Methyl Benzimidazole Carbamate Research Articles

Sensitivity of Monilinia fructicola from Peach Farms in China to Four Fungicides and Characterization of Isolates Resistant to Carbendazim and Azoxystrobin

Brown rot of peach caused by Monilinia fructicola can cause considerable preharvest and postharvest losses in China. Fungicides are increasingly utilized to minimize such losses. Eighty isolates of M. fructicola were collected from commercial peach orchards located in five provinces in China and the sensitivity to carbendazim, azoxystrobin, tebuconazole, and boscalid was determined. Resistance to carbendazim was detected only in the Yunnan province in 15 of 16 isolates. Characterization of carbendazim-resistant isolates revealed stable resistance, no fitness penalty, and negative cross resistance to diethofencarb. Resistant isolates produced disease symptoms on detached fruit sprayed with label rates of formulated carbendazim and possessed the amino acid mutation E198A in β-tubulin. Resistance to azoxystrobin was detected in 3 of 10 isolates from Fujian. In contrast to carbendazim resistance, however, azoxystrobin resistance was unstable, associated with a fitness penalty, and not associated with mutations in the target gene cytochrome b. The concentration at which mycelial growth is inhibited 50% (EC50) values of the azoxystrobin-sensitive isolates were 0.02 to 1.94 μg/ml, with a mean value of 0.54 μg/ml. All isolates were sensitive to tebuconazole, with a mean EC50 value of 0.03 μg/ml. The EC50 values for boscalid were 0.01 to 3.85 μg/ml, with a mean value of 1.02 μg/ml. Our results indicate that methyl benzimidazole carbamates (MBCs), quionon outside inhibitors, demethylation inhibitor fungicides, and succinate dehydrogenase inhibitors are likely to be very effective in controlling brown rot in many peach production areas in China, but that resistance to MBCs is emerging.

Sensitivity towards DMI fungicides and haplotypic diversity of their CYP51 target in the Mycosphaerella graminicola population of Flanders

Septoria leaf blotch, caused by the fungus Mycosphaerella graminicola, is the most important wheat disease in Northwestern Europe, and is currently controlled by fungicide applications. Since the spread of resistance to methyl benzimidazole carbamates (MBCs) and quinone outside inhibitors (QoIs) in European countries, reliable control is mainly dependent upon sterol 14 alpha-demethylation inhibitors (DMIs). In the last decades however, a slow shift towards reduced sensitivity of M. graminicola to DMIs has been observed. This shift is caused mainly by mutations in the CYP51 gene encoding the 14 alpha-demethylase target protein for these fungicides. In this work, M. graminicola isolates were sampled at fields spread over Flanders, Belgium. In vitro assays were used to analyze the sensitivity of the Flemish M. graminicola population towards different DMIs. Sequencing of the CYP51 gene of these isolates allowed us to identify and map the haplotypes in this population. The results showed that there is a large variability in DMI sensitivity between the isolates, even within one field, which is reflected in a high diversity in CYP51 haplotypes within the M. graminicola population in Flanders. Next to some haplotypes that were not described in literature before, we found that the population is dominated by CYP51 haplotypes which were previously associated with increased resistance towards DMIs.

A novel bioassay to monitor fungicide sensitivity in Mycosphaerella fijiensis.

Black leaf streak disease (BLSD) is the most important disease of bananas for export. The successful control of BLSD requires an intensive use of systemic fungicides, leading to the build-up of resistance and failure of control. Early detection of fungicide resistance is crucial to drive rational chemical strategies. Present methods relying on ascospore germination bioassays have several drawbacks that could be overcome using conidia. Generally, a single genotype is present on the conidial population derived from one lesion. Conidial germination tests with thiabendazole (5 mg L(-1)) enable a clear detection of strains resistant to methyl benzimidazole carbamates. Germination bioassays on azoxystrobin (10 mg L(-1)) enable the detection of most QoI-resistant strains, but their proportion might be underestimated with cut-off limits of germ tube length (L > 120 µm) or growth inhibition (GI < 50%). The level of fungicide resistance differs at different canopy levels of a banana tree, which should be considered for sampling. The ascospore germination bioassay provided more variable estimations of the level of resistance by comparison with the new conidial germination bioassay. Germination bioassays performed with conidia obtained from young lesions overcome most drawbacks encountered with ascospore germination bioassays and could be considered as a new reference method for fungicide resistance monitoring in this species. Different steps are proposed, from sampling to microscopic examinations, for the implementation of this technique.

