Patricia K. Bryson

A single gene (yes) controls pigmentation of eyes and scales in Heliothis virescens.

Abstract 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.

Selective inhibitors of methyl parathion-resistant acetylcholinesterase from Heliothis virescens

Abstract Acetylcholinesterase activity from the tobacco budworm, Heliothis virescens , was 22-fold less sensitive to inhibition by methyl paraoxon in larvae or adults of a methyl parathion-resistant Woodrow strain when compared to a susceptible, Florence strain. Resistant acetylcholinesterase was also less sensitive to the closely related structural analogues fenitrooxon and ethyl paraoxon, and to the N -methyl carbamates eserine, propoxur, and methomyl. Methyl paraoxon-resistant acetylcholinesterase was selectively inhibited by the nonanalogous organophosphate inhibitors monocrotophos and dicrotophos. This enzyme was also sensitive to 4-nitrophenyl di-2-thienylphosphinate, 4-nitrophenyl diphenylphosphinate, and N-n -propyl 1-naphthylcarbamate. Individual adult tobacco budworms were diagnosed as SS, RS, or RR genotypes by clusters formed in scatterplots contrasting pairs of inhibitors. Methyl paraoxon-resistant preparations were more active toward both acetylthiocholine and butyrylthiocholine substrates and were not inhibited by high substate concentrations. Inhibition results suggest that there has been a qualitative change in the resistant acetylcholinesterase producing greater sensitivity to certain inhibitors. The resistant strain may also possess a greater quantity of enzyme; however, this is not clear due to substrate inhibition in the susceptible strain and apparent substrate activation in the resistant strain.

Synergism by Propynyl Aryl Ethers in Permethrin-Resistant Tobacco Budworm Larvae, Heliothis virescens

Synergists were used to diagnose possible mechanisms of permethrin resistance in permethrin-selected strains of the tobacco budworm, Heliothis virescens (F.). In addition to permethrin, these strains of the tobacco budworm were resistant to α-cyano-pyrethroid insecticides, organophosphorus insecticides and DDT. The monooxygenase-inhibiting prop-2-ynyl aryl ethers were the only effective synergists of permethrin among 16 candidates tested. The most effective synergist was 1,2,4-trichloro-3-(2-propynyloxy)benzene. Piperonyl butoxide, a common monooxygenase-inhibiting synergist in other species and tobacco budworm strains, was inactive. These results suggested the presence and contribution of an unusual monooxygenase in the enzymatic detoxication of permethrin. DDT cross-resistance, which was not synergized, and broad pyrethroid cross-resistance supported previous evidence for target site insensitivity as a second pyrethroid-resistance mechanism in these strains. The actions of S,S,S-tributyl phosphorotrithioate (TBPT) and triphenyl phosphate (TPP) suggested that hydrolytic detoxication, important in methyl parathion-resistance tobacco budworm strains, had little or no role in conferring pyrethroid resistance in these strains.

Identification and characterization of Armillaria tabescens from the southeastern United States

In the southeastern USA, Armillaria root rot disease on peach (Prunus persica) is caused by Armillaria tabescens, and to a lesser degree by A. mellea. Recent attempts to genetically characterize A. tabescens isolates using rDNA indicated the existence of heterozygosity in diploid isolates. In order to clarify this heterozygosity, DNA from stipe and single spore cultures of A. tabescens isolate SC.MF-1.01 was characterized using RFLP and sequence analysis. Direct sequencing of rDNA amplicons from diploid stipe tissue indicated heterozygosity in the IGS1 and ITS2 regions. IGS1 copies A and B, and ITS copies A and B segregated in a 1:1 ratio in single spore progeny, whereby IGS1 copy A always segregated with ITS copy A and IGS1 copy B always segregated with ITS copy B. The results indicate the existence of divergent haplotypes in the two nuclei of SC.MF-1.01 diploid mycelium and a single locus for rDNA tandem repeats. Additional IGS1 copies, designated IGS1 copy C, D, and E, with variable AluI restriction sites were cloned from SC.MF-1.01 diploid mycelium indicating polymorphism within ribosomal tandem repeats in A. tabescens. IGS1 copies C, D, and E were found once each in 100 clones analyzed, and therefore seemed to be rare compared to copies A (44 clones) and B (53 clones). RFLP and sequence analysis of the ribosomal IGS1 and ITS1 regions in North American A. tabescens isolates indicated that heterozygosity is common in A. tabescens diploid mycelium. A PCR-based molecular technique was developed to distinguish A. tabescens from many other North American Armillaria species, including A. mellea.

