Methyl Benzimidazole Carbamate Fungicides Research Articles

Prevalence of H6Y mutation in β-tubulin causing thiophanate-methyl resistant in Monilinia fructicola from Fujian, China

Brown rot disease broke out in stone fruit orchards of Fujian, China in 2019, despite pre-harvest application of methyl benzimidazole carbamate (MBC). To determine the reason, a total of 44 Monilinia fructicola strains were collected from nectarine, plum and peach fruits in this study, among which 79.5% strains were resistant to thiophanate-methyl, indicated by discriminatory dose of 5 μg/mL. The resistance of these strains was confirmed by treating detached peach fruit with label rates of formulated thiophanate-methyl which only completely inhibit infection of the sensitive strains, but not the resistant strains. Further analysis of the mechanism of MBC resistance revealed that all resistant strains carry a H6Y mutation in β-tubulin protein Tub2, which was only reported previously in the M. fructicola strains from California, USA, and do not display obvious fitness penalties, as no significant defects in mycelial growth rate, sporulation, conidia germination, aggressiveness on detached peach fruit and temperature sensitivity was detected. In addition, we found that diethofencarb, the agent for managing MBC-resistance strains, was unable to inhibit growth of the H6Y strains. Taken together, our study, for the first time, identified a mutation form of H6Y in the β-tubulin protein of M. fructicola in China, rendering the strains wide resistance to thiophanate-methyl. This mechanism of M. fructicola gaining resistance to MBC fungicides needs to be fully considered, when designing management strategies to control brown rot disease in stone fruit orchards.

Emergence of multiple Diaporthe species causing kiwifruit rot and occurrence of resistance to a methyl benzimidazole carbamate fungicide in South Korea

In recent years, new fungal diseases in kiwifruits have emerged worldwide. This study is the first report of a postharvest kiwifruit rot in South Korea caused by Diaporthe eres and related fungal pathogens. After single spore isolation of Diaporthe-like fungi isolated from rotten kiwifruits, isolates were identified based on the multi-locus phylogeny using the nuclear rDNA internal transcribed spacer and three nuclear protein-coding loci. Among the 41 single-spore isolates, a majority of the isolates (75%) were identified as D. eres, followed by D. unshiuensis, D. phaseolorum, D. cf. pseudomangiferae and D. caryae. We evaluated fungicide resistance of the 41 isolates and confirmed that a significant fraction of the isolates (15%) was resistant to thiophanate-methyl, raising a concern on overuse of this type of fungicide in kiwi orchards. By sequencing a region of β-tubulin gene, we identified two point mutations altering amino acid sequences correlated with the observed resistance. For effective diagnosis of incidence of the fungicide resistance in the fields, we developed allele-specific PCR analyses detecting the point mutations that confer reduced sensitivity to thiophanate-methyl. Genetic structure of the 41 isolates suggested that there were at least five independent mutational events for acquiring the fungicide resistance. Unexpectedly, there was no clear distinction in the genetic structure between the 41 kiwifruit isolates and Chinese isolates that caused grapevine dieback disease, suggesting these Diaporthe species are non-host-selective pathogens that could damage different horticultural crops of economic importance. This study will help develop a discerned protection program to cope with the ongoing and upcoming threats of these genetically diverse kiwifruit rot pathogens.

Resistance ofCorynespora cassiicolafrom soybean to QoI and MBC fungicides in Brazil