Thiophanate-methyl sensitivity and fitness in Lasiodiplodia theobromae populations from papaya in Brazil

Stem-end rot, caused by Lasiodiplodia theobromae, is an important postharvest disease of papaya in Brazil. The use of fungicides is one of the main disease management measures. However, there are no data available on the sensitivity of L. theobromae to thiophanate methyl (methyl benzimidazole carbamate), the most common fungicide used in papaya orchards in northeastern Brazil. Thus, the effective concentration that results in 50 % of mycelial growth inhibition (EC50) of 109 isolates, representing five populations of the pathogen was estimated in vitro. Seven components of fitness were measured for the 10 isolates with lower and high values of EC50. Of the 109 isolates, 20.2 % were resistant to the fungicide with EC50 values greater than 300 μg ml−1, whereas the remaining 79.8 % were sensitive with an average EC50 of 1.87 μg ml−1. The EC50 values for the resistant isolates were significantly (P ≤ 0.05) higher than those for the sensitive isolates. When the fitness components were evaluated, only in relation to the spore production was significant difference among sensitive and resistant isolates, and resistant isolates showed sporulation capacity significantly lower than the S isolates, indicating a fitness cost.

Emergence of Prochloraz-Resistant Populations of Calonectria pauciramosa and Calonectria polizzii in Ornamental Nurseries of Southern Italy.

Management of Calonectria spp. infections in nurseries requires scheduled fungicide applications, particularly with methyl benzimidazole carbamates (MBCs) and sterol demethylation inhibitors (DMIs). Due to rising concerns about the occurrence of MBC resistance in different Calonectria populations and variability in prochloraz efficacy in controlling these pathogens, a detailed study on prochloraz sensitivity distributions of Calonectria isolates belonging to the Calonectria scoparia complex was carried out. In total, 105 isolates collected in two distinct periods (1993 to 1996 and 2005 to 2009) were analyzed for prochloraz sensitivity. Based on DNA sequencing and phylogenetic analyses of β-tubulin, histone H3, and translation elongation factor-1α gene sequences, 69 and 36 isolates were identified as C. pauciramosa and C. polizzii, respectively. The isolates collected more recently (group B) had a reduced prochloraz sensitivity, as indicated by greater values for the effective dose to reduce growth by 50% than those collected earlier (group A). The reduced sensitivity detected in vitro corresponded to partial loss of fungicide efficacy in controlling infections in red clover and feijoa under controlled and semi-field conditions, respectively. Frequent prochloraz application in nurseries for controlling Calonectria spp. infections is discouraged.

Resistance of Kansas Sclerotinia homoeocarpa Isolates to Thiophanate-Methyl and Determination of Associated β-Tubulin Mutation

Eighty-two isolates of Sclerotinia homoeocarpa from 12 sites in Kansas were evaluated for in vitro sensitivity to the methyl benzimidazole carbamate (MBC) fungicide thiophanate-methyl at the discriminatory dose of 10 μg/ml. Seventeen isolates were sensitive to thiophanate-methyl and the remaining isolates were resistant. Of the 65 isolates from golf course putting greens, two isolates were sensitive and the remaining 63 isolates were resistant. Six resistant and five sensitive isolates were also evaluated in greenhouse assays on fungicide-treated plants. The isolates that were sensitive to thiophanate-methyl in vitro did not cause any disease on thiophanate-methyl-treated plants, and those that were resistant in vitro caused blighting on treated plants equivalent to the nontreated controls. The entire β-tubulin gene was sequenced for four resistant and four sensitive isolates. The resistant isolates all harbored a substitution of alanine for glutamic acid at codon 198 (E198A). These results provide a starting point for further surveys and monitoring of fungicide sensitivity. Accepted 7 April 2014. Published 6 June 2014.

Field Strains of Monilinia fructicola Resistant to Both MBC and DMI Fungicides Isolated from Stone Fruit Orchards in the Eastern United States.

