Plant–microbe interactions promoting plant growth and health: perspectives for controlled use of microorganisms in agriculture

Plant-associated microorganisms fulfill important functions for plant growth and health. Direct plant growth promotion by microbes is based on improved nutrient acquisition and hormonal stimulation. Diverse mechanisms are involved in the suppression of plant pathogens, which is often indirectly connected with plant growth. Whereas members of the bacterial genera Azospirillum and Rhizobium are well-studied examples for plant growth promotion, Bacillus, Pseudomonas, Serratia, Stenotrophomonas, and Streptomyces and the fungal genera Ampelomyces, Coniothyrium, and Trichoderma are model organisms to demonstrate influence on plant health. Based on these beneficial plant–microbe interactions, it is possible to develop microbial inoculants for use in agricultural biotechnology. Dependent on their mode of action and effects, these products can be used as biofertilizers, plant strengtheners, phytostimulators, and biopesticides. There is a strong growing market for microbial inoculants worldwide with an annual growth rate of approximately 10%. The use of genomic technologies leads to products with more predictable and consistent effects. The future success of the biological control industry will benefit from interdisciplinary research, e.g., on mass production, formulation, interactions, and signaling with the environment, as well as on innovative business management, product marketing, and education. Altogether, the use of microorganisms and the exploitation of beneficial plant–microbe interactions offer promising and environmentally friendly strategies for conventional and organic agriculture worldwide.     

Development of cyanobacterium-based biofilms and their in vitro evaluation for agriculturally useful traits

The ability of cyanobacteria to be useful as matrices for agriculturally important bacteria was evaluated. Biofilms were generated with the selected strain Anabaena torulosa after co-culturing with Azotobacter chroococcum, Pseudomonas striata, Serratia marcescens, and Mesorhizobium ciceri. The biochemical attributes were compared with individual bacterial and cyanobacterial cultures. The biofilms were characterized in terms of proteins, chlorophyll, IAA production, acetylene-reducing activity, phosphate solubilization, and antagonism towards selected phytopathogenic fungi. An enhancement in the population counts was recorded in A. torulosa–S. marcescens and A. torulosa–P. striata biofilms. The A. torulosa–A. chroococcum and A. torulosa–M. ciceri biofilms were also able to utilize new saccharides as compared to the individual cultures. Such novel biofilms with agriculturally useful traits can provide additional advantages including the broader spectrum of activity and the presence or formation of biologically active compounds; they also suggest the way to effective inoculants for sustainable and environment friendly agriculture.     

Evaluating the biochemical traits of novel Trichoderma-based biofilms for use as plant growth-promoting inoculants

Bacteria-mediated plant growth promotion is a well-established and complex phenomenon that is often achieved by the activities of more than one plant growth-promoting (PGP) trait, which may not always be present in a single organism. Biofilms developed using a combination of two organisms with useful plant growth-promoting rhizobacteria (PGPR) traits may provide a definite advantage. In this context, in vitro studies were conducted evaluating the PGP traits of novel biofilms developed using Trichoderma as matrix and agriculturally important bacteria (Azotobacter chroococcum, Pseudomonas fluorescens and Bacillus subtilis) as partners. Such biofilms exhibited higher values for various biochemical attributes as compared to the individual organisms and dual cultures. Trichoderma–Bacillus and Trichoderma–Pseudomonas biofilms exhibited enhanced antifungal activity, ammonia, indole acetic acid (IAA) and siderophore production, as compared to the other treatments. Trichoderma–Azotobacter biofilm recorded the highest nitrogenase activity and 1-aminocyclopropane-1-carboxylic (ACC) deaminase activity. The synergism in terms of the PGP traits in the biofilms revealed their promise as superior PGP inoculants.     

