Involvement of G-alpha protein GNA3 in production of cell wall-degrading enzymes by Trichoderma reesei (Hypocrea jecorina) during mycoparasitism against Pythium ultimum

The involvement of the G-alpha protein GNA3 in the production of cell wall-degrading enzymes (CWDEs) by Trichoderma reesei during antagonism against Pythium ultimum was investigated. cAMP content was 2.8-fold higher in the T. reesei mutant gna3QL than in the parental TU-6. The gna3QL, like TU-6, inhibited the growth of P. ultimum in dual culture assays. Scanning electron microscopy showed that the gna3QL promoted more morphological alterations of P. ultimum cell wall than TU-6. In general, gna3QL produced higher activities of CWDEs than TU-6. We therefore suggest that CWDEs production during mycoparasitism by T. reesei against P. ultimum may be associated with the level of GNA3 activity.     

Influence of Sugar Beet Seed Treatment with Pseudomonas fluorescens and Low Fungicide Doses on Infection with Pythium and Plant Yield and Quality

The effect of sugar beet seed inoculation with the bacterium Pseudomonas fluorescens and treatment with the fungicides Thiram 42‐S and Dithane S‐60 with and without seed inoculation aiming to control the root decay agents Pythium ultimum and Pythium debarianum was studied during a 2‐year trial on two soil types (Mollic Gleysols and Eutric Cambisols). The influence of the treatments on parameters of sugar beet yield and quality such as root yield, sugar content, sugar in molasses, sugar yield as well as percentage of the infected and decayed plants as a consequence of parasitic oomycete infestation will be described.     

Survival, root colonisation and biocontrol capacities of Pseudomonas fluorescens F113 LacZY in dry alginate microbeads

Cells of Pseudomonas fluorescens F113 LacZY were encapsulated in alginate and their survival and ability to colonise sugar beet were evaluated. To assess survival, the formulation, composed of dry alginate microbeads of 300- to 700-μm diameter, was stored 1 year at 28±2 and 4±2°C and then tested against pathogenic fungi Pythium ultimum and Rhizoctonia solani in in vitro inhibition experiments. The same material was also used as inoculant for protection of sugar beet against Py. ultimum in microcosm experiments. The results obtained indicated that, although drying alginate beads resulted in a significant reduction of bacterial viability, the use of microbeads enabled a satisfactory level of root colonisation and protection, at least under microcosm conditions. The capability of the encapsulated cells to produce the antifungal metabolite 2,4-diacetylphloroglucinol (Phl) was not significantly affected by 12 months storage. Journal of Industrial Microbiology & Biotechnology (2001) 27, 337–342. Received 07 September 2000/ Accepted in revised form 08 May 2001     

Thiamine-Auxotrophic Mutants of Pseudomonas fluorescens CHA0 Are Defective in Cell-Cell Signaling and Biocontrol Factor Expression

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 × 10⁻⁸ M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.     

Cloning of Genes Involved in the Synthesis of Pyrrolnitrin from Pseudomonas fluorescens and Role of Pyrrolnitrin Synthesis in Biological Control of Plant Disease

A soil isolate of Pseudomonas fluorescens (BL915) was shown to be an effective antagonist of Rhizoctonia solani-induced damping-off of cotton. Investigation of the biological basis of this antagonism revealed that the strain produces pyrrolnitrin, a secondary metabolite known to inhibit R. solani and other fungi. Mutants of strain BL915 that did not produce pyrrolnitrin and did not suppress damping-off of cotton by R. solani were generated by exposure to N-methyl-N′ -nitro-N-nitrosoguanidine. A gene region that was capable of restoring pyrrolnitrin production to the non-pyrrolnitrin-producing mutants and of conferring this ability upon two other P. fluorescens strains not otherwise known to produce this compound or to be capable of suppressing damping-off caused by R. solani was isolated from strain BL915. The non-pyrrolnitrin-producing strains (mutants of BL915 and the other two P. fluorescens strains) which synthesized pyrrolnitrin after the introduction of the gene region from strain BL915 were also shown to be equal to strain BL915 in their ability to suppress R. solani-induced damping-off of cotton. These results indicate that we have isolated from P. fluorescens BL915 a gene(s) that has a role in the synthesis of pyrrolnitrin and that the production of this compound has a role in the ability of this strain to control damping-off of cotton by R. solani.     

