黄柄钙皮菌无菌培养条件的优化筛选

黄柄钙皮菌是黏菌中的一种重要模式生物,本文筛选了其无菌培养条件,比较了生活史不同阶段的发生时间,同时比较了生活史中孢子萌发、原质团生长及子实体形成3个阶段的培养条件。结果表明,黄柄钙皮菌适宜的培养条件为:在20%水琼脂上于28℃进行孢子萌发;在24–26℃下20%琼脂+40%燕麦培养基上进行原生质团的培养;在24–26℃下1 000Lx光照10–14h时形成子实体。     

光照、湿度和培养基对苎麻疫霉卵孢子产生量的影响

采用3因素随机区组设计研究了光照、湿度和培养基对苎麻疫霉(hytophthoraboehmeriae)卵孢子产生量的影响。结果表明,各因子对卵孢子产生量的影响效应大小次序为培养基>光照>湿度,其中培养基和光照两因子影响均在0.01水平上显着。在供试的4种常用培养基上,卵孢子产生量的大小次序为：SLA培养基(SLA)>利马豆培养基(LBA)>V₆汁培养基(V₆A)>澄清的V₆汁培养基(V₆B).在设置的3种光照条件中,卵孢子产生量以连续黑暗处理最高,连续光照最低,光照与黑暗交替处理居中。3个试验因子间的所有互作对卵孢子产生量均有极显着的影响。在不同光照、湿度、培养基组合中,卵孢子产生量以低湿+连续黑暗+SLA组合最高,低湿+连续光照+V₆B组合最低。     

光照,湿度和培养基对苎麻疫霉卵孢子产生量的影响

采用３因素随机区组设计研究了光照、湿度和培养基对苎麻疫霉（Ｐｈｙｔｏｐｈｔｈｏｒａｂｏｅｈｍｅｒｉａｅ）卵孢子产生量的影响．结果表明,各因子对卵孢子产生量的影响效应大小次序为培养基＞光照＞湿度,其中培养基和光照两因子影响均在０．０１水平上显著．在供试的４种常用培养基上,卵孢子产生量的大小次序为：ＳＬＡ培养基（ＳＬＡ）＞利马豆培养基（ＬＢＡ）＞Ｖ６汁培养基（Ｖ６Ａ）＞澄清的Ｖ６汁培养基（Ｖ６Ｂ）．在设置的３种光照条件中,卵孢子产生量以连续黑暗处理最高,连续光照最低,光照与黑暗交替处理居中．３个试验因子间的所有互作对卵孢子产生量均有极显著的影响．在不同光照、湿度、培养基组合中,卵孢子产生量以低湿＋连续黑暗＋ＳＬＡ组合最高,低湿＋连续光照＋Ｖ６Ｂ组合最低．     

金灰藓(Pylaisiella polyantha)配子体发生的实验观察

金灰藓Pylaisiella polyantha(Hedw.)Grout孢子在光照培养箱中进行培养,实验组为含有Knop培养液的琼脂培养基质,对照组为无营养成分的琼脂培养基质。在光学显微镜下对配子体发生进行了观察、描绘和照相。结果表明:实验组和对照组孢子萌发所需时间相同,均为53.5h,萌发极相为1～4极。4d时,实验组萌发率为85.4%,对照组为54.1%;20d时,开始形成配子体芽原基;40d时,形成具假根、茎、叶的配子体,并形成两性器官。并对配子体发生过程的特点进行了分析和讨论。     

养分浓度和光照强度对泥炭藓有性更新的影响

作为优势植物,泥炭藓(Sphagnum)在泥炭沼泽中缺乏有性更新的原因尚不清楚。针对影响孢子萌发的光强和养分条件,以泥炭藓(S.palustre)为材料,通过室内孢子萌发实验,研究不同光强和养分浓度对孢子萌发率、萌发势及萌发指数的影响。4种培养基中,养分浓度高的营养液培养基中孢子萌发率最高,达到60%,其次为养分浓度与营养液相近的琼脂+营养液培养基,萌发率为48%,再次为养分水平很低的沼泽水培养基,萌发率约为30%,几乎无养分的蒸馏水培养基中萌发率最低,约为5%。萌发势和萌发指数亦呈现相同的规律。琼脂+营养液和营养液培养基较沼泽水和蒸馏水培养基孢子萌发时间提前约3天时间。增加光强使孢子萌发率仅提高10%。研究表明,低养分浓度和弱光照均不利于孢子萌发,相对而言,泥炭沼泽的贫营养特征应是限制泥炭藓有性更新的更重要因素。     

