Broad spectrum and species specificity of plant allelochemicals 1,2-benzenediol and 3-indoleacrylic acid against marine and freshwater harmful algae.

Allelochemicals derived from plants have shown great potential in mitigating harmful algal blooms (HABs), although different algal species can respond differently to these chemicals. Therefore, we first investigated the allelopathic effects of two newly identified plant-derived allelochemicals, 1,2-benzenediol (1,2-BD) and 3-indoleacrylic acid (3-IDC), on six algal species. Then we further evaluated the allelopathic responses of two bloom-forming species, Microcystis aeruginosa FACHB-905 and Heterosigma akashiwo to 1,2-BD. Results showed that 1,2-BD had a broader antialgal spectrum than 3-IDC. Allelopathic response analysis indicated that 1,2-BD consistently and stably inhibit the growth of M. aeruginosa FACHB-905, with inhibitory mechanism being disruption of photosynthetic activity, overwhelming of the antioxidant system and activation of programmed cell death (PCD). H. akashiwo displayed resistance to 1,2-BD during exposure, and the growth inhibition was mainly attributed to PCD. Therefore, the species-specific allelopathic responses provide new insights for controlling HABs using 1,2-BD and 3-IDC.

Changes in Growth, Photosynthesis Performance, Pigments, and Toxin Contents of Bloom-Forming Cyanobacteria after Exposure to Macroalgal Allelochemicals.

Macroalgae can directly restrict the growth of various phytoplankton species by releasing allelopathic compounds; therefore, considerable attention should be paid to the allelopathic potential of these organisms against harmful and bloom-forming cyanobacteria. The main aim of this study was to demonstrate for the first time the allelopathic activity of Ulva intestinalis on the growth, the fluorescence parameters: the maximum PSII quantum efficiency (Fv/Fm) and the effective quantum yield of PSII photochemistry (ΦPSII), the chlorophyll a (Chl a) and carotenoid (Car) content, and the microcystin-LR (MC-LR) and phenol content of three bloom-forming cyanobacteria, Aphanizomenon sp., Nodularia spumigena, and Nostoc sp. We found both negative and positive allelopathic effects of U. intestinalis on tested cyanobacteria. The study clearly showed that the addition of the filtrate of U. intestinalis significantly inhibited growth, decreased pigment content and Fv/Fm and ΦPSII values of N. spumigena and Nostoc sp., and stimulated Aphanizomenon sp. The addition of different concentrations of aqueous extract also stimulated the cyanobacterial growth. It was also shown that the addition of extract obtained from U. intestinalis caused a significant decrease in the MC-LR content in Nostoc sp. cells. Moreover, it the phenol content in N. spumigena cells was increased. On the other hand, the cell-specific phenol content for Aphanizomenon sp. decreased due to the addition of the filtrate. In this work, we demonstrated that the allelopathic effect of U. intestinalis depends on the target species' identity as well as the type of allelopathic method used. The study of the allelopathic Baltic macroalgae may help to identify their possible role as a significant biological factor influencing harmful cyanobacterial blooms in brackish ecosystems.

The current state of knowledge on taxonomy, modulating factors, ecological roles, and mode of action of phytoplankton allelochemicals.

Allelopathy is widespread in marine, brackish, and freshwater habitats. Literature data indicate that allelopathy could offer a competitive advantage for some phytoplankton species by reducing the growth of competitors. It is also believed that allelopathy may affect species succession. Thus, allelopathy may play a role in the development of blooms. Over the past few decades, the world's coastal waters have experienced increases in the numbers of cyanobacterial and microalgal blooming events. Understanding how allelopathy is implicated with other biological and environmental factors as a bloom-development mechanism is an important topic for future research. This review focuses on a taxonomic overview of allelopathic cyanobacteria and microalgae, the biological and environmental factors that affect allelochemical production, their role in ecological dynamics, and their physiological modes of action, as well as potential industrial applications of allelopathic compounds.

Inductive and synergistic interactions between plant allelochemical flavone and Bt toxin Cry1Ac in Helicoverpa armigera.