AbstractFungicide sprays on soybean in Brazil have contributed to the selection of less sensitive isolates ofCorynespora cassiicola. We collected 59 isolates ofC. cassiicolafrom three Brazilian states and two isolates from Paraguay. We investigated their EC50to quinone outside inhibitors (QoI) and methyl benzimidazole carbamate (MBC), any cross‐resistance to compounds within QoI and MBC groups, and characterized the polymorphisms in theircytbandβ‐tubulingenes. Local associations of polymorphisms identified in each gene were statistically correlated with assays results. In total, 79% and 74% of the isolates were classified as resistant to QoI and MBC fungicides, respectively. There was positive cross‐resistance to active ingredients within QoI and MBC groups. For QoI, all isolates presented heteroplasmy in G143A ofcytbgene; the mutations F129L and G137R were not found. For MBC, 63% of isolates possessed E198A and 21% possessed F200Y mutations, associated with reduced control by MBC fungicides. Heteroplasmy was identified in two and one isolates from Brazil with E198A and F200Y mutations, respectively. The resistance factor for isolates with E198A (10.9) was statistically similar to the isolate with F200Y (8.8) mutation. Genic association analysis of the in vitro assays using discriminatory doses proved them to be accurate. Reduced sensitivity ofC. cassiicolato QoI and MBC was also identified in isolates from Paraguay and resistance to QoI and MBC was widely present inC. cassiicolaisolates from the main soybean‐producing states in Brazil. Thus, integrated management measures should be adopted to manage soybean target spot in these countries.

Sensitivity of Cercospora sojina to demethylation inhibitor and methyl benzimidazole carbamate fungicides

Cercospora sojina, the causal agent of frogeye leaf spot (FLS) of soybean (Glycine max), can cause significant yield losses. Foliar fungicide application is a common strategy used to control FLS; however, C. sojina strains with resistance to quinone outside inhibitor (QoI) fungicides have become widespread throughout U.S. soybean production regions. Because of QoI-resistance, demethylation inhibitor (DMI) and methyl benzimidazole carbamate (MBC) fungicides have become more important in managing FLS. Baseline sensitivity assays are important to monitor any shifts in fungicide sensitivity. Sensitivity of the DMI fungicides flutriafol and tetraconazole and the MBC fungicide thiophanate methyl were established for 145 C. sojina isolates collected prior to 2001(baseline isolates) and C. sojina isolates collected from 2007 to 2012 representing 12 states. Bioassays were conducted to assess effective fungicide concentration at which 50% of mycelium growth was inhibited relative to a non-fungicide control (EC50) for each isolate-fungicide combination. Baseline EC50 value means for flutriafol, tetraconazole, and thiophanate methyl were 0.268, 0.153, and 0.521 μg/ml. When means of EC50 values for isolates collected from 2007 to 2012 were compared to baseline sensitivity EC50 means, no significant difference (P < 0.05) was observed for sensitivities to flutriafol and tetraconazole. When this same comparison was done for sensitivity to thiophanate methyl, isolates collected in 2008 and 2012 were significantly more sensitive than the baseline isolates. Overall, these findings indicate no shift towards reduced sensitivity to flutriafol, tetraconazole, and thiophanate methyl. Based on these results, continued monitoring of C. sojina population sensitivity to DMI and MBC fungicides should occur.

Sensitivity to MBC fungicides and prochloraz of Colletotrichum gloeosporioides species complex isolates from mango orchards in Mexico

Anthracnose, caused by Colletotrichum spp., is the most significant field and post-harvest disease of mango worldwide and is mainly controlled through the use of systemic fungicides belonging to the methyl benzimidazole carbamate (MBC) class. In Mexico, this disease in mango has been associated with at least seven cryptic species belonging to the C. gloeosporioides complex. In this study, the distribution of sensitivity to MBC (benomyl and carbendazim) and prochloraz fungicides of 101 Colletotrichum spp. isolates obtained from 101 commercial orchards in Mexico’s most important mango-producing states was determined. All Colletotrichum spp. isolates were obtained from commercial mango orchards with applications of MBC fungicides and that had not been exposed to applications of prochloraz, or any other DMI (demethylation inhibitors) class fungicide. Isolates were evaluated using an in vitro test to determine the effective fungicide concentration at which 50% of mycelial growth is inhibited (EC50) for each isolate-fungicide combination. EC50 values for benomyl and carbendazim ranged from 0.0967 to 0.3995 µg mL−1 (median = 0.1923 µg mL−1) and from 0.0880 to 0.1703 µg mL−1 (median = 0.1171 µg mL−1), respectively, while those for prochloraz ranged from 0.0066 to 0.0813 µg mL−1 (median = 0.0253 µg mL−1). Our results revealed a wide distribution in Mexico of Colletotrichum spp. isolates from mango sensitive to MBC fungicides and prochloraz. Data obtained in this study will serve as a source of comparison of the evolution of the sensitivity of Colletotrichum spp. populations obtained from mango to MBC and DMI fungicides in the future.

