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Keywords:

  • carboxamides;
  • Cucumis sativus;
  • fungicide resistance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

A total of 651 isolates of cucumber corynespora leaf spot fungus (Corynespora cassiicola) collected from cucumber in Japan, either with (438 isolates) or without (213 isolates) a prior history of boscalid use, were tested for their sensitivity to boscalid by using a mycelial growth inhibition method on YBA agar medium. Additionally, seven isolates of C. cassiicola obtained from tomato, soybean, eggplant (aubergine) and cowpea in different locations in Japan were tested before boscalid registration. Minimum inhibitory concentration (MIC) and 50% effective concentration (EC50) values for 220 isolates from crops without a prior history of boscalid use ranged from 0·5 to 7·5 μg mL−1 and from 0·04 to 0·59 μg mL−1, respectively. Two hundred and fourteen out of 438 isolates collected from ten cucumber greenhouses in Ibaraki Prefecture, Japan, which received boscalid spray applications showed boscalid resistance, with MIC values higher than 30 μg mL−1. Moreover, resistant isolates were divided into two groups: a moderately resistant (MR) group consisting of 189 isolates with EC50 values ranging from 1·1 to 6·3 μg mL−1, and a very highly resistant (VHR) group consisting of 25 isolates with EC50 values higher than 24·8 μg mL−1. MR isolates were detected from all ten greenhouses, but VHR isolates were detected from only three. As a result of fungus inoculation tests which used potted cucumber plants, control failures of boscalid were observed against resistant isolates. Efficacy of boscalid was remarkably low against VHR isolates in particular. This is the first known report on boscalid resistance in Japan.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Corynespora leaf spot caused by Corynespora cassiicola is one of the most important foliar diseases of cucumber (Cucumis sativus) in Japan. This disease has been known for a long time in Japan but outbreaks were not very serious until several years ago. However, in recent years, this disease has caused a serious problem on cucumber cultivated in plastic greenhouses, in spite of treatment with various fungicides. It is thought that year-round cropping of cucumber for many years (Hasama et al., 1987), the change of cucumber cultivars (Miyamoto et al., 2006) and occurrence of C. cassiicola isolates resistant to benzimidazoles, dicarboximides, N-phenylcarbamates and QoI fungicides (Hasama, 1991; Hasama & Sato, 1996; Date et al., 2004; Miyamoto et al., 2006; Takeuchi et al., 2006; Ishii et al., 2007) are the reasons for recent heavy occurrence of the disease. Benzimidazoles and QoI fungicides appear to have lost their control efficacies completely against corynespora leaf spot due to the development of a high frequency of resistance in the pathogen.

Boscalid, which was registered commercially in Japan for the control of grey mould and sclerotinia drop of cucumber in January 2005, and corynespora leaf spot of cucumber in July 2006, is a new fungicide in the succinate dehydrogenase inhibitor (SDHI) group, previously known as carboxamides, with a broad spectrum of activity. This fungicide inhibits fungal respiration through its inhibition of the enzyme succinate dehydrogenase (SDH), also known as complex II, in the mitochondrial electron transport chain (Fungicide Resistance Action Committee, 2009). However, as this is a single site mode of action, the risk of pathogen resistance development in the field needs to be considered. SDH consists of four subunits (SdhA, SdhB, SdhC and SdhD). Mutations which lead to amino acid substitutions in the SdhB-, SdhC- or SdhD-subunit of SDH confer laboratory resistance to carboxin, belonging to the SDHI group of fungicides (Keon et al., 1991; Broomfield & Hargreaves, 1992; Matsson et al., 1998; Skinner et al., 1998; Matsson & Hederstedt, 2001; Ito et al., 2004; Li et al., 2006; Shima et al., 2008). Furthermore, boscalid resistance has been reported recently in field isolates of Alternaria alternata on pistachio, Botrytis cinerea on grapevine and strawberry, Podosphaera xanthii on cucurbits and other species in several countries (Avenot & Michailides, 2007; Avenot et al., 2008a,b; McGrath, 2008; McGrath & Miazzi, 2008; Miazzi & McGrath, 2008; Stammler, 2008). As expected, these fungi have the mutations which lead to amino acid substitutions in the SdhB- or C- (Avenot et al., 2008b,c), and SdhB-subunit (Stammler, 2008). Therefore, it is important to monitor shifts in the sensitivity of C. cassiicola to boscalid.

