Laboratory studies on benzimidazole resistance in Septoria tritici1

A technique is described for testing isolates of Septoria tritici from winter wheat for resistance to benzimidazole fungicides. Secondary spores from 23 isolates were tested on Czapek Dox V-8 agar amended with benomyl at 1–10 μg ml. Twenty-one isolates were recovered from eight crops in England in 1984 and two (PBI isolates) were obtained in 1973. Thirteen isolates, including both PBI isolates, were sensitive to benomyl at 1 μg ml^?1 and nine were resistant at 10 μg ml^?1. The remaining isolate had a low proportion (1:3.7 x 10⁴) of resistant spores. The minimum inhibitory concentration for sensitive isolates was 0.2–0.4 μg ml ¹ benomyl and for resistant isolates was more than 1000 μg ml ¹. Benomyl-resistant isolates were cross-resistant to carbendazim, thiabendazole and thiophanate-methyl, but not to 12 other fungicides with different modes of action. The implications of these findings are discussed.     

Failure to detect highly resistant strains in dicarboximide resistant field populations of Botrytis cinerea1

Field isolates of Botrytis cinerea with moderate levels of resistance to dicarboximide fungicides (ED50 1.0–4.9 μg ml^?1) and to dicloran were obtained from glasshouses where vinclozolin and iprodione failed to control grey mould. From sensitive and moderatcly-resistant cultures, laboratory isolates were selected on dicarboximide-amended medium, which were highly resistant to these fungicides (ED50 125->3000 μg ml^?1). Conidia of all the resistant isolates germinated well on media amended with 100 μg ml^?1 of the dicarboximides vinclozolin, iprodione, procymidone and myclozolin and with 5 μg ml^?1 of metomeclan. However, the spores of the moderately resistant isolates did not germinate on 100 μg ml^?1 metomeclan while the spores of the highly resistant isolates germinated well. Using media with 100 μg ml^?1 of metomeclan to distinguish between the two phenotypes, no highly resistant strain was detected among 312 resistant samples from five cucumber glasshouses with a high frequency of moderately resistant strains. From air-borne inoculum of five glasshouses with 100% resistant populations, 1604 colonies were recovered on vinclozolin-amended (100 μg ml^?1) medium and none on metomeclan-amended (100 μg ml^?1) medium. It is concluded that strains of B. cinerea highly resistant to dicarboximides are absent from field populations.     

Chemical control of the olive rot caused by Glomerella cingulata1

The effect of various fungicides on Glomerella cingulata, the causal agent of a serious rot of olive fruits, was evaluated both in vitro and in the field. The following fungicides were tested: copper oxychloride, Bordeaux mixture, ziram, myclobutanil, hexaconazole, metiram, bitertanol and benomyl. Field trials were carried out in Calabria (southern Italy) for 2 years on two olive cultivars, Ottobratica (susceptible) and Sinopolese (resistant). The minimum effective concentrations of bitertanol, hexaconazole, myclobutanil, benomyl, metiram, ziram, and copper oxychloride on mycelial growth in vitro were < 5, < 50, < 50, < 1000, < 1000, < 1000, < 1000 μg ml^?1, respectively. The ED90 values for conidial germination were 0.01, 0.02, 50, 50, 1000, 1000, and 1000 μg ml^?1 of benomyl, ziram, myclobutanil, copper oxychloride, bitertanol, hexaconazole and metiram, respectively. In field trials, Bordeaux mixture and copper oxychloride proved to be the most effective products; however ziram, myclobutanil, bitertanol, metiram, and hexaconazole reduced the incidence of fruit rot significantly.     

Sensitivity of Lasiodiplodia theobromae from Brazilian papaya orchards to MBC and DMI fungicides

