Kerik D. Cox

Monilinia species causing brown rot of peach in China.

In this study, 145 peaches and nectarines displaying typical brown rot symptoms were collected from multiple provinces in China. A subsample of 26 single-spore isolates were characterized phylogenetically and morphologically to ascertain species. Phylogenetic analysis of internal transcribed spacer (ITS) regions 1 and 2, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), β-tubulin (TUB2) revealed the presence of three distinct Monilinia species. These species included Monilinia fructicola, Monilia mumecola, and a previously undescribed species designated Monilia yunnanensis sp. nov. While M. fructicola is a well-documented pathogen of Prunus persica in China, M. mumecola had primarily only been isolated from mume fruit in Japan. Kochs postulates for M. mumecola and M. yunnanensis were fulfilled confirming pathogenicity of the two species on peach. Phylogenetic analysis of ITS, G3PDH, and TUB2 sequences indicated that M. yunnanensis is most closely related to M. fructigena, a species widely prevalent in Europe. Interestingly, there were considerable differences in the exon/intron structure of the cytochrome b (Cyt b) gene between the two species. Morphological characteristics, including spore size, colony morphology, lesion growth rate, and sporulation, support the phylogenetic evidence suggesting the designation of M. yunnanensis as a new species. A new multiplex PCR method was developed to facilitate the detection of M. yunnanensis and differentiation of Monilinia spp. causing brown rot of peach in China.

Instability of Propiconazole Resistance and Fitness in Monilinia fructicola.

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.

Enhancer–promoter interference and its prevention in transgenic plants

Biotechnology has several advantages over conventional breeding for the precise engineering of gene function and provides a powerful tool for the genetic improvement of agronomically important traits in crops. In particular, it has been exploited for the improvement of multiple traits through the simultaneous introduction or stacking of several genes driven by distinct tissue-specific promoters. Since transcriptional enhancer elements have been shown to override the specificity of nearby promoters in a position- and orientation-independent manner, the co-existence of multiple enhancers/promoters within a single transgenic construct could be problematic as it has the potential to cause the mis-expression of transgene product(s). In order to develop strategies with, which to prevent such interference, a clear understanding of the mechanisms underlying enhancer-mediated activation of target promoters, as well as the identification of DNA sequences that function to block these interactions in plants, will be necessary. To date, little is known concerning enhancer function in plants and only a very limited number of enhancer-blocking insulators that operate in plant species have been identified. In this review, we discuss the current knowledge surrounding enhancer–promoter interactions, as well as possible means of minimizing such interference during plant transformation experiments.

Minimizing the unpredictability of transgene expression in plants: the role of genetic insulators

The genetic transformation of plants has become a necessary tool for fundamental plant biology research, as well as the generation of engineered plants exhibiting improved agronomic and industrial traits. However, this technology is significantly hindered by the fact that transgene expression is often highly variable amongst independent transgenic lines. Two of the major contributing factors to this type of inconsistency are inappropriate enhancer-promoter interactions and chromosomal position effects, which frequently result in mis-expression or silencing of the transgene, respectively. Since the precise, often tissue-specific, expression of the transgene(s) of interest is often a necessity for the successful generation of transgenic plants, these undesirable side effects have the potential to pose a major challenge for the genetic engineering of these organisms. In this review, we discuss strategies for improving foreign gene expression in plants via the inclusion of enhancer-blocking insulators, which function to impede enhancer-promoter communication, and barrier insulators, which block the spread of heterochromatin, in transgenic constructs. While a complete understanding of these elements remains elusive, recent studies regarding their use in genetically engineered plants indicate that they hold great promise for the improvement of transgene expression, and thus the future of plant biotechnology.