影响活体内马铃薯晚疫病菌卵孢子产生因素的研究*

研究了不同菌株组合,马铃薯植株茎、叶及接种物中A1和A2菌株孢子囊比例、温度、湿度对卵孢子产生的影响。不同菌株组合产生卵孢子的数量有显著差异;在离体接种情况下,叶片中产生卵孢子数量大于茎中产生卵孢子数量;A1和A2菌株中孢子囊不同比例对卵孢子产生影响很大,当比值为1∶1时卵孢子产生量最大;15℃光照条件下培养,并给侵染叶片持续的水分供应才能产生大量卵孢子;寄主的抗性水平对卵孢子产生有明显的影响,中抗品种上产生卵孢子量最多,高抗品种上产生卵孢子量最少,感病品种上产生卵孢子量居中。     

影响活体内马铃薯晚疫病菌卵孢子产生因素的研究

研究了不同菌株组合,马铃薯植株茎、叶及接种物中A1和A2菌株孢子囊比例、温度、湿度对卵孢子产生的影响。不同菌株组合产生卵孢子的数量有显著差异;在离体接种情况下,叶片中产生卵孢子数量大于茎中产生卵孢子数量;A1和A2菌株中孢子囊不同比例对卵孢子产生影响很大,当比例为1：1时卵孢子产生量最大;15℃光照条件下培养,并给侵染叶片持续的水分供应才能产生大量卵孢子;寄主的抗性水平对卵孢子产生有明显的影响,中抗品种上产生卵孢子量最多,高抗品种上产生卵孢子量最少,感病品种上产生卵孢子量居中。     

金银忍冬花粉离体萌发初探

以金银忍冬[Loniceramaackü(Rupr.)Maxim.]为试材,研究了不同种类糖、硼酸、钙离子、pH值、光照等单因子对花粉萌发的影响.结果表明,培养基中分别含有25%的蔗糖、0.01%的硼离子、0.02%～0.05%的钙离子以及pH6.7时较适合金银忍冬的花粉萌发,萌发率分别为85.63%、59.23%、78.12%和75.27%;培养基中不同种类的糖对花粉萌发的影响不同,以乳糖最适宜,其次是葡萄糖、蔗糖和果糖;在光照(0.5625 μmol m-2s-1)与黑暗下培养,金银忍冬花粉的萌发率没有明显的变化,但黑暗条件更利于花粉管的生长.金银忍冬花粉在含有25%蔗糖、0.01%硼离子、0.02%钙离子、pH为6.7的培养基上光照培养3 d,其花粉萌发率均达到80%以上,最高的萌发率可达88.1%.     

金灰藓(Pylaisiella polyantha)配子体发生的实验观察

金灰藓Pylaisiella polyantha (Hedw．)Grout孢子在光照培养箱中进行培养,实验组为含有Knop培养液的琼脂培养基质,对照组为无营养成分的琼脂培养基质。在光学显微镜下对配子体发生进行了观察、描绘和照相。结果表明：实验组和对照组孢子萌发所需时间相同,均为53．5h,萌发极相为1～4极。4d时,实验组萌发率为85．4％,对照组为54．1％;20d时,开始形成配子体芽原基;40d时,形成具假根、茎、叶的配子体,并形成两性器官。并对配子体发生过程的特点进行了分析和讨论。     

中国石竹离体快繁与试管苗玻璃化研究

研究了培养基组成成分对中国石竹品种Diana F1 White组培程序中种子萌发、诱芽增殖、试管苗玻璃化及生根等重要环节的影响。结果表明:(1)种子适宜的萌发培养基为1/2MS,在此培养基中种子萌发率为83.33%,播种后30d时无菌苗高度达6.99cm,叶片数达26.7。(2)在芽诱导阶段玻璃化苗发生率最高可达53.85%。将光照强度提高至2000lx后,采用培养基MS+6-BA3.0mg/L+NAA0.3mg/L+琼脂8.0g/L+蔗糖8.0g/L进行诱芽培养,玻璃化苗发生率降至3.33%,外植体出芽率达到90%,单个外植体出芽数达到7.2个。(3)适宜的生根培养基为1/2MS+1.0mg/LIBA+琼脂8g/L+蔗糖40g/L,培养30d时生根率为100%,平均根条数达到24.7条,平均根长度达到4.7cm。试管苗在消毒后的腐殖土中移栽,成活率达95%。该研究结果为DianaF1试管苗的快速繁殖提供科学依据。     

Isolation of Oospores of Sunflower Downy Mildew, Plasmopara halstedii, and Microscopical Studies on Oospore Germination

A method was elaborated to isolate oospores of Plasmopara halstedii from tissue of its host, Helianthus annuus . Isolated oospores were studied microscopically and germination was documented with respect to the time course and the mode of germination. Formation of primary sporangia was similarly observed in oospores, harvested from 4- to 6-week-old systemically infected sunflower plants, grown under constant conditions at 16°C, as well as from field plants, harvested late in the season. Pretreatment of oospores with cold temperatures, previously assumed to stimulate the rate and to accelerate the speed of oospore germination, did not result in such effects. Germination usually occurred within 10–30 days of incubation at a highly variable rate of about 1 to 17% (average 6.7%) in deionized water.     

