Plate Bioassay Research Articles

Allelochemicals produced by Caribbean macroalgae and cyanobacteria have species-specific effects on reef coral microorganisms

Coral populations have precipitously declined on Caribbean reefs while algal abundance has increased, leading to enhanced competitive damage to corals, which likely is mediated by the potent allelochemicals produced by both macroalgae and benthic cyanobacteria. Allelochemicals may affect the composition and abundance of coral-associated microorganisms that control host responses and adaptations to environmental change, including susceptibility to bacterial diseases. Here, we demonstrate that extracts of six Caribbean macroalgae and two benthic cyanobacteria have both inhibitory and stimulatory effects on bacterial taxa cultured from the surfaces of Caribbean corals, macroalgae, and corals exposed to macroalgal extracts. The growth of 54 bacterial isolates was monitored in the presence of lipophilic and hydrophilic crude extracts derived from Caribbean macroalgae and cyanobacteria using 96-well plate bioassays. All 54 bacterial cultures were identified by ribotyping. Lipophilic extracts from two species of Dictyota brown algae inhibited >50% of the reef coral bacteria assayed, and hydrophilic compounds from Dictyota menstrualis particularly inhibited Vibrio bacteria, a genus associated with several coral diseases. In contrast, both lipo- and hydrophilic extracts from 2 species of Lyngbya cyanobacteria strongly stimulated bacterial growth. The brown alga Lobophora variegata produced hydrophilic compounds with broad-spectrum antibacterial effects, which inhibited 93% of the bacterial cultures. Furthermore, bacteria cultured from different locations (corals vs. macroalgae vs. coral surfaces exposed to macroalgal extracts) responded differently to algal extracts. These results reveal that extracts from macroalgae and cyanobacteria have species-specific effects on the composition of coral-microbial assemblages, which in turn may increase coral host susceptibility to disease and result in coral mortality.

Evaluation of the toxicity of Streptomyces aburaviensis (R9) extract towards various agricultural pests

The dichloromethane extract of culture filtrate from Streptomyces aburaviensis R9 was evaluated using various rapid bioassays to determine potential inhibitory effects towards phytopathogenic fungi (Colletotrichum acutatum, C. fragariae, C. gloeosoprioids, Botrytis cinerea, Fusarium oxysporum, Phomopsis viticola and P. obscurans), fish bacterial pathogens (Edwardsiella ictaluri and Flavobacterium columnare), a green alga (Selenastrum capricornutum), plant seeds [Bent grass (Agrostis sp.) monocot and lettuce (Lactuca sativa) dicot] and 2-methylisoborneol (MIB)-producing cyanobacteria (Planktothrix perornata and Pseudanabaena sp.). The dichloromethane extract showed selective inhibition against the cyanobacterium P. perornata, with a lowest-complete-inhibition concentration (LCIC) of 10 mg/L and lowest-observed-effect concentration (LOEC) of 10 mg/L while LCIC and LOEC values were 100 mg/L when tested against S. capricornutum. This extract also showed slight meristematic cytogenic necrosis at 200 mg/L towards germinated seeds of both test plants. The compounds were not very toxic towards the channel catfish (Ictalurus punctatus) pathogenic bacteria E. ictaluri and F. columnare. Preliminary evaluation of the extract toward C. acutatum, C. fragariae and C. gloeosoprioids using TLC bioautography revealed moderate activity. However, further evaluation of the extract using a microtiter plate bioassay determined that inhibition was strongest against C. acutatum and C. fragariae, though this inhibitory activity diminished at 72 hours and was moderately less active than the commercial fungicides azoxystrobin and captan when comparing 1 - 100 mg/L levels at 48 hours.