Changes in the physiological and agricultural characteristics of peat-based Bradyrhizobium japonicum inoculants after long-term storage

Commercial soybean inoculants processed with sterilised peat and stored at 20 °C for 1–8 years were used as experimental materials to assess the changes in the physiological activity of Bradyrhizobium japonicum after storage. Viable counts decreased and physiological characteristics of the bacterium changed during storage, with an increase in the time taken for colony appearance on a medium without yeast extract, an increase in the lag time for nodule appearance on soybean grown in glass tubes and a decrease in survival on seeds. All the inoculants produced a significant increase in grain yield in a field experiment. The percentage of efficient cells in the field (relative to the plate counts) decreased as the length of storage increased. These results suggest that the physiological activity of B. japonicum cells changes after storage. Practical implications for inoculant quality control are discussed. Received: 20 September 1999 / Received revision: 3 March 2000 / Accepted: 6 March 2000     

Effects of marine paints on microbial biofilm development on three materials

The development of biofilms of Pseudomonas aeruginosa PAO-1 was studied using modified Robbins devices. Biofilm development was measured using viable counts, acridine orange direct counts (AODC), and a colorimetric method for exopolysaccharide (EPS). Biofilms reached their maximum population 24–72 h after inoculation on coupons with no paint or on coupons coated with marine paint VC-18 without additives. Biofilms on stainless steel contained higher numbers of total cells and of viable cells than biofilms on fiberglass or aluminum. Coating the surfaces with marine paint VC-18 resulted in decreased numbers of cells on stainless steel but had little effect on numbers of cells on fiberglass or aluminum. Addition to the paint of Cu or tributyltin (TBT), the active components in two types of antifouling paints, inhibited the initial development of biofilms. However, by 72–96 h, most biofilms contained the same number of cells as surfaces without additives as shown by both viable counts and AODC. Biofilms that formed on surfaces coated with Cu- or TBT-containing paint did not synthesize more EPS, suggesting that P. aeruginosa PAO-1 does not respond to these compounds by synthesizing more EPS, which could bind the metal and protect the cells. Rather, these biofilms may contain Cu- or TBT-resistant cells. TBT-resistant cells made up 1–10% of the viable counts in biofilms on uncoated stainless steel, but in biofilms on stainless steel coated with marine paint containing TBT, TBT-resistant cells made up as much as 50% of the population. For non-coated stainless steel surfaces, Cu-resistant cells initially made up the majority of the population, but after 48 h they made up less than 1% of the population. On Cu-coated stainless steel, Cu-resistant cells predominated through 48 h, but after 48 h they comprised less than 10% of the population. These results suggest that the growth of TBT-resistant and Cu-resistant cells contributes to biofilms of P. aeruginosa PAO-1 at early stages of development but not at later stages. Received 16 December 1997/ Accepted in revised form 9 March 1998     

The effect of molybdenum on biofilm development

Little is known about the formation and effects of biofilms on stainless steel pipes in freshwater environments, particularly as they are considered as a direct replacement for copper pipes for ‘problem’ water. There is some cause for concern especially as stainless steel cannot claim the inherent biocidal potential of copper. As molybdenum is known to be leached out of stainless steel grade 316, in very small amounts, a study was set up to see if molybdenum could retard the development of biofilms. When a comparison of biofilm viable and total cell counts was made between pure molybdenum metal and stainless steel grade 304, it was found that cell counts were significantly higher (P < 0.05) on grade 304 stainless steel after 5 weeks exposure to flowing water (0.64 m s⁻¹). Molybdenum (above a concentration of 1 g L⁻¹) affected the growth rate of Acinetobacter sp, a pioneering bacterium of biofilms in potable water. Received 18 February 1998/ Accepted in revised form 17 May 1999     

MALDI-TOF MS of <Emphasis Type="Italic">Trichoderma</Emphasis>: a model system for the identification of microfungi

