球囊霉属几种AM真菌孢子表面消毒与萌发的研究

通过对球囊霉属几种AM真菌进行孢子表面消毒与萌发研究,结果表明,采用“差别灭菌法”即先用含2％氯胺 T、0.02％链霉素和0.01％庆大霉素的溶液孢子表面消毒10min,然后在25-27℃下培养3天,再用含2％氯胺 T、0.02％链霉素和0.01％庆大霉素的溶液孢子表面消毒 10min,其消毒效果最好,孢子萌发率较高,污染率较低。同时发现土壤、草炭和寄主植物根的分泌物对孢子萌发有促进作用。培养基 pH 值的变化对孢子萌发也有一定的影响。AM 真菌孢子用“改良 Sandwich 法”进行萌发,其萌发率最高。     

球囊霉属几种AM真菌孢子表面消毒与萌发的研究

通过对球囊霉属几种AM真菌进行孢子表面消毒与萌发研究,结果表明,采用"差别灭菌法"即先用含2%氯胺T、0.02%链霉素和0.01%庆大霉素的溶液孢子表面消毒10min,然后在25～27℃下培养3天,再用含2%氯胺T、0.02%链霉素和0.01%庆大霉素的溶液孢子表面消毒10min,其消毒效果最好,孢子萌发率较高,污染率较低.同时发现土壤、草炭和寄主植物根的分泌物对孢子萌发有促进作用.培养基pH值的变化对孢子萌发也有一定的影响.AM真菌孢子用"改良Sandwich法"进行萌发,其萌发率最高.     

磷和镉对根内球囊霉Glomus intraradices孢子萌发、菌丝生长和外生菌丝内聚磷酸累积的影响

丛枝菌根真菌(AMF)能够显著提高植物对重金属的抗性,菌丝内聚磷酸(Polyphosphate,PolyP)可能参与了这种抗性的形成。试验以Glomus intraradices孢子为试材,对灭菌条件进行了优化,并进一步研究了不同P和Cd2+水平对孢子萌发、菌丝生长、分支和外生菌丝中聚磷酸含量的影响。结果表明,孢子在1%氯胺T+0.02%链霉素+0.01%庆大霉素+1/100(V/V)吐温-20中灭菌5min的效果最好。孢子萌发率、菌丝分支和菌丝长度随着Cd2+浓度的增加不断降低;当Cd2+浓度达到0.1mmol/L时,孢子萌发率降低为0%,表明Glomus intraradices的孢子萌发对Cd2+的耐受极限为0.1mmol/L;1mmol/L的P促进菌丝分支增加,却降低了萌发率,但对菌丝生长没有影响;在培养23d以后,三者基本不再变化。外生菌丝内的聚磷酸含量随着P的升高而增加;在Cd2+胁迫作用下,聚磷酸的含量降低而菌丝密度随着聚磷酸的升高而升高,表明聚磷酸在减弱重金属毒性方面起了重要作用。     

温水浸种和IAA溶液浸种对当归种子萌发特性的影响

为了探索当归种子的休眠机制和提高其萌发率,本文以药用植物当归的种子为研究对象,分析了不同温度的水浸种不同时间和不同浓度3-吲哚乙酸(IAA)溶液浸种不同时间后对当归种子萌发率的影响。结果表明:60℃水浸种2 min萌发率最高,为78%,是对照的2.5倍;0.05 g·L~(-1)IAA溶液浸种3 h萌发率最高,为80%,是对照的1.7倍;用30、35、40、45、50、55、60、65、70℃9个温度的水浸种1、2 min的萌发率与水温的相关性不显著,浸种5、10 min相关性显著,浸种15 min相关性极显著;用0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1 g·L~(-1)10个浓度的IAA溶液浸种1、3、6、12 h的萌发率与溶液浓度的相关性均不显著;水浸种提高种子萌发率可能与改变种子种皮透性有关,IAA溶液浸种提高种子萌发率可能与打破休眠种子的休眠有关;60℃水浸种2 min和0.05 g·L~(-1)IAA溶液浸种3 h均可显著提高当归种子的萌发率;IAA溶液为有机溶剂,污染环境,在实际生产中建议多使用水浸种法为宜。     