Potential for integrated biological and chemical control of damping-off disease caused by Pythium ultimum in tomato

Pythium ultimum (Trow) is one of the main causes of damping-off disease in many parts of the world. Control of the disease depends mainly on application of chemical fungicides. However, soil treatments with fungicides are not always feasible due to economical and ecological reasons. Soil-borne, non-pathogenic bacteria of the genus Pseudomonas fluorescens with the ability to antagonise fungal phytopathogens, represent a realistic alternative to chemical fungicides and show great promise with respect to protect plant roots from fungal-induced diseases. In an attempt to find an integrated control system of damping-off disease in tomato, fungicides including azoxystrobin, metalaxyl-M and pyraclostrobin were applied alone and in combination with P. fluorescens isolate CW2. The fungicides were tested in in vitro for their antagonistic potential against P. ultimum and for compatibility with CW2. It was found that the fungicides were fungitoxic to P. ultimum, but did not inhibit the growth of the P. fluorescens. The efficacy of the fungicides alone and in combination with CW2 was also tested in greenhouse experiments against damping-off disease on tomato. Two concentrations (5 and 10?μg?ml^?1) were applied. Damping-off incidence of tomato seedlings in Humosoil^?:sand mixture infested with P. ultimum was reduced following seed treatment with the fungicides. However, the degree of control obtained varied significantly depending on the fungicide used. Combined seed treatment with P. fluorescens and the fungicides resulted in a significant improvement in disease control and improved plant growth as indicated by shoot and root dry weights. Metalaxyl-M treatment applied alone or in combination with P. fluorescens, significantly protected tomato seedlings against damping-off. Strobilurin fungicides stimulated plant growth compared to metalaxyl-M. Combined treatment of tomato seeds with strobilurin fungicides and CW2 showed a moderate to good disease control and an increase in shoot and root dry weights.     

Compost amendments enhance peat suppressiveness to Pythium ultimum,Rhizoctonia solani and Sclerotinia minor

Peat is the most common organic material used for the preparation of potting mix because of its homogeneous and favorable agronomic characteristics. However, this organic material is poorly suppressive against soilborne pathogens and fungicides are routinely used to manage damping-off diseases. In the present study, we investigated the suppressive capability of five compost – peat mixtures towards the plant pathogens Pythium ultimum, Rhizoctonia solani and Sclerotinia minor – Lepidium sativum pathosystems. For all organic media, 18 parameters were measured including enzymatic activities (glucanase, N-acetyl-glucosaminidase, chitobiosidase and hydrolysis of fluorescein diacetate), microbiological (BIOLOG® EcoPlates?, culturable bacteria and fungi), and chemical features (pH, EC, total, extractable and humic carbon, total and organic N, NH₄–N, total protein and water content). In addition, ¹³C-CPMAS-NMR spectroscopy was used to characterize the organic materials. Peat amended with composts reduced disease damping-off caused by P. ultimum, R. solani and S. minor in 60% of the mixtures and compost derived from animal manure showed the largest and most consistent disease suppression. Sterilization decreased or eliminated suppressiveness of 42.8% of the mixtures. The most useful parameters to predict disease suppression were different for each pathogen: extractable carbon, O-aryl C and C/N ratio for P. ultimum, alkyl/O-alkyl ratio, N-acetyl-glucosaminidase and chitobiosidase enzymatic activities for R. solani and EC for S. minor. Our results demonstrate that the addition of composts to peat could be useful for the control of soilborne pathogens.     

Antibiotic and Biosurfactant Properties of Cyclic Lipopeptides Produced by Fluorescent Pseudomonas spp. from the Sugar Beet Rhizosphere

Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent Pseudomonas spp. To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent Pseudomonas spp. from two different agricultural soils by using three different growth media. CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil. Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups. The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety. Production of specific CLPs could be affiliated with Pseudomonas fluorescens strain groups belonging to biotype I, V, or VI. In vitro analysis using both purified CLPs and whole-cell P. fluorescens preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi. The CLP-producing P. fluorescens strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum.     

