Barry Pryor - Academia.edu (original) (raw)

Papers by Barry Pryor

Frontiers in Cellular and Infection Microbiology, 2021

Alternaria alternata is a ubiquitous fungus and a major allergen associated with the development ... more Alternaria alternata is a ubiquitous fungus and a major allergen associated with the development of asthma. Inhalation of intact spores is the primary cause of human exposure to fungal allergen. However, allergen-rich cultured fungal filtrates are oftentimes used in the current models of fungal sensitization that do not fully reflect real-life exposures. Thus, establishing novel spore exposure models is imperative. In this study, we established novel fungal exposure models of both adult and neonate to live spores. We examined pathophysiological changes in the spore models as compared to the non-exposure controls and also to the conventional filtrate models. While both Alternaria filtrate- and spore-exposed adult BALB/c mice developed elevated airway hyperresponsiveness (AHR), filtrates induced a greater IgE mediated response and higher broncholavage eosinophils than spores. In contrast, the mice exposed to Alternaria spores had higher numbers of neutrophils. Both exposures induced c...

HortScience, 2010

Lettuce drop caused by Sclerotinia spp. is an economically important disease of lettuce (Lactuca ... more Lettuce drop caused by Sclerotinia spp. is an economically important disease of lettuce (Lactuca sativa L.), and cultivars with resistance to mycelial infection by Sclerotinia sclerotiorum (Lib.) de Bary and S. minor Jagger as well as to S. sclerotiorum ascospores are needed. Assessing resistance in field experiments can be complicated by fast bolting or small stature lettuce lines that may escape rather than resist the pathogens. Therefore, methods to select resistant lines from morphologically variable populations are needed. We used S. sclerotiorum and S. minor-infested field experiments, regression analysis, field experiments with artificially high plant densities, and S. sclerotiorum ascospore inoculations to identify lettuce lines with resistance to both pathogens. Three replicated experiments in S. sclerotiorum-infested fields were conducted in Yuma, AZ, and three replicated experiments in a S. minor-infested field were conducted in Salinas, CA, using diverse populations of i...

African Journal of Microbiology Research, Mar 5, 2013

Isothiocyanates (ITCs) is a group of defense related compounds synthesized by Brassicas that have... more Isothiocyanates (ITCs) is a group of defense related compounds synthesized by Brassicas that have positive effects on human health which suggest that they are environmentally friendly compounds to control fungi infections. However, the development of ITC-resistant strains of fungi is a matter of concern. The objective of the present work was to study the response of Alternaria alternata to allylisothiocyanate (AITC) and benzyl isothiocyanate (BITC) chronic exposure and the effect of the treatment on some of the inter simple sequence repeats (ISSR) regions. Five strains of A. alternata isolated from tomato and five isolated from cabbage were independently exposed in vitro to AITC and BITC. Concentrations were increased until it reached 0.08 and 0.6 mg/mL of AITC and BITC, respectively. Genomic DNA from both wild type and isogenic adapted strains to ITCs was isolated and five primers were used for ISSR amplification: (GACA) 4 :5´-GACAGACAGACAGACA-3´; M13:5´-GAGGGTGGCGGCGGTTCT-3´;(AAG) 8 :5´-AAGAAGAAGAAGAAGAAGAAGAAG-3´; (ACA) 5 : 5´-ACAACAACAACAACA-3´ and T3B: 5´-AGGTCGCGGGTTCGAATCC-3´. A lower degree of polymorphism was found to be induced by the treatment in the wild-type strains isolated from cabbage as compared with the wild-type strains isolated from tomato. It can be concluded that ITCs exposure induced random mutations in different ISSR regions of the A. alternata genome which does not lead to the development of strains with a hereditable resistant phenotype.

