STIMULATORY PROPERTIES OF FILTRATE FROM THE GREEN ALGA HORMOTILA BLENNISTA. II. FRACTIONATION OF FILTRATE1,2

This investigation reports on the fractionation of filtrate from the green alga Hormotila blennista known to contain autostimulatory properties. Acid, basic, and volatile acid filtrate extracts reduced the lag time of H. blennista at low concentrations. Whole filtrate did not express those lag time reducing capacities which were demonstrated in filtrate extracts. Glycolic acid was identified in both the acid and volatile acid extracts. Growth rate stimulation could not be demonstrated with any filtrate extract. Stimulatory properties of filtrate were shown to be dialyzable and heat labile. It was suggested that heat-labile low molecular weight organic extracellular products are responsible for the growth rate stimulatory property of filtrate. Although dialysis and heat treatments of filtrate removed growth rate stimulation, filtrate properties capable of extending final population levels were retained. High molecular weight heat-stable extracellular products appear to be at least partially responsible for these extended growth levels.     

Importance of Nutrient Competition and Allelopathic Effects in Suppression of the Green Alga Scenedesmus obliquus by the Macrophytes Chara, Elodea and Myriophyllum

Possible allelopathic effects of substances released from the macrophytes Chara globularis, Elodea canadensis, Myriophyllum spicatum on the common green alga Scenedesmus obliquus were tested in the laboratory with plastic plants and untreated medium as controls. A two-phase approach was used in which first the effects of physical presence of plants was studied (phase I) followed by the effects of plant culture filtrates (phase II). In the presence of plastic plants growth was reduced only marginally, but strong growth inhibition of Scenedesmus occurred in the physical presence of all macrophytes. In contrast, filtrates from Chara had no growth inhibitory effect on Scenedesmus. Myriophyllum filtrate reduced particle-based growth rate by 7% compared to filtration controls, while Elodea culture filtrate reduced volume-based growth by 12%, chlorophyll-based growth by 28% and particle-based growth by 15%. Photosystem II-efficiency of Scenedesmus was reduced in all three macrophyte treatments in phase I, but not in filtrates from macrophyte cultures (phase II). Thus, while enzyme activity or other physiological aspects may have been affected, the current study yielded no proof for allelopathically active compounds being directed at photosynthesis. Mean particle volume (MPV) of Scenedesmus was not influenced by macrophyte exudates and cultures remained dominated by unicells. The strong growth inhibitory effects found for Scenedesmus in the physical presence of macrophytes, but not in plastic controls, and no or weaker response in nutrient-enriched filtrates, suggest nutrient competition was a more powerful driving factor than allelochemicals. However, the experimental design does not exclude disappearance of allelochemicals during the filtration process.     

The possible involvement of cytokinins in the pathogenicity of Helminthosporium maydis

Green islands/infection sites recorded higher cytokinin activity than surrounding tissue as well as non-inoculated tissue. This activity in infected areas increased with time of incubation while in tissue surrounding the green islands and non-inoculated tissue, cytokinin activity decreased with time of incubation. The culture filtrate extracts of H. maydis had cytokinin activity which increased with growth of the fungus. Cytokinin activity of thin-layer Chromatographic fractions from tissue and culture filtrate extracts revealed that a major portion of the activity was confined to Rf zone 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. Presence of zeatin and zeatin riboside in tissue and culture filtrates was confirmed by high performance liquid chromatography. Cytokinin substances, such as zeatin and zeatin riboside, increase at infection sites with growth of the pathogen suggesting they may be involved in the pathogenicity of H. maydis on maize.     

Effect of culture filtrates ofXanthomonas campestris pv.pelargonii and extracts of geranium stems inoculated withX. c. pv.pelargonii on geranium callus and seedlings

Experiments were designed to determine whetherXanthomonas campestris pv.pelargonii produces a toxin which induces symptoms of bacterial blight in geranium, and is active at the cellular level. Culture filtrates ofX. c. pv.pelargonii were prepared by ethyl acetate extraction and ultrafiltration of the aqueous fraction. Culture filtrates adjusted to several pH values induced maximum disease ratings on geranium seedlings in the pH range 7–10. Geranium callus growth was significantly reduced by the filtrate in the same pH range. An active fraction could also be isolated from diseased tissue. A thin-layer chromatography-callus bioassay system detected toxin activity in the culture filtrate and in extracts of geranium stems inoculated withX. c. pv.pelargonii. Callus growth inhibition was located at R_f = 0.2–0.3 for both sources of toxin. These results suggest thatX. c. pv.pelargonii produces a toxin which causes disease symptoms, is present in diseased tissues, and inhibits callus growth. This opens the possibility of developing resistance to this pathogen by selecting cells insensitive to the toxin and regenerating plants from these cells.     

