Susanne Elmholt

Czapek‐Dox agar containing iprodione and dicloran as a selective medium for the isolation of Fusarium species

A new selective medium for Fusarium species has been developed using Czapek‐Dox agar (CZ) containing the fungicides iprodione (3 mg/1) and dicloran (2 mg/1). This new medium (CZID) is selective against numerous species of Alternaria, Epicoccum, Penicillium and mucoraceous fungi. CZID was compared with CZ using samples of barley, malt, sorghum, bean and pea. Fusarium species produced large and easily recognizable colonies on CZID while isolates of Alternaria, Epicoccum and Rhizopus were significantly restricted on CZID compared with their growth on CZ. The use of CZID thus facilitates the isolation and subculturing of Fusarium species.

Suppressiveness of organically and conventionally managed soils towards brown foot rot of barley

Abstract Five sandy loam soils under organic, integrated and conventional management were chosen to investigate the effect of specific agricultural management practices on suppression of brown foot rot of cereals caused by Fusarium culmorum. The relationships between suppressiveness and C and N content of the soil microbial biomass and microbial activity were investigated. Fungistasis tests and plant bioassays were compared. Differences in suppressiveness were most marked in plant bioassays following seed inoculation with F. culmorum. When inoculum was applied to soil as conidia or mycelia, soil-suppressed transmission of the pathogen and disease development. This finding indicates that the competitive saprophytic ability of the isolate of F. culmorum is poor. The microbial biomass and activity of the soils under organic and integrated farming were high. However, high biomass and activity were not always correlated with high disease suppression. Specific organic amendments, such as mulching with straw and the practice of using lucerne as a break-crop in cereal cultivation may influence inoculum potential of F. culmorum, disease outbreak and suppression. The possible significance of soil abiotic factors on disease suppression is discussed.

Phospholipid fatty acid profiles and C availability in wet-stable macro-aggregates from conventionally and organically farmed soils

Abstract Whole soil samples and four aggregate size classes (2–8 mm, 1–2 mm, 0.5–1 mm and 0.25–0.5 mm) from organically or conventionally farmed sandy loam soils were compared with respect to texture, C content and C mineralization potential, microbial biomass C and phospholipid fatty acid (PLFA) composition. The PLFA concentration of organically farmed soils (44–56 nmol g−1 dry wt.) was larger than in soils under conventional management (28–32 nmol g−1 dry wt.) and correlated with biomass C. Principal component analyses demonstrated only minor differences between whole soil samples with respect to PLFA composition. The texture of soil fractions obtained by wet-sieving deviated strongly from the texture of whole soil, paticularly in the 0.25–0.5 mm and 0.5–1 mm size classes. These fractions also appeared to include some non-aggregate particulate organic matter. The C mineralization during a 13-week incubation increased significantly with decreasing aggregate size class in four of the six soils. Biomass C declined during the incubation, and the decline in most cases could account for the C mineralized. No consistent differences were observed between conventionally and organically farmed soils or between aggregate size classes with respect to taxonomic composition or physiological status of the microbial community.

Ecology of the ochratoxin A producing Penicillium verrucosum: Occurrence in field soil and grain with special attention to farming system and on-farm drying practices

ABSTRACT Mycotoxin contamination of organically grown cereals has been a growing concern during recent years, one of the most important being ochratoxin A (OTA). In countries with a temperate, humid climate, OTA is produced by Penicillium verrucosum and it is crucial to focus on the fungus to obviate problems. This paper presents results to elucidate different aspects of the ecology of P. verrucosum. In a survey of differently farmed soils, P. verrucosum was found in 11 out of 65 soils, 35% of the organically and 7% of the conventionally cultivated, these being the first reports of its natural occurrence in soil. Except for two soils, it was found with low frequencies (100–300 cfu g dry soil−1), and the results point to home grown seed as a potential risk of soil contamination. In a survey of organically grown grain, P. verrucosum was found in 51% of the recently combined and not yet dried grain samples; 60% of the rye and 53% of the wheat samples. This confirms that early contamination does take place and increases the demand for proper grain handling during drying and storage. Three case studies were performed at organic farms with different storage facilities. The results showed very clearly that there is no general risk of OTA contamination in organically cultivated grain. Within the same year, major differences between the farms were found and these differences were reproduced during three growing seasons. It therefore seems likely that an organic farming system, as such, does not present problems in relation to OTA contamination. It is, rather, certain management practices that are inappropriate and these management practices may be more prevalent in organic farming. This assumption is discussed in relation to home grown seed, crop rotation, organic fertilizers, exclusion of pesticides, and drying and storage facilities.

Field ecology of the ochratoxin A-producing Penicillium verrucosum: Survival and resource colonisation in soil

A field experiment was conducted to elucidate the survival of P. verrucosum in infested bulk soil(T1) and infested soil with waste grain (T2). The infested soil and reference soil (T3) was filled into steel cylinders, which were buried and sampled 13 times during a period from October 1994 to March 1996.The abundance of P. verrucosum and indigenous soil fungi were assessed by dilution plating on a selective and diagnostic medium (DYSG). Kernel infection was examined in T2. According to our results, P. verrucosum seems well adapted to survival in arable soil and little affected by indigenous fungi. During the first autumn and winter the grain caused a proliferation of P. verrucosum while its abundance in bulk soil was more constant except for a decrease in February 95, which is ascribed to frost/thaw alternations. In T2, P. verrucosum initially infected more than 50% of the kernels but during the first few months it was ousted by other fungi. A hypothesis regarding waste grain as the natural niche for the fungus in the field was therefore partly rejected. A gradual decrease in the abundance of P. verrucosum in soil during spring, a die-off in the dry summer and aproliferation during the second winter were found in both T1 and T2. Our results cannot provide the reason for the increase during the second winter. On an overall basis, however, they show that P. verrucosum can survive in the field, proliferate on soil organic matter and probably become an integral part of the soil ecosystem. This may constitute a risk of grain contamination when given appropriate environmental conditions.