This investigation aimed to assess whether MALDI-TOF MS analysis of the proteome could be applied to the study of Trichoderma, a fungal genus selected because it includes many species and is phylogenetically well defined. We also investigated whether MALDI-TOF MS analysis of peptide mass fingerprints would reveal apomorphies that could be useful in diagnosing species in this genus. One hundred and twenty nine morphologically and genetically well-characterized strains of Hypocrea and Trichoderma, belonging to 25 species in 8 phylogenetic clades, were analyzed by MALDI-TOF MS mass spectrometry. The resulting peak lists of individual samples were submitted to single-linkage cluster analysis to produce a taxonomic tree and were compared to ITS and tef1 sequences from GenBank. SuperSpectra™ for the 13 most relevant species of Trichoderma were computed. The results confirmed roughly previously defined clades and sections. With the exceptions of T. saturnisporum (Longibrachiatum Clade) and T. harzianum (Harzianum Clade), strains of individual species clustered very closely. T. polysporum clustered distantly from all other groups. The MALDI-TOF MS analysis accurately reflected the phylogenetic classification reported in recent publications, and, in most cases, strains identified by DNA sequence analysis clustered together by MALDI-TOF MS. The resolution of MALDI-TOF MS, as performed here, was roughly equivalent to ITS rDNA. The MALDI-TOF MS technique analyzes peptides and represents a rough equivalent to sequencing, making this method a useful adjunct for determination of species limits. It also allows simple, reliable, and quick species identification, thus representing a valid alternative to gene sequencing for species diagnosis of Trichoderma and other fungal taxa.     

Quantification of extraradical soil mycelium and ectomycorrhizas of Boletus edulis in a Scots pine forest with variable sporocarp productivity

The availability of most edible ectomycorrhizal mushrooms depends on their natural fructification. Sporocarp formation of these fungi is linked to habitat characteristics and climate conditions, but these data alone do not explain all the trends of fungal fruiting and dynamics. It could be hypothesized that the amount of soil mycelia could also be related to the production of carpophores. Soil samples (five cylinders of 250 cm³ per plot) were taken monthly, from September to November, in five fenced permanent plots (5 × 5 m) in Pinar Grande (Soria, Spain), a Pinus sylvestris stand situated in the north of the Sistema Ibérico mountain range. Plots were chosen to establish a gradient of Boletus edulis productivity from 0 to 38.5 kg/ha year, according to the mean fresh weight of sporocarps collected during the last 10 years. B. edulis ectomycorrhizal root tips were identified in each soil sample according to its morphology and counted. DNA extractions were performed with the PowerSoil^TM DNA Isolation Kit and quantification of extraradical soil mycelium by real-time polymerase chain reaction using specific primers and a TaqMan? probe. The concentration of soil mycelium of B. edulis (mg mycelium/g soil) did not differ significantly between plots (p = 0.1397), and sampling time (p = 0.7643) within the fructification period. The number of mycorrhizal short roots per soil volume showed significant differences between the plots (p = 0.0050) and the three sampling times (p < 0.0001). No significant correlation between the number of mycorrhizas and the productivity of the plot (kg of B. edulis/ha year) was detected (p = 0.615). A statistically significant positive correlation (p = 0.0481) was detected between the concentration of mycelia of B. edulis in the soil samples and the presence of short roots mycorrhizal with B. edulis in these samples. The productivity of the plots, in terms of sporocarps produced during the last 10 years, was not correlated either with the concentration of soil mycelium or with the presence or abundance of ectomycorrhizas.     

Hydrophobicity and surface electrostatic charge of conidia of the mycoparasitic <Emphasis Type="Italic">Trichoderma</Emphasis> species

