Antifouling activity of six nontoxic chemicals on spore attachment of Ulva fasciata

Marine biofouling cause severe damage to all marine technologies. Indiscriminate use of toxic metallic antifouling (AF) chemicals (cuprous oxide and TBT) to control the biofouling in turn greatly affects the flora and fauna of the marine ecosystem. In search of environmentally safe and effective AF agents, six nontoxic AF candidates were studied against the spore attachment of Ulva fasciata. Spore attachment was inhibited at 100 microg of trans-cinnamic acid (TCA) and benzoic acid (BA). Sulfate-coumaric acid (SCA) had no significant effect on the spore attachment. The release rate of BA exhibited a constant release flow. Varied response of U. fasciata spores to AF chemicals and release rate to the surrounding medium are discussed.     

Evaluation of antifouling activity of eight commercially available organic chemicals against the early foulers marine bacteria and Ulva spores

Environmental impacts caused by tin and copper based commercial antifouling (AF) paints were proved to be detrimental to aquatic ecosystems. Therefore, a search of environmental friendly AF compounds to be used in marine paint to protect the surface of maritime developmental structures from the unwanted biofouling is a burning issue of the present time. Commercially available eight organic chemicals--allyl isothiocyanate, beta-myrecene, cis-3-hexenyl acetate, citral, ethyl heptanoate, eugenol, methyl caproate, and octyl alcohol were evaluated forAF activities using both laboratory and field assays. The test chemicals were found to repel the target motile marine bacteria--Alteromonas marina, Bacillus atrophaeus, Roseobactergallaeciensis and Shewanella oneidensis and motile spores of the green alga, Ulva pertusa. The bacterial and Ulva spore repulsion activities of the test chemicals were measured by chemotaxis and agar diffusion methods respectively interestingly these test chemicals were less toxic to the test fouling species. The toxicity of the test chemicals was measured by using antibiotic assay disks against the bacteria and motility test against Ulva spores. Moreover, in field assay, all test chemicals showed a perfect performance ofAF activity showing no fouling during the experimental period of one year Such results and commercial as well as technical feasibility of the test chemicals firmly showed the possibility of using as alternatives of the existing toxic AF agents.     

Fouling coverage of a green tide alga, Ulva pertusa on some antifouling test surfaces exposed to Ayagin harbor waters, east coast of South Korea

Toxic antifouling chemicals released into the seawaters leads to marine environmental degradation. In order to identify a nontoxic antifoulant, an assessment of antifouling (AF) efficacy of some AF candidates was made at Ayagin harbor, east coast of South Korea. In this static panel study conducted during October 2000-March 2001, some commercial antifoulants, seaweed and seagrass extracts were screened. On panel surfaces coated with a seaweed extract, Ishige okamurae exhibited effective AF activity. Ulva pertusa was encountered as a 'monospecific' fouler with fairly high fouling coverage on many of the test panel surfaces. In recent years the increased influx of inorganic pollutants in the coastal waters causes exorbitant growth of fouling marine algae found all along the Korean peninsula. Especially, a cosmopolitan ship fouling alga U. pertusa occur with high abundance. It was largely suggested that the proposed international ban on the toxic antifoulant tributyltin (TBT) had significant effect on the 'green tide' phenomenon occurring in different parts of the world. However, it appears that Korean scenario of 'green tide' is a localized. Antifouling efficacy of some AF coatings and fouling coverage of a green tide alga, U. pertusa are discussed.     

Biofouling attractants from a brown marine alga Ecklonia cava

In recent years, industrial pollutants and the mountain forest fire ashes released into seawater cause damage to the marine environment, mainly it reduces the algal productivity in the inter tidal region. To get recover from the stress due to pollutants and to increase the growth and development of biofouling algae (benthic organisms), Ecklonia cava extract was investigated for its biofouling attracting efficiency. Bioactive guided fractions of E. cava extract derived from column chromatography were tested against spore attachment of a fouling alga, Ulva pertusa. Fraction B showed increased spore attachment rate with a maximum of 92 +/- 5%. This fraction was further analysed on HPLC, GC-Mass and NMR, deduced as pentadecanoic acid.     

