J. Köhl

Major Changes in Fusarium spp. in Wheat in the Netherlands

The re-emergence of fusarium head blight throughout the world and especially in Western Europe prompted a survey of the situation in the Netherlands. To allow for a high throughput screening of large numbers of samples, a diagnostic PCR method was developed to detect the most common species of Fusarium occurring on wheat. Seven primer pairs were tested for their ability to identify isolates of Fusarium avenaceum, F. culmorum, F. graminearum, F. poae, F. proliferatum and Microdochium nivale var. majus and M. nivale var. nivale. Each primer pair only generated a PCR product with the corresponding Fusarium species and all PCR fragments had different molecular sizes. This allowed the generation of these amplicons using a mixture of all seven primer pairs. The robustness of this multiplex PCR encouraged us to screen a large series of isolates collected in 2000 and 2001. In both years 40 fields were sampled leading to a collection of 209 isolates from 2000 and 145 isolates from 2001. The results of the multiplex PCR demonstrated that F. graminearum was the most abundant species in the Fusarium complex on wheat in both years. This is in sharp contrast to reports from the 1980s and early 1990s, which found F. culmorum as the predominant species. Primers derived from the tri7 and tri13 genes, which are implicated in the acetylation and oxygenation of the C-4 atom of the backbone of the trichothecene molecule, were used to discriminate between deoxynivalenol and nivalenol (NIV) producers. The populations of F. culmorum and F. graminearum both showed a slight increase in NIV-producers in 2001.

Have biopesticides come of age

Biopesticides based on living microbes and their bioactive compounds have been researched and promoted as replacements for synthetic pesticides for many years. However, lack of efficacy, inconsistent field performance and high cost have generally relegated them to niche products. Recently, technological advances and major changes in the external environment have positively altered the outlook for biopesticides. Significant increases in market penetration have been made, but biopesticides still only make up a small percentage of pest control products. Progress in the areas of activity spectra, delivery options, persistence of effect and implementation have contributed to the increasing use of biopesticides, but technologies that are truly transformational and result in significant uptake are still lacking.

Quantitative detection of Fusarium species in wheat using TaqMan

Fusarium head blight (FHB) of wheat and other small-grain cereals is a disease complex caused by several fungal species. To monitor and quantify the major species in the FHB complex during the growing season, real-time PCR was developed. TaqMan primers and probes were designed that showed high specificity for Fusarium avenaceum, F. culmorum, F. graminearum, F. poae and Microdochium nivale var. majus. Inclusion of an internal PCR control and serial dilutions of pure genomic DNAs allowed accurate determination of the concentration of fungal DNA for each of these species in leaves, ears as well as harvested grains of winter wheat. The DNA concentration of F. graminearum in grain samples correlated (r2= 0.7917) with the incidence of this species on the grain as determined by isolation from individual kernels. Application of the TaqMan technology to field samples collected in 40 wheat crops in the Netherlands during the growing season of 2001 revealed that M. nivale var. majus predominated on leaves early in the season (GS 45-65). Ears and harvested grains from the same fields, however, showed F. graminearum as the major species. In 2002, grain samples from 40 Dutch fields showed a much wider range of species, whereas in ears from 29 wheat crops in France, F. graminearum was the predominant species. The concentration of DON correlated equally well with the incidence of the DON-producing species F. culmorum and F. graminearum in the grain samples (r2= 0.8232) as well as with total DNA of both these species (r2= 0.8259). The Fusarium TaqMan technology is an important tool to quantify and monitor the dynamics of individual species of the complex causing FHB in cereals during the growing season. This versatile tool has been applied in a comparison of different genotypes, but can also be applied to other disease management systems, e.g. fungicide treatments.

Potential of fungal antagonists for biocontrol of Fusarium spp. in wheat and maize through competition in crop debris

Abstract Pathogenic Fusarium spp. cause head blight in wheat or ear rot in maize leading to yield losses and also a reduction in quality due to mycotoxin contamination of the grain. Infected crop residues are the main inoculum source for epidemics. Saprophytic fungi, obtained from cereal tissues or necrotic tissues of other crops, were screened for their ability to colonise wheat straw and maize stalks and to suppress sporulation of pathogenic Fusarium spp. Results of bio-assays conducted under controlled conditions were variable among Fusarium spp. and host substrates for most antagonists tested, such as yeasts, Trichoderma spp. and non-pathogenic Fusarium spp. Isolates of Clonostachys rosea consistently suppressed sporulation of F. culmorum and F. graminearum on wheat straw, and of F. culmorum, F. graminearum, F. proliferatum and F. verticillioides on maize stalks. Isolates of C. rosea, C. cladosporioides and F. equiseti were applied to pieces of maize stalks or flowering ears in preliminary experiments conducted under field conditions. The colonisation of stalk pieces by pathogenic Fusarium spp. was assessed after 9 months. Colonisation of stalk pieces by pathogenic Fusarium spp. was significantly reduced at several sampling dates. However, results obtained with the antagonists were not consistent for all sampling dates and between experiments.

