钾与信号抑制剂对外生菌根真菌分泌乙酸的调控作用

外生菌根真菌是森林生态系统中的重要成分,参与树木养分的吸收利用。试验液体培养外生菌根真菌,设置不同供钾水平,添加钙信号抑制剂,研究了它们的有机酸和氢离子分泌,以及乙酸分泌对供钾和信号抑制剂的响应。结果表明,供试菌株的生长,氮、磷、钾含量和吸收量因菌株不同而异,生物量变化于52.91—121.72 mg/瓶之间,相差1倍以上。外生菌根真菌吸收养分的差异可能与它们对土壤养分环境的长期适应、进化、选择有关。在外生菌根真菌的培养液中,分别检测出草酸、乙酸、苹果酸、柠檬酸和丁二酸等,以及大量的氢离子,说明菌根真菌能分解土壤含钾矿物,释放钾离子,改善寄主植物的钾营养。其中,乙酸分泌量较大,具有普遍性,低钾刺激分泌乙酸,高钾时分泌减少,其分泌速率与供钾浓度和菌丝吸钾量之间呈显著负相关(r=-0.734,r=-0.617,n=60)。钾对菌根真菌分泌乙酸的调控作用具有改善森林钾素营养,防止土壤养分淋失的生理和生态意义。此外,在低钾条件下,阴离子通道和钙信号抑制剂抑制外生菌根真菌分泌乙酸。说明钙信号和阴离子通道参与了乙酸分泌,缺钾可能是刺激乙酸分泌的原初信号,通过信号转导和一系列级联反应促进乙酸分泌。     

外生菌根真菌对土壤钾的活化作用

【目的】外生菌根真菌是森林生态系统中的重要组成成分,与木本植物的根系形成菌根,参与树木养分吸收。【方法】试验在液培条件下,以土壤为钾源,利用我国西南地区分离的牛肝菌(Boletnus sp.,Bo 07)、松乳菇(Lactarius delicious,Ld 03)、彩色豆马勃(Pisolithus tinctorius,Pt 715)和内蒙古大青山分离的土生空团菌(Cenococcum geophilum,Cg 04)为材料,研究了它们的生长、钾吸收、氢离子和有机酸分泌,以及土壤钾的变化。【结果】结果表明,Bo 07、Ld 03和Pt 715的生物量、含钾量和吸收量显著高于Cg 04,说明在长期缺钾的环境中,外生菌根真菌经过"物竞天择"可能进化出适应低钾和较强的吸钾能力。以土壤为钾源培养菌根真菌,培养液中的钾浓度显著提高,故外生菌根真菌可促进土壤钾的溶解。在外生菌根真菌的培养液中,分别检测到苹果酸、丁二酸和柠檬酸,均检测到草酸和乙酸。Bo 07、Ld 03和Pt 715显著提高土壤交换性钾含量,Bo 07和Ld 03还显著降低矿物结构钾含量,说明外生菌根真菌不同程度地活化了土壤无效钾,分离自南方的菌株Bo 07、Ld 03和Pt 715总体上大于分离自北方的菌株Cg 04。此外,土壤矿物结构钾分别与总有机酸和草酸分泌量呈极显著负相关(r有机酸=-0.989**,r草酸=-0.950*,n=5),与培养液pH值呈显著正相关(r=0.916*,n=5),故外生菌根真菌分泌的氢离子和有机酸尤其是草酸可能活化土壤无效钾。【结论】供试外生菌根真菌能不同程度的活化土壤无效钾,其活化能力可能与分泌氢离子和有机酸尤其是草酸密切相关。     

磷与信号抑制剂对外生菌根真菌分泌草酸的调控作用

摘要:【目的】磷是树木生长的必需营养元素之一,磷素营养丰缺条件下,研究外生菌根真菌的草酸分泌及调控有益于揭示它们活化利用土壤无机磷的机理。【方法】试验设置低、正常、高3种不同磷浓度,液体培养外生菌根真菌,研究了磷和Ca2+信号/阴离子通道抑制剂对草酸分泌的调控作用。【结果】外生菌根真菌能分泌大量的氢离子和草酸、乙酸、苹果酸、柠檬酸和丁二酸等多种有机酸,对溶解难溶性无机磷有重要作用。在外生菌根真菌分泌的有机酸中,草酸占15.14%－36.01%;低磷促进草酸分泌,正常和高磷则产生抑制作用;培养液磷浓度和菌丝含磷量分别与供试菌种的草酸分泌速率呈显著或极显著负相关,相关系数依次为r=－0.264*和r=－0.349＊＊,n=60,*表示显著(P0.05),＊＊表示极显著(P 0.01),说明磷能调控外生菌根真菌分泌草酸。在低磷胁迫下,钙调蛋白抑制剂、Ca2+通道抑制剂、Ca2+内膜通道抑制剂和阴离子通道抑制剂显著抑制外生菌根真菌分泌草酸。但是,在正常和高磷条件下,草酸分泌速率低,未响应Ca2+信号/阴离子通道抑制剂。【结论】供试外生菌根真菌能分泌大量的氢离子和有机酸(尤其是草酸),有益于溶解土壤无机磷,改善寄主植物的磷营养;供磷水平调控草酸分泌速率;在低磷胁迫下,Ca2+信号是介导外生菌根真菌分泌草酸的信号因子。     