When monitoring fungicide sensitivity, baseline data of the pathogen is needed to explain shifts in sensitivity and to provide evidence that failures in disease control could be due to the emergence of resistant strains in the pathogen populations after the fungicide was used (Justum et al., 1998; Russell, 2004). Baseline sensitivity to boscalid was reported on several important pathogens (Lu et al., 2004; Spiegel & Stammler, 2006; Stammler & Speakman, 2006; Avenot & Michailides, 2007; Stammler et al., 2007; Zhang et al., 2007; Myresiotis et al., 2008; Wise et al., 2008). Recently, methods for testing boscalid sensitivity were developed, and proposed that YBA liquid medium could be used for B. cinerea, Monilinia spp. and Sclerotinia sclerotiorum (Spiegel & Stammler, 2006; Stammler & Speakman, 2006; Stammler et al., 2007). Subsequently, Ishii & Nishimura (2007) reported that the measurement of mycelial growth on boscalid amended YBA agar medium could assess boscalid sensitivity of C. cassiicola.

The objectives of this study were to (i) determine the baseline sensitivity of C. cassiicola isolates to boscalid using mycelial growth tests on YBA agar medium; and (ii) monitor the shifts of boscalid sensitivity of fungal isolates collected from cucumber greenhouses with a history of boscalid treatment.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fungal isolates

To obtain the baseline sensitivity data, 220 isolates were collected from cucumber, tomato, eggplant, soybean and cowpea in different locations in Japan without a prior history of boscalid use (Table 1). Nine isolates designated with MAFF numbers were taken from the National Institute of Agrobiological Sciences Genebank (Tsukuba, Japan). Two isolates from cucumber, three isolates from tomato and one isolate from eggplant were gifts from Okayama Prefectural General Agriculture Center, and one isolate from cucumber was a gift from Chiba Prefectural Agriculture Research Center. Fifty-one isolates were obtained by single spore isolation from cucumber leaves with lesions in Ibaraki, Saga, Okayama and Miyazaki Prefectures, Japan. These isolates were maintained on potato dextrose agar (PDA: Difco Laboratories, Detroit) slants and stored at 5°C until use. One hundred and fifty-three isolates collected from cucumber in Ibaraki Prefecture from 2004 to 2006 were maintained on PDA slants at 5°C or on 10-fold diluted PDA slants at room temperature in the dark after single spore or single mycelium isolation.

Table 1. Corynespora cassiicola isolates used for testing baseline sensitivity to boscalid
Year of isolationLocation of fieldHostNo. of isolates tested
  1. aName of isolate taken from National Institute of Agrobiological Sciences Genebank (Tsukuba, Japan).

unknownIbarakiCucumber1 (MAFF712093)a
1949SaitamaSoybean1 (MAFF305087)
1959ChibaCowpea1 (MAFF305092)
1978HokkaidoSoybean1 (MAFF235139)
1988NaganoCucumber1 (MAFF306176)
1989OitaCucumber1 (MAFF306348)
1995IbarakiCucumber2 (MAFF237272, MAFF237273)
MiyazakiCucumber1
OitaCucumber1
1999OkayamaEggplant1
2000IbarakiCucumber12
OkayamaCucumber1
2001IbarakiCucumber10
OkayamaCucumber2
FukuokaCucumber1 (MAFF744073)
2002IbarakiCucumber14
OkayamaTomato3
 Cucumber1
2004IbarakiCucumber2
ChibaCucumber1
SagaCucumber11
2005IbarakiCucumber121
2006IbarakiCucumber30
Total number  220

For monitoring sensitivity to boscalid after the registration of this fungicide, 438 isolates were collected from 10 commercially grown cucumber greenhouses with a history of boscalid treatment at Kasama, Chikusei and Kasumigaura Cities in Ibaraki. These isolates were obtained by single spore isolation from lesions and maintained as described above.

