Evaluation of plate count methods for determination of maximum specific growth rate in mixed microbial communities, and its possible application for diversity assessment

Two plate count methods are proposed for direct assessment of the maximum specific growth rate (microm) of bacteria in mixed communities. An estimate of microm of individual colonies is obtained by plating samples on an agar medium and determining either the time required to form macroscopically visible colonies, Tv (diameter < or =0.2 mm), or the linear radial growth rate, Kr, of single colonies. In accordance with theoretical models, a linear relationship was found between microm determined in liquid culture and 1/Tv, and between microm and Kr. Empirical relationships were established for these relationships. The time required to form a visible colony was 17 +/- 9 h longer for cells in the stationary growth phase, whereas the linear radial growth rate was not affected by the physiological state of the cells. The proposed plate count methods are simple and applicable for describing the community structure, and for estimating the frequency distribution of maximum specific growth rates in mixed communities. By using this frequency distribution it is possible to calculate diversity indexes and to assign a microbial community a position on an r/K-gradient.     

Antifungal Activities of Four Fatty Acids against Plant Pathogenic Fungi

The effect of the fatty acids linolenic acid, linoleic acid, erucic acid and oleic acid on the growth of the plant pathogenic fungi Rhizoctonia solani, Pythium ultimum, Pyrenophora avenae and Crinipellis perniciosa were examined in in vitro studies. Linolenic and linoleic acids exhibited activity against all of the fungi. However, whereas linolenic acid reduced mycelial growth of R. solani and C. perniciosa at 100 microM, the concentration had to be increased to 1000 microM before any effect on mycelial growth of P. ultimum and P. avenae was observed. Linoleic acid only reduced mycelial growth of R. solani, P. ultimum and P. avenae at 1000 microM, but led to a significant reduction in growth of C. perniciosa at 100 microM. In contrast, oleic acid had no significant effect on growth of R. solani or P. avenae, but gave significant reductions in mycelial growth of P. ultimum at 100 microM and reduced growth of C. perniciosa significantly at 1000 microM. All of the fatty acids reduced biomass production by all of the fungi significantly in liquid culture when added to the media at 100 microM. Erucic acid had no effect on fungal growth at any concentration examined. The antifungal activities exhibited by linolenic, linoleic and oleic acids may be useful in the search for alternative approaches to controlling important plant pathogens, such as those examined in this study.     

Effects of osmotic and matric potential on radial growth and accumulation of endogenous reserves in three isolates of Pochonia chlamydosporia

For the first time, the effects of varying osmotic and matric potential on fungal radial growth and accumulation of polyols were studied in three isolates of Pochonia chlamydosporia. Fungal radial growth was measured on potato dextrose agar modified osmotically using potassium chloride or glycerol. PEG 8000 was used to modify matric potential. When plotted, the radii of the colonies were found to grow linearly with time, and regression was applied to estimate the radial growth rate (mm day^?1). Samples of fresh mycelia from 25-day-old cultures were collected and the quantity (mg g^?1 fresh biomass) of four polyols (glycerol, erythritol, arabitol and mannitol) and one sugar (glucose) was determined using HPLC. Results revealed that fungal radial growth rates decreased with increased osmotic or matric stress. Statistically significant differences in radial growth were found between isolates in response to matric stress (P<0.006) but not in response to osmotic stress (P=0.759). Similarly, differences in the total amounts of polyols accumulated by the fungus were found between isolates in response to matric stress (P<0.001), but not in response to osmotic stress (P=0.952). Under water stress, the fungus accumulated a combination of different polyols important in osmoregulation, which depended on the solute used to generate the stress. Arabitol and glycerol were the main polyols accumulated in osmotically modified media, whereas erythritol was the main polyol that was accumulated in media amended with PEG. The results found that Pochonia chlamydosporia may use different osmoregulation mechanisms to overcome osmotic and matric stresses.     

