RESUMEN

Botrytis cinerea Pers. is the causal agent of gray mold and one of the most economically important plant-pathogenic fungi affecting strawberry (Fragaria × ananassa). Control of gray mold mainly depends on the use of site-specific fungicides, including the phenylpyrrole fludioxonil. This fungicide is currently registered in combination with cyprodinil in form of Switch 62.5WG (Syngenta Crop Protection, Greensboro, NC) for gray mold control of small fruits in the United States. In June 2013, strawberries affected with symptoms resembling gray mold were observed despite the application of Switch in one field located in Federalsburg, MD, and one located near Chesnee, SC. Ten single-spore isolates, each from a different fruit, were obtained from each location and confirmed to be B. cinerea using cultural and molecular tools as described previously (3). In vitro sensitivity to fludioxonil (Scholar SC, 20.4% [v/v] active ingredient, Syngenta Crop Protection, Greensboro, NC) was determined using a conidial germination assay as previously described (4). Eight of the 20 isolates (six from Maryland and two from South Carolina) were moderately resistant to fludioxonil, i.e., they grew on medium amended with 0.1 µg/ml fludixonil and showed residual growth at 10 µg/ml (4). The in vitro assay was repeated obtaining the same results. To assess in vivo sensitivity on fungicide-treated fruit, commercially grown strawberries were rinsed with water, dried, and sprayed 4 h prior to inoculation with either water or 2.5 ml/liter of Scholar SC to runoff using a hand mister. Fruit was stab-wounded with a sterile syringe and inoculated with a 30-µl droplet of conidia suspension (106 spores/ml) of either two sensitive or four resistant isolates (two isolates from Maryland and two isolates from South Carolina). Each isolate/treatment combination consisted of 24 mature but still firm strawberry fruit with three 8-fruit replicates. The fruit were kept at 22°C and lesion diameters were measured after 4 days of inoculation. The sensitive isolates developed gray mold symptoms on nontreated (2.5 cm lesion diameter) but not on Scholar SC-treated fruit. The resistant isolates developed gray mold on both, the water-treated control (2.3 cm lesion diameter), and the fungicide-treated fruit (1.8 cm lesion diameter). The experiment was performed twice. To our knowledge this is the first report of fludioxonil resistance in B. cinerea from strawberry fields in Maryland and South Carolina. Resistance to fludioxonil is still rare in the United States and has only been reported in B. cinerea isolates from a Virginia strawberry field (1). The increase in occurrence of resistance to fludioxonil may be a result of increased use of Switch following reports of resistance to other chemical classes in this pathogen in southern strawberry fields (2). References: (1) D. Fernández-Ortuño et al. Plant Dis. 97:848, 2013. (2) D. Fernández-Ortuño et al. Plant Dis. 96:1198, 2012. (3) D. Fernández-Ortuño et al. Plant Dis. 95:1482, 2011. (4) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