The present investigation was done to understand the fungal-fungal interactions mechanisms based on level of nonspecific adhesion of a potential fungal mycoparasite (Trichoderma) to their fungal host (Macrophomina phaseolina). The relative cell surface hydrophobicity (CSH) and cell surface electrostatic charge (CSEC) of 29 isolates of Trichoderma species, analyzed by bacterial adhesion to hydrocarbon (BATH), hydrophobic interaction chromatography (HIC), microelectrophoresis and contact angle, revealed a large degree of variability. CSH and CSEC of conidia depended on culture age, pH and temperature. Maximum CSH and CSEC were recorded in 25–28 °C range, and both declined significantly with increasing temperature. Isolate Trichoderma hazianum (Th)-23/98 expressed surface hydrophobicity at 25–28 °C and hydrophilicity at 40 °C. Surface hydrophobicity of the isolate was susceptible to various proteases (trypsin, pepsin, proteinase k and a-chymotrypsin) and inhibitors (SDS, mercaptoethanol and Triton X-100) and a significant reduction in CSH was recorded in hydrophobic conidia. Hydrophilic conidia remained more or less unaffected by such treatments. SDS-PAGE analysis of the hydrophobic and hydrophilic conidia exhibited several protein bands in the 25 to 61 kDa range. However, each protein population contained one protein that was not observed in the other population. For hydrophobic conidia, the unique protein had an apparent molecular mass of 49 kDa, while the unique protein associated with hydrophilic conidia had a molecular mass of 61 kDa. Our findings suggest that CSH and CSEC of mycoparasitic Trichoderma may contribute to non-specific adhesion on to the sclerotial surfaces of Macrophomina phaseolina that may be influenced by growth and environmental conditions.     

Evaluating the promise of Trichoderma and Anabaena based biofilms as multifunctional agents in Macrophomina phaseolina-infected cotton crop

The influence of biofilmed formulations composed of Trichoderma viride and Anabaena torulosa as matrices was investigated in Macrophomina phaseolina (Tassi) Goid.-infected cotton crop, in terms of plant growth and biocontrol parameters. Trichoderma based biofilms were developed with Azotobacter chroococcum, Pseudomonas fluorescens and Bacillus subtilis, while A. torulosa biofilms were prepared using B. subtilis and T. viride as components. Scanning electron microscopy revealed significant colonisation of biofilms on the root surface, which could be correlated with lowest mortality of 5.67%, recorded using T. viride–B. subtilis biofilm. An increase of 4–7% in polyphenol oxidase enzyme activity was recorded in all biofilm-treated samples, particularly those in which B. subtilis was present. The highest value of 1360.22 µg microbial biomass carbon g^?1 soil was recorded in Anabaena–B. subtilis biofilm treatment. Significantly higher values of plant and soil nutrient parameters in treatments in which biofilms were used vis-à-vis individual cultures reveal their promise. Such novel biofilmed biofertilizers with multiple useful traits can be beneficial options for effective nutrient and pest management of cotton crop.     

Zn biosorption by Rhizopus arrhizus and other fungi

Biosorption of zinc ions by inactivated fungal mycelia was studied. Of the six fungal species, Rhizopus arrhizus, Mucor racemosus, Mycotypha africana, Aspergillus nidulans, Aspergillus niger and Schizosaccharomyces pombe, R. arrhizus exhibited the highest capacity (Q ^max = 213 μmol g⁻¹ dry weight). Further experiments with different cellular fractions of R. arrhizus showed that Zn was predominantly bound to cell-wall chitin and chitosan (Q ^max = 312 μmol g⁻¹ dry weight). Adsorption data were best modelled by the Langmuir isotherm, although they can be modelled by the Freundlich equation as well at relatively low aqueous concentrations. Biosorption generally decreased with increase in biosorbent particle size and its concentration. Low pH reduced Zn sorption, because of the strong competition from hydrogen ions for binding sites on fungi. The presence of ligands reduced metal uptake, chiefly by forming metal complexes of a less biosorbable nature. Received: 2 November 1998 / Received revision: 12 January 1999 / Accepted: 17 January 1999     

Comparison of conditions for mycelial growth of <Emphasis Type="Italic">Lepista sordida</Emphasis> causing fairy rings on <Emphasis Type="Italic">Zoysia matrella</Emphasis> turf to those on <Emphasis Type="Italic">Agrostis palustris</Emphasis> turf