BMC Genomics, 2015

Background: Alternaria is considered one of the most common saprophytic fungal genera on the plan... more Background: Alternaria is considered one of the most common saprophytic fungal genera on the planet. It is comprised of many species that exhibit a necrotrophic phytopathogenic lifestyle. Several species are clinically associated with allergic respiratory disorders although rarely found to cause invasive infections in humans. Finally, Alternaria spp. are among the most well known producers of diverse fungal secondary metabolites, especially toxins. Description: We have recently sequenced and annotated the genomes of 25 Alternaria spp. including but not limited to many necrotrophic plant pathogens such as A. brassicicola (a pathogen of Brassicaceous crops like cabbage and canola) and A. solani (a major pathogen of Solanaceous plants like potato and tomato), and several saprophytes that cause allergy in human such as A. alternata isolates. These genomes were annotated and compared. Multiple genetic differences were found in the context of plant and human pathogenicity, notably the pro-inflammatory potential of A. alternata. The Alternaria genomes database was built to provide a public platform to access the whole genome sequences, genome annotations, and comparative genomics data of these species. Genome annotation and comparison were performed using a pipeline that integrated multiple computational and comparative genomics tools. Alternaria genome sequences together with their annotation and comparison data were ported to Ensembl database schemas using a self-developed tool (EnsImport). Collectively, data are currently hosted using a customized installation of the Ensembl genome browser platform. Conclusion: Recent efforts in fungal genome sequencing have facilitated the studies of the molecular basis of fungal pathogenicity as a whole system. The Alternaria genomes database provides a comprehensive resource of genomics and comparative data of an important saprophytic and plant/human pathogenic fungal genus. The database will be updated regularly with new genomes when they become available. The Alternaria genomes database is freely available for non-profit use at http://alternaria.vbi.vt.edu.

Phytopathology®, 2007

Fusarium oxysporum f. sp. lactucae, causal agent of Fusarium wilt of lettuce, is a serious pathog... more Fusarium oxysporum f. sp. lactucae, causal agent of Fusarium wilt of lettuce, is a serious pathogen recently reported in Arizona. Sequence analysis of the mitochondrial small subunit (mtSSU), translation elongation factor 1-α (EF-1α) gene, and the nuclear ribosomal DNA intergenic spacer (IGS) region was conducted to resolve relationships among f. sp. lactucae isolates, F. oxysporum isolates from other hosts, and local non-pathogenic isolates. Analysis of mtSSU sequences provided limited phylogenetic resolution and did not differentiate the lactucae isolates from 13 other F. oxysporum isolates. Analysis of EF-1α sequences resulted in moderate resolution, grouping seven formae speciales with the lactucae isolates. Analysis of the IGS region revealed numerous sequence polymorphisms among F. oxysporum formae speciales consisting of insertions, deletions, and single nucleotide transitions and substitutions. Repeat sequence analysis revealed several duplicated subrepeat units that were di...

International Journal of Speleology, 2011

Fungal communities on speleothem surfaces in Kartchner Caverns, Arizona, USA. INTRODUCTION Kartch... more Fungal communities on speleothem surfaces in Kartchner Caverns, Arizona, USA. INTRODUCTION Kartchner Caverns, located in Benson, Arizona, USA (N31° 50' 08", W110° 20' 37"), is a wet, actively forming carbonate cave in the Whetstone Mountains. First discovered in 1974, the cave was kept a closely guarded secret among the cave's discoverers in an effort to preserve the cave from anthropogenic harm. In 1988, the State of Arizona purchased the land surrounding the cave and began development of Kartchner Caverns State Park (Tufts & Tenen, 1999). Special effort was made during the development process to preserve the pristine status of the cave while making portions of it accessible to tourism. In

Plant Disease, 2008

Field experiments were conducted over 2 years in Yuma County, AZ, and Imperial County, CA, to det... more Field experiments were conducted over 2 years in Yuma County, AZ, and Imperial County, CA, to determine the efficacy of several biocontrol agents for the management of lettuce drop caused by Sclerotinia spp. Commercial formulations of Trichoderma harzianum (Plantshield, Supersivit), Gliocladium virens (Soilgard), Coniothyrium minitans (Contans), and Bacillus subtilis (Companion) were evaluated and compared with the chemical fungicide iprodione (Rovral) against Sclerotinia sclerotiorum and S. minor. A single application of biocontrol products or of Rovral did not reduce lettuce drop caused by either Sclerotinia species. However, two applications of Contans, one at planting and one at post-thinning, significantly reduced the incidence of lettuce drop caused by S. sclerotiorum and increased yield but had no effect on S. minor at both locations in both years. Two applications of other biocontrol products did not significantly reduce disease incidence despite medium to high recovery foll...