RESUMEN

Botrytis cinerea Pers. is an important plant-pathogenic fungi responsible for gray mold on more than 230 plant species worldwide, including blackberry (Rubus). One of the main strategies to control the disease involves the application of different classes of fungicides. The phenylpyrrole fludioxonil is currently marketed in combination with the anilinopyrimidine cyprodinil as Switch 62.5WG (Syngenta Crop Protection Inc., Greensboro, NC) for gray mold control. In August 2013, blackberries affected with symptoms resembling gray mold were collected from a field located in Berrien County (Georgia), where Switch 62.5WG had been used extensively over the last 5 years. Three single-spore isolates, each from a different fruit, were obtained and identified as B. cinerea on the basis of morphology and confirmed by a 238-bp PCR amplification product obtained with primer set G3PDH-F1 (5'-GGACCCGAGCTAATTTATGTCACGT-3'), G3PDH-F2 (5'-GGGTGTCAACAACGAGACCTACACT-3'), and G3PDH-R (5'-ACCGGTGCTCGATGGGATGAT-3'). In vitro sensitivity to fludioxonil (Scholar SC, Syngenta) was determined on 1% malt extract agar (MEA) using a conidial germination assay as previously described (4). One isolate was moderately resistant due to growth on medium amended with the discriminatory dose of 0.1 µg/ml fludioxonil and residual growth at 10 µg/ml (4). To assess performance of fludioxonil in detached fruit assays, commercially grown strawberries (24 in total for each isolate and treatment) were rinsed with water, dried, and sprayed 4 h prior to inoculation with either water (control fruit) or 2.5 ml/liter of Scholar SC to runoff using a hand mister. Scholar SC was used because fludioxonil was the sole active ingredient in this product and strawberries were used because latent infections in fresh blackberry fruit interfered with inoculation experiments. This dose reflects the rate recommended for postharvest gray mold control according to the Scholar label. Fruit was stab-wounded with a sterile syringe and inoculated with a 30-µl droplet of conidia suspension (106 spores/ml) of the two sensitive or the resistant isolate. After inoculation, the fruit were kept at 22°C for 4 days. The sensitive isolates developed gray mold on non-treated (2.7 cm lesion diameter) but not on Scholar SC-treated fruit (0.0 cm lesion diameter). The resistant isolate developed gray mold disease on the water-treated control fruit (2.5 cm lesion diameter) and the fungicide-treated fruit (1.8 cm lesion diameter). EC50 values were determined in microtiter assays as described previously (3) using the concentrations of 0.01, 0.04, 0.12, 0.37, 1.1, 3.3, and 10 µg/ml fludioxonil. Values were 0.02 and 0.05 µg/ml for the two sensitive isolates and 3.15 µg/ml for the resistant isolate. All experiments were performed twice. This is the first report of fludioxonil resistance in B. cinerea from blackberry in Georgia. Prior to this study, resistance to fludioxonil in B. cinerea was reported in France, Germany, and only a few states in the United States including Maryland, South Carolina, Virginia, and Washington (1,2). The emergence of resistance to fludioxonil emphasizes the importance of resistance management strategies. References: (1) D. Fernández-Ortuño et al. Plant Dis. 97:848, 2013. (2) D. Fernández-Ortuño et al. Plant Dis. 98:692, 2013. (3) M. Kretschmer et al. PLOS Pathog. 5:e1000696, 2009. (4) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

RESUMEN

Pilidium concavum (Desm.) Höhn. [synanamorph: Hainesia lythri (Desm.) Höhn.] is an opportunistic pathogen that causes leaf spots and stem necrosis in a wide range of hosts, including strawberry (Fragaria ananassa) (1,2). In October 2013, 24 strawberry plug plants (cv. Chandler) with brown to dark brown necrotic lesions on stolons were obtained from a nursery in Easley, SC. The lesions were oval shaped and varied in length from 2 to 8 mm. The tips of stolons with larger spots had died. To isolate the causal agent, 3 to 5 cm of necrotic stolon tissue was surface disinfected for 1 min with 10% bleach, rinsed with sterile distilled water, air dried, and placed on potato dextrose agar (PDA). After 7 days of incubation at 22°C, pink-orange masses of spores emerged. Single spore colonies on PDA produced a gray to brown colony with whitish aerial mycelium. Numerous discoid to hemisphaerical conidiomata (0.3 to 2.2 mm in diameter) developed with a dark base and exuded a pink, slimy mass that contained many conidia. Conidiophores (10.2 to 47.8 × 0.8 to 2.0 µm) were hyaline, unicellular, cylindrical, and filiform. Conidia (3.0 to 8.5 × 1.0 to 2.9 µm) were aseptate, fusiform, hyaline, and canoe-shaped to allantoid. On the basis of morphology, the pathogen was identified as P. concavum (3). The internal transcribed spacer region ITS1-5.8S-ITS2 was amplified by PCR and sequenced with primers ITS1 and ITS4 (4). The sequence was submitted to GenBank (Accession No. KF911079) and showed 100% homology with sequences of P. concavum. Pathogenicity was examined on strawberry fruit and leaves. Our previous efforts to achieve infection without wounding failed, which is consistent with experiences of other scientists (not cited). Thus, 24 strawberry fruit were wounded (1 cm deep) with a needle once, and submerged for 3 min in a conidial suspension (2 × 106 conidia ml-1). Controls were wounded and submerged in sterile water. After 4 days of incubation at 22°C, characteristic symptoms were observed at the wound site only on inoculated fruit. Detached leaves (about 6 cm in diameter) from 3- to 4-week-old strawberry plants cv. Chandler were surface sterilized and placed right side up in petri dishes (one leaf per dish) containing water agar. Leaves were inoculated at one site with a 50 µl conidial suspension (2 × 106 conidia ml-1) after inflicting a scraping-type injury with a needle to the surface at the point of inoculation. Control leaves received just water. After 7 days of incubation at 22°C, only the inoculated leaves showed symptoms similar to those observed on strawberry stolons. The fungus was re-isolated from symptomatic fruit and leaf lesions and identity was confirmed based on morphological features. The experiments were repeated. To our knowledge, this is the first report of P. concavum causing tan-brown rot on strawberry tissue in South Carolina. Prior to this study, the pathogen has been described from different hosts and countries including Belgium, Brazil, China, France, Iran, Poland, and the United States. Contamination of strawberry nursery stock by P. concavum could become a plant health management issue in the United States, especially if the pathogen is transferred to strawberry production areas. Further information on in-field occurrence of P. concacum is needed. References: (1) J. Debode et al. Plant Dis. 95:1029, 2011. (2) W. L. Gen et al. Plant Dis. 96:1377, 2012. (3) A. Y. Rossman et al. Mycol. Prog. 3:275, 2004. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