In 2012, significant brown rot disease was observed on stone fruit in Pennsylvania, Maryland, and South Carolina despite preharvest application of methyl benzimidazole carbamate (MBC) and demethylase inhibitor (DMI) fungicides. In total, 140 Monilinia fructicola isolates were collected from diseased orchards and examined for fungicide sensitivity. In addition to isolates resistant to either the DMI propiconazole or the MBC thiophanate-methyl, 22 isolates were discovered that were resistant to both fungicides, including 4 isolates from peach in South Carolina, 12 isolates from peach and sweet cherry in Maryland, and 6 isolates from sweet cherry in Pennsylvania. Analysis of MBC resistance revealed that dual-resistant isolates from South Carolina carried the β-tubulin E198A mutation, whereas isolates from Maryland and Pennsylvania carried E198 mutations not previously described in the Monilinia genus, E198Q or F200Y. The genetic element Mona, associated with DMI fungicide resistance in M. fructicola, was detected in the dual-resistant isolates from South Carolina but not in the isolates from the two more northern states. An investigation into the molecular mechanism of DMI resistance in the latter isolates revealed that resistance was not based on increased expression or mutation of MfCYP51, which encodes the target of DMI fungicides. Label rates of formulated propiconazole or thiophanate-methyl were unable to control dual-resistant isolates on detached peach fruit, confirming field relevance of dual resistance. The same isolates were not affected by fitness penalties based on mycelial growth rate, ability to sporulate, and virulence on detached peach fruit. The emergence of M. fructicola strains resistant to both DMI and MBC fungicides in multiple states and multiple stone fruit crops is a significant development and needs to be considered when designing resistance management strategies in stone fruit orchards.

Detection and molecular characterisation of Pyrenopeziza brassicae isolates resistant to methyl benzimidazole carbamates

Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes. The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

Characterization of Monilinia fructicola Strains Resistant to Both Propiconazole and Boscalid.

In 2011 and 2012, significant brown rot disease caused by Monilinia fructicola was observed in a peach orchard in Spartanburg County, SC, despite preharvest fungicide applications of demethylation inhibitor (DMI), quinone outside inhibitor (QoI), and succinate dehydrogenase inhibitor (SDHI) fungicides. All 22 isolates obtained in 2011 from this orchard were sensitive to the QoI fungicide, azoxystrobin, and the methyl benzimidazole carbamate (MBC) fungicide, thiophanate-methyl. Five were resistant to the DMI fungicide, propiconazole, and were selected, together with five propiconazole-sensitive isolates, for further investigations. One of the 10 isolates was resistant to propiconazole but sensitive to the SDHI fungicide, boscalid (EC50 = 0.42 μg/ml), 3 were resistant to propiconazole with intermediate sensitivity to boscalid (EC50 0.72 to 2.1 μg/ml); 2 were sensitive to propiconazole with intermediate sensitivity to boscalid; 3 were sensitive to propiconazole but resistant to boscalid (EC50 ≥ 2.1 μg/ml); and 1 (isolate MD22) was resistant to both propiconazole and boscalid. Disease incidence on detached fruit treated with formulated propiconazole or boscalid was significantly higher for MD22 compared to a sensitive control isolate. Continued monitoring of fungicide resistance in the same orchard in 2012 revealed an increase of isolates resistant to propiconazole from 22.7% in 2011 to 34.7%, and an increase of isolates resistant to both propiconazole and boscalid from 4.5% in 2011 to 18.4%. Propiconazole resistance was always associated with the presence of the 'Mona' mobile element located upstream of the sterol 14α-demethylase (MfCYP51) gene. To investigate whether mutations in the subunits of the succinate dehydrogenase enzyme were involved in boscalid resistance, significant portions of the M. fructicola SdhA, SdhB, SdhC, and SdhD genes were cloned and analyzed for 2 sensitive, 2 boscalid-resistant, and 6 dual-resistant isolates. Although sequence variation was found among the isolates, no single change correlated with resistance. Interestingly, analysis of isolates collected from orchards in 2001 and 2002, prior to the registration of boscalid, revealed a range of sensitivities to boscalid (EC50 0.03 to 3.46 μg/ml) including boscalid-resistant isolates. The presence of boscalid-resistant isolates in the baseline population was unexpected and requires further investigation.