Reaction of soybean callus to culture filtrates of Phialophora gregata

Soybean [Glycine max (L.) Merr.] calli derived from susceptible and resistant soybean genotypes were exposed to the culture filtrates of pathogenic and non-pathogenic isolates of Phialophora gregata (Allington and Chamberlain) W. Gams. The rate of browning, growth and viability (measured by 2,3,5-triphenyltetrazolium chloride reduction) of the callus were determined after various exposure times to the fungus culture filtrates. Callus from susceptible Century, Cumberland, Corsoy 79, Harosoy and Clark 63 were sensitive to the culture filtrates of pathogenic isolates of P. gregata. Callus from Plant Introductions 437833 and 84946-2, when treated with fungal culture filtrates, did not develop browning and callus growth and cell viability were not decreased compared to untreated controls. Culture filtrates from non-pathogenic isolates of the fungus did not affect the growth of susceptible and resistant callus. Tobacco (Nicotiana tabacum L.) callus was not sensitive to the culture filtrate of a P. gregata isolate pathogenic to soybean. The fungal culture filtrate, based on limited evaluation, appears to be selective towards soybean callus. Based on this initial work, it appears that soybean callus bioassays have utility for evaluating soybean for resistance to P. gregata as well as assessing pathogenicity of fungus isolates.     

Antifungal potential of extracellular metabolites produced by Streptomyces hygroscopicus against phytopathogenic fungi

Indigenous actinomycetes isolated from rhizosphere soils were assessed for in vitro antagonism against Colletotrichum gloeosporioides and Sclerotium rolfsii. A potent antagonist against both plant pathogenic fungi, designated SRA14, was selected and identified as Streptomyces hygroscopicus. The strain SRA14 highly produced extracellular chitinase and β-1,3-glucanase during the exponential and late exponential phases, respectively. Culture filtrates collected from the exponential and stationary phases inhibited the growth of both the fungi tested, indicating that growth suppression was due to extracellular antifungal metabolites present in culture filtrates. The percentage of growth inhibition by the stationary culture filtrate was significantly higher than that of exponential culture filtrate. Morphological changes such as hyphal swelling and abnormal shapes were observed in fungi grown on potato dextrose agar that contained the culture filtrates. However, the antifungal activity of exponential culture filtrates against both the experimental fungi was significantly reduced after boiling or treatment with proteinase K. There was no significant decrease in the percentage of fungal growth inhibition by the stationary culture filtrate that was treated as above. These data indicated that the antifungal potential of the exponential culture filtrate was mainly due to the presence of extracellular chitinase enzyme, whereas the antifungal activity of the stationary culture filtrate involved the action of unknown thermostable antifungal compound(s).     

Some chemical and biological properties of culture filtrate ofNostochopsis lobatus

The blue-green algaNostochopsis lobatus released amino acids (glycine, serine, arginine and glutamic acid), sugars (glucose, fructose and sucrose), organic acids (oxaloacetic acid and oxalic acid) and protein in the culture medium. The quantity of extracellular amino acids and protein increased with age of culture from 30 to 120 d. All culture filtrates ofN. lobatus were highly toxic to spore formation and germination ofWestiellopsis prolifica and growth and conjugation ofSpirogyra decimina, while old-age culture filtrates inhibited total chlorophyll, heterocyst and akinete formation in the same alga and total chlorophyll and zoospore formation inChaetophora attenuata. The toxicity ofN. lobatus culture filtrate persists following high temperature treatment and at extremes of pH.     