Adulticidal and larvicidal activity of Beauveria bassiana and Metarhizium anisopliae against housefly, Musca domestica (Diptera: Muscidae), in laboratory and simulated field bioassays

The susceptibility of the adult and larval stage of housefly, Musca domestica L. (Diptera: Muscidae), to two entomopathogenic fungi, Metarhizium anisopliae (Metsch.) Sor. and Beauveria bassiana (Bals.) Vuill., was evaluated under laboratory and simulated field bioassays. Bioassays on adult houseflies were carried out at different conidial concentrations ranging from 10(3) to 10(9) conidia/ml in petri plate and minichamber assays. Absolute mortality was observed within 4-5 days at all the concentrations tested. M. anisopliae was found to be more effective with LC(50) of 6.75 × 10(7) conidia/ml compared with 1.21 × 10(8) conidia/ml of B. bassiana in petri plate bioassay. Similar trend was observed in minichamber bioassay. Larvicidal activity evaluated through petri plate bioassay also indicated that M. anisopliae was more effective larvicide with LC(50) of 4.1 × 10(8) conidia/ml as against 3.31 × 10(9) conidia/ml of B. bassiana. Larvicidal activity was further evaluated in simulated field condition of decaying waste matrix using dry conidial formulations (10(8) conidia/g) of both the fungi. Larval mortality obtained in this assay was 43% (B. bassiana) and 63% (M. anisopliae). Remarkably better performance of M. anisopliae as an adulticidal and larvicidal agent over B. bassiana in laboratory bioassays as well as simulated field conditions suggests that it may have good potential to become part of an integrated housefly control program.

Bacteriocin-like substance from Bacillus amyloliquefaciens shows remarkable inhibition of Acanthamoeba polyphaga

The effectiveness of a bacteriocin-like substance (BLS) produced by Bacillus amyloliquefaciens was tested against Acanthamoeba polyphaga strains, and its cytotoxic potential on Vero cells was investigated. Amebicidal activity of the purified BLS was tested by plate bioassays with concentrations ranging from 12.5 to 6,400 AU mL(-1). Damage to A. pholyphaga cells was monitored using an inverted microscope and counted in a Fuchs-Rosenthal chamber after 24, 48, and 72 h. According to the results obtained, the BLS showed remarkable amebicidal and amebostatic effect on A. polyphaga and showed no cytotoxicity on the Vero cells. These results may have great relevance in the development of new acanthamoebicidal compounds.

Phytochemical screening and antibacterial activity of Parinari curatellifolia stem extract

Microorganisms are increasingly developing resistance against commonly used antimicrobial agents, and the use of herbs in the treatment of diseases is becoming widely accepted. The aqueous and organic solvent extract of the stem of Parinari curatelifolia were screened for phytochemical and antibacterial activity by hole in plate bioassay procedure. The bacterial activity of aqueous extract of this plant was carried out against several bacteria Pseudomonas aeruginosa,Salmonella typhi, Klebsella spp., Bacillus Subtilis and Staphylococcus aureus.Phytochemical constituents present in the extract were found to include saponins, balsams, carbohydrate, alkaloids tannins, cardiac glycosides, flavonoids, digitalis glycosides, phenol, terpenes and steroids. Water (w) fraction at 2.8 g/100 ml showed significant (P < 0.05) inhibitory activity against all the species of microorganism tested in this study and most effective against S. aureus andKlebsiella spp. (P < 0.05). Methanol Extract (ME) fraction at the same concentration was effective (P < 0.05) on B. subtilis and P. aeruginosa. Diethyl and n-hexane extract did not show any antimicrobial activity (P < 0.05). The results shows that different solvent extracts of same plant may have different pharmacological properties. Key words: Parinari curatellifolia stem extract, phytochemical constituents, antibacterial activity, zone of inhibition.

Chemical defenses against generalist fish predators and fouling organisms in two invasive ahermatypic corals in the genus Tubastraea