Effect of ultraviolet-induced mutants of Trichoderma harzianum with altered antibiotic production on selected pathogens in vitro

Mutants of Trichoderma harzianum with altered antibiotic production were isolated using ultraviolet light mutagenesis. These included strains whose activity in a Fusarium oxysporum spore germination assay was greater than twice that of the parental strain and one that had no detectable antifungal activity. Characterisation of extracellular metabolites of these strains using thin-layer chromatography and gas-liquid chromatography showed that the strains with high activity produced only elevated levels of a 6-n-pentyl pyrone, the antibiotic produced by the parental strain, but two new antifungal compounds. One of these has been identified as an isonitrile antibiotic. The nature of the interactions of the mutants with Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum was examined in an in vitro dual-plating assay using two media. High antibiotic production by two T. harzianum strains, BC10 and BC63, did increase inhibition of hyphal growth of R. solani and P. ultimum, but there was no correlation between increased antibiotic production and colonisation ability. In some cases the increased antibiotic levels appeared to impede colonisation of F. oxysporum and R. solani by the mutants. Slow growth rate also affected colonising ability. The types of interactions showed great variability depending on the nature of the T. harzianum isolate and on the test fungus.     

Effect of osmotic potential on the growth of Gaeumannomyces graminis and Phialophora spp.

The linear growth of 10 isolates each of Gaeumannomyces graminis var. graminis, G. graminis var. tritici and Phialophora graminicola and five isolates each of G. graminis var. avenae and a lobed-hyphopodiate Phialophora sp. was studied on osmotically adjusted agar at 20 °C. While most isolates of G. graminis var. avenae ceased growing at osmotic potentials of -60 bars (1 bar = 10⁵ Pa), six out of 10 isolates of G. graminis var. tritici grew at that potential. The growth of all isolates of G. graminis var. tritici and var. avenae ceased at -70 bars. In contrast, four out of 10 isolates of P. graminicola grew at -70 bars, but all stopped growing at -80 bars. Most of the isolates of G. graminis var. graminis and the lobed-hyphopodiate Phialophora sp. grew at -70 bars while three out of 10 isolates of G. graminis var. graminis and one out of five isolates of the lobed-hyphopodiate Phialophora sp. were capable of growth at -80 bars. None of the fungi grew at -90 bars. Detailed studies of the growth of two or three isolates each of the five fungi at 10, 20, 30 and 35 °C were carried out on osmotic agar controlled by the addition of either sodium chloride or potassium chloride. In general, similar reductions in growth occurred with decreasing osmotic potential regardless of the solute used. At 10 and 20 °C., all three isolates of P. graminicola showed optimal growth at about -5 bars while the other fungi grew fastest at -1²middot; bars. At 30 °C., one isolate of the lobed hyphopodiate Phialophora sp. and two isolates each of P. graminicola, G. graminis var. tritici and G. graminis var. avenae grew optimally at osmotic potentials of -10 to -15 bars. The other isolate of the Phialophora sp. and two isolates of G. graminis var. graminis studied grew optimally at the highest potential (-1·2 bars). However, at 35 °C the last three fungi exhibited optimal growth at osmotic potentials of-10 to -20 bars. The ecological significance of these results is discussed in relation to cross-protection against the take-all fungi by the avirulent fungi.     

Osmotic, biomass, and oxygen effects on the growth rate of Fusarium oxysporum using a dissolved-oxygen-controlled turbidostat

The system described is a modified Hospodka's turbidostat. This device helps to measure the maximum growth rate of fungi in steady-state aerobic conditions with defined and independent concentration of dissolved oxygen, biomass, and substrate even unlimited. The principle consists of a turbidostat controlled by the dissolved oxygen concentration. The inlet medium pump operates when the dissolved oxygen concentration falls below the set point value. This method allows us to study independently effects of different physical and chemical variables on the maximum specific growth rate of microorganisms. A fungus, Fusarium oxysporum 47 isolated from soil, does not show a depressive effect on growth when dissolved oxygen concentration decreases to 5% and osmotic potential to -25 bars. Increasing biomass concentration in the range 0.1-1.0 g/L appears to depress markedly the maximum growth rate.     

Antifungal activity and enhancement of plant growth by Bacillus cereus grown on shellfish chitin wastes

Bacillus cereus QQ308 produced antifungal hydrolytic enzymes, comprising chitinase, chitosanase and protease, when grown in a medium containing shrimp and crab shell powder (SCSP) produced from marine waste. The growth of the plant-pathogenic fungi Fusarium oxysporum, Fusarium solani, and Pythium ultimum were considerably affected by the presence of the QQ308 culture supernatant. The supernatant inhibited spore germination and germ tube elongation of F. oxysporum, F. solani, and P. ultimum. The increase in the growth time of the fungal culture was associated with a gradual decrease in inhibition. Besides antifungal activity, QQ308 enhanced growth of Chinese cabbage. These characteristics were unique among known strains of B. cereus. To our knowledge, this is the first report on the antifungal and Chinese cabbage growth enhancing compounds produced by B. cereus.     