不同生境外生菌根真菌对铝胁迫的响应

以南北方不同生境下的10株外生菌根真菌为研究对象,采用液体培养的方法,研究了铝对不同菌根真菌的生物量、有机酸分泌及养分含量的影响,以期筛选出抗铝性强的优良菌株,并探讨其抗铝机理。结果表明:外生菌根真菌Sl 08抗铝性最强;Pt 715、Ld 03、Bo 11、Sl 01、Bo 15也具有不同程度的耐铝性;Sl 14、Gc 99、Cg 04抗铝性较差;Sg 11抗铝性最差。来自南方酸性森林土壤的菌株总体抗铝性强于来自北方石灰性土壤的菌株,这表明外生菌根真菌的铝耐受能力与其原始生境有着密切的联系。外生菌根真菌能分泌多种有机酸,且不同菌株分泌的有机酸种类不同。其中,受铝胁迫分泌量增加最多的是草酸。研究中,铝胁迫能增加大多数铝抗性菌株的草酸分泌量,其中铝抗性最强的Sl 08表现最为明显。但铝胁迫并没有促进具备一定铝抗性的Bo 11和Sl 01草酸的分泌量,同时在铝敏感的菌株中均观察到了草酸分泌量的增加。这表明分泌草酸可能并不是外生菌根真菌抵抗铝毒的唯一途径。对各菌株铝胁迫下对氮,磷及钾的吸收研究表明,除铝敏感菌株Sl 14外,铝胁迫均能促进各供试菌株对氮,磷或钾的吸收。综上,在一定铝浓度下,一些外生菌根真菌可通过增加草酸分泌来抵御铝毒。此外,铝胁迫下外生菌根真菌还可通过调控氮、磷、钾等营养元素的吸收来抵抗铝毒,即通过增加对营养元素的吸收来增强其在铝胁迫下的生存能力,这可能是其抵御铝胁迫的应激反应之一。     

磷对外生菌根真菌松乳菇和双色蜡蘑草酸、氢离子和磷酸酶分泌的影响

在液体培养基中设置无磷、低磷、中磷和高磷四种磷浓度,离体培养松乳菇Lactarius deliciosus的3个株系Ld-01、Ld-02、Ld-03和双色蜡蘑Laccaria bicolor S238N,分别测定外生菌根真菌的生长量、草酸、氢离子和酸性磷酸酶分泌速率。结果表明,在中磷的条件下,外生菌根真菌的生长最好,无磷、低磷或高磷对它们的生长产生抑制作用,抑制程度因菌种（株）不同而异。草酸、氢离子和酸性磷酸酶的分泌速率顺序为：无磷（0g NaH2PO4/L）> 低磷（0.229g NaH2PO4/L）     

土生空团菌对白云母的风化作用及解钾特性

摘要:【目的】为了提高森林土壤含钾硅酸盐矿物利用率和改善树木钾素营养,探讨外生菌根真菌对白云母矿物的解钾及风化效应。【方法】通过5种外生菌根真菌在贫钾条件下与白云母的相互作用,考察了3种常用培养基对菌株解钾能力的影响;采用解钾效果最高的土生空团菌在Modified Melin-Norkrans(MMN)培养基条件下,进一步研究了21d浸出过程中主要矿质元素的释放量、生物量、剩余葡萄糖量、产生的小分子有机酸种类及含量、菌丝-矿物微环境的改变,利用扫描电子显微镜观测白云母被风化的痕迹。【结果】培养基对不同外生菌根真菌解钾能力的影响各异,其中土生空团菌在MMN培养基条件下解钾能力最强,其解钾量与生物量、剩余葡萄糖量、pH值显著相关。土生空团菌能够分泌有助于菌丝-矿物粘附的胞外多糖以形成菌丝-矿物微环境,并富集有机酸,促进微环境内钾的释放;菌丝不仅能够粘附在白云母表面产生刻蚀作用,还能破坏矿物深入其内部。【结论】外生菌根真菌具有促进白云母风化并释放钾素的能力,这种能力可能与菌丝、有机酸、多糖的协同作用有关。     