Biological Control of Botrytis cinerea in Cyclamen with Ulocladium atrum and Gliocladium roseum Under Commercial Growing Conditions

ABSTRACT The effect of treatments with conidial suspensions of Ulocladium atrum and Gliocladium roseum on leaf rot of cyclamen caused by Botrytis cinerea was investigated under commercial greenhouse conditions. Spraying U. atrum (1 x 10(6) conidia per ml) or G. roseum (2 x 10(6) conidia per ml and 1 x 10(7) conidia per ml) at intervals of 2 to 3 weeks during the production period and spraying U. atrum (1 x 10(6) conidia per ml) at intervals of 4 to 6 weeks resulted in a significant reduction of natural infections of petioles by B. cinerea. U. atrum or G. roseum (1 x 10(7)conidia per ml) was as effective as the standard fungicide program. B. cinerea colonized senesced leaves within the plant canopy and infected adjacent petioles and leaves later. The antagonists colonized senesced leaves and reduced B. cinerea development on these leaves. Thus, the inoculum potential on petioles adjacent to necrotic leaf tissues was reduced. The fate of U. atrum conidia on surfaces of green cyclamen leaves during a 70-day period after application was studied. The number of conidia per square centimeter of leaf surface remained relatively constant during the entire experiment. Sixty percent of the conidia sampled during the experiments retained the ability to germinate. When green leaves were removed from the plants to induce senescence and subsequently were incubated in a moist chamber, U. atrum colonized the dead leaves. Senesced leaves also were colonized by other naturally occurring fungi including B. cinerea. On leaves treated with U. atrum from all sampling dates, sporulation of B. cinerea was significantly less as compared with the untreated control. Our results indicate that early applications of U. atrum before canopy closure may be sufficient to achieve commercially satisfactory control of Botrytis leaf rot in cyclamen.

Suppression of sporulation of Botrytis spp. as a valid biocontrol strategy

In this study, the hypothesis was tested that removal of substrate for sporulation ofBotrytis spp. may lead to a retardation of an epidemic if the majority of the inoculum is produced inside the treated crop. Suppression of sporulation ofBotrytis spp. could be an attractive option for biological control ofBotrytis leaf spot in onions. In a field experiment, necrotic leaf tissue was removed to simulate the effect of a biocontrol agent. By this means, the amount of substrate on whichBotrytis spp. sporulates was reduced. In the experiment, the spore load above the onion plots was significantly reduced and the epidemic of onion leaf spot was retarded. At the end of the growing season, the number of leaf lesions in the green leaf area was lower in plots with substrate removal than in control plots (0.6 and 1.1 cm−2, respectively). The results demonstrated that an epidemic of onion leaf spot largely depends on the rate of inoculum production inside a crop. Thus, suppression of sporulation on necrotic leaf tissue is a valid control strategy that could be applied by using sporulation suppressing antagonists.

Effect of interrupted leaf wetness periods on suppression of sporulation of Botrytis allii and B. cinerea by antagonists on dead onion leaves

Saprophytic antagonists were evaluated for suppression of sporulation ofBotrytis allii andB. cinerea on artificially killed segments of onion leaves that were pre-inoculated with the pathogens. During incubation of the antagonisttreated leaf segments in moist chambers, periods of leaf wetness and leaf dryness were alternated to simulate conditions in the field. Interruption of humid conditions with dry periods had a differential effect on antagonists.Alternaria alternata, Chaetomium globosum, Ulocladium atrum andU. chartarum suppressed sporulation ofB. allii almost completely under continuously wet conditions, and when the leaf wetness periods were interrupted with drying periods of 9h imposed 16, 40, and 64 h after the antagonists were applied. When leaf wetness was interrupted 16 h after antagonist application, the number of conidia ofB. allii produced cm−2 leaf surface after eight days was under the detection limit of 5.2 × 103 conidia on leaves treated with these antagonists compared to 3.7 × 105 conidia on leaves that were not treated. On the other hand,Gliocladium roseum, G. catenulatum andSesquicillium candelabrum, all highly efficient under continuously wet conditions, were of low to moderate efficiency when leaf wetness periods had been interrupted 16 h after application of the antagonists. The antagonists showed the same differentiation and sensitivity to interrupted wetness periods when tested withB. cinerea.

