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Disease variation and chemical control of Ramularia leaf spot in sugarbeet

Tine Thach a,b,*, Lisa Munk b, Anne Lisbet Hansen c, Lise N. Jørgensen a

aAarhus University, Faculty of Science and Technology, Department of Agroecology, Forsoegsvej 1, 4200 Slagelse, DenmarkbUniversity of Copenhagen, Faculty of Science, Department of Plant and Environmental Sciences, Hoejbakkegaard Allé 13, 2630 Taastrup, DenmarkcNordic Beet Research, Sofiehoej, Hoejbygaardvej 14, 4960 Holeby, Denmark

a r t i c l e i n f o

Article history:Received 24 June 2012Received in revised form5 February 2013Accepted 24 April 2013

Keywords:Ramularia leaf spotRamularia beticolaSensitivity testEpoxiconazolePrecipitationYield

a b s t r a c t

Data from 1999 to 2009 on Ramularia leaf spot caused by Ramularia beticola in sugar beet showed thatit was a serious disease in sugar beet in 5 out of 11 seasons. The severity and significance of the diseasewas found to vary depending on events with precipitation, particularly in two specific weeks in July andSeptember. Several fungicides were found to give effective control, and positive net yield responseswere found in 9 out of 11 seasons. The average sugar yield response varied in individual years between0.7 and 2.2 t ha�1. High levels of control of Ramularia leaf spot was obtained in field trials, a semi-fieldtrial and an in vitro test using the compounds pyraclostrobin, epoxiconazole, difenoconazole andpropiconazole. Dose response trials with epoxiconazole from two seasons showed both reduced effi-cacy and yield responses from low doses. They also proved that the optimal input of fungicides variessignificantly between seasons depending on disease severity. A sensitivity test of R. beticola to differentfungicides showed a normal distribution of sensitivity with no sign of resistance development to eitherstrobilurins or triazoles. Results from a semi-field trial showed both good preventive and curative ef-fects with 84e100% disease control from epoxiconazole, difenoconazole and pyraclostrobin. In order tooptimize an IPM control strategy better forecasting systems are needed along with cultivars providinghigher levels of resistance to the disease.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Ramularia leaf spot caused by Ramularia beticola Fautr. andLamb is a serious foliar disease of sugar beet (Beta vulgaris L.) intemperate crop growing areas, particularly in Northern Europe(Ahrens, 1987; Hestbjerg and Dissing, 1995; Byford, 1996;Märländer et al., 2003). The disease has become of increasingimportance in the Nordic countries, e.g. Denmark, Sweden (Perssonand Olsson, 2006) and Finland (Eronen, 2010, Pers. comm.). Inseasons with severe epidemics, like 2007 in Denmark, infectionleads to early destruction of sugar beet foliage. This may indicatethat R. beticola has become more aggressive or that the fungicideshave been applied too late or that the sensitivity to fungicides couldhave changed.

Ramularia leaf spot is favoured by climatic conditions with highrelative humidity and moderate temperatures of 17e20 �C (Ahrens,1987; Hestbjerg et al., 1994). Symptoms become apparent after

18 days with moderate temperatures and a relative humidity ofmore than 95% (Hestbjerg and Dissing, 1995). The distribution ofthe spores takes place through wind and rain splash. The fungusoverwinters on crop residues, and crop rotation is thereforeimportant in order to minimize the risk of attack (Nielsen, 1991;Persson and Olsson, 2006). Symptoms of Ramularia leaf spot arecircular necrotic lesions of approx. 2e10 mm in diameter (Ahrens,1987; Hestbjerg et al., 1994). At the early stages of symptomdevelopment they can be difficult to distinguish from those ofanother important foliar disease, Cercospora leaf spot (Cercosporabeticola) (Wenzel, 1931). Whitish sporulation of conidia in thecentre of the sunken lesions is a characteristic of R. beticolacompared to the dark-grey sporulation mass produced byC. beticola. Ramularia leaf spot causes premature defoliation of thecrop, which decreases root weight, sugar content and juice quality(Byford, 1975; Nielsen, 1991). Sugar yield loss in the range of 10e24% has been recorded in Germany (Petersen et al., 2001) andDenmark based on data from fungicide trials where treated plotswere compared to untreated.

Chemical control using triazoles or strobilurins is the commonmeans of managing the disease either by applying fungicides once

* Corresponding author. Aarhus University, Department of Agroecology, For-soegsvej 1, 4200 Slagelse, Denmark. Tel.: þ45 87157504.

E-mail address: Tine.Thach@agrsci.dk (T. Thach).

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it appears in the crop or when weather conditions are regarded asfavourable (Ahrens, 1987; Märländer et al., 2003). Triazoles likeepoxiconazole, propiconazole and difenoconazole, which inhibitthe ergosterol biosynthesis in fungi (Yoshida and Aoyama, 1987;Leroux et al., 2008), have provided effective control of Ramularialeaf spot in field trials. Similarly, the strobilurin pyraclostrobinwhich is a Qol-fungicide inhibiting the mitochondrial respiration(Bartlett et al., 2002) has been found to be effective. As both tri-azoles and strobilurins are known to be fully or partly systemic theyhave the potential of providing both preventive and curative con-trol. Fungicide resistance in R. beticola has so far not been reported.

Two other foliar diseases of importance in sugar beet crops inNorthern Europe are powdery mildew (Erysiphe betae) (Francis,2002) and rust (Uromyces betae) (Byford, 1996; Bauer et al., 2011).They are controlled by using partially resistant cultivars or chemicalcontrol (Märländer et al., 2003; Karaoglanidis and Karadimos,2006). In order to obtain good control of all relevant diseasesduring the season growers are commonly applying fungicides onceor twice depending on the time of harvest.