AbstractThe introduction of exotic marine species has caused considerable economic and ecological damage around the world. Some of these species possess adaptations such as secondary metabolites which facilitate their successful invasion into new areas. In this study we tested two hypotheses: (i) crude extracts produced by two invasive hard corals Tubastraea coccinea and Tubastraea tagusensis, introduced to the southwest Atlantic, inhibit predation by generalist fish; (ii) substances found in the crude extracts of both species reduce or enhance the settlement of other organisms. In an in situ palatability bioassay the extract of T. tagusensis reduced predation by generalist fish. The extracts of both species of coral showed species‐specific effects on fouling organisms in in situ gel plate bioassays. Control and extract plates had similar total cover, although plates with extracts had significantly fewer Cladophora sp., and Lithophyllum sp. was only detected on control gels. In contrast, a hydrozoan was only found on plates with extracts and overall community ordination demonstrated clear differences in the fouling communities between treatments. These results demonstrate that both coral species have chemical substances which can bring about a negative interaction with potential fish predators and competitors such as algal and invertebrate foulers and the advantage gained could explain their success in invading new regions to the detriment of local fauna and flora.

Variation inPseudonocardiaantibiotic defence helps govern parasite‐induced morbidity inAcromyrmexleaf‐cutting ants

Host-parasite associations are potentially shaped by evolutionary reciprocal selection dynamics, in which parasites evolve to overcome host defences and hosts are selected to counteract these through the evolution of new defences. This is expected to result in variation in parasite-defence interactions, and the evolution of resistant parasites causing increased virulence. Fungus-growing ants maintain antibiotic-producing Pseudonocardia (Actinobacteria) that aid in protection against specialized parasites of the ants' fungal gardens, and current evidence indicates that both symbionts have been associated with the ants for millions of years. Here we examine the extent of variation in the defensive capabilities of the ant-actinobacterial association against Escovopsis (parasite-defence interactions), and evaluate how variation impacts colonies of fungus-growing ants. We focus on five species of Acromyrmex leaf-cutting ants, crossing 12 strains of Pseudonocardia with 12 strains of Escovopsis in a Petri plate bioassay experiment, and subsequently conduct subcolony infection experiments using resistant and non-resistant parasite strains. Diversity in parasite-defence interactions, including pairings where the parasites are resistant, suggests that chemical variation in the antibiotics produced by different actinobacterial strains are responsible for the observed variation in parasite susceptibility. By evaluating the role this variation plays during infection, we show that infection of ant subcolonies with resistant parasite strains results in significantly higher parasite-induced morbidity with respect to garden biomass loss. Our findings thus further establish the role of Pseudonocardia-derived antibiotics in helping defend the ants' fungus garden from the parasite Escovopsis, and provide evidence that small molecules can play important roles as antibiotics in a natural system.

N-Acylated homoserine lactone production and involvement in the biodegradation of aromatics by an environmental isolate of Pseudomonas aeruginosa

N-Acyl homoserine lactone (AHL) is a widespread quorum sensing signal molecule in Gram-negative bacteria and has an important role in many biological processes. However, it is still poorly understood whether or not AHL is present in pollutant treatment processes and further, what its role is in biodegradation processes. In this work, an environmental isolate of Pseudomonas aeruginosa CGMCC 1.860 that is an aromatic degrader and AHL producer was selected. The AHL plate bioassay indicated that AHL was produced by this strain during biodegradation of aromatic compounds including phenol, benzoate, p-hydroxy-benzoate, salicylate, and naphthalene. The AHLs were identified as N-butyryl- l-homoserine lactone (BHL) and N-hexanoyl- l-homoserine lactone (HHL) by using thin layer chromatography (TLC) and high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (HPLC–APCI-MS/MS) analyses. Furthermore, phenol biodegradation was improved by exogenously added AHL extracts or by endogenously over-produced AHLs, repressed by abolishment of AHLs production, and not affected by the addition of extracts without AHLs. The results indicated that AHL was involved in the process of biodegradation of pollutants.