Biomass Reducing Potential and Prospective Fungicide Control of a New Leaf Blight of Miscanthus × giganteus Caused by Leptosphaerulina chartarum

During a multistate survey (2009) of diseases of perennial grasses under consideration as biofuel feedstocks, a new leaf blight of Miscanthus × giganteus caused by Leptosphaerulina chartarum was observed on 100 % of the plants evaluated in research plots near Lexington, Kentucky. A greenhouse study was conducted to evaluate the effect of L. chartarum on M. × giganteus biomass and to identify effective foliar fungicides against the disease. Eleven broad-spectrum fungicides were tested with rates typically used in agronomic crops. Fungicides tested included active ingredients from five different chemical groups, demethylation inhibitors (prothioconazole, tebuconazole, cyproconazole, propiconazole, tetraconazole, flutriafol, and metconazole), quinone outside inhibitors (pyraclostrobin), succinate dehydrogenase inhibitors (boscalid), methyl benzimidazole carbamates (thiophanate-methyl), and chloronitriles (chlorothalonil). Infection by L. chartarum significantly lowered aboveground biomass by an average of 33 %. Application of cyproconazole, flutriafol, tebuconazole, or prothioconazole significantly reduced disease severity, with cyproconazole and flutriafol ranking best at an average of 29 % disease control. However, no fungicide treatment resulted in biomass loss abatement. Greater rates or a combination of fungicides might be needed to achieve adequate control.

First Report of Thiophanate-Methyl Resistance in Botrytis cinerea on Strawberry from South Carolina.

Botrytis cinerea Pers.:Fr. is the causal agent of gray mold disease and one of the most important plant-pathogenic fungi affecting strawberry (Fragaria× ananassa). Control of gray mold mainly depends on fungicides, including the methyl benzimidazole carbamate (MBC) thiophanate-methyl. In 2011, strawberries with gray mold symptoms were collected from commercial fields near Chesnee, Florence, Lexington, McBee, Monetta, and North Augusta, all in South Carolina. MBC fungicides were used in most of these fields for gray mold control during the last 3 years. A total of 124 single spore B. cinerea isolates were obtained, each from a different fruit. Resistance to thiophanate-methyl (Topsin M 70WP, Cerexagri-Nisso LLC, King of Prussia, PA) was determined using a conidial germination assay as described previously (1). The majority of isolates (81.4%) were resistant; the rest were sensitive. Resistant isolates were found in all locations with some populations (Chesnee, McBee, and Lexington) revealing no sensitive isolates. Genomic DNA from 35 resistant isolates (representing all locations) and 10 sensitive isolates (from Chesnee, Monetta, and North Augusta, SC) was extracted, and the molecular basis of MBC fungicide resistance was determined as described previously (2). All MBC-resistant isolates possessed the E198A mutation known to confer high levels of MBC fungicide resistance in many fungi, including B. cinerea (2,3). Disease was assessed using a detached strawberry fruit assay. Commercially grown strawberry fruit (24 in total for each isolate and fungicide treatment) were rinsed with water, dried, and sprayed 4 h prior to inoculation with either water or 2.4 g/liter of Topsin M to runoff using a hand mister. Fruit was stab-wounded with a sterile syringe and inoculated with a 30-μl droplet of a conidial suspension (106 spores/ml) of either a sensitive or resistant isolate. After inoculation, the fruit were kept at 22°C for 4 days. The sensitive isolate developed gray mold disease in untreated but not Topsin M-treated fruit. The resistant isolate developed gray mold disease of equal severity in both, the control and fungicide-treated fruit. This experiment was repeated once. The results of the study show that resistance to MBC fungicides is common and widespread in B. cinerea from strawberry in South Carolina. Prior to this study, resistance to MBCs has only been reported in B. cinerea from ornamental crops grown in greenhouses in South Carolina (4).

Sensitivity of Monilinia fructicola from Brazil to Tebuconazole, Azoxystrobin, and Thiophanate-Methyl and Implications for Disease Management.

The aim of this study was to investigate the sensitivity of Monilinia fructicola isolates to tebuconazole (demethylation inhibitor [DMI]), azoxystrobin (quinone outside inhibitor), and thiophanate-methyl (methyl benzimidazole carbamate) in 118 isolates collected from four states in Brazil from orchards with different histories of fungicide use. Sensitivity to fungicides was determined by inhibition of mycelial growth and spore germination on fungicide-amended media. Polymerase chain reaction was used to determine the frequencies of M. fructicola genotypes exhibiting high (HR) and low (LR) resistance to thiophanate-methyl among sampled populations. Resistance to tebuconazole was found in 15.8% of isolates collected from São Paulo State. The 50% effective concentration (EC50) values varied from 0.01 to greater than 100 μg/ml. The EC50 values for tebuconazole corresponded to its historic use frequency in the orchard; for instance, isolates from orchards with no DMI fungicide use had the lowest mean EC50 value (0.04 μg/ml), while those collected from orchards where more than five DMI fungicide sprays were applied per season had a mean EC50 value of 21.17 μg/ml. All isolates were sensitive to azoxystrobin but their EC50, based on tests of inhibition of conidial germination, increased from 0.05 in 2002 to 0.44 μg/ml in 2008. The EC50 values based on mycelial growth inhibition for thiophanate-methyl were >162 μg/ml, 1.99 to 12.5 μg/ml, and <1.0 μg/ml for HR, LR, and sensitive isolates, respectively.