Sensitivity of Cercospora spp. from soybean to quinone outside inhibitors and methyl benzimidazole carbamate fungicides in Brazil

Cercospora leaf blight and purple seed stain are caused by Cercospora kikuchii and other Cercospora spp. The fungus can infect leaves and seeds on soybean, and the disease is controlled with fungicides. In Brazil, the intensive use of fungicides on soybean to control other diseases such as soybean rust has exerted resistance selection pressure on all fungal pathogens that attack in the crop. In this study, we evaluated the sensitivity of 56 Cercospora spp. soybean isolates collected during 9 crop seasons in 9 Brazilian states to quinone outside inhibitors (QoI) and methyl benzimidazole carbamate (MBC) fungicides using mycelial growth inhibition in amended media with a discriminatory dose of 10 μg/mL. We also analyzed single polymorphisms in the target genes cytb and β-tubulin using target genotyping by sequencing by Illumina short reads. Genome-local association was used to correlate the point mutations found in the coding sequence with in vitro assays results. For cytb gene, it was observed only the G143A mutation in 73% of the isolates. The mutation G143A was present in 97% of the isolates classified as resistant at least for two QoI fungicides. The mutation E198A was present in 71% of the isolates being significantly associated with the reduction control to MBC fungicides. Cross-resistance was observed into QoI (azoxystrobin, picoxystrobin, and pyraclostrobin) and MBC (carbendazim, and thiophanate-methyl) active ingredients fungicides. About 97% of Cercospora spp. isolates analyzed possessed the double mutations G143A and E198A and resulting in less sensitive to QoI and MBC fungicides, respectively. All isolates showed homozygous variation in the alternative allele in G143A and E198A mutations. Our results suggest that resistance of the Cercospora spp. from soybean to QoI and MBC fungicides has occurred since the 2008 cropping season in Brazil associated with G143A and E198A mutations.

Fungicide resistance in Cercospora species causing cercospora leaf blight and purple seed stain of soybean in Argentina

AbstractCercospora species cause cercospora leaf blight (CLB) and purple seed stain (PSS) on soybean. Because there are few resistant soybean varieties available, CLB/PSS management relies heavily upon fungicide applications. Sensitivity of 62 Argentinian Cercospora isolates to demethylation inhibitor (DMI), methyl benzimidazole carbamate (MBC), quinone outside inhibitor (QoI), succinate dehydrogenase inhibitor (SDHI) fungicides, and mancozeb was determined in this study. All isolates were sensitive to difenoconazole, epoxiconazole, prothioconazole, tebuconazole, and cyproconazole (EC50 values ranged from 0.006 to 2.4 µg/ml). In contrast, 51% of the tested isolates were sensitive (EC50 values ranged from 0.003 to 0.2 µg/ml), and 49% were highly resistant (EC50 &gt; 100 µg/ml) to carbendazim. Interestingly, all isolates were completely resistant to azoxystrobin, trifloxystrobin, and pyraclostrobin, and insensitive to boscalid, fluxapyroxad, and pydiflumetofen (EC50 &gt; 100 µg/ml). The G143A mutation was detected in 82% (53) of the QoI‐resistant isolates and the E198A mutation in 97% (31) of the carbendazim‐resistant isolates. No apparent resistance mutations were detected in the succinate dehydrogenase genes (subunits sdhB, sdhC, and sdhD). Mancozeb completely inhibited mycelial growth of the isolates evaluated at a concentration of 100 µg/ml. All Argentinian Cercospora isolates were sensitive to the DMI fungicides tested, but we report for the first time resistance to QoI and MBC fungicides. Mechanism(s) other than fungicide target‐site modification may be responsible for resistance of Cercospora to QoI and MBC fungicides. Moreover, based on our results and on the recent introduction of SDHI fungicides on soybean in Argentina, Cercospora species causing CLB/PSS are insensitive (naturally resistant) to SDHI fungicides. Insensitivity must be confirmed under field conditions.

