Phenol degradation and colour removal in submerged culture of Pleurotus sajor-caju with paper mill effluents

The fungus Pleurotus sajor-caju secretes phenol-oxidases that enable the use of recalcitrant compounds as substrates. The residues of paper manufacture contain high lignin levels, which gives the effluents a characteristic brownish colour. To test the potential of P. sajor-caju cultures on reducing these parameters, we used 90% of raw effluents from medium consistency oxygen delignification and bleaching stages plus 10% of mineral solution and different levels of glucose (5–15 g L^?1) as substrate. We observed a greater fungal biomass in cultures using effluent than in controls. Cultures containing 10 to 15 g L^?1 of glucose resulted in about 42% colour reduction. The polyphenol content was also reduced by 58.9% by the 13th day of culture. In addition, we observed the secretion of laccases (211.44 U mL^?1 and 45.98 U mL^?1 using ABTS and syringaldazine, respectively) and peroxidases (6.11 U mL^?1-ABTS) both peaking at the 7th day of culture and with similar kinetics of production in different glucose concentrations.     

Influence of culture medium composition, dissolved oxygen concentration and harvesting time on the production of Serratia entomophila, a microbial control agent of the New Zealand grass grub

The bacterium Serratia entomophila (Enterobacteriaceae) has been developed as a commercially available biopesticide for control of the pasture pest Costelytra zealandica. The influence of culture medium composition, dissolved oxygen (DO) concentration and harvesting time were investigated in order to optimise the production of S. entomophila. In batch fermentations, highest yields were achieved using sucrose (40 g L^-1) as the carbon source, followed closely by fructose and molasses. The effect of yeast extract (YE), marmite and bakery yeast as cell growth enhancers was also examined in both batch and fed-batch mode. Culture medium containing 20 g L^-1 of YE (fed-batch) produced the highest cell density. No significant effect on cell yield was detected when cultures were supplemented with bakery yeast or marmite. The DO concentration influenced biomass production: a 5-fold increase in cell density was achieved when the concentration of DO was maintained in the range of 20-50% (5.7×10¹⁰ CFUs mL^-1) in comparison with 1% (1.2×10¹⁰ CFUs mL^-1). In cultures maintained at 1 and 20% DO concentration, cells harvested from the exponential growth phase survived for less than 2 weeks when stored at 4°C. In contrast, high cell survival (85-100%) was achieved when cells were harvested after they had entered the stationary growth phase. Recommendations are provided for the production of robust, high cell density cultures of S. entomophila.     

不同无机碳源对粉核油球藻生长的影响

研究了2种无机碳源对粉核油球藻(Pinguiococcus pyrenoidosus CCMP2078)生长的影响。结果表明,适量添加NaHCO₃和通CO₂对P.pyrenoidosus CCMP2078的生长均有促进作用,NaHCO₃的添加方式对该藻的生长具有显著差异。一次性添加和分批添加NaHCO₃的优化浓度分别为5mmoL·L^-1和20mmoL·L^-1,后者对数生长末期的细胞密度、干重和比生长速率均最高,分别为8.93×10⁶cells·mL^-1,0.247g·L^-1和0.225dL^-1。通入CO₂的体积分数为0.5%时,最有利于藻细胞的生长,对数生长末期的细胞密度、干重和比生长率分别为3.83×10⁷cells·mL^-1、0.42g·L^-1和0.212d^-1。     

怀山药类原球茎的诱导形成与植株再生

为提高山药离体繁殖的速度, 缩短繁殖周期, 以铁棍山药(Dioscorea opposita cv. ‘Tiegun’)带腋芽茎段为材料, 对类原球茎的诱导、增殖、分化与植株再生进行了研究。结果表明, 铁棍山药类原球茎诱导的最适培养基为MS+1.0 mg·L^-1 TDZ+30 g·L^-1蔗糖, 增殖的最适培养基为MS+9 mg·L^-1 6-BA+30 g·L^-1蔗糖, 分化的最适培养基为MS+2 mg·L^-1 KT+0.02 mg·L^-1 NAA+30 g·L^-1蔗糖, 最适生根培养基为1/4MS+0.05 mg·L^-1 NAA+1.0 mg·L^-1 PP333+15 g·L^-1蔗糖, 生根率达80%, 移栽成活率可达85%。类原球茎的诱导形成及植株再生体系的建立为怀山药种苗的快速繁殖提供了一条新途径。     