Minimum effective release rate of antifoulants.1. Effects of zosteric acid at Ford Island,Hawaii (USA)

Minimum effective release rate (MERR) of zosteric acid (ZA) was studied at Ford Island. At laboratory condition, spore attachment in Enteromorpha intestinalis was inhibited significantly at 28.2 microg cm2 of ZA. More than 95% reduction in spore attachment was achieved in Ulva fasciata at 2,000 microM of ZA through membrane diffusion system. At 40 cm depth level larval settlement was high with maximum number of Hydroides elegans settlement. The overall larval settlement was increased with increasing days of exposure. The MERR of 10 microg cm(-2) d(-1) ZA inhibited 50% of the larval settlement. However, the inhibition rate was not proportionate to the antifouling ZA concentration. Using MERR membrane diffusion system an effective antifouling strategy was suggested for Ford Island.     

Allelopathic inhibition on red tide microalgae by five macroalgal extracts

This study aims to identify effective antialgal allelochemicals from marine macroalgae that inhibit the growth of red tide microalgae. Practically, new algicidal agents were developed to control red tide. The growth inhibitory effects of 5 marine macroalgae Porphyra tenera, Laminaria japonica, Ulva pertusa, Enteromorpha clathrata, and Undaria pinnatifida on Skeletonema costatum were evaluated by adding crude seawater extracts of macroalgal dry tissue into the culture medium containing S. costatum. The half-effective concentrations at 120 h (EC_{50, 120 h}) of the seawater extracts were 0.6, 0.9, 1.0, 1.0, and 4.7 g/L for the five macroalgae above, respectively. E. clathrata, L. japonica and U. pertusa showed strong allelopathic effect on the growth of S. costatum. There have been no previous reports with regard to the allelopathic effects of the former two macroalgae so far. The possible allelochemicals of 21 compounds of the E. clathrata were detected using Gas chromatography-mass spectrometry (GC-MS) analysis. Unsaturated fatty acids, acrylic acid (C₃H₄O₂), and linolenic acid (C₁₈H₃₀O₂) were the most likely allelochemicals in E. clathrata.     

Effect of the endectocide ivermectin on filamentous fungi

We investigated the effect of ivermectin, at environmental concentrations and above, on several strains of filamentous fungi. Ivermectin did not show significant short-term effect on biomass production and germination rate of the spores. By contrast, the spore production was stimulated twofold in Phanerochaete chrysosporium and Mucor racemosus cultures, whereas that endpoint was reduced in Fusarium cultures. A long-term exposure of F. oxysporum to ivermectin reduced both production and germination of fungal spores. Ivermectin was not an inducer of exocellular oxidases.     

标准化测试种子发芽和根伸长毒性试验的基质应用研究

种子发芽和根伸长毒性试验是研究和评价新化学物质对陆生生物危害性的重要手段之一。为探究在标准化测试中能否使用非推荐基质,以及使用推荐基质时能否用灭菌法替代酸洗法处理石英砂,选取8种植物种子在5种基质中进行试验。结果表明,不同基质对种子发芽率的影响较小,但基质较低的养分含量和特殊的物理结构易对部分种子根长形成胁迫,甚至直接制约根系发育。生菜、水稻在5种基质中的发芽率和根长无显著性差异;绿豆、甘蓝、西瓜在酸洗石英砂中的根长分别为25.1、24.5、29.0 mm,在其余4种基质中根长显著缩短;青瓜在吸水纸和发芽纸中发芽率偏低,仅为83.3%;玉米在酸洗石英砂和灭菌石英砂中的发芽率和根长无明显差异,为100%/45.7 mm和93.3%/44.8 mm,其他基质中显著偏低。标准化测试条件下,以推荐的酸洗石英砂上各种子的发芽率和根长为评判依据,生菜和水稻试验可使用本研究中任意基质,绿豆、甘蓝和西瓜试验仅可使用酸洗石英砂作为基质,青瓜和玉米试验可使用干热灭菌法替代酸洗法处理石英砂,并且青瓜试验还可使用滤纸基质。以上结果为测试机构在工作实践中优化试验条件提供理论依据。     

草莓灰霉孢子萌发条件研究

研究了温度、pH、金属离子,以及一些激素、维生素等因素对灰霉孢子萌发的影响,发现灰霉孢子萌发的最适pH有两个峰值,且在不同峰值下萌发管数明显不同;Ca2 、Fe3 等离子能有效刺激孢子萌发.通过正交实验对灰霉孢子的萌发条件进行了优化,孢子萌发的最适条件为:激动素0.01mg/mL,赤霉素0.05mg/mL,维生素B20.15mg/mL,FeCl30.15mg/mL,CaCl20.2mg/mL,不添加CoCl2的萌发率达到了93.3%以上.图6参10     