Stem rot caused by Lasiodiplodia theobromae is an important postharvest disease of papaya in Brazil, responsible for reducing the quality and quantity of fruits. Fungicide use is one of the main disease management measures. However, there are no estimates available of pathogen sensitivity to commonly employed fungicides. Therefore, the EC₅₀ from 120 isolates of L. theobromae from northeastern Brazil, representative of six populations of the pathogen, was estimated in vitro for fungicides of the methyl benzimidazole carbamates—MBC (benomyl and thiabendazole) and demethylation-inhibiting—DMI (imazalil, prochloraz, tebuconazole) groups. Mycelial growth on fungicide-free media and virulence on papaya fruits of the MBC-sensitive and non-sensitive isolates were compared. For MBCs, 8.4% of isolates were non-sensitive to fungicides. For the remaining 91.6%, the mean EC₅₀ ranged from 0.002 to 0.13 μg ml⁻¹ and 0.36 to 1.27 μg ml⁻¹ for benomyl and thiabendazole, respectively. For DMIs, the mean EC₅₀ range for imazalil was 0.001 to 2.27 μg ml⁻¹, 0.04 to 1.75 μg ml⁻¹ for prochloraz, and 0.14 to 4.05 μg ml⁻¹ for tebuconazole. The EC₅₀ values of non-sensitive isolates were significantly (P≤0.05) higher those for the sensitive isolates for each of the DMI fungicides. Differences (P≤0.05) were found in the levels of sensitivity to DMI fungicides among the isolate populations associated with orchards. The populations from two orchards were less sensitive to DMIs. No solid evidence was found for fitness costs relating to MBC non-sensitive isolates because mycelial growth in fungicide-free media and virulence on papaya fruits were similar to those of sensitive isolates.     

Uptake and translocation of fungicides in wheat after seed treatment,as measured by disease response to Fusarium culmorum

The toxicities of five systemic fungicides [benomyl, carbendazim, methyl 4-(2- aminophenyl)-3-thioallophanate (NF48), thiabendazole and thiophanate-methyl] and of two non-systemic fungicides (guazatine and phenylmercury acetate) against Fusarium culmorum were compared on agar plates; their performance as seed treatments was measured by inoculating the shoot bases of wheat seedlings with F. culmorum in pot experiments. The two most effective compounds, benomyl and thiabendazole, and the less effective thiophanate-methyl, were evaluated in further seed treatment experiments in which leaf sheaths and roots of slightly older plants were infected. The three fungicides protected the first leaf sheath for more than 5 weeks, but thiophanate-methyl was least effective. Against root disease, they were effective when infection was mild, but only thiabendazole significantly controlled severe infection. Bioautography confirmed that fungicide levels in shoots were greater after benomyl and thiabendazole, than after carbendazim and thiophanate-methyl treatments, and that concentrations of fungicide after benomyl, carbendazim and thiophanate-methyl treatments generally declined between 5-13 weeks after treatment. Thiabendazole produced a second fungitoxic component detectable in plants after 34 days.     

Fungi in stored barley treated with systemic fungicides

Six fungicides (benomyl, carbendazim, fuberidazole, thiabendazole, thiophanate and thiophanate-methyl) were applied as dusts to barley in concentrations of 100, 1000 and 10 000 μg g^?1 and the barley then stored at 20°C and 90% relative humidity for 60, 105 and 150 days, respectively. Fungal development during storage was assessed by counting the visible patches of developing mould and by dilution plating. None of the fungicides completely prevented fungi developing but for the period up to 60 days, all provided a degree of control that was generally greatest for the highest concentration of fungicide. Benomyl was the most effective of the fungicides but no clear pattern was established for the others. Of the fungi that developed, species of Aspergillus and Penicillium accounted for 94% of the total count.     

Sensitivity of populations ofBotrytis cinerea to triazoles,benomyl and vinclozolin

Sensitivity of field isolates (121) ofBotrytis cinerea from France (1992), Germany (1979–1992), Israel (1990) and the Netherlands (1970–1989) to the triazoles tebuconazole and triadimenol, the benzimidazole benomyl and the dicarboximide vinclozolin were tested in radial growth experiments. Resistance to benomyl (in 21 to 100% of isolates tested) and vinclozolin (in 25 to 71% of isolates tested) was common in most countries. EC₅₀s (concentrations of fungicides inhibiting radial mycelial growth ofB. cinerea on B5-agar by 50%) for tebuconazole and triadimenol ranged between 0.01–1.64 and 0.4–32.6g ml^–1, respectively, and were log-normally distributed. The variation factor (ratio between EC₅₀s of the least and most sensitive isolate tested) amounts 164 and 82 for tebuconazole and triadimenol, respectively. These values are comparable to those for azole fungicides applied in control of other pathogens. Hence, variation in sensitivity to triazoles can probably not explain limited field performance of triazoles towardsB. cinerea. Isolates from south west Germany (1992) were significantly less sensitive to tebuconazole than isolates collected earlier in Germany, Israel and the Netherlands. Such less sensitive populations may contribute to the limited field performance of DMI fungicides towardsB. cinerea. The sensitivity of isolates from south west Germany to tebuconazole was similar to that of DMI-resistant mutants generated in the laboratory. These mutants displayed stable resistance with Q-values (ratio between EC₅₀ of resistant mutant and wild type isolate) between 5 and 20. Sensitivity of field isolates and laboratory mutants to tebuconazole and triadimenol was correlated.     