Bioherbicidal effect of fumonisin B1, a phytotoxic metabolite naturally produced by Fusarium nygamai, on parasitic weeds of the genus Striga

Fusarium nygamai has been isolated and identified from diseased Striga hermonthica plants collected from sorghum fields in the Sudan. Fumonisin B₁, produced by this fungus, was isolated and purified. In in vitro experiments, the bioherbicidal potential of this mycotoxin was evaluated against S. hermonthica and S. asiatica. Different concentrations (250, 500 and 1000 μg L^-1 of water) of fumonisin B₁, applied during the conditioning phase of the Striga seeds significantly reduced the germination of both species in a range of 19.8-32.2 and 34.5-47.6%, respectively. In addition, the length of the germ tubes was reduced and the germ tubes became brownish. Fumonisin B₁ slightly delayed the emergence of Striga when 250 mg fumonisin B₁ dissolved in 1 L of water were applied into the soil per pot before sowing sorghum. By the way of contrast, fumonisin B₁ was very phytotoxic when Striga seedlings (leaves and stems) of different heights and ages were sprayed with a concentration series of 250, 500 and 1000 μg fumonisin B₁ mL^-1 until run-off, or when plants were injured with a scalpel before fumonisin B₁ was applied into the injury at concentrations of 250 and 500 μg mL^-1. One day after application, the plants showed symptoms of wilting, first at the top and then moving downwards. Leaves turned black and stems desiccated at the point of injuries. Within 4 days after the application, 55% of the plants tested were completely destroyed. In total, at the end of the experiment, 40 days after the treatment, 85% of the treated plants were killed by fumonisin B_1, regardless of the concentration applied and the application technique used.     

白檀离体快繁技术

以白檀(Symplocos paniculata)幼嫩茎段为实验材料, 通过对启动培养、增殖、生根培养及移栽的影响因子进行研究, 初步建立了白檀的组织培养体系。结果表明: 白檀外植体最适灭菌方案为0.1%升汞3分钟, 无菌苗获得率达81%; 最适初代启动培养基为1/2MS+30 g∙L^-1蔗糖+8 g∙L^-1琼脂, 出芽率达86.83%; 增殖最适培养基为1/2MS+1.0 mg∙L^-1 6-BA+0.02 mg∙L^-1 IBA+30 g∙L^-1蔗糖+8 g∙L^-1琼脂, 增殖系数达3.57; 最适生根培养基为WPM+0.5 mg∙L^-1 IBA+0.5 mg∙L^-1 NAA+20 g∙L^-1蔗糖+2 g∙L^-1 AC+8 g∙L^-1琼脂, 生根率达93%; 炼苗后, 移入园土:草炭土=1:1 (v/v)的基质中, 成活率达83%。     

东北森林两种典型阔叶树种子和幼苗对15N同位素的富集响应

以东北森林两种典型的阔叶树种风力传播种子——花曲柳和色木槭种子为研究对象,通过室内¹⁵N尿素浸泡试验和温室盆栽试验,设置4个浓度(0、0.05、0.1和0.2 g·L^-1)、3个浸泡时间(4、8和12 d)与4个叶期(2、4、6和8叶)处理,研究种子浸泡浓度、浸泡时间和幼苗叶期对种子和幼苗¹⁵N富集的影响.结果表明: 浸泡浓度和浸泡时间对两树种种子δ¹⁵N值均有显著的正反馈作用,高浓度和长时间(0.2 g·L^-1+12 d)更有利于种子¹⁵N总量的富集,花曲柳和色木槭种子¹⁵N同位素最大富集倍数的浸泡浓度和浸泡时间组合分别为0.1 g·L^-1+(4、8 d)和0.05 g·L^-1+(4、8 d);δ¹⁵N值稀释率随幼苗株高的增加先急剧减少(2~6叶)后趋于稳定,幼苗从8叶开始叶片¹⁵N总量降低,表明6叶幼苗更适合追踪幼苗的来源;幼苗叶片δ¹⁵N值与种子浸泡浓度、浸泡时间和种子的δ¹⁵N值呈显著正相关.花曲柳和色木槭种子及幼苗均能成功富集到¹⁵N信号,采用0.1 g·L^-1+8 d+6叶组合最适合追踪花曲柳和色木槭种子和幼苗.     