Today the common European agricultural policy questions theincreasing dependence on pesticides and supports the concept ofintegrated pest management (IPM) by establishing a framework forcommunity action to achieve the sustainable use of pesticides (EUDirective 2009/128/EC). The aim is to reduce the impact and use ofpesticides. Use of thresholds and decision support systems isconsidered an important element in EU’s IPM policy but at presentlittle knowledge is available which can support control of leaf dis-eases in sugar beet based on these principles. Even though much isknown about the epidemiology and cropping factors influencingRamularia leaf spot several areas including climatic conditionsaffecting Ramularia leaf spot, need to be further elucidated tosupport an IPM-related recommendation.

One of the objectives of this study have been to elucidate therisk and severity of Ramularia leaf spot in Denmark based on his-torical data from 1999 to 2009 and to investigate the relationshipbetween precipitation and the severity of Ramularia leaf spot. Asecond objective has been to extract yield responses to fungicidetreatments and the economic output in order to estimate the po-tential losses from the threemain diseases of sugar beet: Ramularialeaf spot, powdery mildew and rust. A third objective has been todetermine the sensitivity of R. beticola isolates to different fungi-cides and the final objective was to test the efficacy of differentfungicides applied preventatively and curatively on Ramularia leafspot in order to test the potential of the products for control atdifferent timings.

2. Materials and methods

2.1. Historical data and precipitation analysis

A data set of Ramularia leaf spot, powdery mildew and rustassessments as well as yield from Danish sugar beet trials in 1999e2009 was supplied by NBR Nordic Beet Research, DK-Holeby (NBR).From each season the treatment which on average provided thebest yield increase was selected for further analysis. With few ex-ceptions the sugar beet crops were sown from late March to earlyApril. Disease severity was assessed on whole plants giving a scorebased on individual plots. The score ranged from 0 to 10 on a linearscale where 0 ¼ no attack and 10 ¼ severe attack. Forty-one fieldsites located in Southern and Eastern Denmark were included inthe analysis. The number of disease assessments varied from one tothree, and only the last disease assessments (conducted betweenlate September and the start of November) were used in the dataanalysis as this was most consistent for all trials. Precipitation datawere extracted from a national meteorological database provided

by Aarhus University and included daily rainfall (mm) from May toSeptember recorded at the individual weather stations closest tothe field sites. Fungicides used in the trials contained either theactive ingredient epoxiconazole alone or in mixture with pyr-aclostrobin (Opus and Opera, BASF A/S). These were chosen as theyare registered fungicides commonly used in Denmark.

2.2. Effects of foliar diseases and chemical control

The economics of chemical control shown as margin overfungicide cost (V ha�1) were calculated based on the increase insugar yield and beet quality in treated plots (fromwhich the costs ofthe fungicide and machinery costs of application were subtracted)compared with untreated plots. The calculations were based onprice agreements for 2011, which were applied for all years.Furthermore, the calculation was based on the assumption that thecontract on quantity and quality of sugar beet was fulfilled takinginto account that surplus is calculated as the value of the bestpossible economic alternative plus added value of the improvedquality of total delivery to the factory. The costs of fungicides in 2011were used to calculate the margin over fungicide cost. Opus cost40 V ha�1 and Opera cost 53 V ha�1 (www.middeldatabasen.dk,2011). The cost of treatment application was set at 9.3 V l�1 ha�1.

2.3. Fungal material, inoculation and isolation of R. beticola

In total, 13 isolates of R. beticola were isolated and used in thestudies. The isolates originated from diseased dried leaves and purecultures from Denmark, Sweden and the Netherlands. Pure cultureR. beticola reference isolates (JF330013 and JF330014) originatingfrom Switzerland and Sweden were obtained from the FungalBiodiversity Centre, the Netherlands (CBS). The identity of theisolates have been verified as R. beticola when aligned with refer-ence R. beticola isolates obtained from CBS using the ITS1 and ITS2sequence. Spore and mycelia fragment suspensions of 5e10 driedleaves (thus bulk isolates of a field) were made by shaking theleaves in 500 ml demineralized water for 10 min. The inoculumconcentration was determined to >1.0 � 10�4 ml�1 using a hae-mocytometer. Seven drops of Tween-20 were added to the sus-pension per 500 ml. Sugar beet plants (cultivar Angus, Maribo SeedInternational Aps) BBCH-18 (Meier et al., 1993) were inoculatedevenlywith a spray atomizer at 10 cm distance from the leaves untilrun-off. The plants were incubated in the dark for 24 h at 17 �C and>95% relative humidity (RH) (procedure modified from Kema et al.(1996) and Makepeace et al. (2008)) following incubation in agrowth chamber under a 16 h lighte8 h dark regime with 20 �Cdaye17 �C night and RH >95%. Isolates were prepared from spor-ulating colonies of R. beticola from leaf lesions cultured onto grassagar (GA) (1000 ml): 65 g grass pellets boiled for 10 min beforetaken off the heat for 10 min. The solid material was sieved away,and the suspensionmixed with 24 g Bacto agar (DIFCO) and 0.050 gnovobiocin prior to autoclaving. The inoculated plates were incu-bated at 17 �C (12 h darke12 h UV light) for a minimum of 4 weeks.Increased sporulation of colonies was achieved by changing to awhite light regime (12 h lighte12 h dark) in the fourth week.