The advantage of tbz + iprodione treatment for control of gray mold decay of celery caused by the heterogenic spore population ofBotrytis cinerea in Israel

A combined TBZ — iprodione treatment was more effective in inhibiting growthin vitro ofBotrytis cinerea isolates obtained from decayed celery than either of the fungicides applied separately. This was exhibited for both TBZ-resistant and TBZ-sensitive isolates. TBZ at 500 (μg ml^-1 plus iprodione at 1000 μg ml^-1 reduced celery decay beyond the reduction obtained by each fungicide alone. When applied prior to inoculation, the combined treatment prevented decay by the TBZ-sensitive/iprodione-resistant isolates and reduced initial decay by the TBZ-resistant/iprodione-sensitive isolates to 3–10% of the level without treatment. Under natural infection conditions iprodione showed better decay control than TBZ, and at 1500 μg ml^-1 it reduced initial decay during prolonged storage to 3% of the no-treatment level. Although TBZ (500 μg ml^-1) or iprodione (1000 μg ml^-1) applied separately reduced decay significantly, the combination of lower concentrations of each fungicide was sufficient to eliminate decay development almost totally. The combined treatment also inhibited decay bySclerotinia sclerotiorum, which contributed 3% of the total soft rot in stored celery.     

Differential responses of germinating Venturia inaequalis conidia to kresoxim-methyl

A new class of agricultural fungicides derived from strobilurins act as respiration inhibitors by binding to mitochondrial cytochrome b. The effects of the strobilurin, kresoxim-methyl, on conidia germination, mycelial growth and the protection of apple leaves from scab development were investigated for two isolates of Venturia inaequalis randomly selected from a culture collection. Inhibition of mycelial growth required relatively high doses of kresoxim-methyl (ED₅₀=1 μg ml^-1) for both isolates. In comparison, germination of conidia was highly sensitive for one of the isolates (ED₅₀=0·005 μg ml^-1), while the level of inhibition achieved for the second isolate was 60-fold less (ED₅₀=0·3 μg ml^-1). As deduced from identical sequences of cytochrome b cDNAs prepared from both isolates, the different responses of germinating conidia to kresoxim-methyl were not caused by differences in the sequence of cytochrome b as the target site for strobilurin action. Strong synergistic effects of salicylhydroxamic acid on kresoxim-methyl inhibitory potency suggested that the reduced kresoxim-methyl sensitivity observed for germinating conidia was caused by interference of the alternative respiratory pathway with inhibitor action. However, this mechanism of target site circumvention in germinating conidia had no adverse effects on the protection of apple leaves from scab infection by kresoxim-methyl. © 1998 Society of Chemical Industry     

In vitro Variability of a number of characteristics ofSclerotinia sclerotiorum

Studies were made of the variability ofSclerotinia sclerotiorum (Lib.) de Bary: (i) growth rate when cultivated on potato-dextrose agar (PDA); (ii) thein vitro resistance of this organism to the fungicides benomyl, vinclozolin and procymidone; and (iii) the size and number of sclerotia formed on PDA. The isolates used were collected from eggplants, French beans and tomatoes cultivated in the same locality, but although marked differences were observed among them, especially in growth rate and formation of sclerotia, it was not possible to establish groups or categories among the isolates studied, or to relate the observed variability to their origins. Temperature was a decisive factor for formation of sclerotia on PDA. No correlation was found between size and number of sclerotia, and growth rate. The ED₅₀ level for both vinclozolin and procymidonein vitro was 2 mg l⁻¹, whereas that for benomyl was below the minimum level used, and therefore could not be established.     