Biological control using invertebrates and microorganisms: plenty of new opportunities

Abstract In augmentative biological control (ABC), invertebrate and microbial organisms are seasonally released in large numbers to reduce pests. Today it is applied on more than 30 million ha worldwide. Europe is the largest commercial market for invertebrate biological control agents, while North America has the largest sales of microbials. A strong growth in use of ABC, particularly of microbial agents, is taking place in Latin America, followed by Asia. The current popularity of ABC is due to (1) its inherent positive characteristics (healthier for farm workers and persons living in farming communities, no harvesting interval or waiting period after release of agents, sustainable as there is no development of resistance against arthropod natural enemies, no phytotoxic damage to plants, better yields and a healthier product, reduced pesticide residues [well below the legal Maximum Residue Levels (MRLs)], (2) professionalism of the biological control industry (inexpensive large scale mass production, proper quality control, efficient packaging, distribution and release methods, and availability of many (>440 species) control agents for numerous pests), (3) a number of recent successes showing how biological control can save agricultural production when pesticides fail or are not available, (4) several non-governmental organizations (NGOs), consumers, and retailers demanding pesticide residues far below the legal MRLs, and (5) policy developments in several regions of the world aimed at reduction and replacement of synthetic pesticides by more sustainable methods of pest management. We are convinced, however, that ABC can be applied on a much larger area than it is today. We plead in the short term for a pragmatic form of agriculture that is adaptable, non-dogmatic and combines the sustainability gain from all types of agriculture and pest management methods. We then propose to move to “conscious agriculture”, which involves participation of all stakeholders in the production and consumer chain, and respects the environment and resource availability for future generations. Were “conscious agriculture” to be considered a serious alternative to conventional farming, ABC would face an even brighter future.

Population Dynamics of Fusarium spp. and Microdochium nivale in Crops and Crop Residues of Winter Wheat

ABSTRACT Naturally occurring populations of Fusarium avenaceum, F. culmorum, F. graminearum, F. poae, and Microdochium nivale were studied in two field experiments from anthesis in June 2003 until harvest in crops of winter wheat, and subsequently during 10 months after harvest until June 2004 on their residues exposed on the soil surface under field conditions. The dynamics of the different pathogens were estimated by quantifying the amount of DNA present in wheat tissues using TaqMan-polymerase chain reaction. While colonization of grain by Fusarium spp. and M. nivale was low, high amounts of DNA of F. avenaceum, F. graminearum, and F. culmorum were found in ear residues, internodes, and nodes of the mature crop. Amounts of DNA of pathogens decreased significantly during the following 10 months in residues of internodes and nodes, but not in residues of stem bases. Knowledge on population dynamics of pathogens will help to develop preventive measures aimed at reduction of inoculum sources of head blight pathogens.

Quantification of Mycelium of Botrytis spp. and the Antagonist Ulocladium atrum in Necrotic Leaf Tissue of Cyclamen and Lily by Fluorescence Microscopy and Image Analysis

ABSTRACT A technique was developed to localize and quantify the internal mycelial colonization of necrotic leaf tissue of cyclamen (Cyclamen persicum) or lily (Lilium) by pathogenic Botrytis spp. and the antagonist Ulocladium atrum. This technique allows investigation of competitive substrate colonization by both fungi, which is a key process for biological control of Botrytis spp. by U. atrum. A combination of differential fluorescent labeling and image analysis was applied on cryostat sections of necrotic leaf tissue. Botrytis mycelium was labeled specifically by indirect immunofluorescence using a monoclonal antibody specific for Botrytis spp. And an antimouse fluorescein conjugate. Wheat germ agglutinin conjugated to the fluorochrome TRITC was used to label mycelium of both fungi. Image analysis was used to measure the relative surface area of the cryostat section covered by fluorescing hyphae of Botrytis spp. and by fluorescing hyphae of both fungi. A mathematical conversion was derived and used to calculate the relative mycelial volume of each fungal species in the necrotic tissue based on the measured relative surface areas. Temporal aspects of substrate colonization were studied in a short time series. An analysis of components of variance provided insight into spatial colonization patterns for the fungal species involved and allowed the design of efficient sampling strategies for future experiments.