Symptoms of fairy rings caused by Lepista sordida have been reported on Zoysiagrass (Zoysia spp.) turf maintained at fairway height (2 cm), but not on bentgrass (Agrostis spp.) maintained at putting green height (0.5 cm). The mycelia of this fungus inhabit primarily the upper 0–2 cm layer of the soil extending into the thatch. To compare conditions for the mycelial growth in Z. matrella turf to those in A. palustris turf, we examined the effects of nutrients, temperature, water potential, and pH in the field as well as in the laboratory. Greater growth of the mycelia was observed in medium that included hot water extracts from soil of the 0–1 cm zone in Z. matrella turf compared to that from A. palustris. The upper soil layer in Z. matrella turf contained more organic matter from clippings than that in A. palustris. The temperature and water potential of the 0–2 cm soil zone in Z. matrella turf were also more favorable for the mycelial growth. The soil pH values of this zone in Z. matrella turf were less favorable compared to A. palustris but within the range for accelerating mycelial growth. Part of this study was presented orally at the 46th meeting of the Mycological Society of Japan in 2002     

Development of <Emphasis Type="Italic">Mesorhizobium ciceri</Emphasis>-Based Biofilms and Analyses of Their Antifungal and Plant Growth Promoting Activity in Chickpea Challenged by Fusarium Wilt

Biofilmed biofertilizers have emerged as a new improved inoculant technology to provide efficient nutrient and pest management and sustain soil fertility. In this investigation, development of a Trichoderma viride–Mesorhizobium ciceri biofilmed inoculant was undertaken, which we hypothesized, would possess more effective biological nitrogen fixing ability and plant growth promoting properties. As a novel attempt, we selected Mesorhizobium ciceri spp. with good antifungal attributes with the assumption that such inoculants could also serve as biocontrol agents. These biofilms exhibited significant enhancement in several plant growth promoting attributes, including 13–21 % increase in seed germination, production of ammonia, IAA and more than onefold to twofold enhancement in phosphate solubilisation, when compared to their individual partners. Enhancement of 10–11 % in antifungal activity against Fusarium oxysporum f. sp. ciceri was also recorded, over the respective M. ciceri counterparts. The effect of biofilms and the M. ciceri cultures individual on growth parameters of chickpea under pathogen challenged soil illustrated that the biofilms performed at par with the M. ciceri strains for most plant biometrical and disease related attributes. Elicitation of defense related enzymes like l-phenylalanine ammonia lyase, peroxidase and polyphenol oxidase was higher in M. ciceri/biofilm treated plants as compared to uninoculated plants under pathogen challenged soil. Further work on the signalling mechanisms among the partners and their tripartite interactions with host plant is envisaged in future studies.     

Microbial biofilm inoculants benefit growth and yield of chrysanthemum varieties under protected cultivation through enhanced nutrient availability

Protected cultivation of ornamental flowers, as a commercial venture, becomes less profitable with excessive use of fertilizers. The present study examined the influence of microbial biofilm inoculants (Anabaena–Azotobacter, Anabaena–Trichoderma and Trichoderma–Azotobacter) on the availability of soil nutrients and structure of rhizosphere microbial communities in three varieties of chrysanthemum (var. White Star, Thai Chen Queen and Zembla). Varietal-specific responses in growth, enzyme activities, flower yield of plants and availability of soil nutrients were recorded. Dehydrogenase activity was highest in var. White Star treated with the Anabaena–Trichoderma biofilm inoculants. The Anabaena–Azotobacter inoculant enhanced the availability of nitrogen, phosphorus and micronutrients in the soil, besides 40–50% increase in soil organic carbon, as compared to carrier alone or no inoculation. PCR-DGGE profiling of the cyanobacterial communities and qPCR quantification of 16S rRNA abundance of bacteria, archaea and cyanobacteria in the rhizosphere soils, revealed the stronger influences of these inoculants, especially in var. Zembla. Principal Component Analysis (PCA) helped to illustrate that the enhanced microbe-mediated availability of soil macro-and micronutrients, except iron content (Fe), was the most influential factor facilitating improved plant growth and yield parameters. The Anabaena–Azotobacter, and Anabaena–Trichoderma biofilm inoculants, proved superior in all three chrysanthemum varieties.     