Cane spot (Elsinoë veneta) in red raspberry: infection periods and fungicidal control

Dichlofluanid controlled cane spot ( Elsinoë veneta ) of raspberry cv. Mailing Jewel when applied before harvest in a four-spray programme, but carbendazim, vinclozolin and iprodione were ineffective. Lesions predominated in the fruit-bearing region of canes in a zone 46-120 cm above soil level. Bait plants exposed for periods of 2 weeks in two infected plantations in north-east Scotland developed lesions, after incubation for 4-6 weeks, only on internodes which were the youngest at the time of exposure. Infection occurred in all periods from mid-April to late July. The infection of bait plants was related to the distribution of lesions on canes in the spray trial.
An isolate of E. veneta obtained from a plantation sprayed with carbendazim and vinclozolin was resistant when tested in vitro to carbendazim, benomyl and thiophanate-methyl. This isolate and another MBC-sensitive strain were both relatively insensitive to the dicarboximide fungicides iprodione and vinclozolin.     

多菌灵等4种杀菌剂对烟草灰霉病菌的室内生物活性

采用菌丝生长速率法和孢子萌发法,分别测定了烟草灰霉病菌对多菌灵、嘧霉胺、异菌脲和丙环唑的敏感性,同时通过离体叶片法评估了这4种杀菌剂对烟草灰霉病的保护和治疗作用。结果表明:4种杀菌剂对烟草灰霉病菌的菌丝生长和孢子萌发均表现出了不同程度的抑制活性,并对灰霉病同时具有保护和治疗作用。其中多菌灵对菌丝生长的抑制活性最强,EC₅₀平均值为0.06 mg/L,其次为丙环唑、嘧霉胺和异菌脲,EC₅₀平均值分别为0.36、0.53和0.60 mg/L;异菌脲和丙环唑对烟草灰霉病菌孢子萌发的抑制活性较强,EC₅₀平均值分别为2.05和2.21 mg/L,其次为嘧霉胺和多菌灵,EC₅₀平均值分别为10.56和131.23 mg/L。异菌脲和多菌灵对灰霉病的保护作用和治疗作用均最强,当药剂质量浓度为200 mg/L时,其对离体叶片的保护和治疗作用防效分别为100%、100%和98.3%、91.8%。研究结果可为烟草灰霉病的科学防治提供依据。     

Degradation of dicarboximidic fungicides in wine

The degradation of some dicarboximidic fungicides (chlozolinate, iprodione, procymidone and vinclozolin) has been studied in wine at pH values of 3.0 and 4.0, at 30°. The kinetic data obtained by observing the disappearance of the active ingredient (3.0 mg kg^?1) showed the pseudo first-order rate constants to be higher at pH 4.0 than at 3.0, with a trend for the values to be in the order: chlozolinate > vinclozolin > procymidone > iprodione. 3, 5-Dichloroaniline was not detected as a degradation product of any of the compounds. Chlozolinate and vinclozolin in wine each yielded a major metabolite, the structures of which are proposed.     

Characterization of a dicarboximide-fungicide-resistant laboratory isolate ofMonilinia laxa

Monilinia laxa, the incitant of blossom blight in stone fruits in Israel, is sensitive to 5μg/ml of the dicarboximide fungicides vinclozolin and iprodione in the growth medium. When a large number of spores, from an isolate never exposed to these fungicides, was seeded on a medium containing 15 μg/ml iprodione, spontaneous resistant mutants appeared at 10^-5 frequency. These mutants showed cross resistance to the dicarboximide fungicides vinclozolin, procymidone, l-(3,5-dichloro-phenyl)-3-propen(2)-pyrrolidin-2,5-dion (Co 4462), l-(3,5-dichlorophenyl)-3-methoxymethyl-pyrrolidin-2,5-dion (Co 6054), and to dicloran. Growth rate on fungicide-free medium was similar to that of the parental sensitive strain but sporulation was much reduced. Growth rate on media supplemented with dicarboximide fungicides decreased gradually with increasing fungicide concentrations. The resistance has been stable for more than one year in the absence of fungicides. Artificial inoculation of cherry, apricot and plum fruits previously treated with 500 μg/ml vinclozolin, iprodione, procymidone or Co 6054, with a resistant strain, resulted in brown rot; similar treatments provided full protection of the fruits against the sensitive strain.     