Effects of water potential on mycelial growth,sclerotial production,and germination of Rhizoctonia solani from potato

The effects of osmotic and matric potential on mycelial growth, sclerotial production and germination of isolates of Rhizoctonia solani [anastomosis groups (AGs) 2-1 and 3] from potato were studied on potato dextrose agar (PDA) adjusted osmotically with sodium chloride, potassium chloride, glycerol, and matrically with polyethylene glycol (PEG) 6000. All isolates from AGs 2-1 and AG-3 exhibited fastest mycelial growth on unamended PDA (−0.4 MPa), and growth generally declined with decreasing osmotic and matric potentials. Growth ceased between −3.5 and −4.0 MPa on osmotically adjusted media, and at −2.0 MPa on matrically adjusted media, with slight differences between isolates and osmotica. Sclerotium yield declined with decreasing osmotic potential, and formation by AG 2-1 and AG-3 isolates ceased between −1.5 and −3.0 MPa and −2.5 and −3.5 MPa, respectively. On matrically adjusted media, sclerotial formation by AG 2-1 isolates ceased at −0.8 MPa, whereas formation by AG-3 isolates ceased at the lower matric potential of −1.5 MPa. Sclerotial germination also declined with decreasing osmotic and matric potential, with total inhibition occurring over the range −3.0 to −4.0 MPa on osmotically adjusted media, and at −2.0 MPa on matrically adjusted media. In soil, mycelial growth and sclerotial germination of AG-3 isolates declined with decreasing total water potential, with a minimum potential of −6.3 MPa permitting both growth and germination. The relevance of these results to the behaviour of R. solani AGs in soil and their pathogenicity on potato is discussed.     

Osmotic adjustment in the filamentous fungus Aspergillus nidulans.

Aspergillus nidulans was shown to be xerotolerant, with optimal radial growth on basal medium amended with 0.5 M NaCl (osmotic potential [psi s] of medium, -3 MPa), 50% optimal growth on medium amended with 1.6 M NaCl (psi s of medium, -8.7 MPa), and little growth on medium amended with 3.4 M NaCl (psi s of medium, -21 MPa). The intracellular content of soluble carbohydrates and of selected cations was measured after growth on basal medium, on this medium osmotically amended with NaCl, KCl, glucose, or glycerol, and also after hyperosmotic and hypoosmotic transfer. The results implicate glycerol and erythritol as the major osmoregulatory solutes. They both accumulated during growth on osmotically amended media, as well as after hyperosmotic transfer, except on glycerol-amended media, in which erythritol did not accumulate. Furthermore, they both decreased in amount after hypoosmotic transfer. With the exception of glycerol, the extracellular osmotic solute did not accumulate intracellularly when mycelium was grown in osmotically amended media, but it accumulated after hyperosmotic transfer. It was concluded that the extracellular solute usually plays only a transient role in osmotic adaptation. The intracellular content of soluble carbohydrates and cations measured could reasonably account for the intracellular osmotic potential of mycelium growing on osmotically amended media.     

Influence of Culture Conditions on Growth and Survival of Conidia of Trichoderma spp. Coated on Seeds

Five Trichoderma strains were grown on rice, on vermiculite plus potato-dextrose broth (PDB), on potato-dextrose agar (PDA) or in liquid cultures supplemented with glycerol, KCl or polyethylene glycol (PEG) at -1 MPa or - 2 MPa. Conidia were coated on seeds through a methyl cellulose coating or through an industrial film-coating process. The conidial yield decreased with glycerol, KCl or PEG compared with PDB alone. The percentage viability was from 23 to 44% after methyl cellulose coating, regardless of the culture conditions for conidial production. In general, the industrial coating resulted in lower numbers of living conidia. The viability during storage was enhanced when vermiculite, rice or PDA were used as substrates for fungal growth. Nevertheless, temperature of storage was found to be more critical to spore survival than the substrate used for spore production; conidial viability on seeds did not exceed 4 months at 15 C. Solid and liquid cultures produced conidia able to control R. solani and P. ultimum when applied to seeds through industrial film coating. The level of disease suppression varied with the number of viable conidia/seed and with the culture medium used for conidial production. The three main conditions for further industrial application-high yields, longevity and biocontrol effectiveness-might be optimized by selecting the appropriate medium (liquid or solid), water potential and solutes used.     