Pyrethroid-resistant Helicoverpa zea and transgenic cotton in South Carolina

Abstract Failures to control Helicoverpa zea (Boddie) on cotton in South Carolina were associated with resistance to pyrethroid insecticides. Resistance to cyhalothrin, cypermethrin, and permethrin was observed in a colony collected as larvae from cotton in Estill, SC, in September 1996. Cyhalothrin resistance was expressed as an incompletely dominant trait. In 1997, resistance to cyhalothrin was observed in males captured in pheromone traps in Estill and nearby Ulmer. Resistance was also observed in Cameron and Holly Hill (Santee), SC, 150 km to the north, in adults reared from larvae collected from cotton. In contrast, all moths trapped in the Pee Dee River drainage 300 km to the north were susceptible. Putative mechanisms for resistance as related to improved monitoring of population genetics and implications for control of pests on transgenic insecticidal cotton are discussed.

First Report of Fludioxonil Resistance in Botrytis cinerea from a Strawberry Field in Virginia

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.

Linkage of acetylcholinesterase insensitivity to methyl parathion resistance inHeliothis virescens

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 thatAceIn is genetically linked to methyl parathion resistance, which is expressed as a dominant gene. Methyl parathionresistant and -susceptible strains were intercrossed and the resulting mixed colony was heterozygous atAceIn. Pair matings from the mixed colony were chosen, on the basis ofAceIn genotype only, to establish strains Ace-S and Ace-R, homozygous forAceInSS andAceInRR, respectively. The Ace-R strain was 15.9-fold resistant compared toAceInSS, while hybrid progeny expressed 24.6-fold resistance, demonstrating dominant inheritance of resistance. When progeny of the backcross (Ace-S×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 theAceInRS genotype, demonstrating a linkage disequilibrium which was highly significant. Assuming that no other resistance genes are linked closely toAceIn, it would appear thatAceIn is a powerful gene for resistance, conferring a resistance proportional to the slower rate of inhibition in the resistant enzyme. The contribution ofAceIn to resistance relative to detoxicative genes and the possible interaction of resistance genes are discussed.

First Report of Brown Rot of Apple Caused by Monilinia fructicola in Germany

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.

First Report of Fludioxonil Resistance in Botrytis cinerea, the Causal Agent of Gray Mold, from Strawberry Fields in Maryland and South Carolina

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.

Effect of Fungicide Applications on Monilinia fructicola Population Diversity and Transposon Movement

In this study, we investigated whether fungicide-induced mutagenesis previously reported in Monilinia fructicola could accelerate genetic changes in field populations. Azoxystrobin and propiconazole were applied to nectarine trees at weekly intervals for approximately 3 months between bloom and harvest in both 2013 and 2014. Fungicides were applied at half-label rate to allow recovery of isolates and to increase chances of sublethal dose exposure. One block was left unsprayed as a control. In total, 608 single-spore isolates were obtained from blighted blossoms, cankers, and fruit to investigate phenotypic (fungicide resistance) and genotypic (simple-sequence repeat [SSR] loci and gene region) changes. In both years, populations from fungicide-treated and untreated fruit were not statistically different in haploid gene diversity (P = 0.775 for 2013 and P = 0.938 for 2014), allele number (P = 0.876 for 2013 and P = 0.406 for 2014), and effective allele number (P = 0.861 for 2013 and P = 0.814 for 2014). Isolates from blossoms and corresponding cankers of fungicide treatments revealed no changes in SSR analysis or evidence for induced Mftc1 transposon translocation. No indirect evidence for increased genetic diversity in the form of emergence of reduced sensitivity to azoxystrobin, propiconazole, iprodione, and cyprodinil was detected. High levels of population diversity in all treatments provided evidence for sexual recombination of this pathogen in the field, despite apparent absence of apothecia in the orchard. Our results indicate that fungicide-induced, genetic changes may not occur or not occur as readily in field populations as they do under continuous exposure to sublethal doses in vitro.