磷对外生菌根真菌松乳菇和双色蜡蘑草酸、氢离子和磷酸酶分泌的影响

在液体培养基中设置无磷、低磷、中磷和高磷四种磷浓度,离体培养松乳菇Lactarius deliciosus的3个株系Ld-01、Ld-02、Ld-03和双色蜡蘑Laccaria bicolor S238N,分别测定外生菌根真菌的生长量、草酸、氢离子和酸性磷酸酶分泌速率。结果表明,在中磷的条件下,外生菌根真菌的生长最好,无磷、低磷或高磷对它们的生长产生抑制作用,抑制程度因菌种（株）不同而异。草酸、氢离子和酸性磷酸酶的分泌速率顺序为：无磷（0g NaH2PO4/L）〉低磷（0.229gNaH2PO4/L）〉中磷（1．147gNaH2PO4／L）〉高磷（5.735g NaH2PO4／L）,表现出明显的种（株）间差异,看来外源磷的供应状况可能有调节外生菌根真菌活化土壤无机磷和有机磷的作用。在缺磷和低磷条件下,外生菌根真菌具有较强的活化作用,在足磷的条件下活化作用减小,由此有效地利用土壤中的磷。     

磷对外生菌根真菌松乳菇和双色蜡蘑草酸、氢离子和磷酸酶分泌的影响

在液体培养基中设置无磷、低磷、中磷和高磷四种磷浓度,离体培养松乳菇Lactarius deliciosus的3个株系Ld-01、Ld-02、Ld-03和双色蜡蘑Laccaria bicolor S238N,分别测定外生菌根真菌的生长量、草酸、氢离子和酸性磷酸酶分泌速率。结果表明,在中磷的条件下,外生菌根真菌的生长最好,无磷、低磷或高磷对它们的生长产生抑制作用,抑制程度因菌种(株)不同而异。草酸、氢离子和酸性磷酸酶的分泌速率顺序为:无磷(0gNaH2PO4/L)>低磷(0.229gNaH2PO4/L)>中磷(1.147gNaH2PO4/L)>高磷(5.735gNaH2PO4/L),表现出明显的种(株)间差异,看来外源磷的供应状况可能有调节外生菌根真菌活化土壤无机磷和有机磷的作用。在缺磷和低磷条件下,外生菌根真菌具有较强的活化作用,在足磷的条件下活化作用减小,由此有效地利用土壤中的磷。     

青蒿素对外生菌根真菌化感效应

青蒿素是治疗疟疾的首选药物,主要从黄花蒿(Artemisia annua L.)中提取,然而黄花蒿在生长过程中会向周围环境分泌青蒿素。为正确评估青蒿素对森林生态系统中的重要成分""外生菌根真菌的影响,试验以重庆地区有代表性的两株外生菌根真菌——褐环乳牛肝菌(Suillus luteus)Sl 8和松乳菇(Lactarius delicious)Ld 3为材料,研究了青蒿素对菌丝生长,H+和有机酸分泌,以及养分吸收的影响。结果表明,在液体培养基中加入青蒿素,外生菌根真菌的生长受到明显抑制,菌丝生物量降幅高达26.89%(Ld 3)和89.13%(Sl 8);Ld 3分泌H+和草酸的能力增强,而Sl 8分泌量下降。随着青蒿素浓度的增加,菌丝的N、P、K含量及吸收量显著减少。当培养基中青蒿素达到80 mg/L时,Ld 3的N、P、K吸收量比不加青蒿素的处理分别降低了50.55%、46.30%和42.28%;Sl 8几乎丧失对N、P、K的吸收能力。说明青蒿素不同程度地抑制了外生菌根真菌的生长和养分吸收,但对H+和草酸的分泌作用因菌株不同而异。     