Multiple resistance of Botrytis cinerea from kiwifruit to SDHIs,QoIs and fungicides of other chemical groups

BACKGROUND: Botrytis cinerea Pers.: Fr. is a high‐risk pathogen for fungicide resistance development that has caused resistance problems on many crops throughout the world. This study investigated the fungicide sensitivity profile of isolates from kiwifruits originating from three Greek locations with different fungicide use histories. Sensitivity was measured by in vitro fungitoxicity tests on artificial nutrient media. RESULTS: Seventy‐six single‐spore isolates were tested for sensitivity to the SDHI fungicide boscalid, the QoI pyraclostrobin, the anilinopyrimidine cyprodinil, the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the dicarboxamide iprodione and the benzimidazole carbendazim. All isolates from Thessaloniki showed resistance to both boscalid and pyraclostrobin, while in the other two locations the fungal population was sensitive to these two fungicides. Sensitive isolates showed EC₅₀ values to boscalid and pyraclostrobin ranging from 0.9 to 5.2 and from 0.04 to 0.14 mg L^?1 respectively, while the resistant isolates showed EC₅₀ values higher than 50 mg L^?1 for boscalid and from 16 to > 50 mg L^?1 for pyraclostrobin. All QoI‐resistant isolates carried the G143A mutation in cytb. Sensitivity determinations to the remaining fungicides revealed in total eight resistance phenotypes. No isolates were resistant to the fungicides fenhexamid and fludioxonil. CONCLUSION: This is the first report of B. cinerea field isolates with resistance to both boscalid and pyraclostrobin, and it strongly suggests that there may be a major problem in controlling this important pathogen on kiwifruit. Copyright © 2010 Society of Chemical Industry     

Resistance to polyoxin AL and other fungicides in Botrytis cinerea collected from sweet basil crops in Israel

A total of 568 B. cinerea isolates were collected from diseased sweet basil plants and the air in 10 sweet basil greenhouses. Mycelial growth tests were used to evaluate the sensitivity of these isolates to benomyl, fenhexamid, iprodione, polyoxin AL and pyrimethanil. EC₅₀ values for polyoxin, the main botryticide on sweet basil in Israel, ranged from 0.4 to 6.5 μg ml^?1 and had a bimodal distribution; the EC₅₀ values for sensitive isolates ranged from 0.4 to 1.5 μg ml^?1 and the EC₅₀ values for low-level resistant isolates ranged from 4 to 6.5 μg ml^?1. Among populations that had not been exposed to polyoxin treatments, 20 to 35 % of the collected isolates were low-level resistant for polyoxin. Polyoxin treatments in an experimental greenhouse shifted the equilibrium in favour of low-level resistant isolates, and the change occurred rapidly: from a frequency of 20 % low-level resistant isolates in the population that had never been treated with polyoxin to a frequency of 72 % after a few treatments over two seasons. Prolonged use of polyoxin in Israeli basil crops (in some sites for more than 10 years) does not appear to have led to the development of high-level resistance, but low-level resistant isolates were found in commercial greenhouses with the frequency of up to 73 %. High-level resistance to benzimidazoles was common (60 to 80 % of isolates) in greenhouses with a history of benzimidazole treatments; whereas 15–25 % of the isolates from greenhouses in which fungicides were not used were resistant. Low-level resistance to dicarboximides was fairly widespread (frequency of 30 to 80 % depending on the greenhouse) and a few cases of moderate resistance to dicarboximides were also noted (frequency of 0 to 9 %). Neither high- nor low-level resistance to anilinopyrimidines was common in sweet basil commercial greenhouses (0 to 7 %). However, 34 % of the isolates were strongly resistant in the experimental greenhouse, following a few treatments with anilinopyrimidine fungicides during the previous season. Before those treatments, the proportion of anilinopyrimidines resistant isolates had been 1 %. About 3 % of the isolates exhibited low-level resistance to fenhexamid and no isolates were found to be strongly resistant to fenhexamid. Low-level resistance to one fungicide was often associated with low-level resistances to other fungicides. Thirty-two phenotypes exhibiting resistance to one or more of the tested fungicides were noted among B. cinerea isolates. Resistant isolates showed similar or reduced fitness parameters in comparison to wild-type isolates.     