Sensitivity of Ascochyta rabiei populations to prothioconazole and thiabendazole

Ascochyta rabiei causes Ascochyta blight, a yield-limiting disease of chickpea ( Cicer arietinum) world-wide. In 2007, fungal populations of A. rabiei resistant to the QoI group of fungicides were detected in the Northern Great Plains of the United States. Assays were conducted to determine fungal sensitivity for two alternative fungicidal modes of action. A total of 78 isolates of A. rabiei collected between 1983 and 2007 were screened to determine baseline sensitivity to the demethylation-inhibiting foliar fungicide, prothioconazole, and 100 isolates collected between 1987 and 2007 were screened for sensitivity to the methyl benzimidazole carbamate (MBC) fungicide, thiabendazole. Isolates were tested using an in vitro mycelial growth assay to determine the effective fungicide concentration at which 50% of fungal growth was inhibited (EC 50) for each isolate–fungicide combination. Baseline EC 50 values of prothioconazole ranged from 0.0526 to 0.2958 μg/ml, with a mean of 0.1783 μg/ml. Isolates of A. rabiei collected from 2007 to 2009 from North Dakota chickpea fields exposed to prothioconazole, were screened for prothioconazole sensitivity using the same assay. Mean EC 50 values for these isolates were 0.3544 μg/ml, 0.3746 μg/ml, and 0.7820 μg/ml, respectively. These values represent an approximate 2.0 (2007–2008) and 4.4-fold (2009) decrease in sensitivity from the baseline mean. EC 50 values of thiabendazole ranged from 1.192 to 3.819 μg/ml, with a mean of 2.459 μg/ml. No significant decrease in fungicide sensitivity was observed for thiabendazole. To date, no loss of Ascochyta blight control has been observed with the use of either prothioconazole or thiabendazole.

First Report of the β-Tubulin E198A Mutation Conferring Resistance to Methyl Benzimidazole Carbamates in European Isolates of Monilinia fructicola.

The causal agent of brown rot on stone and pome fruits, Monilinia fructicola (G. Wint.), is a quarantine pathogen in Europe. It has been detected in Austria (later eradicated), Spain, the Czech Republic, Italy, Germany, and Switzerland (1). In the United States and other countries, M. fructicola isolates were reported to show resistance to different classes of fungicides, including methyl benzimidazole carbamates (MBC) (2). Lichou et al. (2) reported the presence of isolates resistant to the MBC carbendazim in France, but the mechanisms inducing MBC resistance in these isolates were not studied. Ma et al. (3) in California, and more recently, Zhu et al. (4) in South Carolina, demonstrated that the molecular mechanisms accounting for low and high levels of resistance to MBC fungicides in M. fructicola isolates were the mutations H6Y and E198A, respectively, in the β-tubulin gene. Four M. fructicola isolates each from Italy, France, Spain, and Switzerland (16 isolates total), all having an unknown level of MBC resistance, were selected. In each isolate, the section of the β-tubulin gene containing the two potentially mutant codons was PCR-amplified with the primers TubA and TubR1 (3) and the amplicons were sequenced directly. Sequence analysis revealed the amino acid histidine (H) at codon 6 in all the isolates, which would not predict MBC resistance, while alanine (A) at codon 198 (the mutation predictive of a high level of MBC resistance) was found in all isolates from Spain and Switzerland and in three isolates each from France and Italy. A representative sequence of the four identical partial β-tubulin gene sequences from the Swiss isolates was submitted to GenBank under the Accession No. HQ709265. All isolates were tested in a potato dextrose agar (PDA) petri dish assay for resistance to the MBC fungicide thiophanate-methyl (Nippon Soda Co., Ltd., Tokyo, Japan) at the discriminatory dose of 50 μg/ml (4). All isolates with the E198A mutation were able to grow on the media, while the two isolates without the E198A mutation were not able to grow. The result indicated that most isolates had a high level of resistance to the MBC fungicide. To our knowledge, this is the first report of the presence of the E198A mutation conferring resistance to MBC fungicides in European isolates of M. fructicola. As the mutation appears to be widely distributed, we anticipate that MBC fungicides may be ineffective at controlling brown rot in countries with occurrence of M. fructicola.