2.4. Sensitivity test of R. beticola

The sensitivity of R. beticola to fungicides was determined usinga microtitre assay modified from a Syngenta CP Resistance protocolfor Pyrenophora tritici-repentis (www.FRAC.info). A pilot sensitivitytest using three R. beticola isolates tested with propiconazole anddifferent triazoles determined the dilution series. Eleven isolateswere tested with epoxiconazole, propiconazole, difenoconazoleand pyraclostrobin in the concentrations 0.001, 0.01, 0.03, 0.1, 0.31,

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1.00 and 3.16 mg l�1 in DMSO (dimethyl sulfoxide). Controlswithout additional fungicide were included. Inoculum was pre-pared by releasing spores from colonies in 2 ml demineralizedwater using sterilized steel balls and a shaker (Vibrofix VFI Elec-tronics) for 10 min. The concentration of spores and mycelia frag-ments was adjusted to 1.0 � 106 ml l�1 using a haemocytometer.Eachwell contained 10 ml fungicide solution and 100 ml AE-medium(10 g yeast extract, 0.5 g MgSO4*7H2O, 6.0 g NaNO3, 0.5 g KCl, 1.5 gKH2PO4, 20.0 g Bacto agar, 20.0 ml glycerol and demineralizedwater to a total volume of 1000 ml). 10 ml spore suspension wasadded once the AE medium had set. The microtitre plates werewrapped in tinfoil and incubated in a climate chamber at 21 �C indark for 7 days (after Karaoglanidis et al., 2000). The fungicidesensitivity of the isolates was visually assessed by scoring thefungal mycelia coverage in each well as percentage growth in thetreated wells compared to control wells not amended with fungi-cides. Two replicates per isolate and fungicide were performed. Themean inhibition level of the fungicides was estimated using EC50values.

2.5. Efficacy test of different fungicides

2.5.1. Trial layoutSugar beet seed (cultivar Angus, Maribo Seed International Aps)

were sown individually in jiffypots and planted (BBCH-12) in 8-Lpots with Flakkebjerg standard soil containing a 1:1 mixture oflocal standard soil and compacted peat (Kekkilä, Sphagnum fus-cum, Finland). The pots were placed in the semi-field area at AarhusUniversity, Research Centre Flakkebjerg on 21 April 2010. Theplants were exposed to outside temperature, humidity and wind,although shielded from precipitation from above. The trial setupwas a completely randomized block design with five blocks, eachconsisting of 24 treatments and two control pots. The plants wereirrigated and fertilized automatically throughout the trial period. Asprinkler system was set to sprinkle every 12 h for 2 min from 16June to the end of the trial on 28 July to ensure periods of leafwetness, which promoted disease development.

2.5.2. Fungicide treatmentOpus (epoxiconazole, BASFA/S), Comet (pyraclostrobin, BASFA/S),

Opera (epoxiconazoleþpyraclostrobin, BASFA/S) andArmure300EC(propiconazole þ difenoconazole, Syngenta Crop Protection A/S)were prepared corresponding to full, half and quarter rates of anormal field dose (Table 1). The plants were sprayed either preven-tively, 1 day pre-inoculation, or curatively, 5 days post-inoculation.The treatments were applied in a spray cabin with a 50-cm distancefrom spray nozzles to the top of the pots. The spray was calibrated to

spray 204.9 l ha�1 at the speed of 4.1 km h�1 at 3 bar. Control plantswere only inoculated but not treated with fungicides. Five replica-tions per treatment were done.

2.5.3. Inoculation and disease assessmentThe plants were inoculated with pooled inoculum of 12

R. beticola isolates. Colonies were harvested from plates andblended (Waring Commercial, Laboratory blender) with 500 mldemineralized water (1 min at the lowest setting). The concentra-tion of spores andmycelia fragments was adjusted to 1.00� 106 ml-1 using a haemocytometer. Tween-20 equivalent to 0.01% wasadded. The plants were inoculatedwith a spray atomizer (BBCH-19)and placed under a tunnel of white plastic for 72 h to stimulate theinfection process. Each plant was assessed for Ramularia leaf spotseverity (% leaf cover) on the old, middle and young leaf rosetteonce a week between 30 June and 28 July. A visual diseaseassessment guide originally for assessments of Cercospora leaf spotof sugar beet (EPPO Standards, 2004) was used as guideline for thescores.

2.6. Statistical analysis

2.6.1. Historical data and precipitation analysisDifferences in Ramularia leaf spot severity between years were

analysed using a one-way analysis of variance (ANOVA) under thenull hypothesis that each year had the same degree of diseaseseverity. A pairwise test calculating t values and showing significantdifferences at a 5% level was used to compare a particular year withanother year. Both analyseswereperformed in R version 2.8.1 (The RFoundation for Statistical Computing). Daily precipitation valueswere summarized perweek fromMay to September (weeks 18e39).Disease assessments performed in late September before the har-vest of the crop (reflecting the highest disease severity) were usedwhen the correlation betweenprecipitation and Ramularia leaf spotwas analysed. Regression analysis was performed to find the bestcorrelation between disease scores and precipitation using indi-vidual weeks or sum of pooled data from either four or eight weeks.

2.6.2. Effects of foliar diseases and chemical controlDifferences in disease severity and sugar yield between un-

treated field plots and fungicide treatments were calculated using ageneral linear model (GLM) performed in SAS version 9.2 (SASInstitute V9.2).

2.6.3. Sensitivity test of R. beticolaData from the sensitivity test were log transformed. Means and

variances of EC50 values were analysed using a one-way ANOVAcarried out in GraphPad prism 5 (GraphPad Software, Inc). Differ-ences between the fungicides at a 5% level were determined by theLeast Significant Difference (LSD value).

2.6.4. Efficacy test of different fungicidesThe efficiency of dose and timing of the four fungicides on

Ramularia leaf spot were analysed by means, variance and LSDvalues (P ¼ 0.05, StudenteNewmaneKeuls) of disease severityscores. A one-way ANOVAwas carried out in ARM 8.2.3 (29 March,2010. Gylling Data Management, Inc.). The graphs weremade basedon untransformed data. Fungicide efficacy was calculated usingAbbott’s formula: % efficacy ¼ (disease score of untreatedplants � disease score of treated plants) � 100/disease score ofuntreated plants. Differences in Ramularia leaf spot severity be-tween the three leaf rosettes and the five assessment dates wereanalysed using a mixed-model ANOVA performed in SAS (version9.2). Treatment, dose rate and application date together with theirinteractions were considered systemic effects, whereas rows,

Table 1Fungicide treatments applied in the semi-field trial. The treatments were applied atthe timing 1 day pre-inoculation or 5 days post-inoculationwith Opus, Opera, Cometor Armure 300 EC in three doses of normal field rate.