In vitro sensitivity tests

Each isolate was pre-cultured on PDA plates at 25°C for 5–9 days. Sensitivity tests were performed by transferring a 4-mm disk of mycelia taken from a culture of isolates growing on PDA, onto YBA agar medium (yeast extract 1%, Bacto peptone 1%, sodium acetate 2%, agar 1·5%) (Stammler & Speakman, 2006) amended with 0, 0·1, 0·25, 0·5, 0·75, 1, 2·5, 5, 7·5, 10 and 30 μg mL−1 of boscalid as discriminatory concentrations. Technical grade of boscalid (94·4% a.i.) was dissolved in dimethylsulfoxide (DMSO) and added to cooled YBA agar medium after autoclaving. The final concentration of DMSO in the fungicide-amended and unamended media was 0·25% by volume. After incubation at 25°C in the dark for 4 days, the diameter of the mycelial colony was measured and 50% effective concentration (EC50) of boscalid for colony growth inhibition was calculated using log-linear model software kindly supplied by ZEN-NOH (Tokyo). EC50 and minimum inhibitory concentration (MIC) of boscalid were determined for each isolate from two or three independent tests.

Inoculation tests

Two boscalid sensitive (IbCor0008 and IbCor1522), three moderately resistant (MR, IbCor1679, IbCor1481 and IbCor1482) and two very highly resistant (VHR, IbCor1689 and IbCor2036B) isolates were used in the inoculation tests. After each isolate was incubated on PDA plates at 25°C for 7 days in the dark, the aerial hyphae were removed with a sterile spatula and each plate was incubated at 25°C for 3–4 days under a blacklight blue lamp to promote sporulation. Spores thus formed were suspended in distilled water and the suspension adjusted at to 104 spores mL−1. First leaves of 2-week-old potted cucumber plants (cultivar High-green 21, six seedlings per treatment) grown at 25°C in a phytotron were each sprayed with suspensions of boscalid (as Cantus 50 DF) at a concentration of 0·33 mg a.i. mL−1 and mancozeb (as Jimandaisen 75 WP) at a concentration of 1·25 mg a.i. mL−1, which were the manufacturer’s recommended field dosage. Control plants were sprayed with tap water. After being dried in air, the plants were inoculated with the spore suspensions, stored at 28°C for 2 days in a chamber with high humidity (almost 100%), and then maintained at 25°C for 3 days in a phytotron. The number and diameter of lesions of corynespora leaf spot were evaluated 5 days after inoculation. The diameter of the lesions on each leaf was evaluated by calculating a mean of twenty lesions selected at random in boscalid and tap water treatments, and a mean of three to five lesions in mancozeb treatment, where only a few lesions developed.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Baseline sensitivity to boscalid

MIC and EC50 values of boscalid for 220 isolates of C. cassiicola obtained from cucumber, tomato, eggplant, soybean and cowpea in different locations in Japan without a prior history of boscalid use, ranged from 0·5 to 7·5 μg mL−1, with a mean of 5·27 μg mL−1 (Fig. 1), and from 0·04 to 0·59 μg mL−1, with a mean of 0·27 μg mL−1(Fig. 2), respectively. There were no differences in the baseline sensitivity to boscalid with different hosts or geographical locations in Japan (data not shown).

image

Figure 1.  Frequency distribution of minimum inhibitory concentration (MIC) of boscalid for colony growth inhibition against 220 isolates of Corynespora cassiicola isolated from cucumber (213 isolates), tomato (three isolates), soybean (two isolates), eggplant (one isolate) and cowpea (one isolate) without a prior history of boscalid treatment (black bars), and 438 isolates collected from ten cucumber greenhouses with a history of boscalid treatment (white bars), on YBA agar medium after a 4-day incubation at 25°C in darkness.