Agarose Medium for Germination of Oospores of Phytophthora cactorum

When oospores of Phytophthora caetorum from 30-day-old culture were treated with 0.25% KMnO₄ for 20 min and incubated at 24°C under light for 10 days, 65–75% germinated on water agar and water agarose but only 1–21% germinated on V-8 agar and S+L agar. Water agarose was selected because germinated oospores formed restrieted colonies on this medium that could be isolated easily. KMnO₄ treatment killed sporangia, chlamydospores and mycelial fragments present in oospore suspensions. Under the above conditions, approximately 44% of oospores from 10-day-old culture germinated and the optimum germination rate of about 75% was obtained when oospores reached about 20 days old.     

Extraction from Plant Tissue and Germination of Oospores of Peronospora viciae f.sp. pisi

A protocol was developed to extract oospores of Peronospora viciae f.sp. pisi from plant tissue and control bacterial contamination in a germination assay. Oospores were extracted by comminuting infected leaves and pods in water, sonicating the suspension and sieving it through mesh sizes 53 and 20 pm, respectively. Germination of oospores was negatively influenced by addition of chloramphenicol and penicillin. A combination of ampicillin and rifampicin strongly inhibited bacterial growth at 10 C, and did not negatively affect germination. Washing oospores in water or 0.02% Tween-80, and sonication did not influence germination. Treating oospore suspensions with cellulase buffered at pH 4.6 for 2 h digested most plant tissue but did not influence germination. Incubation in 0.05 m acetate buffer delayed germination. Germination was unaffected when oospores were incubated in 0.05 m citrate buffer.     

Germination of Chara vulgaris and Nitella flexilis oospores: What are the relevant factors triggering germination?

Factors governing the germination of Chara vulgaris L. and Nitella flexilis L. were investigated in a series of three experiments. Temperature, light regimes, drying, and changes in oxidation–reduction potential are postulated to be possible triggers working in isolation or in combination on dormant oospores originating in sediments with different physical/chemical characteristics.In Experiment 1, none of the N. flexilis oospores inoculated into Petri plates and culture tubes containing either agar or water and then exposed to ‘normal’ 12 h daylight 12 h dark germinated when the redox of the media remained relatively constant over the 20-day observation period, while all of the oospores in a tube in which the redox of the agar declined to below 200 mV, germinated. Similarly no germination occurred in the dark without a decrease in redox. When redox fell to below 200 mV, 11% of the inoculated oospores germinated.In Experiment 2, N. flexilis and C. vulgaris oospores were subjected to cold pre-treatments and inoculated into either water or agar in both plates and tubes. Of the N. flexilis oospores 53% germinated in the dark, and 31% under normal lighting. Of the C. vulgaris, 3% germinated in the dark and 53% under normal lighting. The stimulation of germination with the decrease in redox in the media did not re-occur.Experiment 3 compared the germination of C. vulgaris oospores from four ponds contaminated by mining wastes and from a comparatively clean wetland. The oospores were again subjected (or not) to cold pre-treatments, and were drawn from both freshly concentrated sediments and air-dried sediments that had been held in storage for either 97 or 376 days. Notably, a consistent reduction in oospore viability occurred in oospores from a particular mine pond, contaminated by nickel tailings.     

STORAGE AND GERMINATION OF CHARA OOSPORES

Germination tests were conducted on 39 collections of Chara oospores stored under 4 different conditions for periods of approximately 4 yr. In general, storage of dried oospores at low temperatures (3 C) provided the most satisfactory means for long-term preservation of viable disseminules. Oospore germination zuas higher in light than in darkness. Effects of temperature and substrate upon germination were explored briefly.     