Fungicide Active ingredient (g l�1) Dose(l ha�1)

Activeingredient(g ha�1)

Opus Epoxiconazole (125) 1.00 125.00.50 62.50.25 31.25

Opera Pyraclostrobin (133) þEpoxiconazole (50)

1.50 199.5 þ 750.75 99.75 þ 37.50.375 49.9 þ 18.75

Comet Pyraclostrobin (250) 1.00 2500.50 1250.25 62.5

Armure300 EC

Propiconazole (150) þdifenoconazole (150)

0.80 120 þ 1200.40 60 þ 600.20 30 þ 30

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columns, plots, assessment date and leaf rosettes were included asrandom effects in the model. Data were transformed beforehand,analysing the third root of the data to making them approx. nor-mally distributed. Disease assessment data are shown untrans-formed in order to provide data which directly relate to farmingpractices, whereas significant differences indicated by differentletters are based on transformed data in order to improve thestatistical analysis.

3. Results

3.1. Frequency and correlation between Ramularia leaf spot andprecipitation

The severity of Ramularia leaf spot varied significantly betweenyears (P < 0.001) in the period 1999e2009 in Denmark (Fig. 1). Theuntreated sugar beet plots were severely attacked by the disease in2002, 2003, 2004, 2005 and 2007. Very low disease levels wererecorded in 2008 and 2009. Moderate disease levels were found in1999 and 2001, followed by the years 2000 and 2006.

The regression analysis of Ramularia leaf spot and precipitationshowed that increasing precipitation in certain periods betweenMay and September (weeks 18e39) had a positive impact on theseverity of the disease (Table 2). It is shown that up to 41%(R2 ¼ 0.41) of the variation in Ramularia leaf spot could beexplained by the precipitation in week 27 (Fig. 2A), and in week 39(Fig. 2B) precipitation explained 71% (R2 ¼ 0.71) of the variation.Precipitation during the four-week period of weeks 24e27 (Fig. 2C)could explain more than one third (R2 ¼ 0.38) of the disease vari-ation over the years. This was also the case in the eight-week periodof weeks 24e31 (R2 ¼ 0.36) (Fig. 2D) or when the period wasextended to include the whole period investigated, i.e. weeks 18e39 (R2¼ 0.33). For some of the regression analyses it was found thatthe disease assessments in 2009 in particular seemed to fall outsidethe trend line (Fig. 2D).When the 2009 assessments were excluded,the best fit of a curve using the logarithmwas found for weeks 24e31. In this case, 68% (R2¼ 0.68) of the variation in the scenario couldbe explained by precipitation during the period (data not shown).

3.2. Effects of foliar diseases and chemical control

The mean sugar yield increases and economic response of dis-ease control during years 1999e2009 in Denmark are listed inTable 3. The yield increases are due to a combination of Ramularia

leaf spot, rust and powdery mildew, the three dominant leaf dis-eases in Danish sugar beet production. A positive mean sugar yieldincrease and mean gross output were achieved in all 11 yearsfollowing either one or two fungicide applications. The fungicideapplications increased the sugar yield by 10% and gave an averagemargin over fungicide cost of 44 V ha�1 across the 11 years. Thehighestmean sugar yield increase of 2.2 t ha�1was obtained in2009.It corresponded to a mean gross output of 129 V ha�1 but only5V ha�1 when the cost of fungicide application had been deducted.The highest mean gross output of 166 V ha�1 was achieved with asugar yield increase of 2.1 t ha�1 in 2007. Themargin over fungicidecost ranged from 99 to �39 V ha�1 over the years and was positivewith the exception of the treatments in 2006 and 2008. One full rateor half rate application (years 1999e2005) gave the best economicresults compared to two full rate applications (years 2006e2009),the latter being too costly an input in most seasons.

There was no relationship between Ramularia leaf spot severityin untreated plots and the sugar yield increase (R2 ¼ 0.004) acrossthe trials from 1999 to 2009. This was similarly the case evenwhentotal disease severity, including also rust and powdery mildew, wascorrelated to yield increase (R2 ¼ 0.06).

The results of the field screening with epoxiconazole (Opus) in2006 and 2007 in sugar beet against Ramularia leaf spot, rust andpowdery mildew are shown in Table 4. Ramularia leaf spot was thepredominant disease in 2007, whereas powdery mildew was pre-dominant in 2006. A doseeresponse from the chemical control wasobserved in both years for all three diseases as increasing dosages offungicide decreased disease severity except for rust in 2006. Themean sugar yield increased significantly (P ¼ 0.05) in both seasonswhen the diseases were chemically controlled. A positive mean

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Fig. 1. Variation in Ramularia leaf spot severity in Denmark in 1999e2009. The diseasewas assessed at sugar beet field trial sites (n ¼ 39) without any chemical treatments.The trials were conducted by NBR Nordic Beet Research, Holeby, Denmark. Diseasescore of 0 ¼ no attack and 10 ¼ severe attack. Different letters represent significantdifference (P ¼ 0.05).

Table 2Relationship between Ramularia leaf spot severity and the weekly sums of precip-itation given by regression analysis (R2). The analysis includes Ramularia leaf spotassessment scores from untreated sugar beet field sites (n ¼ 39) in Denmark from1999 to 2009. Rows in bold show the highest R2 found.

