Inge M.B. Knudsen

Selection of biological control agents for controlling soil and seed-borne diseases in the field

Different screening methods for selection of biological control agents (BCAs), for controlling soil and seed-borne diseases, are discussed. The shortcomings of laboratory methods focused on mechanism of action are discussed and we conclude that these methods should be used with caution if candidates with multifactorial or plant mediated mechanisms of control are to be obtained. In vitro screens may be useful for specific groups of microorganisms, thus, screens for antibiotics may be relevant for Streptomyces spp., and promising results have been obtained using soil plating or precolonized agar methods to screen for mycoparasitism and competitive saprophytic ability. Experience with screening in the Nordic programme ‘Biological control of seed borne diseases in cereals’ is summarized. Research in the four participating countries – Finland, Sweden, Norway and Denmark – followed the same paradigm: that of obtaining antagonists, well adapted to different Nordic environments, and developing them as effective BCAs. Potential antagonists were isolated from different sources and in planta screening methods were developed in order to optimize selection of antagonists effective against a range of seed borne pathogens. Screens in the laboratory or greenhouse were followed by screening in the field. The different screening procedures are compared and evaluated.

Biopriming of Infected Carrot Seed with an Antagonist, Clonostachys rosea, Selected for Control of Seedborne Alternaria spp.

ABSTRACT An ecological approach was used to select fungal antagonists effective against the seedborne pathogens Alternaria dauci and A. radicina on carrot. Twenty-five and 105 isolates originating from cereal and carrot habitats were screened against the pathogens in planta, respectively. Irrespective of isolate origin, fungal isolates belonging to Clonostachys rosea controlled pre- and postemergence death caused by A. dauci and A. radicina as effectively as the fungicide iprodione. Isolate IK726 of C. rosea was used in biopriming a seed lot with 29% A. radicina and 11% A. dauci (highly infected), and a seed lot with 4% A. radicina and 7% A. dauci (low infection). Seeds were primed with water alone (hydropriming) or with addition of C. rosea IK726 (biopriming). The occurrence of A. radicina and A. dauci increased twofold and fivefold, respectively, during 14 days hydropriming, irrespective of the initial infection level. On highly infected seed, biopriming reduced the incidence of A. radicina to <2.3% and that of A. dauci to <4.8% while the level of both pathogens was <0.5% on bioprimed seed with a low initial infection rate. In sand stand establishment tests, hydroprimed seeds had a lower healthy seedling stand than nonprimed seeds, mainly due to a high degree of postemergence seedling death. In contrast, biopriming resulted in a seedling stand that was better than that of both nonprimed and hydroprimed seeds. C. rosea IK726 multiplied fivefold to eightfold, and microscopic observations using C. rosea IK726 transformed with a green fluorescent protein (GFP) reporter gene showed that seeds were covered with a fine web of sporulating mycelium of C. rosea. The positive effect of biopriming on healthy seedling stand remained after 5 months of storage at 4 degrees C and IK726 survived at high numbers on these seed. In this study, we demonstrated that bio-priming with the biocontrol strain C. rosea IK726 facilitates priming of infected seeds without risking adverse effects on seedling establishment.

Identification of a Universally Primed-PCR-Derived Sequence-Characterized Amplified Region Marker for an Antagonistic Strain of Clonostachys rosea and Development of a Strain-Specific PCR Detection Assay

ABSTRACT We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.

GUS and GFP transformation of the biocontrol strain Clonostachys rosea IK726 and the use of these marker genes in ecological studies

Marker genes were introduced in the biocontrol strain Clonostachys rosea IK726 (IBT 9371) as a tool for monitoring the strain in ecological studies. The β-glucuronidase (GUS) reporter gene and a gene encoding the green fluorescent protein (GFP) were, in separate experiments, integrated into the genome of IK726 using the Hygromycin B (HygB) resistance gene as selective marker. In order to select GUS and GFP transformants that resembled the wildtype strain, growth rate, production of enzymes and of metabolites of four GUS positive and six GFP positive transformants were tested in vitro. In addition, the biocontrol efficacy against disease caused by seed-borne Fusarium culmorum was evaluated on barley grown in sand. Compared to the wildtype, two selected GUS and GFP transformants, IK726c5 and IK726d11, did not vary in physiological properties. Both maintained the ability to colonize barley roots, and to reduce efficiently the severity of F. culmorum without affecting plant emergence. Quantification of GUS activity of IK726c5 in peat and vermiculite and on seeds was carried out. The GFP transformant, IK726d11, was visualized by epifluorescence and confocal scanning laser microscopy directly in soil, vermiculite, on carrot seed and roots, and on barley leaves. It was shown that C. rosea can thrive in very different niches. Conidia germination, colonization and conidiogenesis were demonstrated in vivo in all four environments. This is the first report on transformation of Clonostachys rosea with marker genes.

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.

Biological Seed Treatment of Cereals with Fresh and Long-term Stored Formulations of Clonostachys rosea: Biocontrol Efficacy Against Fusarium culmorum

In six field experiments, seed treatment with Clonostachys rosea (IK726) significantly reduced disease caused by Fusarium culmorum. IK726 was active against the pathogen at average soil temperatures at sowing ranging from 6.2 to 12 °C. Both in the field experiments and in growth chamber experiments conducted in sand, dried and stored conidia of IK726 controlled F. culmorum as effectively as freshly harvested conidia. A high correlation was found between disease index ratings from field experiments and from corresponding growth chamber sand tests. Amendment with the stickers Pelgel or Sepiret did not influence control activity. The effective dosages of IK726 (cfu/seed) were estimated in bioassays and were very similar for freshly harvested conidia and for dried conidia. With a density of > 5×103 conidia per seed more than 80% disease control was repeatedly obtained with both types of conidia.