Plant Disease, 2010

Field experiments were conducted over 2 years in Yuma, AZ, and Holtville, CA, to establish the re... more Field experiments were conducted over 2 years in Yuma, AZ, and Holtville, CA, to establish the relationship between soil sclerotium density of Sclerotinia sclerotiorum and the incidence of lettuce drop on different lettuce (Lactuca sativa) types under different irrigation systems, and to determine the efficacy of the biocontrol agent Coniothyrium minitans (Contans) against S. sclerotiorum on crisphead lettuce at varied sclerotium densities under different irrigation systems. There was no significant interaction of irrigation (overhead sprinkler versus furrow) with either sclerotium density or with biocontrol treatment. Lettuce drop incidence was lowest in romaine lettuce compared with crisphead or leaf lettuce at all soil sclerotium densities. There was a significant positive correlation between the sclerotial density and the percent disease incidence. Disease incidence in plots infested with 2 sclerotia/m2of bed was not significantly higher than in control plots regardless of lettu...

Plant Disease, 2010

A nested polymerase chain reaction-based (nPCR) assay was developed and evaluated for the rapid d... more A nested polymerase chain reaction-based (nPCR) assay was developed and evaluated for the rapid detection of Fusarium oxysporum f. sp. lactucae in seed of lettuce. Three primers were designed from sequences of the intergenic spacer region of the rDNA and were used in the PCR amplifications. The first amplification employed the primer pair GYCF1 and GYCR4C and produced a product of 2,270 bp. The second amplification employed the forward primer GYCF1 and the nested primer R943 and produced a single 936-bp PCR product. The nPCR protocol developed successfully detected the target sequence in genomic DNA at 1 fg/μl. A seed assay was tested that included a 4-day incubation step in which seed were maintained under high humidity conditions to increase fungal biomass for DNA extraction. In seed lots prepared by mixing known amounts of F. oxysporum f. sp. lactucae–infested seed with noninfested seed, this assay permitted the detection of the pathogen from lots with infestation rates as low as...

Plant Disease, 2001

A pair of polymerase chain reaction (PCR) primers was developed based upon the sequence of a clon... more A pair of polymerase chain reaction (PCR) primers was developed based upon the sequence of a cloned random amplified polymorphic DNA (RAPD) fragment of Alternaria radicina, and a PCR-based seed assay was developed for the detection of A. radicina from infested carrot seed. The seed assay involved a 5-day incubation step, in which seed was maintained under high humidity conditions in order to increase fungal biomass. Seed was then incubated with lysis buffer, extracted with phenol-chloroform, and DNA was recovered using a silica matrix. PCR amplification of the target A. radicina DNA sequence was enhanced by the addition of skim milk to the PCR reaction mixture. With this PCR-based seed assay, A. radicina was detected from carrot seed lots with natural infestation rates as low as 0.3%. In seed lots prepared by mixing known amounts of A. radicina-infested seed with noninfested seed, this assay allowed for the detection of the pathogen from lots with infestation rates as low as 0.1%.