Weeks in which precipitationis added up

Approx. period R2

18e39 MayeSeptember 0.33a

18 May 0.1819 May 0.0220 May 0.0221 May 0.00422 May/June 0.0723 June 0.00524 June 0.0125 June 0.2126 June/July 0.1227 July 0.4128 July 0.00429 July 0.0130 July 0.2331 July/August 0.0632 August 0.0133 August 0.000234 August 0.1435 August/September 0.00336 September 0.0737 September 0.00838 September 0.00839 September/October 0.71a

20e23 May/June 0.0524e27 June/July 0.38a

28e31 July/August 0.1332e35 August/September 0.0236e39 September/October 0.0418e23 May/June 0.2224e31 JuneeAugust 0.36a

32e39 AugusteOctober 0.001

a R2 value is for a logarithmic regression as opposed to a linear regression.

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gross output and a positive margin over fungicide cost were ob-tained with increasing dosages towards standard dose. The highestmean sugar yield increase was seen in 2007 with 2.4 t ha�1 cor-responding to 17% increase and a net profit of 255 V ha�1 followingapplication of two full rates of epoxiconazole. Two half rate fungi-cide applications gave the highest sugar yield increase of 0.8 t ha�1

(7% increase) and a net profit of 78 V ha�1 in 2006.

3.3. Sensitivity test of R. beticola

The sensitivity test of R. beticola resulted in EC50 values rangingfrom 0.0003 to 0.971 mg l�1 across the tested fungicides

(epoxiconazole, propiconazole, pyraclostrobin and difenoconazole)(Table 5). The four treatments were significantly different(P ¼ 0.006) from each other (LSD value ¼ 0.134). The mean EC50values indicate that R. beticola was significantly more sensitive topyraclostrobin (0.004 mg l�1), difenoconazole (0.046 mg l�1) andepoxiconazole (0.063 mg l�1) than to propiconazole (0.237 mg l�1).The fungicide sensitivity varied among the isolates. In general, oneisolate from Sweden (JF330016) and a reference isolate fromSwitzerland (JF330013) appeared to be the most sensitive of the 11isolates tested. A Danish isolate (JF330019) exhibited the highestinsensitivity towards all tested fungicides (Table 5). In general, EC50values fell within the range of a normal distribution comparable to

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Fig. 2. Relationship between Ramularia leaf spot severity and precipitation in different periods. A. week 27 (start of July), B. week 39 (end of September), C. weeks 24e27 (end ofJune to start of July) and D. weeks 24e31 (from mid-June to start of August). Disease was assessed in untreated sugar beet field sites (n ¼ 39) in 1999e2009 in Denmark. Diseasescore of 0 ¼ no attack and 10 ¼ severe attack.

Table 3The sugar yield increase and economic output in 1999e2009 of Danish field trials (n ¼ 41) when control of foliar diseases of sugar beet with the best possible treatment wastested. Original data derived from fungicide screening trials supplied by NBR.

Year (No.of trials)

Applicationrate (l ha�1)

Fungicide Mean sugar yield increase(t ha�1) (yield of untreated plots)

Mean gross output fromfungicide treatments (V ha�1)

Margin over fungicidecost (V ha�1)

1999 (4) 1 � 1.0 Opus 0.9 (11.4) 102 532000 (4) 1 � 1.0 Opus 1.0 (13.2) 93 442001 (5) 1 � 1.0 Opus 1.0 (11.9) 124 752002 (4) 1 � 1.0 Opus 1.5 (14.1) 149 992003 (3) 1 � 1.0 Opus 1.2 (13.9) 97 472004 (2) 1 � 0.5 Opera 1.2 (13.6) 135 992005 (4) 1 � 1.0 Opera 1.5 (13.5) 129 672006 (4) 2 � 1.0 Opera 0.7 (12.0) 85 �392007 (4) 2 � 1.3 Opera N 2.1 (14.4) 166 422008 (3) 2 � 1.0 Opera 1.6 (15.6) 119 �52009 (4) 2 � 1.0 Opera 2.2 (16.8) 129 5Mean (n ¼ 41) 1.4 (13.6) 121 44

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populations reflecting a natural variation in sensitivity betweenisolates. There were no indications of resistance towards eitherpyraclostrobin or the triazoles (epoxiconazole, difenoconazole andpropiconazole) tested.

3.4. Efficacy test of different fungicides

In the semi-field trial the first Ramularia leaf spots wereobserved on the old leaf rosettes of untreated control plants 19 daysafter inoculation. Symptoms resembled those leaf spot lesionsfound in a naturally infected sugar beet field. The lesions developedsignificantly (P ¼ 0.05) on untreated plants during the trial periodbetween the assessment dates and between the leaf rosettes. Forty-two days after inoculation (21 July), the Ramularia leaf spot scoreon the old leaf rosette was 24%, whereas the middle leaf rosettescored 17% and the young leaf rosette scored 4%, levels being sig-nificant (P ¼ 0.05). One week later the disease severity of themiddle leaf rosette reached 25%, whereas only 11% was found onthe new leaf rosette. All four screened fungicides (Opus, Comet,Opera and Armure 300 EC) were very efficient in controllingRamularia leaf spot both preventively and curatively (Table 6).

Treated plants had significantly less disease, appearing greenand healthy at the end of the trial compared to the untreated plants.The efficacy of disease control was close to 100% in plants treatedpreventively. The plants treated curatively were controlled withalmost as high efficacy, except in one case with a full rate of pyr-aclostrobin (1.0 l ha�1 Comet) in which only 70% control was ach-ieved. On plants treated curatively the first Ramularia leaf spot

symptoms appeared 28 days after inoculation (7 July), whereas theleaf spots were observed a week later on plants treated preven-tively, i.e. 35 days after inoculation (14 July). No doseeresponse ofthe fungicides was observed.