In vitro toxicity of fungicides with different modes of action to alfalfa anthracnose fungus, Colletotrichum destructivum

Sensitivity of 24 isolates of Colletotrichum destructivum O’Gara, collected from alfalfa plants in Serbia, to eight selected fungicides, was investigated in this study. Molecular identification and pathogenicity test of isolates tested were also performed. Fungicide sensitivity was evaluated in vitro, using mycelial growth assay method. All isolates exhibited significant pathogenicity, causing necrosis at the alfalfa seedling root tips two days after inoculation. Using the primer pair GSF1-SR1 and by comparing the amplified fragments of the tested isolates with the marker (M), the presence of the amplicon of the expected size of about 900 bp was determined for all isolates. The isolates tested in this study showed different sensitivity towards fungicides in vitro. Mycelial growth was highly inhibited by QoI (quinone outside inhibitors) fungicide pyraclostrobin (mean EC50=0.39 µg mL−1) and by DMI (demethylation-inhibiting) fungicide tebuconazole (mean EC50=0.61 µg mL−1), followed by azoxystrobin (mean EC50=2.83 µg mL−1) and flutriafol (mean EC50=2.11 µg mL−1). Multi-site fungicide chlorothalonil and MBC (methyl benzimidazole carbamate) fungicide thiophanate-methyl evinced moderate inhibition with mean EC50=35.31 and 62.83 µg mL−1, respectively. Thirteen isolates were sensitive to SDHI (succinate dehydrogenase inhibitors) fungicide boscalid and fluxapyroxad, (mean EC50=0.49 and 0.19 µg mL−1, respectively), while the rest of isolates were highly resistant.

Sensitivity to methyl benzimidazole carbamate fungicides of Botryosphaeriaceae species from mango orchards in the Northeast of Brazil

Thiabendazole and thiophanate-methyl (methyl benzimidazole carbamate group — MBC) are fungicides commonly used in mango orchards in the Brazilian Northeast, although they are not registered to control dieback and stem-end rot. However, populations of Botryosphaeriaceae are exposed to these fungicides, and resistance may evolve. Therefore, the sensitivity of 154 Botryosphaeriaceae isolates was evaluated by analyzing the effective concentration required to inhibit 50% of mycelial growth (EC50). Ten isolates with the lowest (sensitive—S) and highest (less sensitive—LS) EC50 values for thiabendazole and thiophanate-methyl were evaluated for the stability of the sensitivity, the molecular mechanisms behind low sensitivity, the effectiveness of MBCs to control disease in mango fruits and the components of fitness. Isolates exhibited EC50 values ranging from 0.03 to 6.86 μg ml−1 and from 0.0001 to 10.70 μg ml−1 for thiabendazole and thiophanate-methyl, respectively. A significant and positive correlation (P ≤ 0.01; r = 0.82) was detected between the sensitivity of the isolates to both fungicides. The LS isolates were able to maintain this characteristic after 10 generations and did not have reduced fitness compared with the S isolates. Nevertheless, the most common mutation associated with a low sensitivity to MBCs (at codon 198 of the β-tubulin gene) was not found. Both fungicides were able to control the infection caused by S and LS isolates similarly. Thus, thiabendazole and thiophanate-methyl can be used to control dieback and stem-end rot in mango orchards in the Brazilian Northeast.