Genetically engineered crops simultaneously produce defensive allelochemicals and Bacillus thuringiensis (Bt) toxin proteins to kill some of the world's most devastating insect pests. How the two types of toxins, when ingested sequentially or simultaneously, interact at both lethal and sublethal doses in these pests remains underexplored. Here, we examined the toxicological interactions between the Bt toxin Cry1Ac and the flavonoid allelochemical flavone in Helicoverpa armigera. Simultaneous exposure of H. armigera neonates to lethal doses (LC25 ) of Cry1Ac and flavone caused a mortality significantly higher than that of either toxin alone and their expected additive mortality. Preexposure for 24 h to a sublethal dose (LC10 ) of Cry1Ac followed by 6-d simultaneous exposure to the same dose of Cry1Ac plus a lethal dose (1.6 mg/g diets, LC50 ) of flavone resulted in a mortality significantly higher than that of the LC50 dose of flavone alone and the expected additive mortality of the LC50 dose of flavone plus the LC10 dose of Cry1Ac. One-day preexposure to the sublethal dose (LC10 ) of flavone followed by 6-d simultaneous exposure to the LC50 dose (6 ng/cm2 ) of Cry1Ac plus the LC10 dose of flavone yielded a mortality significantly higher than that of the LC50 dose of Cry1Ac but similar to the expected additive mortality of the LC50 dose of Cry1Ac plus the LC10 dose of flavone. The results suggest that Cry1Ac induces and synergizes the toxicity of flavone against H. armigera larvae.

Life-History Traits Response to Effects of Fish Predation (Kairomones), Fipronil and 2,4-D on Neotropical Cladoceran Ceriodaphnia silvestrii.

Aquatic environments are constantly exposed to a cocktail of contaminants mainly due to human activities. As polluted ecosystems may simultaneously present other multiple natural stressors, the objective of the present study was to evaluate joint effect of stressors (natural and anthropogenic) on life history traits of the Neotropical cladoceran, Ceriodaphnia silvestrii. For this purpose, the effects of water conditioned with predator kairomones (fish) and environmental concentrations (sublethal) of two pesticides widely used in sugarcane monoculture in Brazil, the insecticide Regent® 800 WG (active ingredient-a.i. fipronil) and the herbicide DMA® 806 BR (a.i. 2,4-D) were evaluated using chronic toxicity testing, isolated and in mixture, for this cladoceran species. The environmental risks of pesticides for tropical freshwater biota were also estimated from the risk quotient MEC/PNEC. Among the characteristics of the life history of C. silvestrii evaluated after 8 days of exposure, compared with the mean value of control, the age of primiparous females was not affected by any evaluated treatment. However, species average survival decreased in the treatment of kairomones mixed with fipronil (FK) and in the treatment with a mixture of fipronil, 2,4-D, and kairomones (MFKD). The body length of maternal females was shorter than in the control after exposure in treatments with only kairomones (K) and FK. Fecundity of this cladoceran was reduced when exposed to FK and MFKD treatments, and the intrinsic rate of population increase significantly decreased for organisms exposed to treatment with fipronil (F) and to mixtures of fipronil and 2,4-D (MFD), MFDK, and FK. The results indicated that the combination of anthropogenic and natural stressors causes changes in C. silvestrii life history traits, which can contribute to the decline in populations, and our preliminary risk assessment results are a matter of concern regarding biota conservation.

Invasive plants exert disproportionately negative allelopathic effects on the growth and physiology of the earthworm Eisenia fetida.

Exotic invasive plants possess the capacity to disrupt and extirpate populations of native species. Native plants' increased sensitivity to invaders' allelochemicals is a mechanism by which this can occur. However, it is not clear whether and how the allelopathic effects of invasive plants affect members of the soil faunal community - particularly the important functional guild of earthworms. We used the model earthworm Eisenia fetida to investigate the responses to extracts from the widely invasive Asterids (Ageratina adenophora, Bidens pilosa, Erigeron annuus) and closely-related native species in a greenhouse experiment. We observed declines in body mass and respiration, and increases in oxidative and DNA damage biomarkers in the native earthworm E. fetida when grown under root and leaf extracts from these invasive plants. These effects were concentration-dependent, and worm growth and physiology was most negatively affected under the highest concentrations of leaf extracts. Most importantly, extracts from invasive plants caused significantly more negative effects on E. fetida than did extracts from native plant species, indicating allelopathy from invasive plants may inhibit earthworm physiological functioning. These results expand the domain of the novel weapons hypothesis to the earthworm guild and demonstrate the utility of E. fetida as a bioindicator for plant allelochemicals.

Effect of allelochemicals on photosynthetic and antioxidant defense system of Ulva prolifera.