Antagonism of Rhizoctonia spp. to Pythium oligandrum and Damping-Off Fungi

In paired cultures on corn-meal agar, Rhizoctonia solani, R. cerealis and R. fragariae caused vacuolation, disappearance of cytoplasm, and apparent lysis of hyphae of Pythium oligandrum, P. ultimum, and Aphanomyces cochlioides. Hyphae of Phoma betae were not injured by the Rhizoctonia spp. When sugar-beet seeds dressed with mycelium of R. cerealis, R. fragariae, or an isolate of R. solani nonpathogenic to sugar-beet were planted in soil naturally infested with P. ul-timum, the level of biological control of damping-off was similar to that obtained with captan dressing. In soil artificially infested with P. ultimum, biological dressings were slightly less efficacious than the chemical dressing.     

Sugar Beet-Associated Bacterial and Fungal Communities Show a High Indigenous Antagonistic Potential Against Plant Pathogens

The aim of this study was to analyze microbial communities in/on sugar beet with special focus on antagonists toward plant pathogens. For this purpose, the composition of microorganisms isolated from the rhizosphere, phyllosphere, endorhiza, and endosphere of field-grown sugar beet plants was analyzed by a multiphasic approach at three different plant development stages at six locations in Europe. The analysis of microbial communities by Single Strand Conformation Polymorphism (SSCP) of 16S/18S rRNA clearly revealed the existence of discrete microenvironment- and site-specific patterns. A total of 1952 bacterial and 1344 fungal isolates screened by dual testing for antagonism toward the pathogens Aphanomyces cochlioides, Phoma betae, Pythium ultimum, and Rhizoctonia solani resulted in 885 bacterial (=45%) and 437 fungal (=33%) antagonists. In general, the indigenous antagonistic potential was very high and influenced by (a) the location, (b) the plant developmental stage, and (3) the microenvironment. Furthermore, we showed for the first time that the antagonistic potential was highly specific for each target pathogen. The majority of antagonistic microorganisms suppressed only one pathogen (bacteria: 664 = 75%; fungi: 256 = 59%), whereas the minority showed a broad host range (bacteria: 4 = 0.5%; fungi: 7 = 1.6%). The bacterial communities harbored the highest antagonistic potential against P. ultimum, whereas the fungal communities contained more antagonists against A. cochlioides and R. solani. In contrast to their high proportion, only a low diversity of antagonists at genotypic and species level was found. Novel antagonistic species, e.g., Subtercola pratensis or Microbacterium testaceum were found in the internal part of the sugar beet body.     

Effects of Fungal Root Pathogens on the Population Dynamics of Biocontrol Strains of Fluorescent Pseudomonads in the Wheat Rhizosphere

The influences of Gaeumannomyces graminis var. tritici (which causes take-all of wheat), Rhizoctonia solani AG-8 (which causes rhizoctonia root rot of wheat), Pythium irregulare, P. aristosporum, and P. ultimum var. sporangiiferum (which cause pythium root rot of wheat) on the population dynamics of Pseudomonas fluorescens 2-79 and Q72a-80 (bicontrol strains active against take-all and pythium root rot of wheat, respectively) in the wheat rhizosphere were examined. Root infection by either G. graminis var. tritici or R. solani resulted in populations of both bacterial strains that were equal to or significantly larger than their respective populations maintained on roots in the absence of these pathogens. In contrast, the population of strain 2-79 was significantly smaller on roots in the presence of any of the three Pythium species than on noninfected roots and was often below the limits of detection (50 CFU/cm of root) on Pythium-infected roots after 40 days of plant growth. In the presence of either P. aristosporum or P. ultimum var. sporangiiferum, the decline in the population of Q72a-80 was similar to that observed on noninfected roots; however, the population of this strain declined more rapidly on roots infected by P. irregulare than on noninfected roots. Application of metalaxyl (which is selectively inhibitory to Pythium spp.) to soil naturally infestated with Pythium spp. resulted in significantly larger rhizosphere populations of the introduced bacteria over time than on plants grown in the same soil without metalaxyl. It is apparent that root infections by fungal pathogens may either enhance or depress the population of fluorescent pseudomonads introduced for their control, with different strains of pseudomonads reacting differentially to different genera and species of the root pathogens.     