Methods used to study Pythium oligandrum, an aggressive parasite of other fungi

Isolates of Pythium oligandrum Drechsler and P. acanthicum Drechsler behaved similarly in a range of experiments and in a manner consistent with mycoparasitism. They grew on cellulose in association with some fungi - notably Botryotrichum pilulferum Sacc. & March. and Phialophora radicicola Cain var. radicicola (IMI 187786) - and markedly reduced cellulolysis by these species; however, they had little effect on cellulolysis by Gaeumannomyces graminis (Sacc.) Arx & Olivier var. graminis and made little growth on cellulose in the presence or absence of this fungus. Pythium oligandrum and P. acanthicum also grew rapidly across plates of potato-dextrose agar precolonised by Phialophora radicicola var. radicicola, whereas isolates of P. echinulatum Matthews, P. mamillatum Meurs, P. megalacanthum de Bary, P. spinosum Sawada and P. ultimum Trow did not grow under these conditions. Precolonised agar plates were therefore used as a selective medium to isolate Pythium oligandrum and similar fungi from soils. For this, small pieces of soil organic matter were transferred to precolonised agar plates and incubated for 4–6 (-12) days, when the presence of P. oligandrum or similar fungi was assessed by their production of spiny oogonia across the host colony. The technique proved successful in isolating these fungi from a wide range of cultivated soils, even if the soils had been stored air-dry. However, boiled hemp seeds buried in soil and subsequently transferred to precolonised agar plates usually gave rise to phytopathogenic rather than mycoparasitic pythia. Pieces of wheat straw precolonised by P. oligandrum decomposed at the same rate as virgin straws or straws precolonised by P. ultimum or Mucor hiemalis Wehmer, when all were buried in nitrogen-supplemented soil. However, different cellulolytic fungi appeared to colonise straws in these respective treatments, Fusarium spp. being less common and Stachybotrys atra Corda more common in the presence of P. oligandrum than in its absence. When subsequently opposed to P. oligandrum on agar plates, Fusarium spp. appeared to be parasitised by this fungus and made very little growth across the agar, whereas S. atra grew through the colony of P. oligandrum and was clearly unaffected by the presence of this fungus. Our results demonstrate the widespread occurrence of P. oligandrum in cultivated soils in Britain and also suggest that this species might influence the activities of other soil fungi.     

PhoR/PhoP two component regulatory system affects biocontrol capability of Bacillus subtilis NCD-2

The Bacillus subtilis strain NCD-2 is an important biocontrol agent against cotton verticillium wilt and cotton sore shin in the field, which are caused by Verticillium dahliae Kleb and Rhizoctonia solani Kuhn, respectively. A mutant of strain NCD-2, designated M216, with decreased antagonism to V. dahliae and R. solani, was selected by mini-Tn10 mutagenesis and in vitro virulence screening. The inserted gene in the mutant was cloned and identified as the phoR gene, which encodes a sensor kinase in the PhoP/PhoR two-component system. Compared to the wild-type strain, the APase activities of the mutant was decreased significantly when cultured in low phosphate medium, but no obvious difference was observed when cultured in high phosphate medium. The mutant also grew more slowly on organic phosphate agar and lost its phosphatidylcholine-solubilizing ability. The suppression of cotton seedling damping-off in vivo and colonization of the rhizosphere of cotton also decreased in the mutant strain when compared with the wild type strain. All of these characteristics could be partially restored by complementation of the phoR gene in the M216 mutant.     

In vitro effects of water activity, temperature and solutes on the growth rate of P. italicum Wehmer and P. digitatum Sacc

AIMS: To evaluate the effect of water activity (a(w) 0.98-0.89, adjusted with glycerol, sorbitol, glucose, or NaCl) and temperature (5-25 degrees C) on the lag phase and radial growth rate (mm day(-1)) of the important citrus spoilage fungi, such as Penicillium italicum and Penicillium digitatum grown in potato dextrose agar (PDA) medium. To select, among models based on the use of different solutes, a model fitting accurately the growth of these species in relation to a(w) and temperature. METHODS AND RESULTS: Extensive data analyses showed for both Penicillium species a highly significant effect of a(w), temperature, solutes and their interactions on radial growth rate (P < 0.0001). Radial growth rate was inhibited and the lag phase (i.e. the time required for growth) lengthened as the a(w) of the medium decreased. NaCl appeared to causes the greatest stress on growth when compared with other nonionic solutes. Penicillium italicum stopped growing at 0.96 a(w) and P. digitatum at 0.93 a(w). Under the dry conditions where growth was observed, P. italicum grew faster than P. digitatum at low temperature and P. digitatum remained more active at ambient temperature. Multiple regression analysis applied to the square roots of the growth rates observed in the presence of each solute showed that both the 'glycerol model' and the 'sorbitol model' yielded a good prediction of P. italicum growth and the 'sorbitol model' gave an accurate fit for P. digitatum growth, offering high-quality prediction within the experimental limits described. CONCLUSIONS: Mathematical models describing and predicting, as a function of a(w) and temperature, the square root of the radial growth rate of the agents responsible for blue and green decays are important tools for understanding the behaviour of these fungi under natural conditions and for predicting citrus fruit spoilage. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of these results should contribute towards a more rational control strategy against citrus spoilage fungi.     

