THE EFFECT OF COLD STORAGE OF POTTED AZALEA ON BROAD MITE INFECTION.

In potted azalea (Rhododendron simsii hybrids) the broad mite Polyphagotarsonemus latus (Banks) is considered a severe pest with an important economic impact. Although chemical control is available, permitted acaricides are limited and have a restricted number of applications. Therefore, growers have a keen interest in alternative control measures. Recently, research on the behaviour and population dynamics of P. latus on azalea leaf disks stored at different temperatures indicated that survival and reproductive capacity of broad mite is reduced drastically when temperature drops below 7°C. In Flanders, storage of azalea plants at 3°C is common practice to pause flower development (in function of the date that plants have to be ready for sale) before forcing them to flower in a heated greenhouse. Hence, an experiment was set-up to verify and quantify the effect of cold storage of azalea on broad mite infection. Azalea plants were infected with P. latus and stored at 3°C for 2, 3 or 4 weeks. Then, plants were transferred to a heated greenhouse for 2 weeks to check whether surviving female broad mites were still able to reproduce. The number of P. latus on azalea was assessed before cold treatment, immediately after treatment, and 2 weeks after transfer to the heated greenhouse. Results confirmed that cold storage can play a role in broad mite control as the P. latus population was significantly reduced (up to 90%) immediately after treatment. A further decrease in the number of P. latus during storage in the heated greenhouse indicated that cold treatment during 4 weeks had also an effect on the reproduction capacity of P. latus. We conclude that cold storage of azalea plants (at least 4 weeks at maximum 3°C) should be considered as an additional and alternative control method for P. latus at the end of the azalea production cycle.

MULCHES AND OTHER COVER MATERIALS TO REDUCE WEED GROWTH IN CONTAINER-GROWN NURSERY STOCK.

Due to the recent EU-wide implementation of Integrated Pest Management (IPM), alternative methods to reduce weed growth in container-grown nursery stock are needed to cut back the use of herbicides. Covering the upper layer of the substrate is known as a potential method to prevent or reduce weed growth in plant containers. As a high variety of mulches and other cover materials are on the market, however, it is no longer clear for growers which cover material is most efficient for use in containers. Therefore, we examined the effect on weed growth of different mulches and other cover materials, including Pinus maritima, P. sylvestris, Bio-Top Basic, Bio-Top Excellent, coco chips fine, hemp fibres, straw pellets, coco disk 180LD and jute disk. Cover materials were applied immediately after repotting of Ligustrum ovalifolium or planting of Fagus sylvatica. At regular times, both weed growth and side effects (e.g., plant growth, water status of the substrate, occurrence of mushrooms, foraging of birds, complete cover of the substrate and fixation) were assessed. All examined mulches or other cover materials were able to reduce weed growth on the containers during the whole growing season. Weed suppression was even better than that of a chemical treated control. Although all materials showed some side effects, the impact on plant growth is most important to the grower and depends not only on material characteristics (e.g., biodegradation, nutrient leaching and N-immobilisation) but also on container size and climatic conditions. In conclusion, mulches and other cover materials can be a valuable tool within IPM to lower herbicide use. To enable a deliberate choice of which cover material is best used in a specific situation more research is needed on lifespan and stability as well as on economic characteristics of the materials.

CLIMATE CONDITIONS AFFECTING THE WITHIN-PLANT SPREAD OF BROAD MITES ON AZALEA.

The broad mite Polyphagotarsonemus latus (Banks) is considered a major pest in potted azalea, Flanders' flagship ornamental crop of Rhododendron simsii hybrids. In addition to severe economic damage, the broad mite is dreaded for its increasing resistance to acaricides. Due to restrictions in the use of broad spectrum acaricides, Belgian azalea growers are left with only three compounds, belonging to two mode of action groups and restricted in their number of applications, for broad mite control: abamectin, milbemectin and pyrethrin. Although P. latus can be controlled with predatory mites, the high cost of this system makes it (not yet) feasible for integration into standard azalea pest management systems. Hence, a maximum efficacy of treatments with available compounds is essential. Because abamectin, milbemectin and pyrethrin are contact acaricides with limited trans laminar flow, only broad mites located on shoot tips of azalea plants will be controlled after spraying. Consequently, the efficacy of chemical treatments is influenced by the location and spread of P. latus on the plant. Unfortunately, little is known on broad mites' within-plant spread or how it is affected by climatic conditions like temperature and relative humidity. Therefore, experiments were set up to verify whether climate conditions have an effect on the location and migration of broad mites on azalea. Broad mite infected azalea plants were placed in standard growth chambers under different temperature (T:2.5-25°C) and relative humidity (RH:55-80%) treatments. Within-plant spread was determined by counting mites on the shoot tips and inner leaves of azalea plants. Results indicate that temperature and relative humidity have no significant effect on the within-plant spread of P. latus. To formulate recommendations for optimal spray conditions to maximize the efficacy of broad mite control with acaricides, further experiments on the effect of light intensity and rain are scheduled.