Characteristics of Se-Enriched Mycelia by Stropharia rugoso-annulata and its Antioxidant Activities in vivo

Selenium (Se) is an essential trace element for humans and animals. Stropharia rugoso-annulata is a nutritional and functional mushroom containing many kinds of bioactive ingredients. The aims of this study were to investigate the Se-enrichment characteristics of S. rugoso-annulata in submerged culture and evaluate the antioxidant activities of Se-enriched mycelia in vivo in terms of the values of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA). The optimum parameters of Se-enrichment under the optimal Se concentration (150 μg/mL) in media were as follows: biomass 8.11 ± 0.25 g/L, Se content in mycelia 4,727.68 ± 13 μg/g, Se-accumulated rate 24.68 ± 1.67%, and percentage of organic Se 96.27 ± 3.26%. The mainly subsistent forms of selenium in Se-enriched mycelia were selenoprotein and selenium-polysaccharide. The contents of total amino acids (TAA) and essential amino acids (EAA) in Se-enriched mycelia were increased by 13.5 ± 1.09% and 12.8 ± 0.89%, respectively. It was efficient for Se-enriched mycelia to elevate GSH-Px and SOD activities and decrease MDA content. These results indicated that Se-enriched mycelia of S. rugoso-annulata represent a novel dietary source of bioavailable supplemental selenium.     

Role and genetic basis of specialised secondary metabolites in Trichoderma ecophysiology

Species of fungal genus Trichoderma are characterized by a versatile lifestyle, high adaptability to the changing environmental conditions and the ability to establish sophisticated interactions with other organisms. Due to their ability to antagonize plant pathogens and to elicit the plant defence responses against biotic/abiotic stresses, Trichoderma spp. are commonly used as commercially biopesticides and biofertilizers. The Trichoderma success in the rhizosphere is supported by a wide arsenal of specialised metabolites (SMs) providing morphological and physiological autoregulation, self-protection and facilitating fungal communication. This review aims to explore the roles of SMs in the biology of fungi, with special emphasis on the genus Trichoderma and on how divergence in the SMs genetic structure determine Trichoderma lifestyles. Trichoderma genomes are endowed with a high number of SMs biosynthetic genes, and understanding the genetic basis of their biosynthesis is crucial for determining the role of these metabolites in Trichoderma ecophysiology and for expanding their application in crop protection. Recent advances on the characterization of the Trichoderma SMs genetic inventory driven by computational biology are discussed.     

Nitrogen decreases and precipitation increases ectomycorrhizal extramatrical mycelia production in a longleaf pine forest

The rates and controls of ectomycorrhizal fungal production were assessed in a 22-year-old longleaf pine (Pinus palustris Mill.) plantation using a complete factorial design that included two foliar scorching (control and 95% plus needle scorch) and two nitrogen (N) fertilization (control and 5 g N m⁻² year⁻¹) treatments during an annual assessment. Ectomycorrhizal fungi production comprised of extramatrical mycelia, Hartig nets and mantles on fine root tips, and sporocarps was estimated to be 49 g m⁻² year⁻¹ in the control treatment plots. Extramatrical mycelia accounted for approximately 95% of the total mycorrhizal production estimate. Mycorrhizal production rates did not vary significantly among sample periods throughout the annual assessment (p = 0.1366). In addition, reduction in foliar leaf area via experimental scorching treatments did not influence mycorrhizal production (p = 0.9374), suggesting that stored carbon (C) may decouple the linkage between current photosynthate production and ectomycorrhizal fungi dynamics in this forest type. Nitrogen fertilization had a negative effect, whereas precipitation had a positive effect on mycorrhizal fungi production (p = 0.0292; r ² = 0.42). These results support the widely speculated but poorly documented supposition that mycorrhizal fungi are a large and dynamic component of C flow and nutrient cycling dynamics in forest ecosystems.     