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image

Figure 2.  Frequency distribution of 50% effective concentration (EC50) of boscalid for colony growth inhibition against 220 isolates of Corynespora cassiicola isolated from cucumber (213 isolates), tomato (three isolates), soybean (two isolates), eggplant (one isolate) and cowpea (one isolate) without a prior history of boscalid treatment (black bars), and 438 isolates collected from ten cucumber greenhouses with a history of boscalid treatment (white bars), on YBA agar medium after a 4-day incubation at 25°C in darkness.

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Monitoring for boscalid sensitivity

Boscalid sensitivity was tested using 438 isolates collected from cucumber cultivated in ten greenhouses with a prior history of boscalid use in Ibaraki Prefecture. Two hundred and twenty-four of these isolates (51%) were sensitive and 214 (49%) were resistant with MIC values of higher than 30 μg mL−1 (Fig. 1). As a result of calculating the EC50 of boscalid, resistant isolates were divided into 189 moderately resistant (MR) isolates with EC50 values ranging from 1·1 to 6·3 μg mL−1, with a mean of 3·37 μg mL−1, and 25 very highly resistant (VHR) isolates with EC50 values higher than 24·8 μg mL−1 (Fig. 2). Inhibition of mycelial growth by boscalid against MR isolates and VHR isolates ranged from 65–87% (mean 76·3%) and from 31·3–49·2% (mean 40·2%), respectively, with 10 μg mL−1 boscalid, and from 70·2–96·6% (mean 82·1%) and 43·4–58·5% (mean 50·4%), respectively, with 30 μg mL−1 boscalid (Fig. 3). Mycelial growth of MR and VHR isolates on boscalid-free YBA agar medium was equal to sensitive isolates (data not shown). MR isolates were detected from all 10 greenhouses tested, and VHR isolates from four greenhouses (Table 2). MR isolates were first detected in isolates collected on 21 September 2005 in Chikusei-B and -C, and VHR isolates from those collected on 14 September 2006 in Chikusei-E. With the number of boscalid sprays increased, the frequency of resistant isolates was prone to rise. Frequencies of resistant (MR + VHR) isolates exceeded 88% in seven greenhouses. In three of four greenhouses where VHR isolates were detected, the frequency of VHR isolates was much lower than that of MR isolates, whereas the frequency of VHR isolates was slightly higher than that of MR isolates in Chikusei-D from isolates collected on 27 April 2007.

image

Figure 3.  Effect of concentration of boscalid in YBA agar medium on inhibition of mycelial growth from sensitive (S), moderately resistant (MR) or very highly resistant (VHR) isolates of Corynespora cassiicola after a 4-day incubation at 25°C in darkness. Each value was the mean of 224 S, 189 MR and 25 HR isolates calculated from two to three runs of each experiment. Bars denote standard deviation.

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Table 2.   Monitoring of boscalid sensitivity in Corynespora cassiicola isolates collected from cucumber greenhouses with a history of boscalid use in Chikusei and Kasumigaura City, Ibaraki Prefecture, Japan
Location of greenhouseaDate of collectionNo. of isolates testedNo. of resistant isolatesbFrequency of resistant isolates (%)cNo. of boscalid sprays before isolation
MRVHR MR + VHR
  1. aLetters after city name indicates different greenhouses in the same city.

  2. bMR:Moderately resistant, VHR:Very highly resistant.

  3. cFrequency of resistant isolates (%) = (No. of MR isolates + No. of HR isolates)/No. of isolates tested × 100.

Kasama04 April 200723606262<
Chikusei-A29 August 2005500001<
08 February 200610100101004<
Chikusei-B29 August 20051100003
21 September 20053210134
08 February 20062726026965
14 September 20061512012806
01 December 200613909696
11 January 20071814014786
21 February 200766061006
05 April 20079606676
Chikusei-C29 August 2005900002
21 September 20054101253
13 January 20069707784
08 February 20068707885
Chikusei-D07 September 2005500001<
27 April 20071156111006<
Chikusei-E07 September 20053100002
21 September 20051200002
08 February 200611707643
14 September 200615516405
01 December 200676171006
11 January 20079606676
05 April 20071615015947
Chikusei-F08 February 200688081001<
Chikusei-G12 September 200610101101<
Kasumigaura-A11 April 20061700001
09 January 200777071003
10 April 20072921728973
Kasumigaura-B07 March 20079213332
10 April 200724325212
08 May 200718202112

