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     

In vivo-Untersuchungen zur Entstehung und Funktion der Chlamydosporen von Botrytis cinerea Pers. am Wirt-Parasit-System Fuchsia hybrida – B. cinerea

In vivo-investigations on the formation and function of chlamydospores of Botrytis cinerea Pers. in the host-parasite-system Fuchsia hybrida–B. cinerea On naturally with Botrytis cinerea Pers. infected and artificially inoculated outdoor- and greenhouse-plants of Fuchsia hybrida the extra- and intramatrical formation of the B. cinerea- chlamydospores was investigated. The chlamydospores served 1. as structures of survival, which were tested with regard to their tolerance of drought, nutrient- and oxygen-deficiency, attack by bacteria and pH-requirements. 2. The chlamydospores represented dispersal units, which were capable of germination. 3. The chlamydospores could function as structures of infection, because after chlamydospore germination the outgrowing mycelium – either directly or after production of macroconidia – could serve as secondary inoculum and start new infections.     

Susceptibility of Barley Cultivars and Lines to Fusarium Infection and Mycotoxin Accumulation in Kernels

Heads of 12 barley genotypes (8 cultivars and 4 lines) were inoculated with conidial suspension of the following single isolates: F. culmorum no. 3, F. graminearum no. 122 and F. sporotrichioides no. ATCC 62 360. The number of kernels per head. 1000 Kernel weight and yield have been calculated for each genotype. Seed samples collected at harvest were analysed for each genotype. Seed samples collected at harvest were analysed for several trichothecene mycotoxins and zearalenone.The mycotoxin concentrations (mg/kg) in barley kernels inoculated with F. graminearum were as follows. deoxynivalenol (DON) 0.1 to 5.4 (av. 2.3). 3-acetyldeoxy-nivalenol (3-AcDON) 0.0–0.2 (av. 0.1), 15-acetyldeoxynivalenol (15-AcDON) 0.0–0.7 (av.0.2), nivalenol (NIV) 0.0–0.8 (av. 0.3). zearalenone (ZEA) 0.0–0.1 (av. 0.0); F. culmorum: DON 0.6 to 12.0 (av. 5.3), 3-AcDON 0.1 to 1.0 (av. 0.6). 15-AcDON nd. NIV 0.1–0.7 (av. 0.3). ZEA 0.1–0.5 (av. 0.2). F. sporotrichioides T-2 toxin 2.4–13.9 (av. 6.0), HT-2-toxin 0.1–0.8 (av.0.3) and neosolaniol 0.2–1.5 (av.0.7).     

Chlamydospore Production,Inoculation Methods and Pathogenicity of Fusarium oxysporum M12-4A,a Biocontrol for Striga hermonthica

Fusarium oxysporum isolate M12-4A is currently being evaluated for the biological control of Striga hermonthica . Inoculum production, inoculum delivery to the target, chlamydospore germination, and weed growth suppression of this weed-pathogen system were investigated. Liquid fermentation systems using organic material were evaluated for the production of large numbers of chlamydospores. A 1% sorghum straw powder (< 1 mm) substrate, exposed to black light at 21°C for 21 days, yielded 3.23 X 10 8 colony forming units (CFU) l -1 medium. A two-stage fermentation system using 5% w/v straw substrate under black light at 30°C for 14 days yielded 3.5 X 10 8 CFU l -1 medium. In vitro variations in chlamydospore germination were governed by the presence of exogenous carbon, nitrogen, and sorghum root exudates. Ammonium-nitrogen compounds and urea, in combination with glucose had a stronger stimulatory effect on chlamydospore germ tube growth than did potassium nitrate. Maximal germ tube elongation occurred when chlamydospores were exposed to urea at a C/N ratio of 10. Some mineral solutions and sorghum root exudates inhibited chlamydospore germ tube elongation; however, arabic gum, a complex polysaccharide, stimulated chlamydospore germ tube elongation and the production of secondary chlamydospores. In field trials, chlamydospore powder harvested from small-scale fermenters reduced S. hermonthica emergence by 92%. Complete inhibition of S. hermonthica emergence occurred when the chlamydospore powder was added to the soil at sowing and when sorghum seeds coated with chlamydospores were sown. Effective biological control of S. hermonthica was achieved using a simple fermentation system with sorghum straw as the inoculum growth substrate. For inoculum delivery to the farmers' fields, sorghum seeds were coated with the inoculum using arabic gum as the adhesive. This simple delivery system permits a uniform inoculation of the field as well as the proper positioning of the inoculum in the immediate environment of sorghum roots, where S. hermonthica attaches to its host. To facilitate a broad usage of F. oxysporum M12-4A for the biocontrol of S. hermonthica , we propose an inoculum production strategy based on a cottage industry model that utilizes a liquid fermentation process and inexpensive locally-available substrates including sorghum straw and arabic gum.     