Ulva prolifera is a macroalgae that forms massive blooms, negatively impacting natural communities, aquaculture operations and recreation. The effects of the natural products, eugenol, ß-myrcene, citral and nonanoic acid on the growth rate, antioxidative defense system and photosynthesis of Ulva prolifera were investigated as a possible control strategy for this harmful taxon. Negative effects on growth were observed with all four chemicals, due to the excessive production of reactive oxygen species and oxidative damage to the thalli. However, the response of U. prolifera under the four chemicals stress was different at the cellular level. ß-myrcene, the most effective compound in terms of growth inhibition, induced oxidative stress as shown by the damage of total antioxidant capacity (T-AOC) and the downregulation of the glutathione-ascorbate (GSH-ASA) cycle which inhibited the antioxidative system. This chemical also inhibited photosynthesis and photoprotection mechanisms in U. prolifera, resulting in growth limitation. In contrast, U. prolifera was less affected by the second tested chemical, eugenol, and showed no significant change on photosynthetic efficiency in the presence of the chemical. The inhibition effects of the third and fourth tested chemicals, nonanoic acid and citralon, on growth and on the antioxidant defense system in U. prolifera were inferior. These results provide a potential avenue for controlling green tides in the future.

Toxicity and sublethal effects of two plant allelochemicals on the demographical traits of cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae).

Plant allelochemicals are a group of secondary metabolites produced by plants to defend against herbivore. The mortality of two plant allelochemicals (tannic acid and gossypol) on the cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), were investigated using feeding assays and the sublethal effects were evaluated using the age-stage, two-sex life table approach. Tannic acid and gossypol have deleterious effects on A. gossypii, and as the concentrations increased, the mortality of cotton aphid increased. The life history traits of A. gossypii including the developmental duration of each nymph stage, the longevity, oviposition days, total preadult survival rate and adult pre-oviposition period were not significantly affected by sublethal concentration of tannic acid (20 mg/L) and gossypol (50 mg/L), while the population parameters (r, λ and R0) were significantly affected by these two plant allelochemicals. Furthermore, tannic acid can increase the pre-adult duration time and TPOP but reduce the fecundity of A. gossypii significantly compared to the control and gossypol treatment groups. These results are helpful for comprehensively understanding the effects of plant allelochemicals on A. gossypii.

Linalool- and α-terpineol-induced programmed cell death in Chlamydomonas reinhardtii.

Plant allelochemicals effectively inhibit and/ or control algal growth, and have potential to use as algaecide. To uncover the lethal mechanism of 2 anti-algal compounds linalool and α-terpineol identified from Cinnamomum camphora extracts, and promote their development as algaecide, the H2O2 production, photosynthetic abilities, caspase-like activities, nuclear changes and DNA degradation were investigated in Chlamydomonas reinhardtii treated with the 2 compounds. H2O2 content burst in linalool treatment at 0.5 h and in α-terpineol treatment at 1 h, with increases of 2.7 folds and 1.3 folds, respectively, compared to that at 0 h. The photosynthetic pigments gradually degraded, and Fv/Fm gradually declined to zero, indicating that the cell death was not a necrosis due to the gradual disappearance of physiological process. In C. reinhardtii cells, the caspase-9-like and caspase-3-like were activated in the treatments with the 2 compounds for 1 h. With prolonging the treatment time, the fluorescent intensity of the cell nucleuses stained by DAPI gradually enhanced and then faded, and the genomic DNA isolated from the cells gradually degraded. These hallmarks indicated that the death of C. reinhardtii cells in linalool and α-terpineol treatments was a programmed cell death (PCD) triggered by the increased reactive oxygen species (ROS). Compared to α-terpineol treatment, linalool treatment showed stronger promoting effects on PCD at the same time point, which may be caused by the higher ROS content inducing higher caspase-9-like and caspase-3-like activities in a short time.

Involvement of Carnosic Acid in the Phytotoxicity of Rosmarinus officinalis Leaves.