粗茎鳞毛蕨孢子萌发研究

以经过3年低温储藏的粗茎鳞毛蕨孢子为实验材料,从孢子离心、孢子消毒、培养基种类、光质等4方面对孢子萌发进行研究,结果表明:在离心转数≤14 000 r.min-1、离心时间≤30 min条件下,离心处理对孢子萌发基本无影响;对孢子进行1%NaClO水溶液浸泡处理20～30 min为最佳消毒条件;改良Knop’s培养基为最佳孢子萌发培养基;黑暗条件下孢子不能萌发,但是黑暗处理能够明显提高孢子萌发整齐性;红光比白光能促进孢子提早萌发1 d左右,但对提高萌发率效果不显著。     

乌蕨孢子萌发研究

用随机区组试验和方差分析方法,研究了培养温度、贮藏温度、GA处理和光照强度对乌蕨(Sphe-nomeris chinensis)孢子萌发的影响。与室温(20～25℃)相比,培养温度在28℃时,孢子最大萌发率相近而萌发速率明显较高。贮藏温度(A)极显著(P<0.01)影响孢子萌发率,-20℃贮藏降低萌发率;GA(B)对孢子萌发率无显著影响。光照强度(C)极显著(P<0.01)影响孢子萌发率,充足光照和弱光照无显著差异,黑暗处理降低萌发率。A×B,A×C,B×C及A×B×C交互效应不显著。     

8种甜荞的种子消毒及愈伤组织诱导和增殖条件

8 种甜荞种子表面消毒所适用的消毒剂和处理时间有所不同。10%H₂O₂ 溶液处理150min, 对榆3-3、黎麻道、浦江荞、龙山荞和富源红花荞的种子有很好的消毒作用, 并且处理以后种子仍具有很高的萌发率。8621、B 大粒和美国荞种子的消毒以0.1%的HgCl2 处理12min 效果为好。在无菌苗子叶和下胚轴外植体脱分化形成愈伤组织的过程中, 2, 4-D 起主要作用, BA 起独特的作用。BA 与2, 4-D 配合使用对于愈伤组织的诱导和增殖具有良好的促进作用。高浓度的2, 4-D 或低浓度的2, 4-D 与BA 配合使用都可抑制愈伤组织分化根。     

留兰香组织培养及快速繁殖条件的优化

以留兰香(Menthaspicata L.)茎尖为实验材料,对外植体消毒、不定芽增殖和试管苗移栽生根的最佳条件进行研究。结果表明,最佳外植体消毒方法为:用体积分数75%乙醇浸泡30s,再用1.0g·L-1HgCl2浸泡10min,培养7d后外植体生长状况良好。正交实验结果表明,在附加0.2mg·L-16-BA和0.02mg·L-1NAA的MS培养基中,留兰香不定芽的增殖倍数最高,试管苗生长状况最好。在含25mg·L-16-BA和50mg·L-1NAA的混合溶液中浸泡1h,移栽试管苗的生根率可达100%,且根较长。     

感染羽茅的香柱菌属内生真菌对丛枝菌根真菌孢子萌发的影响

内生真菌与丛枝菌根(AM)真菌是构成草原生态系统的重要组成部分.内生真菌会抑制其宿主植物的AM真菌侵染率.本研究以感染2种香柱菌属内生真菌[Epichloё gansuensis(Eg)和E. sibirica(Es)]的天然禾草羽茅为供试材料,进行体外纯培养的内生真菌培养滤液、感染内生真菌的羽茅叶片(包括鲜叶和枯叶)浸提液,以及根系分泌物对摩西球囊霉(Gm)和幼套球囊霉(Ge)2种AM真菌孢子萌发影响试验.结果表明: 香柱菌属内生真菌的培养滤液会显著抑制2种AM真菌孢子的萌发,而感染香柱菌属内生真菌的羽茅根系分泌物只对Ge孢子萌发有显著抑制作用,且上述抑制作用与内生真菌种类无关;鲜叶浸提液对Gm和Ge的孢子萌发率均无显著影响,而枯叶浸提液对Ge的孢子萌发有显著抑制作用.在自然生态系统中,香柱菌属内生真菌通常存在于宿主植物体内,可能通过影响宿主植物的根系分泌物来影响AM真菌孢子的萌发.     