Degradation of 3,9-dimethoxypterocarpan and medicarpin by Fusarium fungi

Fusarium sporotrichioides and Gibberella saubinetti O-demethylate 3,9-dimethoxypterocarpan to 3-methoxy-9-hydroxypterocarpan and then 3,9-dihydroxypterocarpan. F. anguioides Sherb., F. avenaceum and F. graminearum convert the same substrate to a mixture of 3-methoxy-9-hydroxypterocarpan and 3-hydroxy-9-methoxypterocarpan. Induction of pterocarpan degradation by pisatin in F. avenaceum leads to preferential conversion of 3,9-dimethoxypterocarpan to 3-hydroxy-9-methoxypterocarpan (medicarpin) and the isoflavan vestitol. The data are discussed with respect to phytoalexin degradation by phytopathogenic Fusarium fungi.Dedicated to Prof. Dr. H. Holzer, Freiburg, at the occasion of his 60th birthday     

Production of T-2 toxin by a Fusarium resembling Fusarium poae

A Fusarium species with a micro morphology similar to F. poae and a metabolite profile resembling that of F. sporotrichioides has been identified. Like typical F. poae, the microconidia have a globose to pyriform shape, but the powdery appearance, especially on Czapek-Dox Iprodione Dichloran agar (CZID), less aerial mycelium and the lack of fruity odour on Potato Sucrose Agar (PSA) make it different from F. poae. The lack of macroconidia, polyphialides and chlamydospores differentiates it from F. sporotrichioides. All 18 isolates investigated, 15 Norwegian, two Austrian and one Dutch, produced T-2 toxin (25–400 μg/g) on PSA or Yeast Extract Sucrose agar (YES). In addition, neosolaniol, iso-neosolaniol, HT-2 toxin, 4- and 15-acetyl T-2 tetraol, T-2 triol and T-2 tetraol and4,15-diacetoxyscirpenol were formed in variable amounts. Neither nivalenol, 4- or 15-acetylnivalenolor 4,15-diacetylnivalenol were detected in any of the cultures, while these toxins were produced at least in small amounts by all the 12 typical F. poae isolates studied. The question of whether this Fusarium should be classified as F. poae or F. sporotrichioides or a separate taxon should be addressed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth and toxigenicity of Fusaria of the sporotrichiella section as related to environmental factors and culture substrates

Isolates ofFusarium poae, F. sporotrichioides, F. sporotrichioides var.chlamydosporum andF. sporotrichioides var.tricinctum made their best growth on PDA substrates at 24 °C, but good growth was also made at 18 °C and 30 °C. At 35 °C growth made by theF. sporotrichioides var.chlamydosporum was quite good, and superior to that of the other fungi. Moderate growth was made by all fungi at 12 °C and byF. sporotrichioides var.tricinctum also at 6 °C, while growth of the other fungi at that temperature was slight. At low temperatures toxic isolates of all butF. sporotrichioides grew better than non-toxic isolates, and growth of all isolates usually was better in light than in darkness up to temperatures of 18 °C. F. poae andF. sporotrichioides produced highest toxicity on rabbit skins when grown at 5–8 °C,F. sporotrichioides var.tricinctum at 15–20 °C. Darkness always favoured toxin development at all temperatures. In a comparison of 3 liquid substrates, overall toxin production was stronger on a starch substrate than on Czapek's or carbohydrate-peptone substrates. Among grain substrates, barley gave highest overall toxicity, which was again favoured by darkness.F. poae isolates were most toxic when derived from soil,F. sporotrichioides isolates when derived from barley. Further tests with 8 liquid substrates confirmed thatF. poae andF. sporotrichioides produce stronger toxicity at 8 °C than at 25 °C, and substrates favoured toxin production at pH 5.6 more than at pH 3.8 or 7.2. At pH 5.6 the isolates induced marked changes in the pH level of the substrate on which they grew. No relation was found to exist between the vigour of growth made by any of these fungi under various environmental conditions and the severity of the toxiç reaction their extracts produced on rabbit skins.     

Roles of low pH, carbon and inorganic nitrogen source use in chlamydospore formation by Fusarium solani.