STIMULATING EFFECT OF ANABAENA SP. (CYANOBACTERIA) EXUDATE ON PRYMNESIUM PARVUM (HAPTOPHYTA)1

Prymnesium parvum blooms have become more frequent in the south‐central United States, leading to significant ecological and economic impacts. Allelopathic effects from cyanobacteria were suggested as a mechanism that might limit the development of P. parvum blooms. This research focused on the effects of cultured cyanobacteria, Anabaena sp., on P. parvum. Over a 6‐d period, daily additions of filtrate from the senescent Anabaena culture were made to P. parvum cultures growing in log phase. All treatments, including several types of controls, showed reductions in P. parvum biomass over the course of the experiment, but the treatments receiving Anabaena filtrate were reduced to a lesser degree, suggesting that filtrate from the senescent cyanobacteria culture was beneficial to P. parvum in some way. This unexpected outcome may have resulted from stimulation of heterotrophic bacteria by the addition of Anabaena filtrate, which likely contained exudates rich in dissolved organic carbon compounds. P. parvum was then able to supplement its nutritional requirements for growth by feeding on the elevated bacteria population. These findings coupled to previous observations suggest that interactions between cyanobacteria and P. parvum in natural environments are complex, where both allelopathic and growth‐stimulating interactions are possible.     

Bio-activity of fungal culture filtrates against root-knot nematode egg hatch and juvenile motility and their effects on growth of mung bean (Vigna radiata L. Wilczek) infected with the root-knot nematode,Meloidogyne incognita

Culture filtrates of selected soil fungi, namely Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium oxysporum, Penicillium vermiculatum and Rhizopus nigricans exhibited variable response to egg hatching and mortality of the root-knot nematode, Meloidogyne incognita. Higher concentrations of the culture filtrates of all the fungi inhibited egg hatching and proved to be toxic to the juveniles of M. incognita. In addition, development of the gall and multiplication of M. incognita were also found adversely affected in varying degrees on all the plants of Vigna radiata treated with the filtrates. The culture filtrate of A. niger showed highest toxicity to the nematode than those of any other fungus tested. Soil drench application of the culture filtrates gave better seedling growth and least nematode multiplication in comparison to seed soaking treatment.     

Viability staining of soybean suspension-cultured cells and a seedling stem cutting assay to evaluate phytotoxicity of Fusarium solani f. sp. glycines culture filtrates

The phytotoxicity of culture filtrates of Fusarium solani f. sp. glycines, the fungus causing sudden death syndrome (SDS) of soybean (Glycine max), was tested with a viability stain of soybean suspension-cultured cells and a stem cutting assay of soybean seedlings. Suspension-cultured cells from a SDS-susceptible soybean cultivar were exposed to cell-free culture filtrates of F. solani f. sp. glycines or other F. solani isolates for 2, 4, 6, and 8 days and then stained with 0.1% phenosafranin. The percentage of dead soybean suspension-cultured cells was greater (P<0.001) with filtrates prepared from F. solani f. sp. glycines than from other F. solani isolates, and dead cells increased over time and with higher concentrations of culture filtrate. Cuttings of soybean seedlings with their stems immersed in culture filtrates of F. solani f. sp. glycines isolates developed SDS-like foliar symptoms, but not when immersed in filtrates of other isolates. There was a positive correlation (r=0.94, P<0.001) between soybean foliar symptom severity and percentage of stained soybean suspension-cultured cells. Both methods were used to determine the phytotoxicity of fungal culture filtrates. Received: 9 December 1997 / Revision received: 10 August 1998 / Accepted: 28 August 1998     

Influence of Fusarium wilt toxin(s) on carnation cells

The influence of culture filtrates of Fusarium oxysporum f.sp. dianthi which causes Fusarium wilt was investigated on growth and viability of carnation tissue cultures and leaf segments. Culture filtrates of avirulent race 1 of this fungus did not affect calli and leaf segments of cultivars both susceptible and resistant to Fusarium wilt. However, culture filtrates of virulent race 2 decreased viability and suppressed growth of callus of the susceptible cultivar. In contrast, callus of the resistant cultivar showed resistance to the culture filtrates. The results of these experiments may provide information on methods of selection of new wilt resistant carnation varieties.Abbreviations A270 absorbance at 270 nm - 2,4-d 2,4-dichlorophenoxyacetic acid - CF-MCD culture filtrate of 16064 grown in MCD medium - MCD medium modified Czapeck-Dox medium - MS medium basal medium of Murashige and Skoog - MW molecular weight - PD medium potato dextrose medium - TTC 2,3,5-triphenyl tetrazolium chloride     

The Rice Culture Filtrate of Bacillus cereus Isolated from Emetic-Type Food Poisoning Causes Mitochondrial Swelling in a HEp-2 Cell