The Allelopathic Potential of Kudzu (Pueraria montana)

We explored the allelopathic potential of kudzu as a function of its phenolics. Aqueous and methanol extracts of different kudzu organs (leaf, stem, root, and seed) were assayed for allelopathy with the use of lettuce and radish seeds. Both leaf and root extracts significantly inhibited all of the measured germination indices (total germination, speed of germination, and coefficient of the rate of germination) (all P &lt; 0.01). When treated with leaf extract, the total germination of both species was ∼ 20% less than the control. Furthermore, the leaf extract significantly reduced the speed of germination to 38 and 53% that of the lettuce and radish controls, respectively. Lettuce and radish seeds soaked in leaf and root extracts for 24 h imbibed less water (∼ 30% for both species) than those soaked in distilled water (control), suggesting that a reduction of water imbibition might be one of the mechanisms of germination retardation. Stem and seed extracts affected neither the water uptake nor the germination indices of radish and lettuce seeds. Kudzu leaves and roots contain higher amounts of total phenolics (P = 0.001) and soluble phenolics (P = 0.005) than stems and seeds, consistent with the results of the germination bioassays. In agar plate bioassays, both litter and rhizosphere soil had phytotoxic effects on the radicle growth of radish (P = 0.003) and perennial ryegrass (P = 0.001) seedlings. Perennial ryegrass and cobbler's pegs seedlings grown on leaf and root leachate-amended soil gave ∼ 40% shorter roots and shoots and ∼ 50% less dry weight than those grown in leachate-free soil. Kudzu litter was incorporated and incubated in soil for 6 wk, at which point the soluble phenolics in the soil solution were at a level (&gt; 150 ppm) considered to be allelopathic.

Monoamine oxidase inhibition by Rhodiola rosea L. roots

Aim of the study Rhodiola rosea L. (Crassulaceae) is traditionally used in Eastern Europe and Asia to stimulate the nervous system, enhance physical and mental performance, treat fatigue, psychological stress and depression. In order to investigate the influence of Rhodiola rosea L. roots on mood disorders, three extracts were tested against monoamine oxidases (MAOs A and B) in a microtitre plate bioassay. Materials and methods Methanol and water extracts gave the highest inhibitory activity against MAOs. Twelve compounds were then isolated by bioassay-guided fractionation using chromatographic methods. The structures were determined by 1H, 13C NMR and HR-MS. Results The methanol and water extracts exhibited respectively inhibitions of 92.5% and 84.3% on MAO A and 81.8% and 88.9% on MAO B, at a concentration of 100 μg/ml. The most active compound (rosiridin) presented an inhibition over 80% on MAO B at a concentration of 10 −5 M (pIC 50 = 5.38 ± 0.05). Conclusions The present investigation demonstrates that Rhodiola rosea L. roots have potent anti-depressant activity by inhibiting MAO A and may also find application in the control of senile dementia by their inhibition of MAO B.

Occurrence of Saccharomyces cerevisiae killer yeasts in wine-producing areas of Greece

The occurrence of the killer character was studied in 840 Saccharomyces cerevisiae isolates at the end of spontaneous fermentations in four wine-producing regions of Greece, Mantinia and Nemea in Peloponnese and the islands Limnos and Santorini. The incidence of killer strains varied from one region to another. Sensitive and neutral strains were also found among the S. cerevisiae strains. Using the plate bioassay at pH 4.5 two different killer phenotypes were detected among the killer isolates which differed in their degree of killer activity. They were designated as SK (strong killer) and WK (weak killer). The proportion of SK to WK phenotypes differed from one area to another. All killer isolates were assayed for expression of killer activity also at pH 3.5. The lower pH decreased the killer activity of all isolates and changed the proportion of SK to WK phenotypes. The percentage of WK phenotypes increased in all cases and some killer isolates lost their killer activity completely, but generally the killer activity remained significant, especially in the area of Mantinia where the SK phenotype remained dominant at the low pH found in musts and wine.

Rapid quantifiable assessment of nutritional parameters influencing pediocin production by Pediococcus acidilactici NRRL B5627

A direct plate bioassay procedure was applied for rapid and quantifiable assessment of the influence of various nutritional parameters on pediocin production by Pediococcus acidilactici NRRL B5627. Solid-state cultivation of the microorganism was done on MRS-based media over 3-and 6-hours incubation periods. Nutritional parameters assessed included the carbon source (glucose, sucrose, fructose, galactose, glycerol), and various salts (NH 4PO 4, CaCl 2, KH 2PO 4, MnSO 4 · H 2O). Glucose was found to be the optimal carbon source while glycerol exhibited the most suppressive effect. Using glucose as the carbon source, addition of various salts, in amounts used in liquid media commonly applied in the cultivation of the pediococci, was assessed with respect to bacteriocin production on a per cell basis. Experimental data obtained showed that several nutritional parameters repress pediocin production by P. acidilactici, while the direct plate assay proved to be a good pilot assay prior to conducting more intensive kinetic analysis in liquid cultivation.