Inoculation tests

Two sensitive, three MR and two VHR isolates were used for the inoculation tests to examine the control efficacy of boscalid using the first leaves of cucumber plants (Table 3). All seven isolates showed a similar degree of pathogenicity on cucumber. Sensitive isolates were almost completely controlled by boscalid, as well as mancozeb which was used as a reference fungicide. In contrast, low efficacy of boscalid was recorded against MR and VHR isolates. Boscalid still slightly inhibited the number and the diameter of lesions on leaves inoculated with MR isolates, but completely lost its efficacy against VHR isolates.

Table 3.   Efficacy of boscalid against corynespora leaf spot disease of cucumber inoculated with boscalid-sensitive,-moderately resistant and -very highly resistant isolates of Corynespora cassiicola
IsolateSensitivity to boscalida FungicidebAverage no. of lesionscDisease control (%)dAverage diameter of lesions (mm)e
  1. aS:sensitive, MR:moderately resistant, VHR:very highly resistant.

  2. bBoscalid and mancozeb were sprayed at a concentration of 0·33 mg a.i. mL−1 and 1·25 mg a.i. mL−1, respectively.

  3. cAverage number of lesions assessed 5 days after inoculation with spore suspensions (ca. 1 × 104spores mL−1) on first leaves of six seedlings.

  4. dDisease control (%) of individual fungicides was calculated as (No. of lesions on tap water treated leaves - No. of lesions on fungicide treated leaves)/No. of lesions on tap water treated leaves × 100.

  5. eThe diameter of the lesions on each leaf was evaluated by calculating a mean of 20 lesions selected at random in boscalid and tap water treatments, and a mean of three to five lesions in mancozeb treatment.

  6. fNM = not measured.

IbCor0008SBoscalid0.0100NMf
Mancozeb0.0100NM
Tap water46·7-3·8
IbCor1522SBoscalid0·3991·2
Mancozeb1·3984·5
Tap water59·3-4·3
IbCor1679MRBoscalid31·3412·5
Mancozeb1·0984·9
Tap water53·2-4·9
IbCor1481MRBoscalid33·5432·6
Mancozeb1·0984·1
Tap water58·8-4·2
IbCor1482MRBoscalid51·8222·9
Mancozeb0.0100NM
Tap water66·0-4·4
IbCor1689VHRBoscalid53·044·1
Mancozeb1·3983·7
Tap water55·0-4·2
IbCor2036BVHRBoscalid64·024·5
Mancozeb2·0974·1
Tap water65·0-4·7

Survey of spray history of boscalid and other fungicides

In two greenhouses, Chikusei-B and Chikusei-E, a survey of growers was carried out to obtain their spray histories of boscalid and other fungicides. The spray histories before the first detection of boscalid-resistant isolates in both greenhouses are given in Table 4. Cucumber cultivations in both greenhouses were performed twice a year, with a late raising culture from late July to early October, and a forcing culture from early November to early May in the next year. Boscalid had been used 1–3 times per cropping season since February 2005 in both greenhouses. Boscalid-resistant isolates were first detected after four boscalid sprays in Chikusei-B, and after three sprays in Chikusei-E. Many kinds of fungicides with different modes of action from boscalid were used between boscalid sprays. The spray histories after 2005 in Chikusei-B and 2006 in Chikusei-E tended to include similar fungicides to those included before (data not shown).

Table 4.   History of boscalid sprays and other fungicides in two greenhouses in Chikusei City, Ibaraki Prefecture, Japan
Location of greenhouseaCropping typeDate of sprayName of fungicides
  1. aLetters after city name indicates different greenhouses in the same city.