Rice culture filtrates of Bacillus cereus SA-50, an emetic-type strain, produced a toxin which caused cytoplasmic vacuole formation in HEp-2 and HeLa cells. Electron microscopic observation revealed that the apparent vacuoles in HEp-2 cells seen under a light microscope were actually swollen mitochondria. The oxygen consumption of HEp-2 cells was accelerated by the addition of the rice culture filtrate as was measured with a polarographic oxymeter; a respiratory control ratio was 1.0 for control cells, while 1.4 for ones with the filtrates. The culture filtrates showed a similar effect on the isolated mouse liver mitochondria; respiratory control ratios for the mitochondria with and without the filtrates were 3.6 and 1.0, respectively. The affecting manner of the culture filtrates on the oxygen consumption of mitochondria was similar to that of 2,4-dinitrophenol, suggesting that the culture filtrate contains a toxin acting as an uncoupler of oxidative phosphorylation in mitochondria. It is likely that the culture filtrates containing the emetic toxin of B. cereus causes mitochondrial swelling with a close relationship to the uncoupling of the oxidative phosphorylation of mitochondria.     

Antagonism between Gliodadium roseum,Trichoderma harzianum,or Trichothecium roseum and Phytophthora megasperma f. sp. glydnea

The antagonism between Gliodadium roseum, Trichoderma harzianum, or Trichothecium roseum and Phytophthora megasperam f. sp. glycinea (Pmg), cause of Phytophthora rot of soybeans (Glycine max), was studied. G. roseum, T. harzianum, and 17 isolates of T. roseum were grown separately on modified Czapek-Dox medium (MCD) in the dark for 25 days at 25 °C. Culture filtrates of T. roseum at 0.5% and 20.0% concentrations inhibited mycelial growth of Pmg at 3.1% and 90.4%; and those of G. roseum at –0.7% and 44.0%; and of T. harzianum at 0.7% and 46.0%, respectively. Culture filtrates of T. roseum at 0.5% concentration inhibited zoosporangenesis of Pmg at 98.0% and at 1.0% concentration prevented it. Culture filtrates of G. roseum and T. harzianum at 5% concentration significantly (P=0.05) inhibited zoosporangenesis of Pmg, but differences were not significant from that on MCD. Culture filtrates of 17 isolates of T. roseum inhibited mycelial growth and zoosporangenesis of Pmg at different percentages with different concentrations with those of isolate 9 showing the greatest inhibition of both. Mycelial growth of most of 16 races of Pmg was prevented at 10% concentration of the culture filtrates of isolate SS-2 of T. roseum, and all 16 races, except race 6, was prevented at 20% concentration. Zoosporangenesis of all races of Pmg was prevented at 2% the culture filtrate of SS-2. Culture filtrates of SS-2 inhibited zoosporangenesis of Pmg in soil.     

Control of powdery mildew in okra using cultural filtrates of certain bio-agents alone and mixed with penconazole

This study was carried out to evaluate the culture filtrates of certain bio-agents (Epicoccum nigrum, Epicoccum minitans, Epicoccum sp., Trichoderma harzianum, Trichoderma viride and Bacillus pumilus) alone and mixed with penconazole against powdery mildew in okra. The results showed that the culture filtrate of E. nigrum induced the highest efficacy against powdery mildew relative to other treatments in both tested seasons. Moreover, culture filtrate of E.nigrum mixed with the tested fungicide gave higher efficiency against powdery mildew than use of the fungicide alone. The efficacy of the tested culture filtrates against powdery mildew due to the presence of different antifungal compounds identified by (GC–MS) analysis. This study suggests the ability of using the culture filtrates of the tested bio-agents as alternative of fungicides to control powdery mildew in okra. Furthermore, the results implied the possibility to minimise the use of fungicides by mixing with the microbial culture filtrates.     

Presence of a Root Malformation Factor in Culture Filtrates of Fusarium moniliforme var. subglutinans1

The presence of a root malformation factor, different from plant growth substances viz. NAA, GA₃, Kinetin and reduced Glutathion was demonstrated in culture filtrates of Fusarium moniliforme var. subglutinans, a fungus commonly associated with malformed mango tissues. Culture filtrate extracts of the fungus caused curling and stunting of maize and pea roots and formation of flap like growth at the tip of wheat roots, but failed to induce malformation in bean plants as reported, for malformin.     