An N-acetyl-β-d-glucosaminidase gene, cr-nag1, from the biocontrol agent Clonostachys rosea is up-regulated in antagonistic interactions with Fusarium culmorum

Clonostachys rosea is a widely distributed fungus that often acts as a parasite on other soil fungi. This fungus has also been reported as a potential parasite against nematodes and insects. The antagonistic activity is thought to be correlated with the secretion of cell wall-degrading enzymes, including chitinases. In this work, we identified and characterized an N-acetyl-β-d-glucosaminidase-encoding gene, cr-nag1, belonging to glycosyl hydrolase family 20, from the C. rosea strain IK726 using a degenerated primer strategy designed from conserved motifs. The complete gene, including its promoter region, was obtained by genomic walking. Southern analysis showed that cr-nag1 is present as a single copy gene in C. rosea. Phylogenetically, cr-nag1 showed the highest similarity to N-acetyl-β-d-glucosaminidase genes from other mycoparasitic fungi. Enzymatic assays and RT-PCR showed that the NAGase activity of C. rosea is specifically repressed in medium containing a high glucose content and is expressed in media containing chitin or Fusarium culmorum cell walls as sole carbon sources. Macroscopic and microscopic observations indicated that the mycelial growth of F. culmorum and Pythium ultimum were inhibited during interactions with C. rosea. High expression of cr-nag1 was found in interactions between C. rosea and F. culmorum, whereas the expression of cr-nag1 in interactions between C. rosea and P. ultimum was similar to the control. This indicates that although C. rosea secretes chitin-hydrolysing agents in order to target the cell wall of F. culmorum, it seems to use another strategy for controlling the development of the oomycete P. ultimum.     

Isolation and Antifungal and Antioomycete Activities of Aerugine Produced by Pseudomonas fluorescens Strain MM-B16

The bacterial strain MM-B16, which showed strong antifungal and antioomycete activity against some plant pathogens, was isolated from a mountain forest soil in Korea. Based on the physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, the bacterial strain MM-B16 was identical to Pseudomonas fluorescens. An antibiotic active against Colletotrichum orbiculare and Phytophthora capsici in vitro and in vivo was isolated from the culture filtrates of P. fluorescens strain MM-B16 using various chromatographic procedures. The molecular formula of the antibiotic was deduced to be C₁₀H₁₁NO₂S (M⁺, m/z 209.0513) by analysis of electron impact mass spectral data. Based on the nuclear magnetic resonance and infrared spectral data, the antibiotic was confirmed to have the structure of a thiazoline derivative, aerugine [4-hydroxymethyl-2-(2-hydroxyphenyl)-2-thiazoline]. C. orbiculare, P. capsici, and Pythium ultimum were most sensitive to aerugine (MICs for these organisms were approximately 10 μg ml⁻¹). However, no antimicrobial activity was found against yeasts and bacteria even at concentrations of more than 100 μg ml⁻¹. Treatment with aerugine exhibited a significantly high protective activity against development of phytophthora disease on pepper and anthracnose on cucumber. However, the control efficacy of aerugine against the diseases was in general somewhat less than that of the commercial fungicides metalaxyl and chlorothalonil. This is the first study to isolate aerugine from P. fluorescens and demonstrate its in vitro and in vivo antifungal and antioomycete activities against C. orbiculare and P. capsici.     

Untersuchungen zum Vorkommen von Pythium spp. im Boden

Investigations on the occurrence of Pythium spp. in soil: I. The isolation of Pythium spp., their distinction to macroscopically characteristics and their determination The aim of the present paper consisted in detection of Pythium spp. directly in the soil. This was possible by using a selective medium and by crumbling smallest particles of agar-covered soil on its surface. On the basis of simple morphologically criteria (growth patterns) this method allows to decide concerning the presence of high and less pathogen or apathogen Pythium spp. in a soil sample within 48 hours. About 700 isolates have been cultivated from hyphal tips, determinated and about 230 tested for pathogenicity to sugar beet seedlings in vitro. Most of the Pythia pathogen to sugar beet belong to P. ultimum Trow followed by P. paroecandrum Drechsler and P. debaryanum sensu Drechsler non Hesse. The taxonomically characteristics are demonstrated by figures of the three species.     