Chikusei-BForcing13 January 2005Captan
20 January 2005Kasugamycin + copper, Procymidone
28 January 2005Iminoctadine-albesilate, Polyoxins
03 February 2005Boscalid
14 February 2005Diethofencarb + thiophanate-methyl
23 February 2005Kasugamycin + copper
02 March 2005Boscalid
12 March 2005Kasugamycin + copper, Diethofencarb + procymidone
21 March 2005Captan
31 March 2005Kasugamycin + copper, Cyflufenamid + triflumizole
8 April 2005Boscalid, TPN
24 April 2005Captan
Late-raising09 August 2005Polyoxins, Procymidone
14 August 2005Cyazofamid
21 August 2005TPN, Triflumizole
03 September 2005Boscalid
17 September 2005Diethofencarb + procymidone
Chikusei-EForcing06 January 2005Mancozeb
11 January 2005Diethofencarb + procymidone
21 January 2005Iminoctadine-albesilate
01 February 2005Cymoxanyl + TPN
06 February 2005Boscalid
18 February 2005Diethofencarb + procymidone, Triflumizole
25 February 2005Simeconazole + mancozeb
04 March 2005Polyoxins
14 March 2005Diethofencarb + thiophanate-methyl
20 March 2005Polycarbamate
28 March 2005Diethofencarb + procymidone, Polyoxins
02 April 2005Boscalid
15 April 2005TPN
Late-raising02 August 2005Captan
07 August 2005TPN
13 August 2005Polycarbamate
23 August 2005Simeconazole + mancozeb
30 August 2005Diethofencarb + procymidone
10 September 2005Polyoxins
Forcing09 November 2005TPN, Triflumizole
18 November 2005Polycarbamate, Polyoxins
10 December 2005Mancozeb, Mepanipyrim
20 December 2005Diethofencarb + procymidone, TPN
30 December 2005Boscalid, Captan
10 January 2006Mancozeb, Polyoxins
20 January 2006Cyazofamid, Thiophanate-methyl
04 February 2006Diethofencarb + procymidone, Polycarbamate

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cucumber growers in Japan generally apply fungicides for the control of corynespora leaf spot at 7- to 10-day intervals after transplanting or first sign of disease. However, outbreak of the disease starts at early stages of harvest. One of the reasons for this severe outbreak is the occurrence of C. cassiicola strains resistant to various fungicides (Hasama, 1991; Hasama & Sato, 1996; Date et al., 2004; Miyamoto et al., 2006; Takeuchi et al., 2006; Ishii et al., 2007). The introduction of new fungicides with high efficacy against corynespora leaf spot and novel mechanism of action is imperative to successful disease management. However, considering the high disease pressure and occurrence of resistance to fungicides previously described, it is thought that C. cassiicola is a high-risk pathogen for fungicide resistance development. Therefore, development of resistance to other fungicide classes must be carefully checked.

According to FRAC (Fungicide Resistance Action Committee, 2009), the risk for resistance evolution to the SDHI group of fungicides is estimated to be medium, the same as for anilinopyrimidines and DMI fungicides. Immediately after boscalid was registered, it was confirmed that boscalid had high control efficacy against corynespora leaf spot and was introduced into spray programmes applied in commercial cucumber greenhouses. To avoid resistance, growers received guidelines from manufacturers for boscalid and adopted rotation spray with other fungicides having different modes of action. Although these anti-resistance strategies were followed by most growers, a rapid decline in fungicide efficacy against corynespora leaf spot was reported. The results shown in this study clearly indicated the existence of C. cassiicola isolates bearing resistance to boscalid and suggest that the appearance of them play an important role in the control failure of the disease. To the authors’ knowledge, this is the first report of boscalid resistance in Japan.