Local spread of metamitron resistant Chenopodium album L. patches.

Molecular markers can provide valuable information on the spread of resistant weed biotypes. In particular, tracing local spread of resistant weed patches will give details on the importance of seed migration with machinery, manure, wind or birds. This study investigated the local spread of metamitron resistant Chenopodium album L. patches in the southwest region of the province West-Flanders (Belgium). During the summer of 2009, leaf and seed samples were harvested in 27 patches, distributed over 10 sugar beet fields and 1 maize field. The fields were grouped in four local clusters. Each cluster corresponded with the farmer who cultivated these fields. A cleaved amplified polymorphic sequence (CAPS) procedure identified the Ser264 to Gly mutation in the D1 protein, endowing resistance to metamitron, a key herbicide applied in sugar beet. The majority of the sampled plants within a patch (97% on average) carried this mutation. Amplified fragment length polymorphism (AFLP) analysis was performed with 4 primer pairs and yielded 270 molecular markers, polymorphic for the whole dataset (303 samples). Analysis of molecular variance revealed that a significant part of the genetic variability was attributed to variation among the four farmer locations (12 %) and variation among Chenopodium album patches within the farmer locations (14%). In addition, Mantel tests revealed a positive correlation between genetic distances (linearised phipt between pairs of patches) and geographic distances (Mantel-coefficient significant at p = 0.002), suggesting isolation-by-distance. In one field, a decreased genetic diversity and strong genetic relationships between all the patches in this field supported the hypothesis of a recent introduction of resistant biotypes. Furthermore, genetic similarity between patches from different fields from the same farmer and from different farmers indicated that seed transport between neighbouring fields is likely to have an important impact on the spread of metamitron resistant biotypes.

Distribution of metamitron-resistant Chenopodium album L. in Belgian sugar beet.

Chenopodium album L. (fat-hen) with a Ser264-Gly mutation is resistant to photosystem II-inhibiting herbicides like the triazinone metamitron, a key herbicide in sugar beet. In recent years, this resistant biotype may cause unsatisfactory weed control in Belgian sugar beet. However, the dimension of the problem was yet unknown. Therefore, a survey was conducted in 2008 covering the whole Belgian sugar beet area. In randomly selected fields, C. album plants surviving weed control were counted and sampled. First, the number of surviving plants was used to estimate the prevalence of fields with unsatisfactory control and to classify the surveyed fields. Then, the share of the resistant biotype in each field was determined with cleaved amplified polymorphic sequence-analysis (CAPS-analysis) on sampled leaves. Finally, all results were visualised on the map of Belgium. Twenty percent of the fields had more than 500 surviving plants per hectare and were thus classified as fields with unsatisfactory C. album control. The resistant biotype was present in 95% of these fields and even in 74% of the sampled fields with good weed control. No pattern was found during mapping. These results indicate that the metamitron-resistant biotype has spread over the whole sugar beet area but that it is not (yet) causing severe problems in every field. To get a more accurate estimation of the portion of resistant plants in the field and the effect of herbicide treatment on this biotype, an elaborate survey will be conducted in 2010 on fields that have both untreated and treated plots installed.

Chlorophyll fluorescence protocol for quick detection of triazinone resistant Chenopodium album L.