RESUMEN

Gray mold caused by Botrytis cinerea Pers.:Fr. is one of the most economically important diseases of cultivated strawberry (Fragaria × ananassa) worldwide. Control of gray mold mainly depends on fungicides, including the phenylpyrrole fludioxonil, which is currently marketed in combination with cyprodinil as Switch 62.5WG (Syngenta Crop Protection, Research Triangle Park, Raleigh, NC). In 2012, 790 strains of B. cinerea were collected from 76 strawberry fields in eight states, including Arkansas, Florida, Georgia, Kansas, Maryland, North Carolina, South Carolina, and Virginia. Strains were collected from sporulating flowers and fruit and sensitivity to fludioxonil was determined using a conidial germination assay as previously described (2). Only one isolate from a farm located in Westmoreland County, Virginia, grew on medium amended with the discriminatory dose of 0.1 µg/ml fludioxonil and was therefore considered low resistant. The isolate did not grow on 10 µg/ml. All other 789 isolates did not grow at either of the two doses. This assay was repeated twice with a single-spore culture of the same strain. In both cases, residual growth was observed on the fludioxonil-amended medium of 0.1 µg/ml. The single spore isolate was confirmed to be B. cinerea Pers. using cultural and molecular tools as described previously (1). To assess resistance in vivo, commercially grown ripe strawberry fruit were rinsed with sterile water, dried, placed into plastic boxes (eight strawberries per box for each of the three replicates per treatment), and sprayed 4 h prior to inoculation with either water or 2.5 ml/liter of fludioxonil (Scholar SC, Syngenta) to runoff using a hand mister. This dose reflects the rate recommended for gray mold control according to the Scholar label. Each fruit was stabbed at three equidistant points, each about 1 cm apart and 1 cm deep using a syringe tip. Wounds were injected with a 30-µl droplet of conidia suspension (106 spores/ml) of either 5 sensitive or the resistant isolate. Control fruit were inoculated with water. After inoculation, the fruit were kept at 22°C for 4 days. In two independent experiments, sensitive and low resistant isolates were indistinguishable in pathogenicity on detached, unsprayed fruit. The low resistant isolate developed gray mold disease on all treated and untreated fruit (100% disease incidence) as determined by the absence or presence of gray mold symptoms. The sensitive isolates only developed disease on untreated fruit. The EC50 values, determined in microtiter assays with concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µg/ml fludioxonil, were 0.01 µg/ml for the sensitive isolates and 0.26 µg/ml for the resistant isolate. To our knowledge, this is the first report of fludioxonil resistance in B. cinerea from strawberry in North America. Our monitoring results indicate that resistance is emerging 10 years after the introduction of fludioxonil and stress the importance of chemical rotation for gray mold control. References: (1) X. P. Li et al. Plant Dis. 96:1634, 2012. (2) R. W. S. Weber and M. Hahn. J. Plant Dis. Prot. 118:17, 2011.