Impact of allelopathic rice seedlings on rhizospheric microbial populations and their functional diversity

Field performance of rice allelopathic potential is indirectly regulated by the microflora in the rhizosphere. The present study aimed to investigate the dynamics of microbial populations and their functional diversities in the seedling rhizospheres of rice cultivars with varied allelopathic activities by employing agar plate bioassay, fumigation and BIOLOG analysis. Rice cultivars significantly affected the microbial carbon content in their associated rhizospheric soil. The microbial carbon contents were ranked in a decreasing order as Iguape Cateto (441.0 mg·kg –1) > IAC47 (389.7 mg·kg –1) > PI312777 (333.2 mg·kg –1) > Lemont (283.8 mg·kg –1) with the nil-rice control soil of 129.3 mg·kg –1. Similarly, the respiration rate of the soils was 1.404, 1.019, 0.671 and 0.488 μgC·g –1· h –1 for PI312777, Iguape Cateto, IAC47 and Lemont, respectively. The respiration rate was only 0.304 μ gC·g –1·h –1 for the control soil. The microbial flora in the rhizospheric soil of different rice cultivars was dominated by bacteria (58.4%–65.6%), followed by actinomycete (32.2%–39.4%) and fungi (2.2%–2.8%). BIOLOG analysis showed that the value of Average Well Color Development (AWCD) differed significantly among rice cultivars. It was always the highest in the rhizospheric soil of the strongly allelopathic rice cv. PI312777, and the lowest in the rhizospheric soil of the poorly allelopathic rice cv. Lemont. The AWCD value reached the maximum in all the sampled soils after 144 hours of incubation. The AWCD values from the rhizospheric soils of PI312777, IAC47, Iguape Cateto and Lemont were 1.89, 1.79, 1.60 and 1.43 times higher than that of the control soil. Principal Component Analysis (PCA) identified 3 principal component factors (PCF) in relation to carbon sources, accounting for 70.1%, 11.3% and 7.0% of the variation, respectively. 19 categories of carbon sources were significantly positively correlated to the 3 principal components. Phenolic acids, carbohydrates, amino acids and amides were significantly correlated to the principal component 1, phenolic acids, carbohydrates and fatty acids to the principal component 2, and carbohydrates and hydroxylic acids to the principal component 3. Amino acids and amides were the two main carbon sources separating the 3 principal component factors. In addition, the total microbial population in the rhizospheric soil was significantly positively correlated with AWCD, microbial biomass carbon, microbial respiration and Shannon index. There was a significantly positive correlation between the total microbial population and the inhibition rate (IR) on the root length of lettuce owing to the different allelopathic activities of the rice cultivars. These results suggest that changes in microbial population, activity and functional diversity in the rhizospheres are highly cultivar-dependent. These changes might play an important role in governing the rice allelopathic activity in the field.

Analysis of Bioavailable Arsenic in Rice with Whole Cell Living Bioreporter Bacteria

A multiwell plate bioassay was developed using genetically modified bacteria (bioreporter cells) to detect inorganic arsenic extracted from rice. The bacterial cells expressed luciferase upon exposure to arsenite, the activity of which was detected by measurement of cellular bioluminescence. The bioreporter cells detected arsenic in all rice varieties tested, with averages of 0.02-0.15 microg of arsenite equivalent per gram of dry weight and a method detection limit of 6 ng of arsenite per gram of dry rice. This amounted to between approximately 20 and 90% of the total As content reported by chemical methods for the same sample and suggested that a major proportion of arsenic in rice is in the inorganic form. Calibrations of the bioassay with pure inorganic and organic arsenic forms showed that the bacterial cells react to arsenite with highest affinity, followed by arsenate (with 25% response relative to an equivalent arsenite concentration) and trimethylarsine oxide (at 10% relative response). A method for biocompatible arsenic extraction was elaborated, which most optimally consisted of (i) grinding rice to powder, (ii) mixing with an aqueous solution containing pancreatic enzymes, (iii) mechanical shearing, (iv) extraction in mild acid conditions and moderate heat, and (v) centrifugation and pH neutralization. Detection of mainly inorganic arsenic by the bacterial cells may have important advantages for toxicity assessment of rice consumption and would form a good complement to total chemical arsenic determination.