Citrate and malate were poorer sources of exogenous carbon than several hexose, pentose, or disaccharide sugars for supporting macroconidial germination by Fusarium solani at high conidial density (1 X 10(5) condia/ml). Only citrate, however, failed to block chlamydospore morphogenesis to a degree comparable to glucose or other readily used sugars. Mostly immature chlamydospores were formed in the presence of citrate. At low conidial density (5 X 10(3) conidia/ml), exogenous carbon-independent macroconidial germination and subsequent rapid chalmydospore formation on germ tubes was not inhibited by ammonium or nitrate nitrogen. The citrate-phosphate buffered, low pH (4.0) medium of Cochrane induced more immature chlamydospore formation by F. solani than a pH 6.0 medium, but few mature chlamydospores were formed in either medium. Condensation of hyphal cytoplasm into developing chlamydospores, a character typical of chlamydospore formation, did not occur extensively and macroconidia, hyphae, and immature chlamydospores stained deeply with Sudan III, suggesting lipid biosynthesis. This inhibition of chlamydospore maturation may be due partly to nitrogen deficiency imposed by the high C:N ratio of the medium and to the presence of citrate. Only vesiculate hyphal cells were formed by F. solani f. sp. phaseoli in both media. Field soils to which the clone of F. solani used is indigenous had mean pH values ranging from 5.2 to 6.0.     

Temperature-dependent germination,growth and co-infection of Beauveria spp. isolates from different climatic regions

Biological control agents based on entomopathogenic fungi traditionally contain a single strain that is efficient under certain biotic and abiotic conditions. Since particularly abiotic conditions vary, biological control efficiency may become more resilient at extreme temperatures if two or more fungal strains are combined based on their adaptations to their original environment. Here we evaluated the in vitro temperature-dependent germination and growth rate for six Beauveria spp. isolates originating from either arctic or tropical regions. Isolates of arctic origin showed higher germination and growth rate at 8°C and 12°C than isolates from the tropics while the latter group showed highest germination and in vitro growth at 32°C. Three of the isolates belonging to Beauveria bassiana were further tested in vivo for temperature-dependent infection in the mealworm beetle Tenebrio molitor both individually and combined. The same amounts of conidia were used in all bioassays. Virulence was isolate dependent at all temperatures with no additional effect at the low (12°C) and high (32°C) temperatures of combinations of arctic and tropical isolates. The results therefore indicate that adaptations to abiotic conditions in the natural environment do not directly reflect the effect of biotic environment (such as host infection) under similar conditions. Selection of isolates for biocontrol agents should not be based solely on in vitro experiments, while isolate selection based on virulence should also include considerations of the abiotic conditions the isolates are expected to function.     

Mycotoxin production of <Emphasis Type="Italic">Fusarium langsethiae</Emphasis> and <Emphasis Type="Italic">Fusarium sporotrichioides</Emphasis> on cereal-based substrates

The present study investigated and compared the mycotoxin production of two Fusarium species, F. sporotrichioides and F. langsethiae, isolated from grain samples. Fusarium strains were cultivated at 25°C for 7 days on two types of solid media, i.e. rice-flour and cereal-flour agar. Toxins produced were measured after the incubation period with a multi-mycotoxin method based on liquid chromatography–tandem mass spectrometry (LC-MS/MS). Both F. sporotrichioides and F. langsethiae synthesised type-A trichothecenes, i.e. T-2 and HT-2 toxins, diacetoxyscirpenol (DAS) and neosolaniol (NEO). In addition, both species could be verified as beauvericin producers. The toxin production occurred in both cereal-based assays but was more predominant on the carbohydrate-rich rice-flour medium. The two species were potent producers of T-2 toxin, the highest amounts measured being at a level of 20,000 μg/kg after 7 days’ incubation. Differences between the species were observed regarding the quantitative production of the other trichothecenes: F. sporotrichioides was a more prolific producer of HT-2 toxin and beauvericin, whereas F. langsethiae produced higher amounts of DAS and NEO. On rice-flour assay, the toxin production was monitored during the growth period. The production started rapidly at an early growth phase and several toxins could be detected already after the 1st day of incubation, the highest concentrations being at mg/kg level. The results also indicated that the biosynthesis by F. sporotrichioides and F. langsethiae shifted towards the other type-A trichothecenes at the expense of T-2 toxin at the end of the cultivation.     