Both the constitutive Cauliflower Mosaic Virus 35S and tissue-specific AGAMOUS enhancers activate transcription autonomously in Arabidopsis thaliana

The expression of eukaryotic genes from their cognate promoters is often regulated by the action of transcriptional enhancer elements that function in an orientation-independent manner either locally or at a distance within a genome. This interactive nature often provokes unexpected interference within transgenes in plants, as reflected by misexpression of the introduced gene and undesired phenotypes in transgenic lines. To gain a better understanding of the mechanism underlying enhancer/promoter interactions in a plant system, we analyzed the activation of a β-glucuronidase (GUS) reporter gene by enhancers contained within the AGAMOUS second intron (AGI) and the Cauliflower Mosaic Virus (CaMV) 35S promoter, respectively, in the presence and absence of a target promoter. Our results indicate that both the AGI and 35S enhancers, which differ significantly in their species of origin and in the pattern of expression that they induce, have the capacity to activate the expression of a nearby gene through the promoter-independent initiation of autonomous transcriptional events. Furthermore, we provide evidence that the 35S enhancer utilizes a mechanism resembling animal- and yeast-derived scanning or facilitated tracking models of long-distance enhancer action in its activation of a remote target promoter.

Genetic Diversity of Prunus necrotic ringspot virus Isolates Within a Cherry Orchard in New York

A survey for Prunus necrotic ringspot virus (PNRSV) in an orchard of Prunus cerasus cv. Montmorency and Prunus avium cv. Hedelfingen in New York by enzyme-linked immunosorbent assay indicated an eightfold higher infection rate in sour cherry (33%, 32 of 96) than in sweet cherry (4%, 6 of 136) trees. The presence of PNRSV was confirmed by reverse transcription-polymerase chain reaction and amplification of the coat protein (CP) gene in total RNA from infected leaf tissue. Latent infection was prevalent in the majority of trees infected (87%, 33 of 38), while a few of them exhibited shock symptoms or had severely reduced growth (13%, 5 of 38). Asymptomatic PNRSV-infected trees clustered in spatial proximity to symptomatic trees. Sequence analysis of the CP gene (675 bp) indicated a population structure consisting of one predominant molecular variant for 10 isolates and six minor molecular variants for seven isolates. A high sequence identity was found between the CP gene of PNRSV isolates from cherry trees and other isolates from diverse hosts and various geographic origins at the nucleotide and amino acid levels (88 to 100%). Phylogenetic analyses showed a clustering of PNRSV isolates from cherry trees in New York in the predominant group PV-96.

Characterization of the cytochrome b (cyt b) gene from Monilinia species causing brown rot of stone and pome fruit and its significance in the development of QoI resistance

BACKGROUND Quinone outside inhibitor (QoI) resistance as a consequence of point mutations in the cytochrome b (cyt b) gene has been reported in numerous plant pathogenic fungi. To examine the potential for QoI resistance development in those Monilinia species causing brown rot of stone and pome fruits [Monilinia fructicola (G Winter) Honey, M. laxa (Aderhold & Ruhland) Honey and M. fructigena (Aderhold & Ruhland) Honey], an examination was made of the sequence and exon/intron structure of their cyt b genes for the presence of any point mutations and/or introns commonly associated with resistance to QoIs in fungal plant pathogens. RESULTS None of the point mutations typically linked to QoI resistance was present in any of the Monilinia isolates examined. Furthermore, the cyt b genes from M. fructicola and M. laxa, but not M. fructigena, possessed a group-I-like intron directly after codon 143. Based on the results obtained, a simple PCR assay using a single primer pair was developed, allowing discrimination between the three Monilinia species without the need for culturing. CONCLUSIONS Results suggest that resistance to QoI fungicides based on the G143A mutation is not likely to occur in M. fructicola or M. laxa. Conversely, M. fructigena may be at higher risk for developing QoI resistance owing to the absence of a G143-associated intron.