First Report of the β-tubulin E198A Allele for Fungicide Resistance in Monilinia fructicola from South Carolina.

Resistance to methyl benzimidazole carbamates (MBCs) in Monilinia fructicola, the causal agent of brown rot of stone fruits, is known to be present in South Carolina peach orchards, but the molecular mechanism of resistance has not been investigated. Nine isolates were collected from peach in five counties in South Carolina and examined in petri dish assays on potato dextrose agar (PDA) for resistance to the MBC fungicide thiophanate-methyl (Topsin-M 70WP; Ceraxagri, King of Prussia, PA) at the discriminatory dose of 50 μg/ml. Isolates that grew on the fungicide-amended medium were considered highly resistant (HR). The β-tubulin gene from four sensitive (S) and five HR M. fructicola isolates was PCR-amplified with primer pair TubA and TubR1 as described previously (1). Sequence analysis revealed several silent mutations in introns and exons in S and HR isolates and the presence of the previously described E198A allele in HR but not S isolates (1). Nucleotide sequences of the β-tubulin gene from three S (BS, S2, MfEgpc1) and two HR isolates (MfPdt6 and BR2) were submitted to GenBank under accession numbers HM051379, HM051380, HM051381, HM051382, and HM051383, respectively. To our knowledge, this is the first report of the E198A in M. fructicola isolates from South Carolina and the East Coast. This allele is responsible for high levels of MBC resistance in M. fructicola (1). A previously reported PCR-based method using primers HRF+HRR designed to detect the E198A mutation in M. fructicola HR isolates (1) was improved by adding primer TR739 (5'-TCA CGA CGA ACA ACA TCA AGA-3') to the PCR cocktail. This additional internal primer amplified a 222-bp fragment from all S and HR isolates and therefore provided a useful, additional control. The confirmation of the E198A allele in M. fructicola isolates provides another useful tool to detect MBC resistance in commercial peach orchards in South Carolina. Reference: (1) Z. H. Ma et al. Appl. Environ. Microbiol. 69:7145, 2003.

Resistance of storage rot fungi Neofabraea perennans, N. alba, Glomerella acutata and Neonectria galligena against thiophanate-methyl in Northern German apple production

Fungi causing apple storage rots in the Lower Elbe region were examined for resistance against the methyl benzimidazole carbamate (MBC) fungicide thiophanate-methyl. In Neofabraea perennans, the most important post-harvest pathogen, strains with sensitivity (EC50 = 0.07 ppm), intermediate resistance (EC50 = 1.0–3.4 ppm) or high resistance (EC50 > 400 ppm) were found. A similar pattern was observed for N. alba in the Lower Elbe region, and for additional isolates of both species collected from the East German state of Brandenburg. In contrast, Neonectria galligena showed a uniform response of intermediate resistance to thiophanate-methyl (EC50 = 1.0–1.7 ppm). Glomerella acutata isolates were partially inhibited by low fungicide concentrations (EC50 = 0.2–0.6 ppm) but showed residual (20–40%) mycelial growth even at 400 ppm. Since Neofabraea strains with intermediate and high resistance were distributed throughout the Lower Elbe region, pre-harvest applications of thiophanate- methyl against storage rots cannot be recommended at present for this important centre of German apple production.

Effects of fungicides onin vitrospore germination and mycelial growth of the phytopathogensLeptosphaeria maculansandL. biglobosa(phoma stem canker of oilseed rape)

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 ED(50) values were calculated. 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 ED(50) values for mycelial growth for all fungicides tested than isolates of L. biglobosa. These results suggest that fungicide applications might affect the structure of L. maculans/L. biglobosa populations in UK winter oilseed rape crops.