烟气对泥炭藓孢子萌发影响的模拟研究

适量烟气能促进种子萌发,但对苔藓植物孢子的作用尚不清楚.选取采自长白山区泥炭地的粗叶泥炭藓和中位泥炭藓的孢蒴为试验材料,通过燃烧泥炭地植物产生烟气,制备烟溶液,分别与不同大小(大:直径为2.10～2.50 mm;小:直径为1.50～1.90 mm)以及不同保存时长(旧:4.3和6.3年;新:0.3年)的孢蒴进行两组双因素试验,经不同时长的烟溶液浸泡和萌发试验,模拟研究烟气、孢蒴大小和保存时长对苔藓植物孢子萌发的影响.结果表明: 烟溶液浸泡影响孢子萌发,培养10 d时,不同时长的烟溶液浸泡均可使孢子的萌发率提高5倍以上,小孢蒴孢子的萌发率高;培养21 d时,仅适度浸泡(3 d)表现出促进萌发的作用,孢蒴大小对孢子萌发率无影响;烟溶液浸泡对长时间保存(4.3和6.3年)的孢蒴孢子萌发无促进作用.研究表明,适量烟气可加速新泥炭藓孢子以及小孢蒴孢子的萌发.在存在不定期火烧干扰的泥炭地中,与对种子植物的作用类似,烟气可能在苔藓植物种群的有性更新和种群维持中发挥重要作用.     

缩球囊霉孢子萌发及细胞核在孢子和芽管中分布的研究*

本文研究了从本校分离的丛枝菌根真菌缩球囊霉Glomus constrictum的孢子萌发和萌发孢子的细胞核DAPI(46-diamidino-2-phenylindol)染色。结果显示,G. constrictum孢子直径为179.5-198.7μm ,顶生于产孢菌丝上。经表面消毒处理后,孢子在水琼脂平板上7天开始萌发。DAPI染色后,经稀释荧光计数,单个孢子细胞核数目约为5300,在孢子中无序分布, 细胞核直径约为9.9-11.2μm 。孢子萌发过程中,细胞核总数无明显变化,只是部分细胞核从孢子流向了萌发伸长的菌丝。     

Surface-sterilization of Glomus mosseae sporocarps for studying endomycorrhization in vitro

 The effects of sterilization time, sterilizing agents (ethanol, Chloramine T, calcium hypochlorite) and antibiotics (streptomycin and gentamycin) on Glomus mosseae (BEG 12) sporocarp germination and contamination were evaluated. Incubation for 10 s in 96 % ethanol, followed by 10 min in a solution of 2% Chloramine T, 0.02% streptomycin, 0.01% gentamycin and Tween 20, and then 6 min in 6% calcium hypochlorite greatly reduced fungal and bacterial contamination from sporocarps and caused little change in germination rate in water agar medium. Accepted: 4 March 1999     

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid

High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrient-induced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis. When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study (D_120°C, 1.9 ± 0.2 and 1.3 ± 0.1 min; z value, 12.2 ± 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients.     

Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis

Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of β-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.     