Use of Trichoderma harzianum and Gliocladium vitens for the Biological Control of Post-emergence Damping-off and Root Rot of Cucumbers Caused by Pythium ultimum

The antagonist strains Gliocladium virens G2 and Trichoderma harzianum T3 originally isolated from Pythium suppressive peat, and two benomyl-resistant strains of T. harzianum, T12B and T95, were evaluated as biological control agents of damping-off and root rot of cucumbers in sphagnum peat caused by Pythium ultimum. All strains were equally effective when applied as 1 % peat-bran preparations, whereas the effectiveness of disease control was reduced at higher concentrations of the antagonists. The two wild-type strains were also found to be effective when applied as conidial suspensions, and in this case no reduction in disease control was seen at higher concentrations. G. virens G2 and T. harzianum T12B showed antibiotic activity against P. ultimum in in vitro tests; however there were no signs of mycoparasitism of P. ultimum by any of the antagonist strains.     

Taxonomic classification of asexual isolates ofPythium ultimum based on cultural characteristics and mitochondrial DNA restriction patterns

Taxonomic characteristics of 8 isolates ofPythium ultimum and 11 isolates ofPythium in the spherical hyphal swelling (HS) group of Van der Platts-Niterink were compared. Isolates in the two groups had identical temperature growth responses and morphological features of hyphal swellings. All isolates were pathogenic on sugar beet. Attempts to cross HS isolates among themselves and with opposite mating types ofP. heterothallicum andP. sylvaticum failed. HS isolates were not induced to form antheridia when paired with isolates ofP. ultimum using a polycarbonate membrane sandwich technique. In single culture, some HS isolates formed low numbers of spherical structures encompassed by swollen hyphal masses resembling antheridia. Comparisons of restriction banding patterns ofHindIII-digested mitochondrial DNA revealed that all but 1 isolate ofP. ultimum were identical; however, the variable isolate shared 80% of the bands in common with the others. Seven of 11 HS isolates had banding patterns identical to the predominantP. ultimum pattern and 1 isolate shared 96% comigrating bands. On the basis of the number of shared characteristics, these isolates appear to beP. ultimum which have lost the ability to reproduce sexually.     

Selection of available suicide vectors for gene mutagenesis using chiA (a chitinase encoding gene) as a new reporter and primary functional analysis of chiA in Lysobacter enzymogenes strain OH11

Here, three different suicide vectors were evaluated for the possibility of performing gene mutagenesis in strain OH11 using the chiA gene (accession number: DQ888611) as a new reporter. Suicide vector pEX18GM was selected, and it was successfully applied for disruption and in-frame deletions in the chiA gene in strain OH11, which was confirmed by PCR amplification and Southern hybridization. The chiA-deletion mutant OH11-3 did not have the ability to produce chitinase on chitine selection medium. Interestingly, the chiA-deletion mutants displayed wild-type antimicrobial activity against Saccharomyces cerevisiae, Magnaporthe grisea, Phytophthora capsici, Rhizoctonia solani, Sclerotinia sclerotiorum and Pythium ultimum. Our data suggest that chitinase might not be a unique lytic enzyme in controlling S. cerevisiae, M. grisea, P. capsici, and P. ultimum. R. solani, S. sclerotiorum. Also, suicide vector pEX18GM might be explored as a potential tool for gene deletions in L. enzymogenes, which will facilitate the molecular study of mechanisms of biological control in L. enzymogenes.     

Survival and Ecological Fitness of Pseudomonas fluorescens Genetically Engineered with Dual Biocontrol Mechanisms