Nature of Resistance to Methyl Benzimidazole Carbamate Fungicides in Fusarium oxysporum f.sp. lilii and F. oxysporum f.sp. gladioli in Taiwan

Abstract Fusarium oxysporum f.sp. gladioli and F. oxysporum f.sp. lilii cause corm rot and yellowing of gladiolus and lilies respectively. Resistance among isolates of these two pathogens to two benzimidazole (methyl benzimidazole carbamate: MBC) fungicides, benomyl and thiabendazole, has been identified. However, sensitivity to other benzimidazole fungicides among isolates of F. oxysporum f.sp. gladioli and F. oxysporum f.sp. lilii remains unclear. Sensitivity among isolates of these two pathogens to benomyl, thiophanate‐methyl, carbendazim and thiabendazole were evaluated in this study. Results showed that among six F. oxysporum f.sp. gladioli isolates, one was highly resistant, four were moderately resistant and one was highly sensitive to benomyl, five were highly resistant to thiophanate‐methyl, four were sensitive and one was highly sensitive to carbendazim and all six isolates were sensitive to thiabendazole. Among the 13 F. oxysporum f.sp. lilii isolates tested, three were moderately resistant and 10 were highly sensitive to benomyl, and five were highly resistant, three were moderately resistant and five were sensitive to thiophanate‐methyl. While all isolates were highly sensitive or sensitive to carbendazim and thiabendazole. These results strongly suggest that the mode of fungicide resistance in these two pathogens varies among members of the benzimidazole fungicide group and that cross resistance does not always occur. Sequence analysis of the partially amplified β‐tubulin gene indicated that mutation in codons 198 and 200 had not occurred in the benzimidazole resistant isolates. It was concluded that resistance found in Taiwan isolates of F. oxysporum f.sp. gladioli and F. oxysporum f.sp. lilii to these fungicides may be due to some other mechanism.

PIRA‐PCR for detection of Fusarium graminearum genotypes with moderate resistance to carbendazim

PIRA‐PCR (primer‐introduced restriction analysis PCR) was developed to detect isolates of Fusarium graminearum with moderate resistance to carbendazim, a methyl benzimidazole carbamate (MBC)‐group fungicide. Two primer pairs were designed and synthesized according to the nucleotide sequence of the β2‐tubulin gene from F. graminearum. Fragments of 164 bp were amplified by nested PCR from isolates differing in carbendazim sensitivity. A HindIII restriction enzyme recognition site was introduced artificially by inner primers to detect a mutation at codon 167, and TaaI (Tsp4CI) restriction enzyme was used to detect a mutation at codon 200. The sensitivity of isolates to carbendazim was determined by analyzing electrophoresis patterns of the resulting PCR products after simultaneous digestion with both HindIII and TaaI. Results from PIRA‐PCR and a conventional method (mycelial growth on agar) were identical but PIRA‐PCR required only 7–8 h while the conventional method required 5–7 days. This study demonstrates that PIRA‐PCR not only monitors the appearance of moderately resistant isolates, but can be useful for detecting genotypes of F. graminearum with moderate resistance to carbendazim.

Changes in fungicide sensitivity and relative species abundance in Oculimacula yallundae and O. acuformis populations (eyespot disease of cereals) in Western Europe

Changing fungicide sensitivities in populations of Oculimacula yallundae and O. acuformis, the species responsible for cereal eyespot in Western Europe, were determined over a 17 year period between 1984 and 2000. The data were collected by Aventis Crop Science as part of their long‐term survey to monitor changes in sensitivity to prochloraz and the methyl benzimidazole carbamate (MBC) fungicides in eyespot populations. The results show evidence for reduced sensitivity to both fungicides over the period of the survey. The decline in MBC sensitivity is in agreement with reports of practical resistance (a detectable loss of disease control in the field) to this fungicide which were widely reported from the mid 1980s onward. Prochloraz sensitivity was more complex, with the emergence of a higher resistance category of isolates in the late 1980s and early 1990s which then decreased in frequency towards the end of the survey. This may be partly explained by the introduction and increased use of cyprodinil in the mid 1990s. Although all trends were similar across Europe, differences were observed between the two eyespot species. A higher frequency of O. yallundae isolates showed decreased sensitivity to MBC, whereas decreased sensitivity to prochloraz was at a higher frequency in O. acuformis populations. The relative abundance of the two eyespot species was influenced by their differential levels of fungicide sensitivity, with the ratio increasing toward the species with the highest level of resistance to the prevailing fungicide.