铝对外生菌根真菌草酸分泌及磷、钾、铝吸收的影响

试验研究了在铝胁迫条件下,6种(株)外生菌根真菌(ECMF)的生长、草酸分泌,以及磷、钾、铝的吸收状况。结果表明,铝对抗(耐)型菌种Pt715、HrSp、CgSIV的生长无抑制作用,但显著抑制敏感型菌株LbS238N、LbS238A和Lb270的生长,说明ECMF对铝胁迫的生长反应可能是筛选抗(耐)铝的指标之一。在铝胁迫条件下,无论是抗(耐)型还是敏感型菌种(株),都会发生一系列有益于抗(耐)铝的生化反应,包括草酸分泌量、菌丝磷和钾含量增加,H+分泌改变等。在培养液中,草酸电离产生的H+仅占H+总浓度的少数,说明溶液中H+的主要来源不是ECMF所分泌的草酸,而是菌丝细胞为保持吸收阳离子的电荷平衡排出的H+或分泌的其它有机酸。     

Influence of potassium and calcium ions on the phosphatidylinositol and phosphatidylcholine metabolism in rat fibroblasts after growth stimulation by calf serum.

In secondary cultures of embryonic rat fibroblasts which were arrested in G1 (G0) by serum depletion and subsequently triggered into the cell cycle by readdition of growth factors isolated from fetal calf serum the influence of the potassium and calcium concentrations in the medium on phosphatidylinositol and phosphatidylcholine metabolism was investigated. The incorporation of inorganic [32P]phosphate into phosphatidylinositol is dependent on the potassium content of the culture medium. The specific activity of 32P in phosphatidylinositol is increased at K+ concentrations between 0.1 and 1 mM. Also calcium (between 0.01 and 2 mM) slightly stimulates phosphatidylinositol metabolism. Also the incorporation of myo-[3H]inositol is increased at potassium concentrations between 0.2 and 1 mM, whereas calcium is slightly inhibitory. The labelling of phosphatidylcholine with either [32P]phosphate or [3H]choline is not dependent on the potassium and calcium concentrations of the culture medium. Moreover, the phospholipid metabolism of permanently growing epithelioid and fibroblastoid cells lines, which were investigated, is considerably less dependent on the K+ and Ca2+ ions.     

实验室薄口螨的大量培养及休眠体的诱导

用玉米粉培养基、BY(牛肉膏+酵母膏)软琼脂培养基、BY培养液3种培养基对实验室薄口螨(Histiostoma laboratorium)进行了培养。其最适培养基是玉米粉培养基;该螨在BY软琼脂培养基上也能生长,但生长速度比较缓慢,经过BY软琼脂培养基的培养,能够收集到大量干净的个体,为DNA提取和分子生物学研究提供了方便;在BY培养液中,实验室薄口螨不能进行继代生长,但能够产生大量的卵,可收集卵做更进一步的深入研究。休眠体是该螨生活史中的重要阶段,是借助携播者进行传播的特殊形式。对孳生于培养有果蝇的玻璃指管中的实验室薄口螨产生的休眠体及其在果蝇体表的吸附状况进行了观察,利用较高温度(30～35℃)培养基逐步干燥、较低温度(10～15℃)、BY液体培养3种方法,可以诱导该螨休眠体集中大量地形成,方便收集休眠体,是对其进行生理生化、基因表达、疾病传播机理等方面研究的先决条件。     

钙、磷、镁和钾对雪莲细胞悬浮培养的影响

考察了钙、磷、镁和钾等大量营养元素对雪莲细胞TUIP-8悬浮培养的影响,确定了培养雪莲细胞的钙、磷、镁、钾的最佳浓度范围。低于以上4种大量营养元素的最适浓度范围,TUIP-8细胞的生长和黄酮合成都将明显受到抑制。该细胞对Ca^2 和Mg^2 具有较好的浓度耐受性,而对PO4^3-和K^ 的耐受性较差。     

Organic anion exudation by ectomycorrhizal fungi and Pinus sylvestris in response to nutrient deficiencies

Low molecular weight organic anions (LMWOA) can enhance weathering of mineral grains. We tested the hypothesis that ectomycorrhizal (EcM) fungi and tree seedlings increase their exudation of LMWOA when supply of magnesium, potassium and phosphorus is low to enhance the mobilization of Mg, K and P from mineral grains. Ectomycorrhizal fungi and Pinus sylvestris seedlings were cultured in symbiosis and in isolation on glass beads with nutrient solution or with sand as a rooting medium, with a complete nutrient supply or with Mg, K, P or N in low supply. Concentrations of all dicarboxylic LMWOA in the rooting medium were measured. Nonmycorrhizal seedlings released predominantly malonate. Colonization with Hebeloma longicaudum decreased the amount of organic anions exuded, whereas Paxillus involutus and Piloderma croceum increased the concentration of oxalate but not the total amount of LMWOA. Phosphorus deficiency increased the concentration of LMWOA by nonmycorrhizal and EcM seedlings. Magnesium deficiency increased the concentration of oxalate by nonmycorrhizal and EcM seedlings, but not the concentration of total LMWOA. Paxillus involutus grown in pure culture responded differently to low nutrient supply compared with symbiotic growth. Ectomycorrhizal fungi did not increase the total concentration of LMWOA compared with nonmycorrhizal seedlings but, depending on the fungal species, they affected the type of LMWOA found.     

