1.14倍、1.2倍;与香菇多糖代谢相关的UDP-葡萄糖焦磷酸化酶（UGPase）、葡萄糖磷酸异构酶（PGI）、α-葡萄糖磷酸变位酶（α-PGM）活性增加,为对照组的1.58、1.13、3.21倍;其基因相对表达量较对照组也显著上调,为对照组的5.73、1.4、1.77倍;香菇多糖合成量显著增加,为对照组1.58倍。10μmol/L的MeJA处理后,香菇多糖代谢关键酶活性及基因相对表达量与多糖含量间存在显著的正相关关系,表明添加适宜浓度的茉莉酸甲酯会影响香菇菌丝的生长及多糖合成。     

4种虫草相关真菌菌丝体粗多糖的生物活性评价

本研究以细脚棒束孢、蛹虫草、蝉棒束孢和球孢白僵菌的菌丝体粗多糖为对象,分析4种虫草相关真菌菌丝体粗多糖含量与生物量的相关性,并进一步对其抗氧化能力和抗肿瘤活力进行评价。液体发酵结果表明,蝉棒束孢MF12、MF13和蛹虫草MF27、MF1的菌丝体粗多糖含量（>40mg/g）显著高于其他菌株,蝉棒束孢MF11、MF13和蛹虫草MF27菌丝体生物量（>12g/L）显著高于其他菌株,但相关性分析表明,4种虫草相关真菌10个菌株菌丝体的多糖含量与生物量之间没有显著相关性;抗氧化活性表明,蛹虫草MF27、MF1和球孢白僵菌MF10菌丝体粗多糖具有良好的体外抗氧化活性,其EC₅₀均小于0.9mg/mL;抗肿瘤活性表明,蛹虫草MF1、MF28、MF27和球孢白僵菌MF10菌丝体粗多糖在体外能有效抑制HepG-2细胞增殖,其IC₅₀均小于1.5mg/mL。综上,蛹虫草MF27、MF1和球孢白僵菌MF10虫草菌株具有良好的开发和应用潜力。     

Optimizing edible fungal growth and biodegradation of inedible crop residues using various cropping methods

Long-term manned space flights to Mars require the development of an advanced life support (ALS) ecosystem including efficient food crop production, processing and recycling waste products thereof. Using edible white rot fungi (EWRF) to achieve effective biomass transformation in ALS requires optimal and rapid biodegradative activity on lignocellulosic wastes. We investigated the mycelial growth of Lentinula edodes and Pleurotus ostreatus on processed residues of various crops under various cropping patterns. In single cropping, mycelial growth and fruiting in all strains were significantly repressed on sweet potato and basil. However, growth of the strains was improved when sweet potato and basil residues were paired with rice or wheat straw. Oyster mushroom (Pleurotus) strains were better than shiitake (L. edodes) strains under single, paired, and mixed cropping patterns. Mixed cropping further eliminated the inherent inhibitory effect of sweet potato, basil, or lettuce on fungal growth. Co-cropping fungal species had a synergistic effect on rate of fungal growth, substrate colonization, and fruiting. Use of efficient cropping methods may enhance fungal growth, fruiting, biodegradation of crop residues, and efficiency of biomass recycling.     

Characterization of a respiratory mutant of Escherichia coli with reduced uptake of aminoglycoside antibiotics

A strain of Escherichia coli (NSW77) which is partially resistant to streptomycin was isolated by selecting for growth on plates supplemented with 12.5 μg/ml streptomycin, a concentration which completely inhibits growth of wild-type strains. The low-level resistance of the mutant appears to result from a reduced ability to accumulate streptomycin intracellularly. In addition, the mutant strain is unable to use succinate for growth because of a defective respiratory chain. Thus, membranes of the mutant strain were found to have approximately half the NADH and D-lactate oxidase activity of the parent strain. Succinate oxidase activity was reduced more drastically, to a level of 7% that of the parent strain. Moreover, membranes of the mutant were found to contain demethyl-menaquinone and, in place of ubiquinone, a structural analogue, 2-octaprenyl-3-methyl-6-methoxy-1,4 benzoquinone. The mutation responsible for both the Suc⁻ phenotype and partial resistance to streptomycin was found to be located near minute 15 on the bacterial chromosome. Both the biochemical and genetic evidence suggests that the mutation in strain NSW77 resides in the ubi F gene. Another previously characterized ubi F strain was also found to have a reduced capacity to take up an aminoglycoside antibiotic (gentamicin). These results suggest that the respiratory defects in ubi F strains are responsible for the reduced capacity of such strains to accumulate aminoglycosides.     

Nitrogen fixation and biomass production in symbioses between Alnus incana and Frankia strains with different hydrogen metabolism

Three different strains of Frankia , the pure cultures AvcI1 and CpI1 and a local strain (crushed nodule inoculum), were compared in symbiosis with one clone of Alnus incana (L.) Moench. Hydrogen metabolism, nitrogenase (EC 1.7.99.2) activity and relative efficiency of nitrogenase were studied as well as growth and nitrogen content of the plants. The local Frankia strain showed no measurable hydrogen uptake but high H₂-evolution. No H₂-evolution was detected in Frankia AvcI1 because of its hydrogenase activity. CpI1 also had hydrogenase, although only a very small H₂-evolution was detected at the end of the growth period. Hydrogenase activity was detected both in pure cultures and nodule homogenates of CpI1 and AvcI1. Growth, biomass production and nitrogen content were highest in alders inoculated with Frankia AvcI1 while the lowest values were found for alders living in symbiosis with the local Frankia strain. The presence of hydrogenase in Frankia seemed to be benefical for growth and biomass production in the alders. However, the strains also differed with respect to spore formation. The local strain, but not AvcI1 and CpI1, formed spores in the root nodules.     

苹果连作生防细菌B6对平邑甜茶幼苗生物量及连作土壤环境的影响

以苹果连作障碍病原真菌层出镰刀菌、串珠镰刀菌、尖孢镰刀菌和腐皮镰刀菌为靶标菌,通过平板对峙法对分离自苹果根际土壤的细菌进行反复筛选比较,对筛选出的拮抗效果最优的菌株进行形态学、生理生化特征和16S rDNA序列分析鉴定,并于盆栽条件下探讨其菌肥对平邑甜茶幼苗生长及连作土壤环境的影响.结果表明: 菌株B6对上述4种病原真菌的抑菌率最高,分别达到71.8%、70.1%、72.6%、91.5%.经鉴定,菌株B6为甲基营养型芽孢杆菌.盆栽试验表明,与连作处理(CK₁)相比,B6菌肥处理(T)可以不同程度地促进平邑甜茶幼苗生物量的增加,其中地径、鲜质量和干质量分别显著增加18.3%、51.2%;显著提高连作土壤中可培养细菌和放线菌数量,使真菌数量下降为连作土壤的37.7%,促使土壤类型向细菌型转化;显著提高连作土壤中的蔗糖酶、磷酸酶、脲酶和过氧化氢酶的活性,增长率分别为37.3%、24.0%、42.9%、49.4%.表明B6菌肥可以优化苹果连作障碍土壤中可培养微生物群落结构,提高土壤酶活性,增加平邑甜茶幼苗生物量.