Comparative degradation of oomycete, ascomycete, and basidiomycete cell walls by mycoparasitic and biocontrol fungi

Fourteen fungi (primarily representing mycoparasitic and biocontrol fungi) were tested for their ability to grow on and degrade cell walls (CWs) of an oomycete (Pythium ultimum), ascomycete (Fusarium equisetii), and basidiomycete (Rhizoctonia solani), and their hydrolytic enzymes were characterized. Protein was detected in the cultural medium of eleven of the test isolates, and these fungi significantly degraded CWs over the 14-day duration of the experiment. In general, a greater level of CW degradation occurred for F. equisetii and P. ultimum than for R. solani. Fungi that degraded F. equisetii CWs were Coniothyrium minitans, Gliocladium roseum, Myrothecium verrucaria, Talaromyces flavus, and Trichoderma harzianum. Taxa degrading P ultimum CWs included Chaetomium globosum, Coniothyrium minitans, M. verrucaria, Seimatosporium sp., Talaromyces flavus, Trichoderma hamatum, Trichoderma harzianum, and Trichoderma viride. Production of extracellular protein was highly correlated with CW degradation. Considerable variation in the molecular weights of CW-degrading enzymes were detected among the test fungi and the CW substrates in zymogram electrophoresis. Multivariate analysis between CW degradation and hydrolysis of barley beta-glucan (beta1,3- and beta1,4-glucanases), laminarin (beta1,3- and beta1,6-glucanases), carboxymethyl cellulose (endo-beta1,4-glucanases), colloidal chitin (chitinases), and chitosan (chitosanases) was conducted. For F. equisetii CWs, the regression model accounted for 80% of the variability, and carboxymethyl cellulases acting together with beta-glucanases contributed an R2 of 0.52, whereas chitinases and beta-glucanases alone contributed an R2 of 0.11 and 0.12, respectively. Only 61% of the variability observed in the degradation of P. ultimum CWs was explained by the enzyme classes tested, and primarily beta-glucanases (R2 of 0.53) and carboxymethyl cellulases (R2 of 0.08) alone contributed to CW break down. Too few of the test fungi degraded R. solani CWs to perform multivariate analysis effectively. This study identified several fungi that degraded ascomyceteous and oomyceteous, and to a lesser extent, basidiomycetous CWs. An array of enzymes were implicated in CW degradation.     

Intraspecific variability in growth response to cadmium of the wood-rotting fungus Piptoporus betulinus

The intraspecific variability in growth response to cadmium (Cd) on agar media and in liquid culture was studied among fourteen strains of a wood-rotting fungus Piptoporus betulinus. The variability of Cd tolerance was found to be very high. The ED(50) ranged from 6.8 μM Cd in the most sensitive strain, up to 255.1 μM in the most resistant one. On agar media the addition of Cd to nutrient media resulted in reduction of relative growth rate and increased lag time. While the reduction of growth rate was already apparent at 10 μM Cd, the lag time was significantly increased in higher Cd concentrations. Five strains of P. betulinus failed to grow at 250 μM Cd and none grew at 500 μM metal. Biomass production in liquid culture was less sensitive to addition of Cd than the growth rate on solid media. At 100 μM Cd the radial growth rate of the mycelium was reduced to 27%, whereas the dry mass of mycelium was 77% of the respective control value. A group of four Cd-sensitive strains was found, showing low metal tolerance both on solid media and in liquid cultures. Although the isolates originated from sites with different Cd-pollution level, no correlation between level of Cd-pollution and resistance (ED(50)) was found. The growth rate of fourteen tested strains displayed lower variability than biomass production, showing that radial growth rate is more species-specific and therefore more valuable for interspecific comparisons of growth response.     