Identification of antifungal substances secreted by Bacillus subtilis Z-14 that suppress Gaeumannomyces graminis var. tritici

Bacillus subtilis strain Z-14 has biological control activity against the take-all fungus Gaeumannomyces graminis var. tritici (Ggt). In Petri dishes, the crude extract from B. subtilis Z-14 culture filtrate reduced take-all severity in roots of wheat seedlings by 91.3% and potted plants by 69.8% compared to the Ggt-inoculated control. Treatment with the crude extract also significantly (P?<?.05) increased growth of roots’ average length, and fresh weight in comparison with those of the Ggt-inoculated control. B. subtilis Z-14 culture filtrate was relatively thermally stable with 88.2% of the antifungal activity being retained after being heated at 100°C for 30?min. Meanwhile, the antifungal activity remained almost unchanged (>95%) when the culture filtrate was exposed to a pH ranging from 3 to 8, but significantly reduced in basic conditions. This activity was not transferred to the organic solvent phase after treatment with organic extraction agents. B. subtilis Z-14 culture filtrate exhibited a broad spectrum of antifungal activities against various phytopathogenic fungi. Three homologs of iturin A (C14–16) were characterised by liquid chromatography-mass spectroscopy (LC-MS) and electrospray ionisation mass spectrometry/mass spectrometry collision-induced dissociation (ESI-MS/MS CID).     

Enhancement of biodegradation of 2,7-dichlorodibenzo-p-dioxin by addition of fungal culture filtrate

The hydrophilicity of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), a model dioxin compound, increased when incubated with the culture filtrates of several strains of fungi. The possibility that the addition of these filtrates could enhance the biodegradation of 2,7-DCDD by the white-rot basidiomycetous fungus Phanerochaete sordida YK-624 was examined. The decrease of 2,7-DCDD after 3 weeks incubation in a YK-624 culture containing these filtrates was greater (30%) than that in the culture of YK-624 alone (15%). This is the first report describing the enhancement of dioxin decrease by the addition of a fungal filtrate.     

RECOVERY RESPONSE OF HETERORHABDITIS BACTERIOPHORA AND STEINERNEMA CARPOCAPSAE TO DIFFERENT NON‐SYMBIOTIC MICROORGANISMS*

Abstract Axenic Steinernema carpocapsae Agriotos (A24) and Heterorhabditis bacteriophora H06 dauer juveniles were exposed to Spodoptera litura insect cell cultures, and the cell‐free filtrates or cells of different non‐symbiotic microorganism cultures, including Bacillus subtilis, B. thuringiensis, Pseudomonas fluorescens, Micromonospora purpurea, Rhizopus delemar, Pseudomonas aeruginosa, Streptomyces venezuelae, Streptomyces antibioticus, Penicillium citrnum, Ganoderma lucidum, Agaricus bisporus, Pleurotus ostreatus, Rhizobium legumiunosarum, and Photobacterium phosphoreum. None of these cell‐free filtrates or cultures, or insect cell culture triggered recovery of H. bacteriophora H06. However, cell‐free filtrate of P. phosphoreum induced recovery of S. carpocapsae A24, although the cell culture of this bacterium kill the A24 dauer juveniles before recovery. S. litura insect cells provided the nutrients for axenic S. carpocapsae A24 nematode growth and next generation of dauer juveniles were observed. These results further demonstrated that food signals were much more specific to H. bacteriophora than to S. carpocapsae.     

Pathogenesis of barley leaves by Helminthosporium teres I: Green island formation and the possible involvement of cytokinins

Infection sites/green islands were formed in host leaf tissue infected with drops of H. teres. They exhibited higher cytokinin-like activity, sugar and starch than their surrounding tissue and tissue under water drops. The cytokinin-like activity at the infection sites increased from 24 to 72 h of incubation. However, the cytokinin-like activity of the tissue surrounding the infection drops and the tissue under water drops fell from 24 to 72 h incubation. The culture filtrate extracts of the fungus also produced cytokinin-like activity which increased from 1 to 10 days incubation. Application of this culture filtrate extract evoked green island formation. Application of kinetin to host leaves duplicated the green island effect. Thin-layer chromatographic fractions of the tissue extracts and the culture filtrate extracts revealed that a major portion of cytokinin-like activity corresponded to zeatin and zeatin riboside. The presence of zeatin and zeatin riboside (both in tissue and culture filtrate extracts) was confirmed by high performance liquid chromatography. Increases in the amounts of cytokinin-like substances, particularly zeatin and zeatin riboside, attributed to pathogen influence are suggested to be involved in infection and pathogenicity of H. teres.