Nutrient stimulation of spore settlement in the kelpsPterygophora californica andMacrocystis pyrifera

FertilePterygophora californica Rupr. andMacrocystis pyrifera (L.) C.Ag. were collected in California, USA, from 1987 to 1989. Settlement activity of the spores was stimulated by nutrients, but this was not constant over time. Nutrients had no effect on the settlement ofP. californica spores between 2 and 18 h after sunrise, but settlement activity was stimulated by a nutrient mixture between 20 and 24 h after sunrise (14 to 18 h after release). Settlement activity inM. pyrifera spores was unaffected at 2 to 3 h after release, but settlement activity was significantly stimulated from 5 to 12 h after release. A variety of individual nutrients significantly stimulated settlement in 8 to 9 h oldM. pyrifera spores: ammonium, nitrate, glycine, phosphate, manganese-EDTA (18µM), borate, ferrous iron-EDTA, and ferric iron-EDTA. Spores also settled readily in unenriched artificial seawater, and nutrient-stimulated settlement rates were usually 150% of the unenriched control levels. Neither EDTA alone, cobalt-EDTA, nor manganese-EDTA (2µM) had significant effects onM. pyrifera spore settlement. The effects of time and of several individual nutrients on spore settlement activity are different from previously reported chemotactic effects of nutrients onP. californica andM. pyrifera spores. It is suggested that nutrient settlement-stimulation is mechanistically different from nutrient chemotaxis. However, like chemotaxis, settlement stimulation is probably an adaptation which increases the likelihood of spore settlement in microhabitats suitable for subsequent growth and reproduction of gametophytes.     

Effects of sediments on the development of Macrocystis pyrifera gametophytes

Rocky ocean bottoms are covered from time to time with small amounts of fine sediments. This material may interfere with the development of germling stages of Macrocystis pyrifera. Tests were performed by introducing sediments before and after spores were dispersed in culture dishes. 10 mg cm^-2 of sediment, enough to occlude the surface, prevents spore attachment, greatly reducing the probability of survival. Smothering of established germlings was severe at 108 mg sediment cm^-2. Water motion further reduced spore success where sediments were present, probably because of abrasive scour. Mechanisms of natural and human interference with M. pyrifera reproduction are suggested.     

Minimum effective release rate of antifoulants. 2. Effects of TBTCI, 2-furyl-n-pentyl ketone and coumaric acid at Snug harbor,Hawaii

Minimum effective release rate (MERR) of three antifoulants was examined at Snug harbor, Hawaii using a dynamic diffusion system. Among the three antifoulants tested TBTCI was found to be effective in preventing the settlement of Hydroides elegans and Crisea sp at 0.5 microg cm(-2) d(-1) flux rate. At a maximum flux rate of 10 microg cm(-2) d(-1) of 2-furyl-n-pentyl ketone larval settlement of both the test species were 32-36% reduced. More or less similar effect was seen at 10 microg cm(-2) d(-1) of coumaric acid. Non-toxic antifoulants, 2-furyl-n-pentyl ketone and coumaric acid exhibit specific activity against target species. MERR obtainedfor the three antifoulants is discussed.     

7种酚类化学物质对活性污泥的呼吸抑制作用

苯酚类化学物质是废水中常见的有机污染物,其对活性污泥的毒性数据对于污水处理厂稳定运行和化学品危害性评估具有重要意义。本研究采用活性污泥呼吸抑制试验(209)测定7种酚类化学物质对活性污泥的呼吸抑制作用。结果显示,2,6-二叔丁基苯酚和对特辛基苯酚对活性污泥未产生明显的毒性效应;2,4-二氯酚、2-苯基苯酚、4-硝基酚、4-氯酚和对甲酚等5种化学物质都对活性污泥呼吸有不同程度的抑制效应,3 h-EC50值分别为49.7、77.6、102、150.1和462 mg·L-1,构效关系分析结果表明-Cl、-NO2等官能团是导致活性污泥呼吸抑制效应增强的关键因素。在化学品生物降解性测试研究中,要确保有毒化学物质的测试浓度低于EC50值的1/10。     

Effect of arbuscular mycorrhizal (AM) fungus and plant growth promoting rhizomicroorganisms (PGPR's) on medicinal plant Solanum viarum seedlings