东北森林两种典型阔叶树种子和幼苗对15N同位素的富集响应

以东北森林两种典型的阔叶树种风力传播种子——花曲柳和色木槭种子为研究对象,通过室内¹⁵N尿素浸泡试验和温室盆栽试验,设置4个浓度(0、0.05、0.1和0.2 g·L^-1)、3个浸泡时间(4、8和12 d)与4个叶期(2、4、6和8叶)处理,研究种子浸泡浓度、浸泡时间和幼苗叶期对种子和幼苗¹⁵N富集的影响.结果表明: 浸泡浓度和浸泡时间对两树种种子δ¹⁵N值均有显著的正反馈作用,高浓度和长时间(0.2 g·L^-1+12 d)更有利于种子¹⁵N总量的富集,花曲柳和色木槭种子¹⁵N同位素最大富集倍数的浸泡浓度和浸泡时间组合分别为0.1 g·L^-1+(4、8 d)和0.05 g·L^-1+(4、8 d);δ¹⁵N值稀释率随幼苗株高的增加先急剧减少(2~6叶)后趋于稳定,幼苗从8叶开始叶片¹⁵N总量降低,表明6叶幼苗更适合追踪幼苗的来源;幼苗叶片δ¹⁵N值与种子浸泡浓度、浸泡时间和种子的δ¹⁵N值呈显著正相关.花曲柳和色木槭种子及幼苗均能成功富集到¹⁵N信号,采用0.1 g·L^-1+8 d+6叶组合最适合追踪花曲柳和色木槭种子和幼苗.     

Pectinase and cellulase enhance the control of Abutilon theophrasti by Colletotrichum coccodes

Inundative mycoherbicidal biocontrol agents are typically insufficiently virulent to be commercially competitive with herbicides in row crop agriculture, and require enhancement. Pectinase and cellulase are typically used by pathogens during infection. Thus, it was hypothesized that adding exogenous cell wall degrading enzymes might enhance fungal infection. Pectinase or cellulase was added to inocula of aqueous chopped mycelial suspensions of a strain of Colletotrichum coccodes for control of Abutilon theophrasti. Plants treated with 5.3×10⁶ C. coccodes propagules mL^-1 and 1.65 U mL^-1 pectinase had more rapid and complete disease development. Similar trend was achieved when 10 U mL^-1 of cellulase were added to 2.2×10⁶ C. coccodes propagules mL^-1. Adding pectinase or cellulase did not increase the host range of the wild-type fungus. The results suggest that there might be value to transforming biocontrol agents to overproduce these enzymes.     

Production of Chlamydospores and Conidia in Submerged Culture by Rhynchosporium alismatis, a Mycoherbicide of Alismataceae in Rice Crops

The objective of this work was to evaluate the production of chlamydospores and conidia of Rhynchosporium alismatis in a liquid Czapex-Dox based medium supplemented with increasing concentrations of sodium nitrate and malt extract. In addition, the germination of chlamydospores was evaluated. A high concentration of malt extract (4.4 g L^-1) as the sole carbon source and a high level of sodium nitrate as the sole nitrogen source (3.3 g L^-1) were shown to increase chlamydospore production while agitation (150 rpm) enhanced conidial yields. Maximum chlamydospore production (2.03×10⁵±0.7 total chlamydospores mg DW^-1) was achieved in cultures grown in a medium supplemented with 8.8 g L^-1 malt extract and 5.74 g L^-1 sodium nitrate. Two days growth was required for maximum chlamydospore and conidial production, while 6 days was necessary to obtain maximum dry weight accumulation (350 mg per flask). Germination of chlamydospores (90%) was significantly higher than germination of conidia (47%) after 2 days growth.     