Characterization of microbial communities and fungal metabolites on field grown strawberries from organic and conventional production.

The background levels of culturable indigenous microbial communities (microbiotas) on strawberries were examined in a field survey with four conventional and four organic growers with different production practise and geographic distribution. The microbiota on apparently healthy strawberries was complex including potential plant pathogens, opportunistic human pathogens, plant disease biocontrol agents and mycotoxin producers. The latter group was dominated by Penicillium spp. and Aspergillus niger was also isolated. As expected, bacteria were the most abundant and diverse group of the strawberry microbiota followed by yeasts and filamentous fungi. No obvious correlation between grower practice and the strawberry microbiota was observed. Differences between microbiotas on strawberries from conventional systems with up to 10 fungicide spray treatments and organic production systems were insignificant. Mycotoxins were not detected in mature strawberries from any of the eight different growers neither in additional samples of low quality berries. However, isolates of Penicillium expansum and A. niger produced high amounts of mycotoxins when incubated on strawberries at 25°C. Penicillium polonicum produced cyclopenol, cyclopenin, and viridicatin on the artificially infected berries, while Alternaria arborescens produced tenuazonic acid, Alternaria tenuissima produced altertoxin I and altenuene, and Trichoderma spp. produced several peptaibols. In conclusion, native strawberry microbiotas are highly diverse both in terms of taxonomic groups and functional traits that are important in relation to plant and human health.

Development of a biocontrol agent for plant disease control with special emphasis on the near commercial fungal antagonist Clonostachys rosea strain ‘IK726’

Numerous experiments demonstrating potential biocontrol effects on soilborne diseases have been reported in the scientific literature. However, from the lists of approved and registered biocontrol agents, it is striking how few have been commercialised and are used in practise for plant disease control. The main hindrances are often claimed to be legislative aspects and the costs involved in the registration. Although this is in many respects true, there is a range of both biological and technical problems which must be considered when developing an effective biocontrol agent for commercial use.Among the success stories for control of seed- and soilborne diseases are fungal biocontrol agents based on Trichoderma harzianum, Clonostachys rosea and Conithyrium minitans, and bacterial biocontrol agents based on strains of Agrobacterium, Pseudomonas and Streptomyces. We have developed C. rosea strain ‘IK726’, which has proved to be an effective antagonist in several crops against seed- and soilborne diseases. Although a biocontrol agent based on C. rosea ‘IK726’ is not yet commercialised, this paper will be used to address some of the biological and technical aspects that must be dealt with in such a development.

Survival of Conidia of Clonostachys rosea on Stored Barley Seeds and Their Biocontrol Efficacy Against Seed-borne Bipolaris sorokiniana

Survival and biocontrol activity of Clonostachys rosea (isolate IK726) conidia during storage on barley seeds were investigated. The initial density of colony forming conidia on seed was 4 2 10 3 to 9 2 10 4 colony forming units (cfu)/seed. After 5 months storage at 4°C, the density decreased by less than one order of magnitude and the biocontrol efficacy against seedling blight caused by seed-borne Bipolaris sorokiniana was maintained at a significantly high level ( > 80% disease reduction) for > 5 months. Conidial survival on seeds stored at 20°C declined more rapidly than at 4°C, and biocontrol efficacy was significantly reduced after 3-5 months. However, conidia produced on solid media over 20 days survived better than conidia produced in liquid culture and conidia from solid media produced over 12 days. In contrast, when seeds treated with conidia were packed with silica gel and stored at 20°C, the cfu density decreased by less than one order of magnitude after 5 months and the biocontrol efficacy was still high after 6 months. A dose-response curve revealed that 103 cfu/seed were needed for 80% control of seedling blight. Similar control was obtained in storage experiments when approximately 103 cfu/seed were recovered from seed, indicating that conidia which survived also retained a high ability to control disease.

Potential suppressiveness of different field soils to Pythium damping-off of sugar beet

A highly reproducible, low resource requiring disease severity bioassay for testing suppression of Pythium damping-off in sugar beet caused by Pythium ultimum in soil is described. Changing of soil characteristics and the native microflora are avoided and standardization of the assay is carried out by testing infested soils on evenly germinating plants growing in an inert medium. The disease suppressiveness of the test soils is compared to standard, conducive soil, and quantified by calculation of symptoms on the hypocotyls. The reliability of the assay was investigated in nine well characterised organically or conventionally managed soils. In the standard, conducive soil it was clearly demonstrated that disease expression is correlated with available nutrient supply (oatmeal). Addition of oospores to the test soil had no effect on disease severity and the role of inoculum level is discussed. There was a clear tendency for low clay content to result in higher disease suppression. Partial sterilisation of the selected soils by γ-irradiation and steaming at 54 °C indicated that suppression was of microbial origin. Although texture may play a role in suppressiveness no clear effect, related to any management practice, was found. Tests on 64 soils confirmed that sandy soils tend to be more suppressive than clayey soils.