Impact of nutritional conditions on yields,germination rate and shelf-life of Plectosporium alismatis conidia and chlamydospores as potential candidates for the development of a mycoherbicide of weeds in rice crops

The effect of nutritional conditions on spore qualities was investigated in order to select which propagules, conidia or chlamydospores, would be most suitable for mycoherbicide development. Plectosporium alismatis was grown in a liquid basal medium supplemented with glucose and a mineral nitrogen source (sodium nitrate) or an organic nitrogen source (casamino acids). Conidial and chlamydospore yields, germination rate and shelf-life were compared. Two growth models were developed: on one hand, sodium nitrate added as the sole nitrogen source was partially utilised (8%), resulting in poor growth (1.77±0.02 mg mL^?1; 6±1.7×10⁵ conidia mL^?1). Under these conditions, P. alismatis produced dense, melanised-like aggregates that contained chlamydospores (12.4±0.7×10⁴ chlamydospores mL^?1). Germination rates of chlamydospores and conidia produced under these conditions was high (80%). Twenty percent of chlamydospores were able to germinate after 4 months storage at 25°C, while survival of conidia declined rapidly (<2%). When casamino acids were added to the liquid medium as the sole nitrogen source, P. alismatis produced sparser pellets resulting in high dry weights (5.37±0.09 mg mL^?1 and high conidia numbers (9.6±1.5×10⁶ conidia mL^?1), while no chlamydospore were observed. The germination rate of conidia produced in casamino acids was low (33±13%) after 8 h incubation and microcycle conidiation occurred. Five percent of these conidia germinated after 4 months storage. These data indicate that chlamydospores may be suitable for mycoherbicide development, provided further optimisation of yields is achieved.     

Characteristics of Inhibition of a Taiwanese Soil Suppressive to Fusarium oxysporum f. sp. raphani

Germination of nutrient-amended chlamydospores of the three formae speciales of Fusarium oxysporum tested were inhibited in a suppressive soil collected from central Taiwan. The suppressive soil released a volatile substance when moistened with alkaline solution. The inhibition spectrum of the volatile substance was different from that of the suppressive soil. The inhibitory effect of the suppressive soil was greatly reduced when it was heat-treated for 30 min at 40°C or higher. The inhibitory effect of the heat-treated suppressive soil was restored after infestation with 1% conducive or suppressive soil for 14 days. However, infestation of heat-treated conducive soil even with 1% suppressive soil did not render it suppressive. Amendment of suppressive soil with rose bengal, streptomycin or Rubigan completely or partially reduced the inhibitory effect. Increasing the total population of indigenous microorganisms in conducive soil by amendment with rice germ or soybean meal to about the same level as that in suppressive soil did not render it suppressive. Results suggest that a combination of biotic and abiotic factors is responsible for the inhibitory effect of the suppressive soil.     

Enhanced Recovery of Phytophthora ramorum from Soil Following 30 Days of Storage at 4°C

Chlamydospores of Phytophthora ramorum were used to infest field soil at densities ranging from 0.2 to 42 chlamydospores/cm³ soil. Recovery was determined by baiting with rhododendron leaf discs and dilution plating at time 0 and after 30 days of storage at 4°C, as recommended by USDA‐APHIS. Baiting was slightly more sensitive than dilution plating in recovering P. ramorum immediately following infestation of soil and allowed detection from samples infested with as little as 0.2 chlamydospores/cm³ compared with 1 chlamydospore/cm³ for dilution plating. After 30 days of infested soil storage at 4°C, P. ramorum was detected at significantly (P = 0.05) higher levels than at time 0 with both recovery methods. The results indicate that storage of P. ramorum‐infested soil at 4°C may allow for pathogen activity, such as sporangia production, which may enhance recovery from soil.     

Immunochemical analysis of trichothecenes produced by various fusaria

The production of type A trichothecene mycotoxins by 19 Fusaria, including 12Fusarium sporotrichioides, 4F. chlamydosporum and 3F. graminearum at 15°C and 25°C over a 35-day period was analyzed by ELISA using antibodies cross-reactive with most type A trichothecenes after conversion to T-2 tetraol tetraacetate. The toxin production peaked at 20–25 days of incubation with maximum yield between 4–6 mg type A trichothecene/ml of culture medium for 5F. sporotrichioides cultures and between 1 to 2 mg/ml for 6F. sporotrichioides cultures. OneF. sporotrichioides produced 700 µg type A trichothecenes/ml of culture medium. Detectable type A trichothecene was also found in the culture extracts ofF. chlamydosporum andF. graminearum, but the yield was very low (less than 100 µg/ml). Quantitative determination of individual trichothecenes was achieved by separation of different toxin in HPLC and followed by ELISA analysis. Eight to 10 immunoreactive peaks, corresponding to various type A trichothecenes, were detected in all the fungal extracts. T-2 tetraol (T-2-4ol), 4-acetyl-T-2 tetraol (4-Ac-T-2-4ol), neosolaniol (NEOS), diacetoxyscirpenol (DAS), HT-2 and T-2 toxin accounted for more than 85% of the total toxins. In general, low temperature was preferred for total type A trichothecene production. More T-2-4ol, 4-Ac-T-2-4ol, HT-2 and DAS were produced at 25°C. In contrast, more T-2 toxin and NEOS were produced at 15°C. Transformation of T-2 toxin and NEOS to polar metabolites such as T-2-4ol, 4-acetyl-T-2-4ol and HT-2 by various strains were observed at both temperatures after 25 days incubation.     