Effect of hydrogen peroxide on morphological characteristics and resistance of wheat calluses to the stinking smut fungus

We studied the effect of hydrogen peroxide on morphological characteristics and resistance of common wheat calluses (Triticum aestivum L.) to Tilletia caries Till. The induction of the defense response and morphogenesis in calluses depended on H2O2 concentration. A correlation was revealed between the elevated concentration of hydrogen peroxide in wheat calluses and high activity of oxalate oxidase in the cell wall. Administration of H2O2 into the callus culture medium was followed by rhizogenesis, induced the formation of dense regions, and inhibited fungal growth on calluses. Hydrogen peroxide at high concentrations was less potent in inhibiting the growth of fungi. A relationship was found between oxalate oxidase activity, H2O2 concentration, and morphogenetic and defense responses of calluses induced by exogenous hydrogen peroxide. These data suggest that the induction of H2O2 generation is one of the approaches to increase callus resistance.     

Oxalate production by Basidiomycetes,including the white-rot species Coriolus versicolor and Phanerochaete chrysosporium

Oxalate was found to accumulate in liquid culture media from the growth of the white-rot basidiomycetes Coriolus versicolor, Heterobasidion annosum, Pleurotus florida and Phanerochaete chrysosporium. Whereas little oxalate accumulated during active growth, millimolar concentrations of oxalate were detected in culture media during the stationary phase. The basidiomycete Agaricus bisporus, the cultivated mushroom, also accumulated oxalate in its culture medium in the stationary phase. In comparison, the brown-rot fungi Amyloporia xantha, Coniophora marmorata, C. puteana and Poria vaporaria accumulated oxalate in the primary metabolic phase and throughout growth up to 35 days. Oxalate accumulation (0.04–10.0 mm) in white-rot cultures did not lower the pH of the medium during growth, whereas in brown-rot cultures oxalate (2.0–20.0 mm) reduced the media pH during growth. Cultures of Agaricus bisporus, C. puteana and Coriolus versicolor grown on solid media containing high levels of calcium (50 or 100 mm calcium chloride) produced calcium oxalate crystals to varying extents on the surface of the hyphae. Correspondence to: C. S. Evans     

Glutathione and the gated potassium channels of Escherichia coli

Glutathione-deficient mutants of Escherichia coli were found to require high potassium concentrations for growth, unless supplemented with glutathione. The unsupplemented mutants exhibited a rapid leak of potassium when transferred to a K+-free medium and a fast K+ turnover at the steady state of K+ accumulation, contrasting with the slow rate of the same processes in the wild-type. The steady-state level of K+ accumulation in low potassium medium increased immediately upon addition of glutathione, even in the absence of protein synthesis. K+-independent revertants were found to possess restored glutathione synthesis. Many properties of the glutathione-deficient mutants were identical with those of the potassium leaky K-B- and K-C- mutants, which, however, have a normal glutathione content. Both types of mutants differ from the wild-type in their response to thiol reagents in that no rapid loss of K+ is observed: they have, however, clear-cut differences under these circumstances. These results suggest that the products of trkB and trkC genes are essential for the formation of the potassium channel and glutathione plays an important role in the gating process.     

Influence of Ca2+ ions on metabolism of active oxygen species in combined cultures of wheat calluses with the fungus Tilletia caries

The effect of Ca2+ on morphophysiological parameters of calluses of wheat Triticum aestovum L., the level of active oxygen species, and the activity of oxalate oxidase, peroxidase, and catalase is investigated in the case of infestation with the fungus Triticum aestivum causing ball smut. The concentration of O2-, H2O2, and activity of oxidoreductases (oxalate oxidase, peroxidase, and catalase) depends on the content of Ca2+ ions in the culture medium of calluses. The increase in the concentration of Ca2+ ions in the culture medium led to higher structuring of calluses, induction of activity of oxalate oxidase and of some forms of peroxidase, and to accumulation of active oxygen species. These changes contributed to inhibition of development of the fungus. Discovery of such dependence agrees with the role of calcium as the intermediary in biochemical reactions related to the formation of the protective response of plant cells in case of infestation.