RESUMEN

Monilinia fructicola (G. Wint.) Honey is a causal agent of brown rot of stone fruits but may also affect pome fruits. M. fructicola is common in North America, Oceania, and South America as well as in Asia, but it is listed as a quarantine pathogen in Europe (3). Since its first discovery in Europe in 2001 (France), it has been reported in Spain, Slovenia, Italy, and Switzerland. Recently, the fungus was also detected in orchards of blackberries and plums in the State of Baden-Württemberg, Germany (4). In July 2010, apples (Malus domestica Borkh.) of the cultivar Jonagold were found in a residential backyard in Fronhausen an der Lahn located in the State of Hessen, Germany with symptoms resembling brown rot caused by Monilinia species. Affected apples were at or near maturity with brown decay that had spread throughout the fruits. On the surface of the decaying apples was tan to white zones of sporulation. Upon isolation, the mycelium grew at a linear rate of 9.2 mm per day at 22°C on potato dextrose agar forming branched, monilioid chains of grayish colonies with concentric rings and little sporulation. The lemon-shaped spores had an average size of 14 × 9 µm, a shape and size consistent with M. fructicola. The ribosomal ITS1-5.8S-ITS2 region was PCR-amplified from genomic DNA obtained from mycelium using primers ITS1 and ITS4. A BLAST search in GenBank revealed highest similarity (99%) to M. fructicola sequences from isolates collected in China, Italy, and Slovenia (GenBank Accession Nos. FJ515894.1, FJ411109.1, GU967379.1). The M. fructicola sequence from the apple isolate was submitted to GenBank (Accession No. JF325841). The pathogen was also identified to the species level and confirmed to be M. fructicola using two novel PCR techniques based on cytochrome b sequences (1,2). Pathogenicity was confirmed by inoculating three surface-sterilized, mature apples cv. Gala with a conidial suspension (105 spores/ml) of the apple isolate. Fruit were stab inoculated at three equidistant points to a depth of 10 mm using a sterile needle. A 30-µl droplet was placed on each wound; control fruit received sterile water without conidia. After 5 days of incubation at room temperature in air-tight plastic bags, the inoculated fruits developed typical brown rot symptoms with sporulating areas (as described above). The developing spores on inoculated fruit were confirmed to be M. fructicola. All control fruits remained healthy. To our knowledge, this is the first report of M. fructicola on apple in Germany and more indication of further geographical spread of the quarantine disease in Germany. References: (1) J.-M. Hily et al. Pest Manag. Sci. Online publication. doi 10.1002/ps.2074, 2011. (2) S. Miessner and G. Stammler. J. Plant Dis. Prot. 117:162, 2010. (3) OEPP/EPPO. EPPO A2 list of pests recommended for regulation as quarantine pests. Version 2009-09. Retrieved from http://www.eppo.org/QUARANTINE/listA2.htm , September 22, 2010. (4) OEPP/EPPO. Reporting Service. No. 1, January 2010. Retrieved from http://archives.eppo.org/EPPOReporting/2010/Rse-1001.pdf , September 22, 2010.

RESUMEN

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 ß-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 ß-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.