Study of the Vapor Phase Over Fusarium Fungi Cultured on Various Substrates

The compositions of volatile organic compounds (VOCs) emitted by Fusarium fungi (F. langsethiae, F. sibiricum, F. poae, and F. sporotrichioides) grown on two nutritive substrates: potato sucrose agar (PSA) and autoclaved wheat kernels (WK) were investigated. The culturing of fungi and study of their VOC emissions were performed in chromatographic vials at room temperature (23 – 24 °C) and the VOCs were sampled by a solid‐phase microextraction on a 85 μm carboxen/polydimethylsiloxane fiber. GC/MS was performed using a 60‐m HP‐5 capillary column. Components of the VOC mixture were identified by electron impact mass spectra and chromatographic retention indices (RIs). The most abundant components of the VOC mixture emitted by Fusarium fungi are EtOH, AcOH, ⁱBuOH, 3‐methylbutan‐1‐ol, 2‐methylbutan‐1‐ol, ethyl 3‐methylbutanoate, terpenes with M 136, sesquiterpenes with M 204 (a total of about 25), and trichodiene. It was found that the strains grown on PSA emit a wider spectrum and larger amount of VOCs compared with those grown on wheat kernels. F. langsethiae strain is the most active VOC producer on both substrates. The use of SPME and GC/MS also offers the potential for differentiation of fungal species and strains.     

Über Entstehung und Keimung der Chlamydosporen von Botrytis cinerea Pers.

On the genesis and germination of the chlamydospores of Botrytis cinerea Pers. The chlamydospores of Botrytis cinerea are hyaline single cells of extremely variable form and size. They are formed under conditions unfavourable for growth as terminal or intercalary cells by transformation of vegetative mycelium parts and are liberated by hyphal disintegration. The chlamydospore genesis in vitro in aging malt agar cultures began about after one month. But the chlamydospore formation could also be initiated earlier by different conditions of culture. The chlamydospores germinated either with hypha or by microconidia — a herewith first described mode of germination. Intermediates of these both modes of chlamydospore germination could be regulated very differentiatedly by transferring the chlamydospores into malt solution (2%)and/or destilled water and by changing the duration of stay in the individual media. Under adverse external conditions no germination occurred. The three Botrytis cinerea-isolates did not show any differences in habitus, genesis and germination of their chlamydospores. Also in vivo on outdoor- and greenhouse-tomatoplants the occurrence of chlamydospores was no rarity. Since the chlamydospores are produced under very different adverse conditions of growth but cannot survive a period of drought lasting longer than three months without damage, they do not represent long-termed resistant perennating structures, but temporary stages of the fungus for intervening periods.     

Seasonal variations of Fusarium species in wheat fields in Upper Egypt

Abstract

Populations of the genus Fusarium in wheat fields were studied within the crop-growing season at Qena area (Upper Egypt) using two different types of media (DCPA and DRBA) at 25°C. Fourteen Fusarium species were isolated during this study, namely F. anthophilum, F. aquaeductuum, F. chlamdosporum, F. dimerum, F. merismoides, F. moniliforme, F. oxysporum, F. poae, F. proliferatum, F. sambucinum, F. scripi, F. solani, F. sporotrichioides and F. subglutinans. Fusarium merismoides, F. oxysporum and F. sambucinum were the most common Fusarium species isolated from different wheat plant parts (rhizosphere and rhizoplane) as well as from the wheat fields (soil and air). Fusarium spp. rarely appeared at the beginning of the season and increased sharply between January to March and decreased slightly or sharply at the end of the season according to the type of media and isolation source.     

Effect of NH3 on Chlamydospore Formation of Fusarium oxysporum f. sp. dianthi in an NH3-Flow System

Conidia of Fusarium oxysporum on membrane filters on a sand bed saturated with a TRIS-buffer in Conway vessels were exposed to different concentrations of NH₃. NH₃ was generated by bubbling air through NH₄OH-containing cylinders and pumped through the Conway vessels. Low concentrations of NH₃ (15 ppm) stimulated chlamydospore formation in both germinated and ungerminated conidia. Higher concentrations of NH₃ (150 ppm) inhibited chlamydospore formation. Ammonia had no apparent effects on the quality of the produced chlamydospores as measured by determining the persistence in soil.