Analysis of \u03b2-tubulin-carbendazim interaction reveals that binding site for MBC fungicides does not include residues involved in fungicide resistance

Methyl benzimidazole carbamate (MBC) fungicides are fungicidal compounds that exert their biological activities by preventing cell division through the inhibition of tubulin polymerization, which is the major component of microtubules. Several mutations in the β-tubulin gene contribute to MBC resistance, the most common and significant of which occur at residues 198 and 200. Despite nearly 50 years of agricultural use, the binding site of MBCs and the precise mechanism by which those mutations affect fungicide efficacy have not been determined. The aim of this work was to clarify the mode of action and the mechanism of resistance to MBC fungicides in Podosphaera xanthii, the primary causal agent of cucurbit powdery mildew, using a combination of biochemical, biophysical and computational approaches. The results allow us to propose an MBC binding site in β-tubulin that lies close to the GTP binding site and does not include residue 198 involved in MBC resistance.

Nucleic adaptability of heterokaryons to fungicides in a multinucleate fungus, Sclerotinia homoeocarpa

Sclerotinia homoeocarpa is the causal organism of dollar spot in turfgrasses and is a multinucleate fungus with a history of resistance to multiple fungicide classes. Heterokaryosis gives rise to the coexistence of genetically distinct nuclei within a cell, which contributes to genotypic and phenotypic plasticity in multinucleate fungi. We demonstrate that field isolates, resistant to either a demethylation inhibitor or methyl benzimidazole carbamate fungicide, can form heterokaryons with resistance to each fungicide and adaptability to serial combinations of different fungicide concentrations. Field isolates and putative heterokaryons were assayed on fungicide-amended media for in vitro sensitivity. Shifts in fungicide sensitivity and microsatellite genotypes indicated that heterokaryons could adapt to changes in fungicide pressure. Presence of both nuclei in heterokaryons was confirmed by detection of a single nucleotide polymorphism in the β-tubulin gene, the presence of microsatellite alleles of both field isolates, and the live-cell imaging of two different fluorescently tagged nuclei using laser scanning confocal microscopy. Nucleic adaptability of heterokaryons to fungicides was strongly supported by the visualization of changes in fluorescently labeled nuclei to fungicide pressure. Results from this study suggest that heterokaryosis is a mechanism by which the pathogen adapts to multiple fungicide pressures in the field.

β1 Tubulin Rather Than β2 Tubulin Is the Preferred Binding Target for Carbendazim in Fusarium graminearum.

Tubulins are the proposed target of anticancer drugs, anthelminthics, and fungicides. In Fusarium graminearum, β2 tubulin has been reported to be the binding target of methyl benzimidazole carbamate (MBC) fungicides. However, the function of F. graminearum β1 tubulin, which shares 76% amino acid sequence identity with β2 tubulin, in MBC sensitivity has been unclear. In this study, MBC sensitivity relative to that of a parental strain (2021) was significantly reduced in a β1 tubulin deletion strain but increased in a β2 tubulin deletion strain, suggesting that β1 tubulin was involved in the MBC sensitivity of F. graminearum. When strain 2021 was grown in a medium with a low or high concentration of the MBC fungicide carbendazim (0.5 or 1.4 μg/ml), the protein accumulation levels were reduced by 47 and 87%, respectively, for β1 tubulin but only by 6 and 24%, respectively, for β2 tubulin. This result was consistent with observations that MBC fungicides are more likely to disrupt β1 tubulin microtubules rather than β2 tubulin microtubules in GFP-β tubulin fusion mutants in vivo. Furthermore, sequence analysis indicated that a difference in tubulin amino acid 240 (240L in β1 versus 240F in β2) may explain the difference in MBC binding affinity; this result was consistent with the result that an F240L mutation in β2 tubulin greatly increased sensitivity to carbendazim in F. graminearum. We suggest that β1 tubulin rather than β2 tubulin is the preferred binding target for MBC fungicides in F. graminearum.

Fungicide Resistance in Cercospora kikuchii, a Soybean Pathogen.