A green house nursery study was conducted to assess the interaction between arbuscular mycorrhizal (AM) fungus, Glomus aggregatum and some plant growth promoting rhizomicrooganisms (PGPR's), Bacillus coagulans and Trichoderma harzianum, in soil and their consequent effect on growth, nutrition and content of secondary metabolities of Solanum viarum seedlings. Triple inoculation of G. aggregatum+B. coagulans+T. harzainum with Solanum viarum in a green house nursery study resulted in maximum plant biomass (plant height 105 cm and plant dry weight 12.17 g), P, Fe, Zn, Cu and Mn and secondary metabolities [total phenols (129.6 microg g(-1) f.wt.), orthodihydroxy phenols (90.6 microg g(-1) f.wt.), flavonoids (3.94 microg g(-1) f.wt.), alkaloids (5.05 microg g(-1) f.wt.), saponins (5.05 microg g(-1) f.wt.) and tannins (0.324 microg g(-1) f.wt.)] of S. viarum seedlings. The mycorrhizal root colonization and spore numbers in the root zone soil of the inoculated plants increased. The enzyme activity namely acid phosphatase (53.44 microg PNP g(-1) soil), alkaline phosphatase (40.95 microg PNP g(-1) soil) and dehydrogenase (475.5 microg PNP g(-1) soil) and total population of B. coagulans (12.5x10(4) g(-1)) and T. harzianum (12.4 x 10(4) g(-1)), in the root zone soil was found high in the triple inoculation with G. aggregatum+B. coagulans+T. harzianum that proved to be the best microbial consortium.     

Survival and recovery of resting spores and resting cells of the marine planktonic diatom Chaetoceros pseudocurvisetus under fluctuating nitrate conditions

Of the two resting life-forms of the planktonic diatom Chaetoceros pseudocurvisetus Mangin formed during periods of nitrate depletion, resting spores survived at least 1 month after spore formation at 24 °C, while resting cells survived only for about 10 d at the same temperature. Under nitrogen limitation, resting cells exhibited higher specific death rates than resting spores at temperatures ranging from 5 to 30 °C. After nitrogen replenishment, resting spores required a certain lag period of about 1 d to initiate vegetative growth at levels of nitrate supply from 0.5 to 20 M, while resting cells initiated vegetative growth almost immediately. Resting spores exhibited an intracellular accumulation of the supplied nitrate during germination and initial vegetative growth. The resting cells, however, exhibited more active vegetative growth, closely coupled with the uptake of the supplied nitrate. The resting spores and resting cells appear to play different roles in the maintenance of populations under nutrient fluctuations depending on the interval length between nutrient fluxes in natural waters. Received: 27 April 1998 / Accepted: 1 March 1999     

Neutral lipids in macroalgal spores and their role in swimming

We followed changes in the neutral lipid content of actively swimming zoospores of the palm kelp Pterygophora californica in a laboratory experiment to investigate the degree to which spore swimming is fueled by endogenous lipid reserves. The neutral lipid content of individual zoospores during the experiment was measured by flow cytometry using Nile Red, a fluorescent stain that is specific for neutral lipid. Results showed that photosynthesis greatly influenced lipid consumption during zoospore swimming. We found no detectable change in the neutral lipid content of zoospores after 30 h of swimming under conditions where light was near the optimum for photosynthesis. By contrast, neutral lipid declined by ≃43% over 30 h in zoospores kept in the dark. To evaluate whether lipid reserves are generally related to spore motility in macroalgae, we surveyed spore lipid-content and composition in species with motile spores and non-motile spores using thin-layer chromatography (TLC), and flame-ionization detection (FID). We observed substantial differences in lipid content and composition among the 20 species examined. Spores high in total lipid (as estimated by the ratio of lipid:carbon) generally had a large amount of neutral lipid; motile spores had significantly more lipid and a significantly larger neutral lipid fraction than non-motile spores. The kelps as a group had the highest total lipid content and the largest neutral lipid fraction, while non-motile spores of red algae were generally low in total lipid and in the proportional abundance of neutral lipid. Phospholipids accounted for more than half of all lipid in 14 of the 20 species examined, while neutral lipid accounted for the majority of lipid in all five species of kelp examined. Triacylglycerols, which function primarily in energy storage, were the primary form of neutral lipid in all but one species of kelp (Agarum fimbriatum), whereas free fatty acids were the dominant form of neutral lipid in most red algae and in brown algae that had a small neutral lipid fraction. Our results are largely consistent with the hypothesis that macroalgae use endogenous lipid reserves to fuel spore-swimming. The small amounts of triacylglycerols observed in the motile spores of several species of brown and green algae, however, indicate that the amount of lipid reserves needed to fuel spore-swimming may be influenced by a variety of factors including swimming behavior, photosynthetic efficiency, and the light environment inhabited by spores. Received: 8 September 1998 / Accepted: 8 January 1999     