Simultaneous determination of benzimidazole fungicides by HPLC on apples,pears and their pulps

The simultaneous HPLC determination of carbendazim, thiabendazole and thiophanate-methyl in apples, pears and their pulps is described. The method is based on a clean-up procedure carried out on an Extrelut 20 cartridge followed by HPLC analysis on a Diol column using hexune+isopropanol as mobile phase. Average recoveries of 83.8% for carbendazim, 82.9% for thiabendazole and 68.8% for thiophanate-methyl on apple matrix were obtained; recoveries on pear matrix were in the same range. Detection limits with UV detection at 285 nm of 100 ng ml^?1 for carbendazim, 140 ng ml^?1 for thiabendazole and 500 ng ml^?1 for thiophanate-methyl were achieved.     

Effects of fungicides on in vitro spore germination and mycelial growth of the phytopathogens Leptosphaeria maculans and L. biglobosa (phoma stem canker of oilseed rape)

BACKGROUND: Phoma stem canker, caused by the coexisting related fungal pathogens Leptosphaeria maculans (Des.) Ces. & de Not and L. biglobosa Shoemaker & H Brun, is a major disease of winter oilseed rape in the UK. Annually, over 90% of UK crops receive at least one foliar application of fungicide, but little is known about the sensitivity of the more damaging L. maculans and the less damaging L. biglobosa to these fungicides. The effects of flusilazole, tebuconazole and Methyl Benzimidazole Carbamate (MBC) fungicides (benomyl and carbendazim) on the germination of ascospores, conidia and germ tube growth of both species were examined. Isolates collected from different oilseed rape crops in England and Wales were assessed for their mycelial growth on fungicide‐amended medium, and ED₅₀ values were calculated. RESULTS: Leptosphaeria maculans and L. biglobosa differed in their sensitivity to fungicides. Conidial germination of L. maculans was more sensitive to these fungicides than that of L. biglobosa. Isolates of L. maculans had smaller ED₅₀ values for mycelial growth for all fungicides tested than isolates of L. biglobosa. CONCLUSION: These results suggest that fungicide applications might affect the structure of L. maculans/L. biglobosa populations in UK winter oilseed rape crops. Copyright © 2009 Society of Chemical Industry     

Enhanced degradation of iprodione and vinclozolin in soil

Residues of iprodione and vinclozolin were measured following repeated application of the fungicides to a sandy loam soil in the laboratory. There was a progressive increase in rates of degradation with successive treatments. With iprodione, for example, the times for 50% loss of the first and second applications were about 23 and 5 days respectively. When treated for the third time, less than 10% of the applied dose remained in the soil after just 2 days. Similar results were obtained with vinclozolin in the same soil, and with both compounds in a second soil. In a third soil, which had relatively low pH, degradation of both compounds occurred only slowly and the rate of degradation of a second application was the same as that of the first. Degradation rates in this soil were increased by addition of 100 g kg^?1 of a soil in which degradation occurred more readily, and they were markedly increased by addition of 100 g kg^?1 of a soil in which enhanced degradation had been previously induced. Residues of both fungicides were also measured following repeated application in the field. When iprodione was applied to previously untreated plots, about 3% of the initial dose remained in the soil after 77 days. When applied to plots treated once before, less than 1% remained after 18 days, and when applied to plots treated twice previously less than 1% remained after 10 days. Similar results were obtained with vinclozolin. Enhanced degradation of subsequent soil treatments was also observed following a sequence of low-dosage sprays in the field.     

桃褐腐病菌(Monilinia fructicola)对3种杀菌剂的敏感性

采用生长速率法测定了采自北京平谷区3个桃园的125株桃褐腐病菌对甲基硫菌灵、戊唑醇和异菌脲3种杀菌剂的敏感性,发现甲基硫菌灵对桃褐腐病菌的EC50主要分布在1.0×10^-5～0.2μg/mL,戊唑醇对桃褐腐病菌的EC₅₀主要分布在0.006～0.022μg/mL之间。异菌脲对桃褐腐病菌的EC₅₀主要分布在0.15～0.55μg/mL之间。研究结果表明,北京地区的桃褐腐病菌对这3种杀菌剂都比较敏感,未产生明显的抗药群体。建立了褐腐病菌对异菌脲抗药性的敏感基线。而且,数据分析表明:甲基硫菌灵、戊唑醇和异菌脲之间均不存在交互抗性。