4. Discussion

Investigated over a 10-year period (1999e2009) Ramularia leafspot has proved to be a very significant disease in sugar beet,although the disease can vary significantly from season to season.Ramularia leaf spot is favoured by high relative humidity andmoderate temperatures (Ahrens, 1987; Hestbjerg et al., 1994). Thepresent investigation on the relationship between precipitationand the disease severity supports previous findings and specifiesthat the amount of precipitation in certain periods during thegrowing season fromMay to September plays an important role forthe severity of Ramularia leaf spot. The deviation seen for 2009could be ascribed to particular weather conditions as April andAugust/September that year were very dry compared to otherseasons (www.DMI.dk, 2010). Excluding the 2009 data improvedthe correlation between precipitation and disease severity fromR2 ¼ 0.36 to R2 ¼ 0.68 in the period of JuneeAugust (weeks 24e31).If 2009 assessments were excluded for week 39, 71% of the varia-tion explained by precipitation changed to 60% instead (data notshown), indicating that 2009 mainly are outliers in relation toweeks 24e31.

In Germany, Ahrens (1987) observed high incidences of Ramu-laria leaf spot in 1984, which were related to high amounts of

Table 4Control of foliar diseases (Ramularia leaf spot, rust, powdery mildew) of sugar beet in 2006 and 2007 with epoxiconazole. Data represent mean severity of four trials and theeffects of treatments are shown by the mean sugar yield increase, the mean gross output and the margin over fungicide cost.

Year (No.of trials)

Opus applicationrate (l ha�1)

Ramularia(0e10)

Rust(0e10)

Powdery mildew(0e10)

Mean sugar yieldincrease t ha�1 (%)

Mean gross output oftreatment (V ha�1)

Margin over fungicidecost (V ha�1)

2006 (4) Untreated 3.5 1.2 5.5 12.0 0 02 � 1.0 0.5 0.1 3.7 0.6 (5) 115 162 � 0.5 1.1 0.1 4.0 0.8 (7) 137 782 � 0.25 2.0 0.1 4.1 0.3 (3) 58 19

LSD 1e4 1.2 1.2 2.0 0.4LSD 2e4 1.2 ns ns ns2007 (4) Untreated 8.1 1.7 4.2 14.4 0 0

2 � 1.0 2.8 0.2 0.5 2.4 (17) 354 2552 � 0.5 4.3 0.5 1.9 1.7 (12) 249 1902 � 0.25 5.6 1.0 2.9 1.6 (11) 235 196

LSD1e4 0.9 ns 1.8 1.1 (8)LSD2e4 0.7 ns ns ns

ns ¼ not significant.

Table 5Mean EC50 values of epoxiconazole, propiconazole, pyraclostrobin and difenoconazole tested against R. beticola isolates (n ¼ 11) in vitro. Two replicates performed.

Isolate accession numberin GenBank

Origin (year) Fungicide (mg l�1)

Epoxiconazole Propiconazole Pyraclostrobin Difenoconazole

JF330015 Denmark (2009) 0.022 0.067 0.0008 0.016JF330016 Sweden (2009) 0.001 0.010 0.0003 0.0003JF330017 Sweden (2009) 0.038 0.054 0.0003 0.015JF330013 (reference) Switzerland (1967) 0.004 0.019 0.005 0.001JF330014 (reference) Sweden (1985) 0.015 0.032 0.010 0.005JF330018 Denmark (2008) 0.164 0.432 0.001 0.100JF330019 Denmark (2008) 0.174 0.971 0.012 0.158JF330020 Denmark (2008) 0.048 0.137 0.004 0.034JF330021 Sweden (2009) 0.038 0.178 0.004 0.010JF330023 Denmark (2002) 0.164 0.527 0.003 0.145JF330024 The Netherlands (2009) 0.028 0.178 0.004 0.025Mean of isolatesa 0.063b 0.237a 0.004b 0.046bVariance 0.005 0.088 1.45 � 10�5 0.003

a Different letters indicate significant difference (P ¼ 0.006).

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precipitation, close to 100 mm in September and October. Ahrens(1989) also reported mass spore production of R. beticola after pe-riods with humid conditions. The present investigation did notinclude temperature as an explaining parameter as previous papershave stated that the Ramularia leaf spot can easily develop underthe temperature regime common to Danish summers (Hestbjerg,1993).

In a future forecasting model for Ramularia leaf spot the specificrelationship between precipitation and the disease needs to befurther investigated. The data should be extended to include pa-rameters like temperature, leaf wetness and relative humidity asthese parameters are known to influence spore production andspore germination (Ahrens, 1989). An extended climate model hasalready been investigated for Cercospora leaf spot (C. beticola)(Wolf and Verreet, 2005). This has led to a German predictionmodel for Cercospora leaf spot, called CERCBET 3 (Racca and Jörg,2007), which is supported by the advisory services (ISIP). AsRamularia leaf spot and Cercospora leaf spot are alike in many re-spects, this model could possibly be adjusted to Ramularia leaf spot.The preliminary model RAMUBET is such a model for Ramularialeaf spot which was developed in collaboration between theGerman institutions ZEPP, IfZ and ISIP (Kleinhenz et al., 2010). Thispreliminary model has been validated from 2004 to 2009 givingcorrect recommendations in individually years varying between 61and 91% of the tested cases. Successful forecasting against Ramu-laria leaf spot requires both an estimate of the presence of inoculumas well as a climate model. Presence of inoculum could possibly bemeasured using for example spore trapping (Hestbjerg and Dissing,1995; Khan et al., 2009). Modern molecular techniques like qPCRcould help intensify the detection of spores, as experienced forpathogens like Monilinia fructicola (Luo et al., 2007) and Botrytissquamosa (Carisse et al., 2009). If combined with a climatic modelfor disease development, a threshold based forecasting systemcould be developed.