Isolates of Cercospora kikuchii, a soybean (Glycine max) pathogen causing Cercospora leaf blight and purple seed stain, were tested to determine baseline sensitivities (n = 50) to selected quinone outside inhibitor (QoI) fungicides by conducting radial growth assays on fungicide-amended media. Baseline effective fungicide concentration to inhibit 50% of fungal radial growth (EC50) values were compared with EC50 values for isolates collected in 2011 (n = 50), 2012 (n = 50), and 2013 (n = 36) throughout soybean-producing areas in Louisiana. Median EC50 values for isolates subjected to QoI fungicides were significantly (P = 0.05) higher across all 3 years. Cross-resistance to QoI fungicides was observed in resistant isolates collected in 2011 to 2013. Discriminatory doses were developed for QoI fungicides to distinguish between sensitive and resistant isolates. On average, 89% of all isolates screened in 2011 to 2013 were resistant to QoI fungicides. At a discriminatory dose of thiophanate methyl (TM), a methyl benzimidazole carbamate (MBC) fungicide, at 5 μg/ml, resistance was detected in the 2000, 2011, 2012, and 2013 collections at 23, 38, 29, and 36%, respectively. Isolates exhibiting multiple resistance to QoI fungicides and TM also were detected in 2011, 2012, and 2013 at frequencies of 34, 26, and 31%, respectively. Based on these results, Cercospora leaf blight management strategies in Louisiana using solo applications of QoI or MBC fungicides in soybean should be reconsidered.

Benomyl treatment decreases fecundity of ant queens

Methyl benzimidazole carbamate fungicides, including benomyl, are widely used in agriculture, and to eliminate entomopathogenic infections. We treated queens of Myrmica rubra (Hymenoptera:Formicidae) infected or not by Rickia wasmannii (Laboulbeniales:Laboulbeniaceae) with benomyl, 1mg/ml p.o. for six weeks. Benomyl did not treat the infection, and the treatment alone caused strong decrease in the fecundity of control healthy queens from 18.0±8.4 to 3.7±5.2eggs per healthy queen. This is the first evidence on severe adverse effects of methyl benzimidazole carbamate fungicide on the fecundity of insects, which might be responsible for altered species composition of ant assemblages in the cultural landscape.

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.

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).

Analytical Method for Determining the Total Content of Methyl Benzimidazole Carbamate Fungicides in Water by Column Switching Liquid Chromatography Mass Spectrometry

An analytical method for determining the total content of fungicides carbendazim (MBC, methyl benzimidazole-2-1yl-carbamate), benomyl (methyl 1-(butylcarbamoyl)benzimidazole-2-yl-carbamate) and thiophanate-methyl (dimethyl 4, 4′-(o-phenylene)bis(3-thioallophanate)) in water has been developed. The latter two are known to be converted to MBC in the environment. A water sample (100ml) was mixed with potassium dihydrogenphosphate and disodium hydrogenphosphate (pH 6.8±0.2) and then refluxed for 1h. By this reaction the fungicides were quantitatively converted to MBC. An aliquot (10ml) of the reaction mixture was injected into a high-performance liquid chromatograph equipped with a column-switching apparatus and a thermospray mass spectrometer. The detection limit was 0.02ppb equivalent as MBC. The recoveries of benomyl, carbendazim and thiophanate-methyl fortified in water at a fortification level of 0.05ppb ranged from 85 to 95%.

Transformation of Pseudocercosporella herpotrichoides using two heterologous genes

The plant pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed using two positive selection systems, one based on the Escherichia coli hygromycin B phosphotransferase gene (hph) and the other on the Neurospora crassa β-tubulin gene bml which encodes resistance to the methyl benzimidazole carbamate fungicides. Both selection systems gave a transformation frequency of 1–20 transformants μg−1 DNA. The vector DNA was integrated into the genome and the number and sites of integration varied among the transformants. The hph transformants were mitotically stable and the transformed gene was transmitted through spores. In contrast the bml transformants were less stable.