Weeds are rapidly developing resistance to synthetic herbicides, and this can pose a threat to the ecosystem. Exploring allelopathic species as an alternative weed control measure can help minimize the ecological threat posed by herbicide-resistant weeds. In this study, we aimed to evaluate the contribution of some polyphenols to the allelopathy of rosemary (Rosmarinus officinalis L.). The phytotoxic effects of rosemary (leaves, roots, inflorescences, and stems) crude extracts were tested on lettuce (Lactuca sativa L.). Soils incorporated with dried rosemary leaves were also tested on test plants. Reversed-phase high-performance liquid chromatography (HPLC) analysis was used to determine the content of some polyphenols (caffeic, ferulic, gallic, rosmarinic, carnosic, and chlorogenic acids) in rosemary. The specific activity and total activity of crude extracts and individual compounds were evaluated using lettuce. The crude extract of rosemary leaves showed the highest growth inhibitory effect among the rosemary organs tested. Soil amended with rosemary leaf debris reduced the dry matter and seed emergence of lettuce. Carnosic acid was the main compound detected in rosemary leaves and had a high specific activity when tested on lettuce. During the seed germination period, there was observed filter paper coloration among the test plants treated with carnosic acid (250 µg/mL). The high concentration and strong inhibitory effect of carnosic acid could explain the inhibitory activity of the rosemary leaf extract. Hence, we conclude based on the total activity estimation that carnosic acid among the other tested compounds is the major allelochemical in rosemary leaves.

The stresses of allelochemicals isolated from culture solution of diatom Phaeodactylum tricornutum Bohlin on growth and physiology of two marine algae.

The allelopathic effects of extracts isolated from the culture filtrate of diatom Phaeodactylum triconutum Bohlin on typical marine microalgae Prorocentrum donghaiense Lu and Dunaliella salina Teodoresco were investigated by determining different physiological and biochemical parameters, such as growth rate, membrane systems and esterase activity under controlled laboratory conditions. The growth of P. donghaiense was significantly inhibited immediately after exposure to the allelochemicals, while the algae density of D. salina was less sensitive. Chlorophyll-a content, membrane systems, as well as esterase activity were simultaneously investigated by flow cytometry with particular fluorescent markers and exhibited changeable sensitivities. The results demonstrated that the membrane systems of P. donghaiense were suppressed by the allelochemicals directly, causing loss of integrity and membrane penetration. Esterase activity was the most sensitive indicator as that of P. donghaiense cells significantly increased in short time and was inhibited subsequently. However, the membrane of D. salina remained intact still after exposure to the extracts and the esterase activity was only inhibited on last day during experiment period. Membrane potential and chlorophyll-a content of the two marine algae also showed somewhat different changes, as that of P. donghaiense cells were impaired after 5 day exposure to all volume conditions while these two characteristics of D. salina was only suppressed by exposure to high volume of the allelochemicals on day 6. The present results indicated that the inhibition of culture filtrate of P. triconutum on P. donghaiense was algicidal whereas the effect on D. salina appeared to be algistatic.

The Consistency Between Phytotoxic Effects and the Dynamics of Allelochemicals Release from Eucalyptus globulus Leaves Used as Bioherbicide Green Manure.

In the worldwide search for new strategies in sustainable weed management, the use of allelopathic plants incorporated into the soil as green manure can help control weeds by releasing allelochemicals into the environment. In previous experiments, Eucalyptus globulus leaves incorporated into the soil as green manure were shown to have a notable potential for weed control. But, 'what was exactly happening at chemical level?' and 'which were the compounds potentially responsible for the phytotoxic effects observed during those greenhouse assays?' In the present study, in-vitro phytotoxicity bioassays and chemical analysis of eucalyptus leaves were carried out in order to explore the relationship between the temporal phytotoxic effects and the dynamics of chemical composition. For that, eucalyptus leaves were removed from soil at different sampling times during 30 days and analyzed for phenolic and volatile organic compounds (VOCs) by HPLC and HS-SPME/GC-MS, respectively. The phytotoxic potential of the aqueous extract and the volatile fraction was tested on the germination and early growth of Lactuca sativa. Eucalyptus leaves incorporated into the soil as green manure showed a continuous release of different phenolic and volatile compounds during a 30-day period of decomposition. Both fractions had phytotoxic effects during the time assayed; however, the target process of phytotoxicity was different: phenolic compounds being the factor causing germination inhibition and VOCs responsible for growth reduction. The dynamics of release of this cocktail of allelochemicals into the soil environment may be the responsible for the phytotoxicity observed in our previous works.

Interaction between the barley allelochemical compounds gramine and hordenine and artificial lipid bilayers mimicking the plant plasma membrane.