During the course of this study, the baseline sensitivity of C. cassiicola to boscalid was established using mycelial growth tests on YBA agar medium. Growth tests for carboxin, which belongs to the same fungicide group as boscalid, showed a 10-fold increase in toxicity when acetate replaced glucose as the carbon source (Ragsdale & Sisler, 1970). Similarly, it was reported that mycelial growth of C. cassiicola isolates was almost inhibited on YBA agar medium amended with 1 μg mL−1 of boscalid, whereas the isolates were hardly inhibited on PDA amended with 100 μg mL−1 of this fungicide (Ishii & Nishimura, 2007). In this study, baseline sensitivity data obtained from C. cassiicola isolates showed the narrow range of sensitivity and clearly differentiated sensitive from resistant isolates which caused control failure of boscalid in inoculation tests. These data confirmed that YBA agar could be used as a testing medium for monitoring the shift of boscalid sensitivity in C. cassiicola populations.

According to the frequency distribution of EC50 values determined for boscalid, C. cassiicola isolates collected from cucumber plants previously sprayed with boscalid were divided into three separate groups of sensitive, MR and VHR isolates. VHR isolates were detected one year after MR isolates were first detected, but at a lower frequency. These differences between MR and VHR isolates might have been due to the different history of boscalid use in cucumber greenhouses. Resistance factors (RF, EC50 value for a resistant isolate/mean EC50 value for sensitive isolates) for MR and VHR isolates ranged from 4·1 to 23·3, and higher than 91·6, respectively. RF for MR isolates (from 3·3 to 20·1) was similar to that for resistant mutants derived from UV mutagenesis in B. cinerea (Zhang et al., 2007). In A. alternata from pistachio, RF for resistant isolates exceeded 1562·5 (Avenot et al., 2008b), higher than VHR isolates of C. cassiicola. Methods for testing the boscalid sensitivity in both reports (Zhang et al., 2007; Avenot et al., 2008b) were different from that used in the current study. However, in isolates of both fungi, the difference in the levels of resistance to boscalid has not been reported. This paper presents the first description of different levels of field resistance to boscalid.

When considering high disease pressure of corynespora leaf spot in practice, results from inoculation tests in this study suggest that boscalid might have almost lost its control efficacy against the disease in cucumber greenhouses, where resistant strains were distributed regardless of the resistance levels. Additionally, the frequencies of C. cassiicola isolates resistant to boscalid increased rapidly after only 2–3 uses of boscalid. Therefore, it is recommended that farmers withdraw the use of boscalid in regions where resistant isolates have already occurred. It is also advised that the use of boscalid should be limited to during the early stage of cucumber cultivation, such as before transplanting or early harvesting time, as a preventive treatment if resistant isolates have not yet spread. Because severe outbreaks of corynespora leaf spot disease begin at early harvesting time of cucumber in greenhouses, most fungicide applications were sprayed regularly during the period when pressure of this disease was high. Accordingly, it is likely that the present spray programmes are at a higher risk of developing pathogen resistance to fungicides. However, it is difficult to change the cultivars and cropping types, major reasons for recent heavy occurrence of the disease, because cucumber growers need high quality and quantities of cucumber fruit. Consequently, to prevent the heavy occurrence of corynespora leaf spot disease, further studies are needed to develop the schedule of fungicide applications in cucumber cultivation based on seasonal prevalence of the disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We express our thanks to M. Tanina (Okayama Prefectural General Agriculture Center) and T. Takeuchi (Chiba Prefectural Agriculture Research Center) for providing Corynespora cassiicola isolates for this work and Y. Kamihara and M. Kaito (Ibaraki Agriculture Center) for their help in collecting samples in the field.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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  • Avenot HF, Sellam A, Karaoglanidis G, Michailides TJ, 2008b. Characterization of mutations in the iron-sulphur subunit of succinate dehydrogenase correlating with boscalid resistance in Alternaria alternata from California pistachio. Phytopathology 98, 73642.
  • Avenot HF, Sellam A, Morgan DP, Michailides TJ, 2008c. A single amino-acid change in the cytochrome b560 subunit of succinate dehydrogenase complex (SdhC) correlates with boscalid resistance in Alternaria alternata isolates from California pistachio. Phytopathology 98, S16 (abstract).
  • Broomfield PLE, Hargreaves JA, 1992. A single amino-acid change in the iron-sulphur protein subunit of succinate dehydrogenase confers resistance to carboxin in Ustilago maydis. Current Genetics 22, 11721.
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