毛报春(Primula × pubescens)腋芽再生组织培养体系的建立

以毛报春(Primula × pubescens)无菌腋芽为外植体, 分析不同浓度激素配比对愈伤组织诱导和分化以及不定芽增殖和生根的影响, 筛选出不同阶段的最适培养基, 优化毛报春的组织培养再生体系。结果表明, 毛报春腋芽愈伤组织诱导及分化的最适培养基为MS+0.2 mg∙L^-1 NAA+1.0 mg∙L^-1 6-BA, 诱导率达84%, 出芽率达67%; 不定芽增殖最适培养基为MS+0.5 mg∙L^-1 NAA+0.2 mg∙L^-1 6-BA, 增殖率可达67%, 苗绿且健壮; MS+0.2 mg∙L^-1 NAA培养基最有利于组培苗的生根及伸长, 平均单株生根数为9条, 生根率高达70%。该研究建立了毛报春的组织培养再生体系, 可为报春属其它植物的遗传研究及种质创新提供参考。     

Sorbitol and Gluconate Production in a Hollow Fibre Membrane Reactor by Immobilized Zymomonas Mobilis

This study describes the results of a hollow fibre membrane reactor with immobilized treated cells of Zymomonas mobilis which produced sorbitol and gluconic acid continuously from fructose and glucose respectively. A productivity of 10-20 g sorbitol · L^-1 · h^-1 and 10-20 gluconate · L^-1 · h^-1 (based on total bioreactor volume) from a feed of 100 g · L^-1 each of glucose and fructose was possible at high dilution rates. Kinetic parameters describing the reaction rate of treated cells in batch reactors were used to analyse the performance of the hollow fibre membrane reactor employing significant convective mass transfer. No significant mass transfer limitation was apparent.     

Media and Fermentation Processes for the Rapid Production of High Concentrations of Stable Blastospores of the Bioinsecticidal Fungus Paecilomyces fumosoroseus

In shake flask and fermentor studies, various media components and culture inocula were tested to improve P. fumosoroseus spore production rates, yield and stability. To evaluate inoculum potential and inoculum scale-up for fermentor studies, conidia and liquid culture-produced spores of various strains of P. fumosoroseus were compared as inoculum. Inoculation of liquid cultures with blastospores at concentrations of at least 1×10⁶ spores mL^-1 resulted in the rapid production of high concentrations of blastospores (∼1×10⁹ spores mL^-1, 48 h fermentation time) for all strains tested. The rapid germination rate of blastospores (90% after 6 h incubation) compared to conidia (>90% after 16 h incubation) and the use of higher inoculum rates reduced the fermentation time from 96 to 48 h for maximal spore yields. A comparison of various complex nitrogen sources showed that liquid media supplemented with acid hydrolyzed casein or yeast extract supported the production of high concentrations of blastospores that were significantly more desiccation-tolerant (79-82% survival after drying) when compared to blastospores produced in media supplemented with other nitrogen sources (12-50% survival after drying). For rapid spore production, requirements for trace metals and vitamin supplementation were dependent on the type of hydrolyzed casein used in the medium. Fermentor studies with two strains of P. fumosoroseus showed that high concentrations (1.3-1.8×10⁹ spores mL^-1) of desiccation-tolerant blastospores could be produced in 48-h fermentations. These studies have demonstrated that the infective spores of various strains of the fungal bioinsecticide Paecilomyces fumosoroseus can be rapidly produced using deep-tank, liquid culture fermentation techniques.     

黑曲霉解磷能力的影响因素及培养条件优化

本文探讨了培养条件下黑曲霉解磷能力的主要影响因素。通过单孢株筛选得到解磷能力较强的菌株Xj-2,其在液体培养基中的解磷能力达到539.90 mg·L^-1。解磷发酵动力学模型模拟发现,其解磷能力在培养的第4 天达到平稳,可作为终止发酵时间。不同磷源培养液中菌株Xj-2的解磷量依次为磷酸钙(539.90 mg·L^-1)>磷酸锌(238.45 mg·L^-1)>磷酸铁(182.64 mg·L^-1)>磷矿粉(71.80 mg·L^-1)>磷酸铝(24.40 mg·L^-1)。通过单因素试验并结合响应面优化,研究了其解磷最佳条件。结果表明: 碳源对Xj-2的解磷能力影响最大,其次是菌群密度和培养液pH。当培养温度35 ℃、转速160 r·min^-1、培养液pH 6.0、氮源(尿素)浓度0.79 g·L^-1、碳源(葡萄糖)浓度10.00 g·L^-1、菌群密度3.8%、培养时间为4 d时,Xj-2的解磷能力最高,为616.81 mg·L^-1。     

Effect of growth hormone and γ-aminobutyric acid on Brachionus plicatilis (Rotifera) reproduction at low food or high ammonia levels