Some plants affect the development of neighbouring plants by releasing secondary metabolites into their environment. This phenomenon is known as allelopathy and is a potential tool for weed management within the framework of sustainable agriculture. While many studies have investigated the mode of action of various allelochemicals (molecules emitted by allelopathic plants), little attention has been paid to their initial contact with the plant plasma membrane (PPM). In this paper, this key step is explored for two alkaloids, gramine and hordenine, that are allelochemicals from barley. Using in vitro bioassays, we first showed that gramine has a greater toxicity than hordenine towards a weed commonly found in northern countries (Matricaria recutita L.). Then, isothermal titration calorimetry was used to show that these alkaloids spontaneously interact with lipid bilayers that mimic the PPM. The greater impact of gramine on the thermotropic behaviour of lipids compared to hordenine was established by means of infrared spectroscopy. Finally, the molecular mechanisms of these interactions were explored with molecular dynamics simulations. The good correlation between phytotoxicity and the ability to disturb lipid bilayers is discussed. In this study, biophysical tools were used for the first time to investigate the interactions of allelochemicals with artificial PPM.

Identification of phytotoxins in different plant parts of Brassica napus and their influence on mung bean.

Plants in Brassica genus have been found to possess strong allelopathic potential. They may inhibit seed germination and emergence of subsequent crops following them in a rotation system. Series of laboratory and greenhouse experiments were conducted to determine the allelopathic impacts of Brassica napus L. against mung bean. We studied (1) the effects of aqueous extract (5%) of different plant parts (root, stem, leaf, flower, and whole plant) of B. napus, (2) the effects of leaf and flower extracts of B. napus at 0, 1, 2, 3, and 4% concentrations, and (3) the effect of residues of different B. napus plant parts and decomposition periods (0, 7, 14, and 21 days) on germination and seedling growth of mung bean. Various types of phenolics including quercitin, chlorogenic acid, p-coumeric acid, m-coumaric acid, benzoic acid, caffeic acid, syringic acid, vanillic acid, ferulic acid, cinamic acid, and gallic acid were identified in plant parts of B. napus. Among aqueous extracts of various plant parts, leaf and flower were found to have stronger inhibitory effects on germination and seedling growth traits of mung bean, higher concentrations were more toxic. The decomposition period changed the phtotoxic effect of residues, more inhibitory effect was shown at 14 days decomposition while decomposition for 21 days reduced inhibitory effect. The more total water-soluble phenolic was found in 5% (w/v) aqueous extract and 5% (w/w) residues of B. napus flowers at 14 days of decomposition (89.80 and 10.47 mg L-1), respectively. The strong inhibitory effects of B. napus should be managed when followed in rotation.

Effect of propionamide on the growth of Microcystis flos-aquae colonies and the underlying physiological mechanisms.

Reducing the formation and growth of Microcystis colonies is an important prerequisite for the effective prevention and treatment of cyanobacterial blooms. Microcystis flos-aquae colonies was selected to investigate the potential of propionamide for use in controlling cyanobacterial blooms. Propionamide, one of the major allelochemicals in the root exudates of E. crassipes, was tested using different concentrations (0, 0.2, 1, and 2mgL-1) and dosing methods (one-time addition, semi-continuous addition, and continuous addition) to assess its effect on the growth of M. flos-aquae colonies. The results showed that in the presence of different concentrations of propionamide, the growth of M. flos-aquae colonies followed a logistic growth model, with a higher degree of fit at lower propionamide concentrations. With the semi-continuous addition of 2mgL-1 propionamide, the growth of M. flos-aquae colonies was markedly inhibited; the relative inhibition ratio of algal cells reached >90% at day 7 of co-culture, and the colonial form gradually disintegrated, transforming mainly into unicellular and bicellular forms and small colonies (average diameter<50µm). Following the semi-continuous addition of 2mgL-1 propionamide, the exopolysaccharide content, the chlorophyll-a concentration, and the maximum photochemical efficiency of photosystem II (Fv/Fm) trended downward in M. flos-aquae colonies, whereas the relative expression of the microcystin (MC) biosynthetic genes, mcyA and mcyH, was upregulated overall. Importantly, the synthesis of intracellular microcystin-LR (MC-LR) was decreased after an initial increase, and the extracellular MC-LR concentration did not differ significantly from that in the control group (p>0.05). Moreover, an acute toxicity test showed that 2mgL-1 propionamide was generally non-toxic to Daphnia magna. In conclusion, appropriate use of propionamide could effectively control the expansion of M. flos-aquae colonies without potential risks to the ecological safety of aquatic environments; therefore, propionamide can actually be used to regulate cyanobacterial blooms in natural waters.

Attractive toxic sugar baits for controlling mosquitoes: a qualitative study in Bagamoyo, Tanzania.