The sucrose synthase-1 promoter from Citrus sinensis directs expression of the β-glucuronidase reporter gene in phloem tissue and in response to wounding in transgenic plants

Interest in phloem-specific promoters for the engineering of transgenic plants has been increasing in recent years. In this study we isolated two similar, but distinct, alleles of the Citrus sinensissucrose synthase-1 promoter (CsSUS1p) and inserted them upstream of the β-glucuronidase (GUS) gene to test their ability to drive expression in the phloem of transgenic Arabidopsisthaliana and Nicotiana tabacum. Although both promoter variants were capable of conferring localized GUS expression in the phloem, the CsSUS1p-2 allele also generated a significant level of expression in non-target tissues. Unexpectedly, GUS expression was also instigated in a minority of CsSUS1p::GUS lines in response to wounding in the leaves of transgenic Arabidopsis. Deletion analysis of the CsSUS1p suggested that a fragment comprising nucleotides −410 to −268 relative to the translational start site contained elements required for phloem-specific expression while nucleotides −268 to −103 contained elements necessary for wound-specific expression. Interestingly, the main difference between the two CsSUS1p alleles was the presence of a 94-bp insertion in allele 2. Fusion of this indel to a minimal promoter and GUS reporter gene indicated that it contained stamen and carpel-specific enhancer elements. This finding of highly specific and separable regulatory units within the CsSUS1p suggests that this promoter may have a potential application in the generation of constructs for the use in the development of transgenic plants resistant to a wide variety of target pests.

A rapid method to quantify fungicide sensitivity in the brown rot pathogen Monilinia fructicola.

Management of brown rot of stone fruit relies upon the application of effective fungicides that may be compromised by the development of fungicide resistance. We evaluated fungicide resistance in the brown rot pathogen, Monilinia fructicola, using Alamar blue (AB) dye, or resazurin, a chromogenic substrate that can be used as an indicator of respiration, in a 96-well microtiter format. We compared the AB method to traditional mycelial growth assays for resistance screening using 10 isolates of M. fructicola that represented a range of sensitivities to fenbuconazole. Using traditional mycelial growth assays, isolate sensitivity ranged from 17.7 to 115.3% growth on medium amended with fenbuconazole at 0.03 μg/ml relative to that on nonamended medium. Concordant results between both assays were obtained (R2 = 0.9943, P < 0.0001), but the AB method provided results within 24 h, as opposed to the 3- to 5-day period required for mycelial growth assays. We found that sensitive isolates reduced AB less than resistant isolates in the presence of fungicide. Spore density influenced the reduction of AB by M. fructicola; spectrophotometric discrimination of fungicide sensitivity was best achieved at a density of 105 spores/ml.

Prevalence of Streptomycin-Resistant Erwinia amylovora in New York Apple Orchards

Resistance to streptomycin in Erwinia amylovora was first observed in the United States in the 1970s but was not found in New York until 2002, when streptomycin-resistant (SmR) E. amylovora was isolated from orchards in Wayne County. From 2011 to 2014, in total, 591 fire blight samples representing shoot blight, blossom blight, and rootstock blight were collected from 80 apple orchards in New York. From these samples, 1,280 isolates of E. amylovora were obtained and assessed for streptomycin resistance. In all, 34 SmR E. amylovora isolates were obtained from 19 individual commercial orchards. The majority of the resistant isolates were collected from orchards in Wayne County, and the remaining were from other counties in western New York. Of the 34 resistant isolates, 32 contained the streptomycin resistance gene pair strA/strB in the transposon Tn5393 on the nonconjugative plasmid pEA29. This determinant of streptomycin resistance has only been found in SmR E. amylovora isolates from Michigan and the SmR E. amylovora isolates discovered in Wayne County, NY in 2002. Currently, our data indicate that SmR E. amylovora is restricted to counties in western New York and is concentrated in the county with the original outbreak. Because the resistance is primarily present on the nonconjugative plasmid, it is possible that SmR has been present in Wayne County since the introduction in 2002, and has spread within and out of Wayne County to additional commercial growers over the past decade. However, research is still needed to provide in-depth understanding of the origin and spread of the newly discovered SmR E. amylovora to reduce the spread of streptomycin resistance into other apple-growing regions, and address the sustainability of streptomycin use for fire blight management in New York.