RESUMEN

ABSTRACT The fitness and the dynamics of demethylation inhibitor fungicide (DMI) sensitivity in isolates of Monilinia fructicola sensitive (no growth at 0.3 mg/liter propiconazole) and resistant (>/=50% relative growth at 0.3 mg/liter propiconazole) to propiconazole were investigated. Overall, there was no considerable compromise in the fitness of resistant isolates compared to sensitive isolates of M. fructicola at the time of collection. Resistant and sensitive isolates differed in their sensitivity to propiconazole (P < 0.001) and incubation period (P = 0.044), but not in latent period, growth rate, spore production, and spore germination frequency (P > 0.05). Consecutive transferring on potato dextrose agar had an impact on conidia production, conidial germination, and growth rate (P < 0.0001). Consecutive transferring also had an impact on propiconazole sensitivity in resistant isolates. In the resistant isolates, sensitivity to propiconazole increased (R(2) = 0.960, P = 0.0034) within the first eight transfers. Similarly, sensitivity to propiconazole increased by 273% over the course of 34 months in cold storage in propiconazole-resistant isolates. Our results show that propiconazole resistance is unstable in vitro and that standard subculturing and cold storage procedures impact propiconazole sensitivity of resistant isolates. The instability of propiconazole resistance in M. fructicola may have important implications for disease management in that a reversion to propiconazole sensitivity could potentially occur in the absence of DMI fungicide pressure in the field.

RESUMEN

A Pindo palm tree (Butia capitata) declined in a commercial landscape setting in Georgetown, SC during June of 2005. In the spring, after looking healthy the previous year, the entire canopy collapsed with leaves attached. The canopy defoliated shortly thereafter. A cross section through primary and secondary roots of the wilting plant revealed necrotic areas with the presence of white mycelial fans. Diseased roots containing mycelial fans were collected and small sections were transferred to benomyl dichloran streptomycin (BDS) selective medium. Fungal cultures grew approximately 1 mm in diameter per day and developed aeriel mycelium first. Later, cultures developed mainly crustose mycelium with some parts being aerial. Ribosomal internal transcribed spacer (ITS) regions 1 and 2 were polymerase chain reaction amplified with universal primers ITS1-F and ITS4 and sequenced (GenBank Accession No. DQ109806). A BLAST search in GenBank of the ITS1-5.8S-ITS2 region identified the pathogen as Armillaria tabescens (Scop.) Emel (synonym Clitocybe tabescens (Fr.) Bres) with 98% sequence homology to A. tabescens single-spore isolate ss23 from South Carolina (Accession No. AY695408). The diploid culture from the roots of the Pindo palm was compatible with haploid tester strains of A. tabescens as indicated by diploidization of the haploid mycelium. The disease has been reported on Pindo palm in Florida (1), but to our knowledge, this is the first report of A. tabescens causing disease on Butia species in South Carolina. Our findings indicate that Pindo palm trees could be at risk for infection and should not be cultivated in soils containing pathogenic Armillaria species. Reference: (1) S. A. Alfieri, Jr. et al. Index of Plant Diseases in Florida (Revised). Fla. Dep. Agric. Consum. Serv. Div. Plant Ind. Bull. 11:389, 1984.

RESUMEN

Daylily (Hemerocallis sp.) plants declined in a homeowner's backyard in Walhalla, SC in June 2004. The backyard in northwestern South Carolina contained multiple, hardwood tree stumps, was surrounded by mature hardwood trees, and contained a dogwood tree showing symptoms of Armillaria root rot. Daylily plants were stunted and necrosis of leaves began at the leaf tips. A cross section through the crown of the wilting plants revealed necrotic areas with the presence of white mycelial fans. Rhizomorphs were found in the direct vicinity of the daylily root system, on the roots of the dogwood, and throughout surrounding soil. Diseased daylily crowns, rhizomorphs, and dogwood bark containing mycelial fans were collected. Small sections of white mycelial fans from daylily crowns and the dogwood sample were transferred to benomyl dichloran streptomycin (BDS) selective medium. Rhizomorph pieces were surface sterilized in a 0.6% sodium hypochlorite solution for 10 min and rinsed with sterile water before being transferred to BDS selective medium. Fungal cultures from all three sources looked similar on BDS medium and developed mainly crustose mycelium with some parts being aerial. After 1 week of incubation at room temperature in the dark, all cultures developed nonmelanized, mycelial fans that initiated from the center of the colony. The nucleotide sequences of internal transcribed spacer regions 1 and 2 and the intergenic spacer region 1 were identical for all isolates, and a BLAST search in GenBank of these sequences confirmed the identity of the pathogen as A. gallica (Marxmueller & Romagnesi) for both loci. To our knowledge, this is the first report of A. gallica causing disease on Hemerocallis spp. Our findings indicate that daylilies might be at risk for infection and should not be cultivated in soils containing rhizomorphs from pathogenic Armillaria species.