IMP-4 and OXA β-lactamases in Acinetobacter baumannii from Singapore

To compare the incidence of carbapenemase genes in Acinetobacter baumannii between two time periods. We studied 114 isolates of imipenem-resistant A. baumannii collected over two 5 month periods (in 1996 and 2001). Isolates showing carbapenemase activity by plate bioassay were screened for carbapenemase genes using PCR. Chromosomal DNA from strains carrying carbapenemase genes was subjected to PFGE after digestion with ApaI. The incidence of imipenem-resistant A. baumannii in our hospital rose from 1.1 per 1000 admissions in 1996 to 2.3 per 1000 admissions in 2001. However, the number of carbapenemase-producing A. baumannii rose only slightly in 2001 (0.8 per 1000 admissions) compared to 1996 (0.5 per 1000 admissions). Of 44 isolates with carbapenemase activity, 4 isolates carried bla(IMP-4), 5 carried bla(OXA-58), and 40 carried bla(OXA-23). In addition, most isolates carried a bla(OXA-51)-type beta-lactamase gene. All strains with bla(IMP-4), also carried bla(OXA-58) and bla(PSE-1), but not bla(OXA-51)-type beta-lactamase genes. PCR analysis repeated on seven recent isolates of susceptible A. baumannii showed only the presence of bla(OXA-51)-type beta-lactamase genes. A total of five novel bla(OXA-51)-type beta-lactamase genes (bla(OXA-88),-91,-93,-94, and -95) and one new bla(OXA-58)-type beta-lactamase gene (bla(OXA-96)) were found. The incidence of carbapenemase genes did not vary significantly between the two study periods. There is a wide diversity of OXA genes in A. baumannii in Singapore. The most common carbapenemase gene found in our study was bla(OXA-23).

Two neuropeptides from synganglia of the hard tick, Ixodes sinensis (Acari: Ixodidae)

Two neuropeptides were isolated from synganglia (central nervous system) of the hard tick, Ixodes sinensis. Their primary sequences were established as Leu-Val-Val-Tyr-Pro-Trp-Thr-Lys and Trp-Glu-Lys-Leu-Gly-Ser-Met-Glu-Thr-Leu. By hot plate bioassay, neuropeptide a displayed strong antinociceptive effect in mice by a dose-dependent behavior, while neuropeptide b had some relaxant effects on the isolated rat strip. These neuropeptides might be involved in down-regulating the host’s defensive reaction.

Impacts of the pesticides imidacloprid, propargite, and pymetrozine on Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae): IOBC and life table assays

The effects of imidacloprid, propargite, and pymetrozine on the common green lacewing, Chrysoperla carnea (Stephens) were investigated in the laboratory, using the IOBC-system and the life table response experiment. Residual glass plate bioassays were carried out using two-day-old larvae at the Iranian maximum field recommended rate of each commonly used pesticide. All three tested pesticides produced significant adverse effects on pre-imaginal survival (p < 0.01). Imidacloprid had no significant effect on fecundity, but propargite and pymetrozin caused significant reductions (p < 0.05). According to the IOBC classification, imidacloprid was found to be harmless (E = 27.44%); propargite (E = 49.78%) and pymetrozine (E = 66.9%) were slightly harmful. Life table assays revealed that imidacloprid and propargite had no significant effects on the intrinsic rate of natural increase, while pymetrozine caused a 34% reduction in rm value (p < 0.05). Propargite was non-toxic to C. carnea under the tested conditions. In the case of imidacloprid, using a glass plate as test substrate led to␣underestimation of its effects. The life table assay showed more adverse effects of pymetrozine than the IOBC method.