The effect of copper on the growth of wood-rotting fungi and a blue-stain fungus

The effect of copper (II) ions on the growth of three brown-rot fungi, six white-rot fungi and one blue-stain fungus in solid medium was evaluated. The fungi were grown in malt extract agar with different concentrations of copper added, and the radial growth rate was determined. At the end of the incubation period, the mycelial biomass and the media pH were determined. The white-rot and blue-stain fungus grew up to 3 mM and 6 mM copper, respectively and the brown-rot fungi were the only ones that grew up to 10 mM, with higher growth rates than those shown by the other fungi. In general, the brown-rot fungi produced greater acidification in the culture media than the white-rot fungi and blue-stain fungus, and the acidification increased when the amount of copper was increased. The biomass production for the different species, in the absence or presence of copper, was not related to the radial growth rate, and the fungal species that produced the greatest biomass amounts did not correspond to those that presented the highest growth rates. The brown-rot fungi Wolfiporia cocos and Laetiporus sulfureus and blue-stain fungus Ophiostoma sp. demonstrated greater tolerance to high copper concentrations in solid medium than the white-rot fungi, determined as radial growth rate. On the other hand, the highest biomass producers in solid medium with copper added were the white-rot fungi Ganoderma australe and Trametes versicolor and the brown-rot fungus Gloeophyllum trabeum.     

Growth responses of two phytopathogenic fungi to fernasan in culture media

The toxic effect of fernasan (containing 25% thiram) was tested on Rhizoctonia solani and Fusarium solani in liquid and agar media. The fungicide was more effective in liquid (at 100 p.p.m.) than in solid media, where 400 p.p.m. checked the growth of R. solani, but was ineffective against F. solani. Both fungi exhibited a zone of inversion growth in liquid culture whereby certain intermediate concentrations were less inhibitory than lower or subsequently higher doses. Similar reaction was recorded for F. solani alone in fungicide-containing agar media. Sclerotium formation by R. solani was highly significant, in relation to controls, at 40 p.p.m. The abundance of chlamydospores of F. solani was coupled with cessation of conidium formation increasing fernasan doses.     

Antagonism of Pythium blight of zucchini by Hypocrea jecorina does not require cellulase gene expression but is improved by carbon catabolite derepression

Toward a better understanding of the biochemical events that lead to biocontrol of plant pathogenic fungi by Hypocrea/Trichoderma spp., we investigated the importance of carbon catabolite (de)repression and cellulase formation in the antagonization of Pythium ultimum by Hypocrea jecorina (Trichoderma reesei) on agar plates and in planta. Hypocrea jecorina QM9414 could antagonize and overgrow P. ultimum but not Rhizoctonia solani in plate confrontation tests, and provided significant protection of zucchini plants against P. ultimum blight in planta. A carbon catabolite derepressed cre1 mutant of H. jecorina antagonized P. ultimum on plates more actively and increased the survival rates of P. ultimum-inoculated zucchini plants in comparison with strain QM9414. A H. jecorina mutant impaired in cellulase induction could also antagonize P. ultimum on plates and provided the same level of protection of zucchini plants against P. ultimum as strain QM9414 did. We conclude that cellulase formation is dispensable for biocontrol of P. ultimum, whereas carbon catabolite derepression increases the antagonistic ability by apparently acting on other target genes.     

萝卜抗真菌蛋白1（Rs-AFP1)在大肠杆菌中的融合表达 及其对大丽轮枝菌抑制活性的研究

The Raphanus sativus L. antifungal protein 1 (Rs-AFP1) gene was isolated by polymerase chain reaction (PCR). The complete open reading frame and the fragment encoding the putative mature protein were inserted into the prokaryotic expression vector pET-32b(+), respectively. Subsequent expression showed that the Rs-AFP1 was produced in E. coli as a 27 kD fusion protein only when the N-terminal signal peptide was removed. After treatment with thrombin to remove part of the N-terminal His.tag sequence, the bacterially expressed Rs-AFP1 was used for fungal growth inhibition assay which was conducted on Verticillium dahliae Kleb., a soil-born fungus causing the cotton wilt disease. Results showed that, in the liquid medium, the Rs-AFP1 fusion protein at a concentration of 0.3 g/L clearly inhibited the growth of V. dahliae and the germination of spores. Thus the bacterially expressed fusion protein had the antifungal activity against V. dahliae.