Growth hormone (GH, 0.0025 and 0.025 I.U. ml⁻¹) and γ-aminobutyric acid (GABA, 50 μg ml⁻¹) enhance rotifer population growth in batch cultures. In order to further understand the mechanism of their actions, we conducted experiments culturing isolated females at low food and high free ammonia levels. At an optimum food level of 7×10⁶ Nannochloropsis oculata cells ml⁻¹ or at low free ammonia level of 2.4 μg ml⁻¹, the F₁ offspring of rotifers treated with GH at 0.0025 I.U. ml⁻¹ had significantly higher population growth rate (r) and net reproduction rate (Ro), and shorter generation time than untreated rotifers. At a lower food level of 7×10⁵ cells ml⁻¹ or at high free ammonia level of 3.1 μg ml⁻¹, rotifers treated with GABA at 50 μg ml⁻¹ had significantly higher r and Ro, and shorter generation time. These results indicate that GABA is effective in enhancing rotifer reproduction when rotifers are cultured under stress whereas GH enhances rotifer reproduction when culture conditions are optimal. Significant effects were also observed in F¹ and F² generations which were not treated with hormones. These data may be useful for treating rotifer mass cultures to mitigate the effects of stress caused by high population densities.     

Hg2+浓度和藻类食物密度对多刺裸腹溞生命表统计学参数的影响

为了比较不同食物密度下污染物浓度对受试生物的慢性毒性,以多刺裸腹溞作为受试生物,研究了斜生栅藻密度较低(0.5×106 cells·mL^-1)、中等(1.0×10⁶ cells·mL^-1)和较高(2.0×10⁶ cells·mL^-1)条件下,不同Hg²⁺浓度(0、0.4、1.1、1.8、2.5、3.2和3.9 μg·L^-1)对多刺裸腹溞生命表统计学参数的影响.结果表明: 在较低和较高的食物密度下,一定浓度的Hg²⁺对多刺裸腹溞的存活、生殖乃至种群增长具有不利的影响;但中等食物密度下其不利影响消失.与对照相比,较低食物密度下,0.4~2.5和3.9 μg·L^-1的Hg²⁺显著缩短了多刺裸腹溞的生命期望,0.4、1.1、2.5和3.9 μg·L^-1的Hg²⁺显著缩短了世代时间,1.1、2.5和3.9 μg·L^-1的Hg²⁺显著降低了净生殖率,3.9 μg·L^-1的Hg²⁺显著降低了总生殖率.在较高的食物密度下,1.1 μg·L^-1的Hg²⁺显著降低了多刺裸腹溞的种群内禀增长率;3.9 μg·L^-1的Hg²⁺显著降低了多刺裸腹溞的生命期望和净生殖率.但在中等食物密度下,3.2 μg·L^-1的Hg²⁺显著延长了多刺裸腹溞的生命期望.在较低的食物密度下,Hg²⁺浓度与多刺裸腹溞的生命期望、世代时间和总生殖率之间均具有显著的剂量-效应关系;但在中等和较高的食物密度下,Hg²⁺浓度与多刺裸腹溞的生命表统计学参数间均无显著的剂量-效应关系.     

Hg2+浓度和藻类食物密度对多刺裸腹溞生命表统计学参数的影响

为了比较不同食物密度下污染物浓度对受试生物的慢性毒性,以多刺裸腹溞作为受试生物,研究了斜生栅藻密度较低(0.5×106 cells·mL^-1)、中等(1.0×10⁶ cells·mL^-1)和较高(2.0×10⁶ cells·mL^-1)条件下,不同Hg²⁺浓度(0、0.4、1.1、1.8、2.5、3.2和3.9 μg·L^-1)对多刺裸腹溞生命表统计学参数的影响.结果表明: 在较低和较高的食物密度下,一定浓度的Hg²⁺对多刺裸腹溞的存活、生殖乃至种群增长具有不利的影响;但中等食物密度下其不利影响消失.与对照相比,较低食物密度下,0.4~2.5和3.9 μg·L^-1的Hg²⁺显著缩短了多刺裸腹溞的生命期望,0.4、1.1、2.5和3.9 μg·L^-1的Hg²⁺显著缩短了世代时间,1.1、2.5和3.9 μg·L^-1的Hg²⁺显著降低了净生殖率,3.9 μg·L^-1的Hg²⁺显著降低了总生殖率.在较高的食物密度下,1.1 μg·L^-1的Hg²⁺显著降低了多刺裸腹溞的种群内禀增长率;3.9 μg·L^-1的Hg²⁺显著降低了多刺裸腹溞的生命期望和净生殖率.但在中等食物密度下,3.2 μg·L^-1的Hg²⁺显著延长了多刺裸腹溞的生命期望.在较低的食物密度下,Hg²⁺浓度与多刺裸腹溞的生命期望、世代时间和总生殖率之间均具有显著的剂量-效应关系;但在中等和较高的食物密度下,Hg²⁺浓度与多刺裸腹溞的生命表统计学参数间均无显著的剂量-效应关系.     