Control of fusarium wilt of <Emphasis Type="Italic">Solanum melongena</Emphasis> by <Emphasis Type="Italic">Trichoderma</Emphasis> spp.

Biological control of wilt of egg plant (Solanum melongena L.) caused by Fusarium solani was made with the application of five Trichoderma species, T. harzianum, T. viride, T. lignorum, T. hamatum and T. reesei. The effect of volatile and non-volatile antibiotics of Trichoderma origin on growth inhibition of the wilt pathogen was studied. T. harzianum showed maximum growth inhibition (86.44 %) of the pathogen through mycoparasitism. The non-volatiles produced by the Trichoderma species exhibited 100 % growth inhibition of the pathogen under in vitro condition. Production of siderophores and fungal cell wall degrading enzymes, chitinase and β-1,3-glucanase were found. Treatments with two most efficient Trichoderma species, T. harzianum and T. viride resulted in the decreasing population of Fusarium solani in soil thereby deterring disease incidence in field condition.     

The effect of turbulent flow and surface roughness on biofilm formation in drinking water

There is considerable interest in both Europe and the USA in the effects of microbiological fouling on stainless steels in potable water. However, little is known about the formation and effects of biofilms, on stainless steel in potable water environments, particularly in turbulent flow regimes. Results are presented on the development of biofilms on stainless steel grades 304 and 316 after exposure to potable water at velocities of 0.32, 0.96 and 1.75 m s⁻¹. Cell counts on slides of stainless steel grades 304 and 316 with both 2B (smooth) and 2D (rough) finishes showed viable and total cell counts were higher at the higher flow rates of 0.96 and 1.75 m s⁻¹, compared to a flow rate of 0.32 m s⁻¹. Extracellular polysaccharide levels were not significantly different (P< 0.05) between each flow rate on all stainless steel surfaces studied. higher levels were found at the higher water velocities. the biofilm attached to stainless steel was comprised of a mixed bacterial flora including Acinetobacter sp, Pseudomonas spp, Methylobacterium sp, and Corynebacterium/Arthrobacter spp. Epifluorescence microscopy provided evidence of rod-shaped bacteria and the formation of stands, possibly of extracellular material attached to stainless steel at high flow rates but not at low flow rates. Received 04 February 1998/ Accepted in revised form 12 February 1999     

Liquid fermentation to produce biomass of mycoherbicidal strains of Fusarium oxysporum

Conditions for optimizing spore production, especially chlamydospores, by host-specific mycoherbicidal strains of Fusarium oxysporum causing vascular wilts in coca (Erythroxylum coca) and poppy (Papaver somniferum) were studied in 2.5-1 fermentors. The fermentor dissolved oxygen and pH had significant effects on the growth characteristics of F. oxysporum strains. The effect of the fungal strain, however was not significant for most of the variables studied except for chlamydospore formation. After 14 days of fermentation, the spore types produced were microconidia and chlamydospores, with very little production of macroconidia. While the total viable counts were significantly higher under high than under low dissolved O₂, the chlamydospore counts were significantly higher under low than under high dissolved O₂. The percentage of chlamydospores obtained, as a proportion of total viable was significantly higher when the fermentor pH was increased, than when it was not. Scaling-up the liquid fermentation to 20 l, yielded log₁₀ c = 6.8 (where c = chlamydospores ml⁻¹) after 14 days' fermentation, with biomass viable counts of log₁₀ v∼8.0 (where v = viable counts g⁻¹ air-dried biomass). A single-step liquid fermentation reported in this study increased chlamydospore yields and reduced the time required for their production with techniques currently available from 5 weeks to less than 2 weeks. Received: 24 April 1997 / Received revision: 6 August 1997 / Accepted: 29 August 1997