The antibiotic 2,4-diacetylphloroglucinol (Phl) is produced by a range of naturally occurring fluorescent pseudomonads. One isolate, Pseudomonas fluorescens F113, protects pea plants from the pathogenic fungus Pythium ultimum by reducing the number of pathogenic lesions on plant roots, but with a concurrent reduction in the emergence of plants such as pea. The genes responsible for Phl production have been shown to be functionally conserved between the wild-type (wt) P. fluorescens strains F113 and Q2-87. In this study the genes from F113 were isolated using an optimized long PCR method and a 6.7-kb gene cluster inserted into the chromosome of the non-Phl-producing P. fluorescens strain SBW25 EeZY6KX. This strain is a lacZY, km^R marked derivative of the wt SBW25 which effects biological control against the plant pathogen Pythium ultimum by competitive exclusion as a result of its strong rhizosphere-colonizing ability. We describe here the integration of the Phl antifungal and competitive exclusion mechanisms into a single strain, and the impact this has on survival and plant emergence in microcosms. The insertion of the Phl biosynthetic genes from the F113 into the SBW25 chromosome gave a Phl-producing transformant (strain Pa21) able to suppress P. ultimum through antibiotic production. The growth of Pa21 was not reduced in flask culture at 20°C compared with its parent strain. When inoculated on pea seedlings, the strain containing the Phl operon behaved similarly to the SBW25 EeZY6KX parent but did not show the tendency of the wt Phl producer F113 to cause lower pea seed emergence. Pea roots inoculated with SBW25 EeZY6KX have significantly lower indigenous populations than with F113 and the control. This is indicative of this strains strong colonising presence. Pa21, the Phl-modified strain, is able to exclude the resident population from roots to the same degree as the SBW25 EeZY6KX from which it is derived. This suggests that it has maintained its competitiveness around the root systems of plants even with the introduction of the Phl locus. Thus, strain Pa21 possesses the qualities necessary to provide effective integrated biocontrol, through maintaining both its wt trait of competitive exclusion on the plant roots, while also expressing the genes from the F113 biocontrol strain for Phl production. Interestingly, however, an additional beneficial trait appears to emerge with the strain Pa21s lowered survival competence compared with SBW25 EeZY6KX in the rhizosphere soil. With fears of the spread of genetically modified organisms and persistence in the soil, this trait may be of some ecological and commercial benefit and becomes a candidate for further investigation and possible exploitation.     

Conversion of crude chitosan to an anti-fungal protease by <Emphasis Type="Italic">Bacillus cereus</Emphasis>

Bacillus cereus AU004, isolated from soil samples, secreted a complex of hydrolytic enzymes into the culture broth when it was grown aerobically in a medium containing crude chitosan flakes. The presence of the AU004 culture supernatant substantially influenced the growths of the plant-pathogenic fungi Fusarium oxysporum, F. solani and Pythium ultimum in terms of dry weight. AU004 excreted a protease when cultivated in a medium that contained 4% (w/v) chitosan as the major nutritional source. The protease was purified by sequential chromatography and characterized as a novel extracellularly neutral protease. The protease had an Mr of 28.8 kDa. The optimal pH and temperature for protease activity were 7 and 50°C, respectively. Antifungal activity of the protease was observed using an assay based on the inhibition of spore germination and hyphal extension of the fungal Pythium ultimum. This investigation is the first report of the production of an anti-fungal protease from Bacillus spp.     

Isolation and Characterization of Actinomycete Antagonists of a Fungal Root Pathogen

By use of selective media, 267 actinomycete strains were isolated from four rhizosphere-associated and four non-rhizosphere-associated British soils. Organic media with low nutrient concentrations were found to be best for isolating diverse actinomycetes while avoiding contamination and overgrowth of isolation media by eubacteria and fungi. While all isolates grew well at pHs 6.5 to 8.0, a few were unable to grow at pH 6.0 and a significant number failed to grow at pH 5.5. Eighty-two selected isolates were screened for in vitro antagonism towards Pythium ultimum by use of a Difco cornmeal agar assay procedure. Five isolates were very strong antagonists of the fungus, four were strong antagonists, and ten others were weakly antagonistic. The remaining isolates showed no antagonism by this assay. Additional studies showed that several of the P. ultimum antagonists also strongly inhibited growth of other root-pathogenic fungi. Twelve isolates showing antifungal activity in the in vitro assay were also tested for their effects on the germination and short-term growth of lettuce plants in glasshouse pot studies in the absence of pathogens. None of the actinomycetes prevented seed germination, although half of the isolates retarded seed germination and outgrowth of the plants by 1 to 3 days. During 18-day growth experiments, biomass yields of some actinomycete-inoculated plants were reduced in comparison with untreated control plants, although all plants appeared healthy and well rooted. None of the actinomycetes significantly enhanced plant growth over these short-term experiments. For some, but not all, actinomycetes, some correlations between delayed seed germination and reduced 18-day plant biomass yields were seen. For others, plant biomass yields were not reduced despite an actinomycete-associated delay in seed germination and plant outgrowth. Preliminary glasshouse experiments indicated that some of the actinomycetes protect germinating lettuce seeds against damping-off caused by P. ultimum.