碳源对迷迭香悬浮培养细胞的生长、迷迭香酸积累及抗氧化酶活性的影响

以迷迭香悬浮培养细胞为材料,详细研究了基本培养基中添加蔗糖、麦芽糖和葡萄糖对细胞生长及次生代谢产物积累的影响,同时对不同蔗糖浓度处理的悬浮培养细胞抗氧化酶活性进行了研究。研究结果表明：在不同的糖处理中,30 g·L^-1的蔗糖、70 g·L^-1的麦芽糖及40 g·L^-1的葡萄糖最有利于迷迭香悬浮培养细胞生长。30 g·L^-1蔗糖和70 g·L^-1麦芽糖处理中悬浮培养细胞的生长率分别为74.08%和72.33%,高出40 g·L^-1葡萄糖处理接近3倍之多。30 g·L^-1蔗糖处理的悬浮培养细胞迷迭香酸含量高出70 g·L^-1麦芽糖处理228倍,略低于40 g·L^-1葡萄糖处理。在不同蔗糖的处理中,随着蔗糖浓度的增加,迷迭香酸含量均呈现增加趋势,表明高浓度的蔗糖有利于悬浮培养细胞迷迭香酸的积累。在高浓度的蔗糖处理中,悬浮培养细胞H₂O₂和MDA含量明显增加,同时抗氧化酶SOD、POD及CAT的活性也明显增强,表明高浓度的蔗糖产生了渗透胁迫,这种渗透胁迫虽不利于迷迭香悬浮培养细胞的生长,但有利于次生代谢产物的积累。综合迷迭香悬浮细胞的生长率和迷迭香酸的含量,我们最终得出30 g·L^-1的蔗糖最有利于迷迭香悬浮细胞的培养。     

粉美人萱草的快繁技术和大田种植

以粉美人萱草(Hemerocallis fulva cv. ‘Fenmeiren’)的花茎为外植体进行离体培养, 该研究成功建立了粉美人萱草组培快繁技术。结果表明, 6月获得的外植体用浓度为15% (v/v)的次氯酸钠溶液消毒8分钟, 外植体存活率达95%; 最佳增殖培养基为MS+1.0 mg·L^-1 6-BA+0.004 mg·L^-1 TDZ+0.1 mg·L^-1 NAA, 培养30天后, 月增殖系数达2.9; 壮苗培养基为MS+0.1 mg·L^-1 6-BA+0.1 mg·L^-1 IBA, 在该培养基中, 组培苗不再分化, 长势健壮; 最佳生根培养基为1/2MS+0.4 mg·L^-1 IBA+20 g·L^-1蔗糖, 生根率达95%; 移栽基质采用珍珠岩:草炭=1:2 (v/v), 通过精细化管理, 成活率可达85%, 出圃合格率为75%。目前已实现规模化繁殖, 并生产组培苗2.0×10⁵株, 大田种植表现良好。     

铁皮石斛愈伤组织诱导研究

研究不同培养基和光照条件对铁皮石斛愈伤组织诱导的影响。结果表明,外植体直接接种于培养基上,最适宜培养条件是MS 5.92 g·L^-1＋2,4-D 5 mg·L^-1＋IAA 1.5 mg·L^-1＋KT 0.62 mg·L^-1＋蔗糖37.5 g·L^-1＋琼脂0.8% (pH 5.9～6.0),暗培养15 d后再光培养;外植体捣碎后平铺于培养基上,最适宜培养条件是MS 4.74 g·L^-1＋2,4-D 1 mg·L^-1＋IAA 1.5 mg·L^-1＋KT 0.25 mg·L^-1＋蔗糖30 g·L^-1＋琼脂0.8% (pH 5.9～6.0),25 ℃持续光培养。