BACKGROUND: Malaria elimination is unlikely to be achieved without the implementation of new vector control interventions capable of complementing insecticide-treated nets and indoor residual spraying. Attractive-toxic sugar baits (ATSBs) are considered a new vector control paradigm. They are technologically appropriate as they are simple and affordable to produce. ATSBs kill both female and male mosquitoes attracted to sugar feed on a sugary solution containing a mosquitocidal agent and may be used indoors or outdoors. This study explored the views and perceptions on ATSBs of community members from three Coastal Tanzanian communities. METHODS: Three communities were chosen to represent coastal urban, peri-urban and rural areas. Sensitization meetings were held with a total of sixty community members where ATSBs were presented and explained their mode of action. At the end of the meeting, one ATSB was given to each participant for a period of 2 weeks, after which they were invited to participate in focus group discussions (FGDs) to provide feedback on their experience. RESULTS: Over 50% of the participants preferred to use the bait indoors although they had been instructed to place it outdoors. Participants who used the ATSBs indoors reported fewer mosquitoes inside their homes, but were disappointed not to find the dead mosquitoes in the baits, although they had been informed that this was unlikely to happen. Most participants disliked the appearance of the bait and some thought it to be reminiscent of witchcraft. Neighbours that did not participate in the FGDs or sensitizations were sceptical of the baits. CONCLUSIONS: This study delivers insight on how communities in Coastal Tanzania are likely to perceive ATSBs and provides important information for future trials investigating the efficacy of ATSBs against malaria. This new vector control tool will require sensitization at community level regarding its mode of action in order to increase the acceptance and confidence in ATSBs for mosquito control given that most people are not familiar with the new paradigm. A few recommendations for product development and delivery are discussed.

Effect of allelochemical tricin and its related benzothiazine derivative on photosynthetic performance of herbicide-resistant barnyardgrass.

Despite increasing knowledge of allelochemicals as leads for new herbicides, relatively little is known about the mode of action of allelochemical-based herbicides on herbicide-resistant weeds. In this study, herbicidal activities of a series of allelochemical tricin-derived compounds were evaluated. Subsequently, a benzothiazine derivative 3-(2-chloro-4-methanesulfonyl)-benzoyl-hydroxy-2-methyl-2H-1,2-benzothiazine-1,1-dioxide with 4-hydroxyphenyl-pyruvate dioxygenase (HPPD) inhibiting activity was identified as a target compound on photosynthetic performance of penoxsulam-resistant versus -susceptible barnyardgrass (Echinochloa crus-galli). Regardless of barnyardgrass biotype, the benzothiazine derivative greatly affected chlorophyll fluorescence parameters (Fv/Fm, ETR1min and NPQ1min), reduced the chloroplast fluorescence levels and expression of HPPD gene. In particular, the benzothiazine derivative interfered with photosynthetic performance of resistant barnyardgrass more effectively than the allelochemical tricin itself. These results showed that the benzothiazine derivative effectively inhibited the growth of resistant barnyardgrass and its mode of action on photosynthesis system was similar to HPPD-inhibiting sulcotrione, making it an ideal lead compound for further development of allelochemical-based herbicide discovery.

Effects of an allelochemical in Phaeodactylum tricornutum filtrate on Heterosigma akashiwo: Morphological, physiological and growth effects.

The effects of an allelochemical extracted from the culture filtrate of diatom Phaeodactylum tricornutum on the raphidophyte Heterosigma akashiwo were investigated using a series of morphological, physiological and biochemical characters. Growth experiments showed that H. akashiwo was significantly inhibited immediately after exposure to the allelochemical, with many cells rapidly dying and lysing based on microscopic observation. The effects of the allelochemical on the surviving cells were explored using Scanning Electron Microscopy (SEM) and Flow cytometry (FCM), the latter by examination of a suite of physiological parameters (membrane integrity, esterase activity, chlorophyll-a content, membrane potential). The results demonstrate that the membrane of H. akashiwo was attacked by the allelochemical directly, causing cell membrane breakage and loss of integrity. Esterase activity was the most sensitive indicator of the impacts of the allelochemical. Membrane potential and chlorophyll-a content both showed significant decreases following exposure of the Heterosigma cells to high concentrations of the allelochemical for 5 and 6 days. Both were affected, but the membrane potential response was more gradual compared to other effects. The cell size of H. akashiwo did not change compared with the control group. The surviving cells were able to continue to grow and in a few days, re-establish a successful culture, even in the presence of residual allelochemical, suggesting either development of cellular resistance, or the degradation of the chemical.