RESUMEN

A yellow-eyed mutant was discovered in a strain of Heliothis virescens, the tobacco budworm, that already exhibited a mutation for yellow scale, y. We investigated the inheritance of these visible mutations as candidate markers for transgenesis. Yellow eye was controlled by a single, recessive, autosomal factor, the same type of inheritance previously known for y. Presence of the recombinant mutants with yellow scales and wild type eyes in test crosses indicated independent segregation of genes for these traits. The recombinant class with wild type scales and yellow eyes was completely absent and there was a corresponding increase of the double mutant parental class having yellow scales and yellow eyes. These results indicated that a single factor for yellow eye also controlled yellow scales independently of y. This gene was named yes, for yellow eye and scale. We hypothesize that yes controls both eye and scale color through a deficiency in transport of pigment precursors in both the ommochrome and melanin pathways. The unlinked gene y likely controls an enzyme affecting the melanin pathway only. Both y and yes segregated independently of AceIn, acetylcholinesterase insensitivity, and sodium channel hscp, which are genes related to insecticide resistance.

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RESUMEN

Resistance to methyl parathion insecticide has evolved in the tobacco budworm, Heliothis virescens, and several biochemical mechanisms have been identified in various strains. Reduced sensitivity of acetylcholinesterase to inhibition by methyl paraoxon, the active metabolite of the insecticide, is controlled by a single autosomal locus, AceIn. We report that AceIn is genetically linked to methyl parathion resistance, which is expressed as a dominant gene. Methyl parathion-resistant and -susceptible strains were intercrossed and the resulting mixed colony was heterozygous at AceIn. Pair matings from the mixed colony were chosen, on the basis of AceIn genotype only, to establish strains Ace-S and Ace-R, homozygous for AceInSS and AceInRR, respectively. The Ace-R strain was 15.9-fold resistant compared to AceInSS, while hybrid progeny expressed 24.6-fold resistance, demonstrating dominant inheritance of resistance. When progeny of the backcross (Ace-S x Ace-R) to Ace-S were exposed to a discriminating dose of methyl parathion, 24.5% survived as predicted by the model of a single resistance gene. Survivors displayed only the AceInRS genotype, demonstrating a linkage disequilibrium which was highly significant. Assuming that no other resistance genes are linked closely to AceIn, it would appear that AceIn is a powerful gene for resistance, conferring a resistance proportional to the slower rate of inhibition in the resistant enzyme. The contribution of AceIn to resistance relative to detoxicative genes and the possible interaction of resistance genes are discussed.

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RESUMEN

Porcine liver carboxylester hydrolase (EC 3.1.1.1; carboxylesterase) was rapidly inhibited by 4-nitrophenyl organophosphinates containing aryl or heteroaryl groups directly bound to phosphorus. The most potent inhibitor was 4-nitrophenyl di-2-thienylphosphinate for which the median inhibitory concentration was 7.4 X 10(-9) M. Rabbit liver monomeric carboxylester hydrolase was inhibited, separated from excess inhibitor by gel permeation chromatography, and observed for spontaneous or 1,1'-trimethylene-bis(4-formylpyridinium bromide) dioxime (TMB-4)-induced reactivation. Recovery was most rapid (k = 4 to 7 X 10(-4) min-1) from phosphinyl groups containing one alkyl substituent smaller than isopropyl and one aryl or heteroaryl group smaller than naphthyl. The di-2-thienylphosphinylated enzyme was an exception since it recovered rapidly while lacking an alkyl substituent. Oxime reactivation by TMB-4 doubled rates of recovery.

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