Sugar beet growers in Europe are more often confronted with an unsatisfactory control of Chenopodium album L. (fat-hen), possibly due to the presence of a triazinone resistant biotype. So far, two mutations on the psbA-gene, i.e. Ser264-Gly and Ala251-Val, are known to cause resistance in C. album to the photosystem II-inhibiting triazinones metamitron, a key herbicide in sugar beet, and metribuzin. The Ser264-Gly biotype, cross-resistant to many other photosystem II-inhibitors like the triazines atrazine and terbuthylazine, is most common. The second resistant C. album biotype, recorded in Sweden, is highly resistant to triazinones but only slightly cross-resistant to terbuthylazine. Since farmers should adapt their weed control strategy when a resistant biotype is present, a quick and cheap detection method is needed. Therefore, through trial and error, a protocol for detection with chlorophyll fluorescence measurements was developed and put to the test. First, C. album leaves were incubated in herbicide solution (i.e. 0 microM, 25 microM metribuzin, 200 microM metamitron or 25 microM terbuthylazine) during three hours under natural light. After 30 minutes of dark adaptation, photosynthesis yield was measured with Pocket PEA (Hansatech Instruments). In Leaves from sensitive C. album, herbicide treatment reduces photosynthesis yield due to inhibition of photosynthesis at photosystem II. This results in a difference of photosynthesis yield between the untreated control and herbicide treatment. Based on the relative photosynthesis yield (as a percentage of untreated), a classification rule was formulated: C. album is classified as sensitive when its relative photosynthesis yield is less than 90%, otherwise it is resistant. While metribuzin, and to a lesser extent, metamitron treatment allowed a quick detection of triazinone resistant C. album, terbuthylazine treatment was able to distinguish the Ser264-Gly from the Ala251-Val biotype. As a final test, 265 plants were classified with the protocol. Simultaneously, a CLeaved Amplified Polymorphic Sequence (CAPS)-analysis was conducted on the same plants to verify the presence of the Ser264-Gly mutation. Only one mismatch was found when results of both detection methods were compared. The test results illustrate that this protocol provides a reliable, quick and cheap alternative for DNA-analysis and bio-assays to detect the triazinone resistant C. album biotypes.

Resistance of Chenopodium albumto photosystem II-inhibitors.

Chenopodium album L. (fat-hen), a highly competitive and very prolific species, is a common weed in most spring- and summer-sown crops such as maize, sugar beet and vegetables. In the late seventies, C. album stepped into the limelight as a problem weed in maize. Frequent use of atrazine in maize monoculture did select for plants having a Ser-264-Gly mutation on the psbA gene resulting in atrazine-resistance and cross-resistance to other Photosystem (PS) II-inhibitors. The psbA gene encodes the D1 protein of PS II which is the target site of PS II-inhibitors. Introduction of new herbicides made it possible to control this atrazine-resistant biotype in maize, which allowed C. album to fade into the background again until it resurfaced some years ago as a problem weed in European sugar beet (Belgium, France, The Netherlands and Sweden). Greenhouse bioassays at Ghent University revealed that the unsatisfactory control of C. album in sugar beet is due to resistance to the triazinone metamitron, a key herbicide in sugar beet. The expected cross-resistance to atrazine and metribuzin was found in all populations except for a Swedish one, which is highly resistant to metamitron and metribuzin but not to atrazine. DNA sequence analysis confirmed the presence of a Ser-264-Gly mutation for all populations that are both metamitron- and atrazine-resistant. The Swedish population has an Ala-251-Val mutation on the psbA gene explaining its aberrant (cross)-resistance profile. The occurrence of C. album biotypes with resistance to metamitron but different genotypes and cross-resistance profiles could raise the question which herbicide(s) did select for the resistance. In Sweden, having no history of atrazine use, the triazinones metamitron, used in sugar beet, and metribuzin, used in rotational potato, could have selected for resistance. In Belgium, at least three different herbicides and/or crop rotations could have contributed to resistance development: (1) a record of continuous use of atrazine in maize resulting in triazine-resistant C. album in the seed bank, (2) metamitron use in sugar beet and (3) metribuzin use in potato.

Chlorophyll fluorescence tests for monitoring triazinone resistance in Chenopodium album L.

Recently, fat-hen (Chenopodium album L.) biotypes resistant to metamitron, a key herbicide in sugar beet, were recorded. Pot experiments revealed that these biotypes showed cross-resistance to metribuzin, a triazinone used in potato. Greenhouse and laboratory experiments were performed to develop resistance monitoring tests, so that resistant biotypes can be detected quickly and farmers may adapt their weed management. Resistant and susceptible biotypes were grown in a greenhouse under conditions of natural and artificial light at an intensity of 100 micromol photons m(-2) s(-1). Leaves were collected and, immersed in a solution of 1000 microM metamitron and 500 microM metribuzin, exposed to natural and artificial light (1000, 750 and 100 micromol photons m(-2) s(-1) respectively). After this, chlorophyll fluorescence measurements were carried out. The results revealed that the photosynthetic electron transport of metamitron- and metribuzin-incubated leaves of resistant biotypes decreased less than that of the incubated Leaves of susceptible biotypes. The differences between the metribuzin-incubated leaves of the susceptible and resistant biotypes were larger than those observed with the metamitron-incubated leaves. The aim of the experiments was to optimise the chlorophyll fluorescence test and to find a sufficiently high correlation between the results of the pot experiments and the chlorophyll fluorescence measurements.