Homothallic sexual reproduction of Pustula helianthicola and germination of oospores

Sunflower white blister rust has become an important disease in many countries with intensive cultivation of the important oil crop. The biology of the pathogen is still partly unclear, particular with respect to its sexual reproduction and primary mode of infection. Zoospores released from sporangia of Pustula helianthicola were isolated individually and used for the inoculation of sunflower in order to generate unithallic, genetically homogenous infections. Single zoospore inoculation of young seedlings resulted in mitotic sporulation within subepidermal blisters on cotyledons and true leaves after approximately 2 weeks. Three weeks postinoculation, the infected plants started forming oospores, hence indicating homothallic sexual reproduction of the pathogen. The development of oogonia and antheridia was studied using light and fluorescence microscopy. Oospores were isolated from infected plant tissue and used for infection and germination studies. Microscopic observation of isolated oospores showed germination that formed sessile vesicle-like structures, germ sporangia or only germ tubes. The rate of germination reached approximately 40 %. Germination was not dependant on a resting phase after oospore formation. Oospores applied to the above ground parts of sunflower seedlings lead to infections within a similar time frame as was achieved with mitotic sporangia. The results underline the importance of oospores for primary infection at the beginning of the season and for long-distance dispersal of the pathogen with sunflower seeds contaminated by oospores.     

Oospores progenies from Phytophthora ramorum

Oospores of Phytophthora ramorum were produced from intraspecific pairings between a European A1 and European or American A2 strains. Their viability was evaluated through colouration with tetrazolium bromide. The distribution of oospores in the different classes of colouration was similar to that found in other Phytophthora species (homothallic and heterothallic): most of the oospores stained purple, which corresponds to spores in dormancy. In order to produce single-oospore cultures, a method was developed to separate oospores from mycelium and chlamydospores. Germination of oospores was observed in 110, 250, 350 and 500-d-old cultures at a low proportion. Microsatellite marker analyses on oospore progenies revealed that the oospores resulted from hybridisation. More than 50 oospore progenies were characterised in terms of mating type, aggressiveness on Rhododendron leaves, and growth rate on two different media. The results are discussed in the context of pest risk analysis.     

Phytophthora kernoviae oospore maturity, germination, and infection

Limited information is known on the basic biology of the recently described Phytophthora kernoviae that produces homothallic oospores. In this study, different P. kernoviae isolates were used to investigate oospore maturity, germination, and infection. All isolates produced oospores in V8 broth at 20°C in the dark by 6d. Oospores also formed at 10 and 15°C, but did not form at 25 and 28°C. Continuous light inhibited oospore production of some isolates but had no negative effect on others. Maturation time of the oospores, as noted by germination and staining with tetrazolium bromide, was not much different among the isolates between 2 and 14 weeks. Oospore germination was optimal at 18 and 20°C, and did not occur at 5, 25, and 30°C. Oospore germination under continuous light was higher than in the dark, but individual isolates showed variable results. Rhododendron leaf disks inoculated with oospores and maintained in the dark at 20°C were necrotic after 1 week, while those kept under continuous light did not develop necrosis. The percentage of leaf disks infected with P. kernoviae was lower in the leaves exposed to continuous light (40%) compared to those kept in the dark (100%).     

Generality of the prerequisite of conidium attachment to a hydrophobic substratum as a signal for germination among Phyllosticta species

It has been shown that conidia of Phyllosticta ampelicida require attachment to a substratum to initiate germination. Furthermore this attachment occurs only on hydrophobic surfaces. This study was initiated to ascertain the breadth of this phenomenon among other species of the genus Phyllosticta. We tested 23 isolates of Phyllosticta representing at least 14 named species. These isolates were collected from North America, Asia and Africa. For 22 of the 23 isolates tested spore attachment occurred at a rate of 60-100% on hydrophobic polystyrene but at 0-5% on hydrophilic polystyrene. The one exception to the preference for a hydrophobic substratum for attachment was an unnamed species of Phyllosticta from Rhus glauca that attached less than 10% on either surface. A similar response was observed when assaying germination and appressorium formation for 17 isolates. Germination and appressorium formation for these isolates proceeded on hydrophobic polystyrene but not on nutrient agar, which is hydrophilic. In five of the tested isolates germination was high on both hydrophobic polystyrene and hydrophilic nutrient media. The isolate from Rhus glauca did not germinate appreciably on either surface. Taken together these results suggest that the requirement for conidium contact/attachment to trigger germination is pervasive to the genus Phyllosticta.     

Phytochrome-mediated germination of very sensitive oospores

The light receptor and its mode of operation were studied in photosensitive oospores of Nitella furcata subsp. megacarpa (Allen emend. Wood). Brief pulses of light activated maximal germination of post-secondary dormant oospores removed from lake sediments. Fluence response data at 12 wavelengths were used to construct an action spectrum for germination. The shape of the action spectrum with its maximum at 669 nm provides evidence for the involvement of phytochrome. Germination was induced with photon fluences that established as little as 0.01% of the phytochrome in the far red-absorbing form, which suggests that phytochrome was operating in the very low-fluence response mode. The functioning of phytochrome in the very low-fluence response mode in Nitella is similar to that in higher plants.