Resting spore formation of the marine planktonic diatom Chaetoceros anastomosans induced by high salinity and nitrogen depletion

The marine planktonic diatom Chaetoceros anastomosans, which was isolated from Sagami Bay, was used for a study of resting spore formation mechanisms in batch culture experiments. Vegetative cells could grow at salinities ranging from 20.7 to 45.5‰, and resting spore formation was enhanced significantly in nitrate-depleted, high salinity media (40.0 to 45.5‰). The rate of resting spore formation (1.9 d⁻¹) was comparable to the specific growth rate (1.8 d⁻¹) of vegetative cells in the exponential growth phase in normal salinity medium. The size of resting spores formed under high salinity conditions was smaller than that of spores formed in normal salinity media. Unlike vegetative cells, resting spores seemed to possess some mechanisms to survive over a wider range of salinities by resisting bacterial attacks on their cell walls. Received: 4 August 1996 / Accepted: 27 August 1996     

拮抗菌对黄瓜枯萎病菌的室内生物活性

测定了从沤肥浸渍液和土壤中分离并筛选得到对黄瓜枯萎病菌的室内生物活性的6个菌株,编号为94－I、94－Ⅱ、94－Ⅲ、96－Ⅰ和98－Ⅱ,测定其孢子悬浮培养16h时对黄瓜枯萎菌的抑制率分别为48．29％、0．00％、60．69％、39．28％、61．29％和72．32％,孢子和菌丝有的变畸形并产生大泡囊;平皿拮抗试验表明,其活体浓度为50％时对黄瓜枯萎病菌的抑制率最大分别为74．52％、83．27％、90．60％、88．59％、89．35％和94．30％;而其相同浓度高压蒸汽灭活后的代谢产物对黄瓜枯萎病菌的抑制率最大分别为33．215、52．22％、62．84％、46．57％、47．61％和70．34％。     

Spore inhibition-based enzyme substrate assay for monitoring of aflatoxin M1 in milk

A spore germination-based concept and its transformation into a field level prototype for monitoring aflatoxin M₁ (AFM₁) in milk was developed. Initially, 15 strains of Bacillus spp. procured from different culture collection were screened for AFM₁ sensitivity using spot assay and marker strain showing inhibition at 0.5 ppb was selected based upon maximum zone of inhibition. The selected strain B. megaterium 2949 was further screened for different enzymes activities and subsequently its spores were produced to an extent of 73.13% ± 3.197% in newly developed sporulation medium containing beef extract (0.0075% ± 0.0004%), yeast extract (0.015% ± 0.001%), peptone (0.0375% ± 0.0016%), and sodium chloride (0.0375% ± 0.0018%). A spore germination-based concept/ assay was optimized by immobilizing spores in eppendorf with pretreated milk (80°C/15 min) containing germinant and chromogenic substrate followed by incubation at 37°C. The appearance of sky blue color within real time of 45 min indicated spores germination and release of specific marker enzyme such as acetyl esterase and its specific action on chromogenic substrate which demonstrates absence of AFM₁ in milk. However, if there was no color change, presence of AFM₁ at 0.5 ppb MRL was denoted by Codex. The developed concept on AFM₁ detection was validated and a correlation of 0.97 was established with AOAC approved Charm 6602 and ELISA at Codex MRL with minimal false positive and negative results. The cost effective test has potential application in dairy farms, manufacturing, and R&D units for routine monitoring of AFM₁ in milk.     

Spore germination and early stages of development in Hypnea musciformis (Rhodophyta,Gigartinales)

The early stages of development of the carpospores and tetraspores of Hypnea muscilormis (Wulfen) Lamouroux have been investigated. Both types of spores germinated immediately after liberation. The spores segmented repeatedly into, 2, 3, 4 cells, etc. until a multicellular ball of cells was produced. The germination pattern was thus of the discal type, i.e., Typus discalis mediatus (Inoh, 1947) or the Dumontia-type in the terminology of Chemin (1937). Subsequently, a relatively expansive attachment holdfast was produced from each sporeling. This was followed by the development of up to 4 or more shoot axes arising from the basal disc. These findings are discussed with reference to an earlier study by the senior author on the developmental biology of other species of Hypnea Lamouroux.