Nematicidal activity of medicinal plant extracts and two cinnamates isolated from Kaempferia galanga L. (Proh Hom) against the pine wood nematode, Bursaphelenchus xylophilus

Abstract Extracts from 40 medicinal plant species in 27 families were tested for their nematicidal activity against the pine wood nematode, Bursaphelenchus xylophilus, in 96-well plate bioassays. Responses varied with plant material and concentration. Extracts of Acorus gramineus (Araceae), Asiasarum sieboldi (Aristolochiaceae), Illicium verum (Illicaiceae) and Kaempferia galanga (Zingiberaceae) showed nematicidal activity against male, female and juvenile nematodes of B. xylophilus at 2000 μg ml−1. Among test plant species, Kaempferia galanga showed the most potent nematicidal activity. As K. galanga was the most nematicidally active of the 40 species, the extracts were chromatographically fractionated and two cinnamates, ethyl trans-cinnamate and ethyl p-methoxycinnamate, were discovered to be responsible for much of the activity. The nematicidal activity of ethyl trans-cinnamate and ethyl p-methoxycinnamate was 100% at 60 μg ml−1.

Fusarium root rot of coneflower seedlings and integrated control using Trichoderma and fungicides

Fusarium root rot (Fusarium spp.) is one of the most important seedling diseases of coneflower (Echinacea spp.) in Alberta greenhouses. Effects of microbial antagonists (Trichoderma spp.) and fungicides, including difenoconazole, fludioxonil, and a mixture of fludioxonil, metalaxyl and difenoconazole, on the management of this disease, were investigated in Alberta. Twenty Trichoderma isolates demonstrated antagonistic activity to Fusarium in agar plate bioassays, with inhibition rates ranging from 44 to 65%. Some Trichoderma isolates significantly ( p < 0.05) reduced disease incidence and severity on seedlings in greenhouse experiments. An in vitro bioassay indicated that difenoconazole and the mixture equally inhibited the growth of both Fusarium and Trichoderma, but, while fludioxonil strongly inhibited the growth of Fusarium, it had little effect on Trichoderma, according to the dose--response models developed ( p < 0.01, R2= 0.902-0.998). Two Trichoderma isolates, T1 and T13 were applied singly or in combination with a low rate of fludioxonil in greenhouse evaluations. The results suggested that fludioxonil and Trichoderma could be integrated into a disease management program for fusarium root rot in coneflower.

Tandemly Duplicated Acyl Carrier Proteins, Which Increase Polyketide Antibiotic Production, Can Apparently Function Either in Parallel or in Series

Polyketide biosynthesis involves the addition of subunits commonly derived from malonate or methylmalonate to a starter unit such as acetate. Type I polyketide synthases are multifunctional polypeptides that contain one or more modules, each of which normally contains all the enzymatic domains for a single round of extension and modification of the polyketide backbone. Acyl carrier proteins (ACP(s)) hold the extender unit to which the starter or growing chain is added. Normally there is one ACP for each ketosynthase module. However, there are an increasing number of known examples of tandemly repeated ACP domains, whose function is as yet unknown. For the doublet and triplet ACP domains in the biosynthetic pathway for the antibiotic mupirocin from Pseudomonas fluorescens NCIMB10586 we have inactivated ACP domains by inframe deletion and amino acid substitution of the active site serine. By deletion analysis each individual ACP from a cluster can provide a basic but reduced activity for the pathway. In the doublet cluster, substitution analysis indicates that the pathway may follow two parallel routes, one via each of the ACPs, thus increasing overall pathway flow. In the triplet cluster, substitution in ACP5 blocked the pathway. Thus ACP5 appears to be arranged "in series" to ACP6 and ACP7. Thus although both the doublet and triplet clusters increase antibiotic production, the mechanisms by which they do this appear to be different and depend specifically on the biosynthetic stage involved. The function of some ACPs may be determined by their location in the protein rather than absolute enzymic activity.