Detection and estimation of conidial abundance of Penicillium verrucosum in soil by dilution plating on a selective and diagnostic agar medium (DYSG)

Penicillium verrucosum is one of the main producers of ochratoxin A (OA) in agricultural commodities. To forecast the risk of OA contamination, there is a need to improve our knowledge on the ecology of P. verrucosum in the field. Dilution plating on ‘dichloran yeast extract sucrose 18% glycerol agar’ (DYSG) offers a simple and very sensitive method of detecting P. verrucosum propagules in soil. The properties of DYSG are illustrated in a suspension mixture experiment and confirmed in a soil mixture experiment. In the latter, P. verrucosum could be detected in conidial concentrations below 200 colony forming units (cfu) g −1 soil even when it constituted no more than 0.3% of the cfu. Furthermore, the DYSG method can be used to estimate the abundance of P. verrucosum propagules in soil with good precision. In some of the analysed cases, however, it was necessary to use appropriate mathematical models to treat results with high numbers of cfu on the Petri dishes.

Distribution of ergosterol in organically and conventionally cultivated agricultural soils.

ABSTRACT Ergosterol is generally assumed to be one of the best indicators of fungal biomass and soil quality. The present case studies describe variations in ergosterol content in soil, sampled at three Danish farms. One is organically cultivated according to the biodynamical principles (ORG). The other two are conventionally cultivated, one with animal husbandry (CONV-H) and one with plant production (CONV-P). The analytical method combined extraction in a microwave oven, clean-up with a new SPE sorbent material (OasisTM), and quantification by HPLC. The average estimated concentrations of ergosterol were 1.66 μg g−1 in ORG, 2.35 μg g−1 in CONV-H and 1.13 μg g−1 in CONV-P. These results correlated well with some of the important variables in the management systems of the soils (frequency of grass and clover, input of animal manure, total-C, frequency of ploughing). However, the exact causal relationships cannot be revealed using the case-study approach. The statistical analysis of the spatial variation in ergosterol across the field demonstrated very clearly that in some soils, small-scale (few cm2) variations in ergosterol concentrations may be quite large in comparison with large scale (few hundred m2) variations. This should be taken into account when planning the sampling strategy.

A resource-saving method for isolation of Fusarium and other fungi from individual soil particles

Planting of individual soil particles onto an agar medium, poured into Petri dishes, is a method of studying fungal distribution between microhabitats in soil. The substitution of Petri dishes with well plates reduced both the required amount of agar medium and labour. A microshovel prepared from an injection needle facilitated the handling of individual, small size, soil particles (0.25–0.50 mm). The described method was evaluated in a study of the distribution of Fusarium within two wheat field soils from As, Norway, and from Ostermarie, Denmark. Four types of organic particles were distinguished: light coloured root pieces, dark coloured root pieces, straw pieces, and miscellaneous organic pieces. The dominating species were F. culmorum, F. oxysporum and F. avenaceum . Aspects on the value of the method with regard to fungal substrate preference are discussed.

Mycotoxins in the Soil Environment

The paper outlines the current knowledge concerning fate of mycotoxins in the soil environment, including - outline of mycotoxins addressed (trichothecenes, zearalenone, fumonisins, aflatoxins, ochratoxins and patulin) - routes by which the mycotoxins enter the soil environment - routes by which they are immobilised or removed from the soil environment - mycotoxigenic fungi and mycotoxins in the soil environment

Strategies to reduce mycotoxin and fungal alkaloid contamination in organic and conventional cereal production systems

Mycotoxins are toxic metabolites formed by specific fungi that grow on living plants and their residues under favourable conditions. They are undesirable ingredients of food and feed. Risks are also posed by the spores and toxin-contaminated raised dusts. Contamination by mycotoxins is a severe problem in food security. More than 300 species of fungi with the ability to form mycotoxins have been identified. More than 400 metabolites are assigned to the group of mycotoxins. Fortunately, only about 20 mycotoxins produced by five genera of fungi (Fusarium, Penicillium, Claviceps, Alternaria and Aspergillus) are found regularly or periodically in food and feed at levels which might have an impact on human and animal health (Gareis, 1999a). Since synthetic fungicides are not allowed in Organic Agriculture, fungal populations are often presumed to be higher in this farming system, and a higher frequency of mycotoxin contamination of organic food and feed has frequently been postulated by Tinker (2001). Others argued that omitting fungicides might lead to higher diversification of microbial populations and therefore limit growth of specific mycotoxin producers. There are controversial reports on varying mycotoxin contamination levels of grains produced organically and in mainstream farming (Marx et al., 1995, Berleth et al., 1998, Birzele et al., 2002, Meier et al., 1999, Stahle et al., 1998, Doll et al., 2002). Thus, the first aim of this contribution is to analyse whether a higher occurence of mould on organically grown crops and, as a result, a higher risk of mycotoxin impact due to specific organic farming as well as low input farming methods is confirmed by the reviewed literature. Secondly, factors that may influence mycotoxin contamination in the different cereal production systems are highlighted. Finally, strategies to reduce mycotoxin contamination are described. The recommendations given are limited to the conditions of the temperate European climate.