Factors such as variety susceptibility, crop rotation, sowing date,the length of cropping season and soil management should also betaken into consideration as part of an IPM strategy having animpact on the incidence and severity of the disease (Nielsen, 1991;Adams, 1998; Persson and Olsson, 2006). In the present study noneof the cultural factors were included in the analyses as the data setwas too limited in numbers and variation. Screening for varietyresistance to Ramularia leaf spot has shown differences in sus-ceptibility (Ahrens, 1987; Adams, 1998; Petersen et al., 2001), butno varieties have been reported to be completely resistant to thedisease (Draycott, 2006; Nielsen et al., 2011). A study of Cercospora

leaf spot showed that the variety susceptibility clearly had animpact on the disease epidemiology (Kaiser et al., 2010). The studyalso showed that Cercospora leaf spot resistant varieties reacheddisease thresholds for fungicide treatments later than susceptiblevarieties, which also led to reduced loss of white sugar yield. Dis-ease resistance breeding in sugar beet has so far mainly focused onresistance to C. beticola (Taguchi et al., 2011), beet necrotic yellowvein virus causing rhizomania (Biancardi et al., 2002; Panella et al.,2008), nematode resistance/tolerance (Niere, 2009) and Rhizoc-tonia solani (Behn et al., 2012). New efforts to strengthen the area ofresistance to Ramularia leaf spot would be very useful as part of anIPM strategy.

The importance of soil inoculum of R. beticola is an area whichshould be looked into in the future. The quantity of soil inoculum islikely to be important as R. beticola is a soil dwelling pathogen ableto survive for 4e5 years (Persson and Olsson, 2006). Residues ofsugar beet leaf material are currently not removed from fields afterthe harvest of sugar beets and could increase the risk of disease. Aspart of an IPM concept it is important to minimize primary inoc-ulum by having a crop rotationwith a minimum of 5 years betweensugar beet crops. However, crop rotation does not protect the cropfrom secondary inoculum in the air mainly coming from otherinfected sugar beet fields.

Continuous awareness of fungicide efficacy and fungicidesensitivity is crucial to ensure that only efficient fungicides areapplied for control of Ramularia leaf spot. The actual need forapplication of chemicals against plant disease in sugar beet crop isimportant to consider, but as presented in this paper data haveshown that treatments have been economically justified in nearlyall seasons. In general, foliar disease control in Denmark haveproved to increase the sugar yield by 10% and providing a net in-come of 44 V ha�1 as a mean of 41 trials over 11 years if treatmentsare timed right. No significant correlation was found between theseverity of the diseases present in untreated plots of the trials atBBCH-40 and the yield response from the best fungicide treatments(data not shown). Particularly, the responses from 2008 to 2009conflicted as yield responses were high, even though diseasepressure from Ramularia leaf spot, powdery mildew and rust wasrelatively low. The lack of a good correlation corresponds withstudies from cereal crops in which significant yield increases fromfungicides were also found despite low disease levels, which isdescribed to be partly caused by fungicides having positive physi-ological effects on the crop (Wu and von Tiedemann, 2001; Bartlettet al., 2002). However, high yield increases found in 2002, 2005 and2007 corresponded well with high Ramularia leaf spot severities.Ahrens (1987) also found a good correlation between severity ofRamularia leaf spot and sugar yield in trials in which fungicideswere applied.

Effective fungicides for control of Ramularia leaf spot are avail-able; both the field trials and the semi-field confirmed it. The semi-field trial indicates that with respect to optimal timing of applica-tion the chemicals offer some flexibility as also the curative treat-ments gave good control. Although the semi-field trial providedvery high control using low fungicide rates, ordinary field trialshave indicated that 50% of standard rates may give insufficientcontrol in some seasons. The lower dosages are less persistent andwill in many cases need to be repeated, in particular when beets areharvested late in the season.

In the semi-field trial the incubation period for R. beticolawas 19 days, which correlates well with the 2e3 weeksreported by Hestbjerg et al. (1994) under growth chamber condi-tions. Epoxiconazole and pyraclostrobin and the mixturesepoxiconazoleþpyraclostrobinanddifenoconazoleþpropiconazoleall provided good preventive and curative control when applied fivedays after inoculation. Further delayed applications are found to

Table 6Ramularia leaf spot severity in the semi-field was assessed on the old leaf rosettes(49 days post-inoculation) with application of different fungicides either preven-tively (1 day pre-inoculation) or curatively (5 days post-inoculation) at the normalfield rates of full, half and quarter rates ha�1. Significant difference is based ontransformed data.

Timing Dose (% of standarddose ha�1)

Fungicidea

Opus Opera Comet Armure 300 EC

Preventive Untreated 16.0a 16.0a 16.0a 16.0a100 0.0c 0.0c 0.02c 0.0c50 0.0c 0.0c 1.0c 0.0c25 0.0c 0.0c 0.4c 0.0c

LSD 2.25Curative Untreated 16.0a 16.0a 16.0a 16.0a

100 1.0c 0.6c 4.8b 0.0c50 2.0bc 0.7c 1.6c 0.02c25 0.0c 0.03c 2.5bc 1.4c

LSD 2.35

a Different letters indicate significant difference (P ¼ 0.05, StudenteNewmaneKeuls).

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generally give amuch reduced efficacy as seen from other pathogenslike Mycosphaerella graminicola (Jørgensen and Nielsen, 2003),because the disease at these stages is too advanced in itsdevelopment.