Inhibition of germinant binding by bacterial spores in acidic environments

Commitment to germinate occurred in both Clostridium botulinum and Bacillus cereus spores during 0.5 min of exposure to 100 mM L-alanine or L-cysteine, measured by the inability of germination inhibitors (D form of amino acid) to inhibit germination. Spore germination at pH 4.5 was inhibited because the germinant did not bind to the trigger sites. C. botulinum spores exposed to 100 mM L-alanine or L-cysteine at pH 4.5 remained sensitive to D-amino acid inhibition at pH 7, indicating that no germinants had bound to the trigger site at pH 4.5. Inhibition of germinant binding at pH 4.5 was reversible but lagged in commitment to germinate upon transfer to pH 7. Spores sequentially exposed to pH 4.5 buffer and pH 7 buffer with the germinant also demonstrated a lag in commitment to germinate. The pH at which binding was inhibited was not significantly affected by composition of the buffer or by reduced germinant concentrations (10 mM). Nonspecific uptake of L-[3H]alanine by C. botulinum spores was not inhibited at pH 4.5. Inhibition of germinant binding in acidic environments appeared to be due to protonation of a functional group in or near the trigger site. This may represent a general mechanism for inhibition of spore germination in acidic environments.     

Inhibition of germinant binding by bacterial spores in acidic environments.

Commitment to germinate occurred in both Clostridium botulinum and Bacillus cereus spores during 0.5 min of exposure to 100 mM L-alanine or L-cysteine, measured by the inability of germination inhibitors (D form of amino acid) to inhibit germination. Spore germination at pH 4.5 was inhibited because the germinant did not bind to the trigger sites. C. botulinum spores exposed to 100 mM L-alanine or L-cysteine at pH 4.5 remained sensitive to D-amino acid inhibition at pH 7, indicating that no germinants had bound to the trigger site at pH 4.5. Inhibition of germinant binding at pH 4.5 was reversible but lagged in commitment to germinate upon transfer to pH 7. Spores sequentially exposed to pH 4.5 buffer and pH 7 buffer with the germinant also demonstrated a lag in commitment to germinate. The pH at which binding was inhibited was not significantly affected by composition of the buffer or by reduced germinant concentrations (10 mM). Nonspecific uptake of L-[3H]alanine by C. botulinum spores was not inhibited at pH 4.5. Inhibition of germinant binding in acidic environments appeared to be due to protonation of a functional group in or near the trigger site. This may represent a general mechanism for inhibition of spore germination in acidic environments.     

GerN, an antiporter homologue important in germination of Bacillus cereus endospores

A homologue of the grmA spore germination gene of Bacillus megaterium and of a NaH-antiporter gene (napA) of Enterococcus hirae has been identified in Bacillus cereus 569 (ATCC 10876). The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively. Insertional inactivation of this B. cereus gene, named gerN, did not affect vegetative growth or sporulation. The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to L-alanine (10 mM). The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed. At a suboptimal germinant concentration (50 microM), inosine germination was completely blocked in the mutant, while the rate of germination in 50 microM L-alanine was reduced to one-third of that of the wild type. The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN. Since germination in suboptimal concentrations of L-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations.     

Effects of moderately high pressure plus heat on the germination and inactivation of Bacillus cereus spores lacking proteins involved in germination

Aims:  To determine the germination and inactivation of Bacillus cereus spores lacking various germination proteins using moderately high pressure (MHP) and heat.
Methods:  The inactivation and germination of wild-type B. cereus spores in buffer by MHP (150 MPa) at various temperatures, as well as the MHP inactivation and germination of B. cereus spores lacking individual germinant receptors and monovalent cation antiporters, was determined.
Results:  Loss of individual germinant receptors had no large effects on spore inactivation or germination, although germination of receptor-deficient spores was generally slightly decreased. Loss of the GerN in particular the GerN and GerT antiporters also decreased spore germination by MHP, especially at 40 and 50°C.
Conclusions:  Both inactivation and germination of B. cereus spores by MHP increased with rise of temperature; however, mutant strains lacking individual germinant receptor had similar levels of germination as compared to wild-type spores. To evaluate the role of germinant receptors in MHP, a strain lacking a large number of germinant receptors is needed.
Significance and Impact of the Study:  The results of this work may lead to a better understanding of how MHP causes germination of spores of B. cereus .