Effect of selected sugar beet herbicides on germination of various Chenopodium album populations.

Seeds of various fat-hen populations (Chenopodium album L.), mostly originating from sugar beet fields, were subjected to treatments with the following herbicides: metamitron, acetochlor, dimethenamid-P and S-metolachlor. Herbicides were applied either incorporated into a sandy Loam soil (2005-2007) and/or on filter paper in Petri dishes (2006-2007). Results between experiments were highly contrasting. Soil applications of metamitron, acetochlor and S-metolachlor were stimulating germination in the 2005 experiments, whereas in the 2006-2007 experiments effects were ranging from slightly stimulating to highly inhibitory.

Metamitron-resistant Chenopodium album from sugar beet: cross-resistance profile.

In recent years, in several of the Belgian sugar beet growing regions, farmers have been confronted with unsatisfactory control of fat hen (Chenopodium album L.). Greenhouse bioassays conducted on reference C. album populations and on "suspected" populations from sugar beet fields where poor fat hen control had been observed, revealed that all "suspected" populations were resistant to metamitron, a key herbicide in the modern low rate weed control programs in sugar beet. These metamitron-resistant biotypes were all cross-resistant to atrazine. Since cross-resistance, particularly negative cross-resistance or reversed resistance, is known to play a major role in resistance management, other herbicides used in sugar beet and/or in rotational crops were tested to determine the cross-resistance profile of metamitron-resistant biotypes. Greenhouse bioassays were conducted using herbicides from different chemical families representing different modes of action. Cross-resistance was found for metribuzin, lenacil and chloridazon, all HRAC Group C1 herbicides that inhibit photosynthesis at PS II. The metamitron-resistant C. album populations examined showed negative cross-resistance to S-metolachlor (HRAC Group K3: inhibition of cell division), prosuifocarb (Group N: lipid synthesis, not AC-Case, inhibition), aclonifen and clomazone (both Group F3: inhibition of carotenoid biosynthesis).

Resistance to metamitron in Chenopodium album from sugar beet. a tale of the (un)expected?

Metamitron is a key herbicide in modern low rate weed control programs in sugar beet. Fat hen (Chenopodium album, CHEAL) is a common, highly competitive, weed in sugar beet and one of the targets of metamitron. Recently, unsatisfactory control of fat hen has been reported in several sugar beet fields situated in various regions in Belgium. Weather conditions as well as the mere fact of using low rate systems have been blamed for these poor performances. To address the question "Is the recently recorded poor control of C. album due to decreased sensitivity to metamitron", greenhouse bioassays were carried out. A first experiment was conducted applying metamitron (0, 350, 700 and 1,400 g ai/ha) postemergence to three "suspected" C. album populations originating from sugar beet fields with unsatisfactory control by standard metamitron based treatment schemes ('Ligne', 'Outgaarden' and 'Boutersem I' respectively) and to one sensitive population originating from an untreated garden site ('Gent'). In a second experiment seven population, five "suspected" fat hen populations from sugar beet fields ('Boutersem I', 'Boutersem II', 'Postel', 'Vissenaken' and 'Kortessem' respectively), one sensitive reference population 'Herbiseed' and one atrazine-resistant reference population 'ME.85.01', were submitted to metamitron (0, 1, 2 and 4 mg ai/kg air-dry soil) and atrazine (1.5 mg ai/kg air-dry soil) preplant incorporated. All "suspected" C. album populations displayed a significantly lower sensitivity to metamitron compared to the sensitive populations ('Gent' and 'Herbiseed') that never had been exposed to this herbicide. As target site cross-resistance of atrazine-resistant C. album, selected by atrazine in maize, to metamitron has been known for a long time, cross-resistance of C. album populations in sugar beet grown on fields with "maize - atrazine" containing rotations might be expected to appear. The outcome of the experiment with atrazine preplant incorporated was the confirmation of resistance in all "suspected" populations ('Boutersem I', 'Boutersem II', 'Postel', 'Vissenaken' and 'Kortessem'). However, some "suspected" populations came from fields with no background of cropping with maize and use of atrazine. So, the question remains whether these triazine-resistant C. album had been imported, e.g. with slurry, or the rather unexpected possibility that metamitron itself did select for metamitron-resistant biotypes bearing cross-resistance to atrazine, had become reality.