Ramularia leaf spot development was delayed by one week inthe semi-field when treated preventively compared to curativeapplication. This indicates an increased strength of the productswhen these are used preventively. Based on trials and experiencesin Danish fields, the effect of a quarter rates of epoxiconazole-basedfungicides was declining approx. three weeks after applicationwhen the first leaf spots became visible. Similarly, fungicide trialsconducted in 1966e1971 in England showed a delay of Ramularialeaf spot by two and a half weeks when treated preventively(Byford, 1975). National field trials from 2010 showed that twoapplications of full rates of epoxiconazole alone orpropiconazole þ difenoconazole provided at least 82% control ofRamularia leaf spot five weeks after the last application (Jørgensenand Thach, 2010). It is important to consider that the duration ofdisease control apart from depending on the applied dose of thefungicide also depends on disease pressure and the climatic con-ditions. Based on field trials, it has been verified across several yearsthat one or two applications with 25e50% of the normal field rateprovide the highest economic net income (Hansen, 2007). How-ever, the data provided in this paper also indicate that two treat-ments with full rates may also be economical in seasons withsevere epidemics as observed in 2007 where double treatmentwith full rate provided the best control and margin over fungicidecost.

To our knowledge there have been no reports on sensitivitydifferences of R. beticola isolates to various fungicides. Testing forfungicide sensitivity is relevant because of the importance ofRamularia leaf spot, increasing use of fungicides and the findingsof other economically important pathogens developing reducedsensitivity or resistance to common fungicides like strobilurinsand/or triazoles (Gisi et al., 2000; Karaoglanidis et al., 2000;Jørgensen and Thygesen, 2006; Fountaine and Fraaije, 2009). Inthe present sensitivity test the mean EC50 values of the R. beticolaisolates ranged from 0.0003 to 0.971 mg l�1 across epox-iconazole, propiconazole, difenoconazole and pyraclostrobin. Theresults gave no indication of a shift in fungicide sensitivity ordevelopment of resistance to strobilurins or triazoles. Some dif-ferences were observed between the isolates originating from1967 to 2009. It is believed that these differences are part of anormal distribution of sensitivity and too small to actuallygive variation in field performances. However, it can be notedthat a clear cross resistance between the three triazoles was seen(R2 varying from 0.83 to 0.94 for the three combinations)(data not shown). Similarly, larger differences in EC50 values inM. graminicola isolates (0.007e1.15 mg l�1) have not givendetectable variation in the field performance (Thygesen et al.,2009). Based on the present study a connection between lowfield performances from fungicides in 2007 could not be linked tochanges in sensitivity as the number of isolates analysed were toolimited. The low field performances were more likely reflectingan unusually fast epidemic development due to favourable con-ditions causing too late timing of fungicide application. A recentstudy by Hanse et al. (2011) showed a major variation in sugarbeet yields partly due to farmer’s skills and management of pestand diseases indicating that poor application timing is not anunlikely event. Field performances from epoxiconazole/epoxiconazole þ pyraclostrobin solutions applied in 2010 and2011 provided satisfactory, high disease control, indicating nomajor problems regarding resistance to triazoles and strobilurins.It is still important to be aware that reduced sensitivity to stro-bilurins in particular is not unlikely to develop if there is a

continuous use of fungicides belonging to this group. Reducedsensitivity and resistance have been reported for related fungi,e.g. Ramularia collo-cygni (Ramularia leaf spot in barley)(Fountaine and Fraaije, 2009) and M. graminicola (Fraaije et al.,2007; McCartney et al., 2007). In a recent paper by Birla et al.(2012) widespread resistance to strobilurins has been reportedin populations of C. beticola from Italy following intensive use ofstrobilurins over several years.

Propiconazole has been used since the late 1980s. Opus andOpera were not introduced in Denmark until 2003 and 2008,respectively. Although significantly lower than for the three othercompounds, the efficacy of propiconazole in the in vitro test is stillhigh with a mean EC50 value of 0.237 mg l�1. Even so, the intrinsiceffect of propiconazole under field conditions was found to beinferior to the two other triazoles. Experiences fromGreece showedthat C. beticola had acquired reduced sensitivity to propiconazoleafter more than 15 years of use (Karaoglanidis et al., 2000).

In order to minimize the risk of resistance development inR. beticola, it remains important to follow the general recommen-dations for lowering the risk of resistance build-up. This includes theuse of fungicides with different modes of action in alternation or inmixtures, reducing the number of treatments per season, and onlyapplying fungicides according to diseasemonitoring and thresholds(www.FRAC.info). It is also important to apply fungicides at lowdisease severity as selection is believed to increase if the pathogenpopulation is high (Brent and Hollomon, 2007). Furthermore, thenumber of generations of fungi per season has an influence on thelikelihood of resistance development (Brent and Hollomon, 2007).In the future it is important to be aware of possible shifts takingplace in the population of R. beticola, and the level of field perfor-mances should be kept under constant attention.

A good IPM strategy for minimizing disease risks in sugar beetstarts by ensuring a minimum of 4e5 years between sugar beetcrops to lower the amount of primary inocula. A regionally basedmonitoring of Ramularia leaf spot is run by NBR and helps thefarmers to time the first treatment once the first symptoms havebeen observed. A weather-based risk model could similarly help tooptimize the timing of the first application. It appears that thecurrent chemical control strategies against Ramularia leaf spot (andother main foliar diseases) in sugar beet in Denmark are efficienteven at reduced dosages when applied timely, not only providing ayield increase but also importantly optimizing the margin overfungicide cost.

Acknowledgements

The research was carried out in collaboration between theUniversity of Copenhagen, Faculty of Science, Department of Plantand Environmental Sciences, Aarhus University, Faculty of Scienceand Technology, Department of Agroecology and NBR Nordic BeetResearch, Holeby, Denmark. The authors wish to thank all thecontributors for donations of biological material. We also wish tothank BASF A/S for financial contribution for fields trials, and bothBASF A/S and Syngenta Crop Protection A/S for their financialcontribution to the semi-field trial. Finally we thank Kristian Kris-tensen, Aarhus University for his statistical expertise in the SAS andR programs, and Jens Nyholm Thomsen, NBR, for calculations of andassisting with the economics of yield.

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