Phytopathology®, 2012

Since 1999, a disease of apple caused by an Alternaria sp. has been affecting orchards in norther... more Since 1999, a disease of apple caused by an Alternaria sp. has been affecting orchards in northern Italy resulting in necrotic spots on leaves and on fruit. Forty-four single-spored isolates were obtained from diseased plant materials to investigate the diversity of this fungus in Italy and to compare these isolates to isolates of Alternaria associated with apple disease in previous studies, including A. mali, causal agent of apple blotch. All isolates, including the reference strains, were tested for pathogenicity utilizing in vitro bioassays on detached leaf or on fruit (‘Golden Delicious’). In addition, morphological characterizations were conducted describing both the three-dimensional sporulation pattern and the colony morphology of each isolate. In order to assess the genetic diversity within the Italian Alternaria population, sequence characterization of specific loci and anonymous regions (endoPG, OPA1-3, OPA2-1, and OPA10-2) and genetic fingerprinting based on amplified fra...

Phytopathology®, 2002

Alternaria isolates were obtained from various pistachio tissues collected in five orchards in Ca... more Alternaria isolates were obtained from various pistachio tissues collected in five orchards in California. For all isolates, morphological characteristics of the colony and sporulation apparatus were determined and compared with those of representative isolates of A. alternata, A. tenuissima, A. arborescens, and A. infectoria. A selection of the pistachio isolates and the representative Alternaria isolates were evaluated for pathogenicity to pistachio. Molecular characteristics of these isolates were determined using random amplified polymorphism DNA (RAPD) analysis, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of nuclear intergenic spacer rDNA, and sequence analysis of nuclear internal transcribed spacer (ITS) rDNA. Based on morphological characteristics, the pistachio isolates were grouped as identical or very similar to either A. alternata, A. tenuissima, A. arborescens, or A. infectoria. Isolates from the alternata, tenuissima, and arbor...

Mycological Progress, 2011

Previous phylogenetic analyses revealed that species within the genera Nimbya and Embellisia resi... more Previous phylogenetic analyses revealed that species within the genera Nimbya and Embellisia reside within a large monophyletic clade that also includes the genera Alternaria, Ulocladium, Undifilum, Sinomyces, and Crivellia with Stemphylium as the sister taxon. This study expands upon previous work by including many contemporary species of each genus and utilizes molecular and morphological characters to further examine relationships. Maximum parsimony and Bayesian analysis reveals that Nimbya is not a monophyletic genus but is split into two phylogenetically distant clades, which have different and distinct conidial morphologies. One of these clades resides completely within Alternaria. Phylogenetic analyses also reveals that Embellisia does not form a monophyletic genus but is split into four monophyletic lineages. Moreover, several species of Embellisia cluster individually with clades that are predominantly Alternaria, Ulocladium, or Stemphylium, yet these Embellisia spp. possess morphological characters that are diagnostically Embellisia. Thus, these data reveal that both Nimbya and Embellisia are polyphyletic as currently defined and taxonomic restructuring is necessary in order to resolve conflict between historical morphological and contemporary molecularbased phylogenies.

Mycological Progress, 2008

The genus Ulocladium represents phaeodictyosporic Hyphomycetes that produce conidia that are esse... more The genus Ulocladium represents phaeodictyosporic Hyphomycetes that produce conidia that are essentially obovoid in shape. Previous molecular studies that included Ulocladium and related taxa in Alternaria, Embellisia, and Stemphylium revealed a conflict between morphology and phylogeny, and Ulocladium was supported as polyphyletic with a paraphyletic core group. Moreover, the genus consistently resolved within a larger Alternaria/ Ulocladium clade, resulting in paraphyly of Alternaria and questions as to the taxonomic status of Ulocladium. In the present study, 13 Ulocladium species and three genetic loci were included for a more comprehensive systematic analysis of the genus than had previously been conducted. Total genomic DNA was extracted from representative taxa and sequences were determined for the nuclear internal transcribed spacer region, including the 5.8S rDNA gene, and the protein-coding genes glyceraldehyde-3-phosphate dehydrogenase and Alt a1. Subsequent phylogenetic analyses based on maximum parsimony and Bayesian methods included related Alternaria, Embellisia, and Stemphylium spp. Results supported previous findings of polyphyletic and paraphyletic relationships of Ulocladium among other taxa. Ten Ulocladium species clustered into a core Ulocladium clade and all taxa possessed the key diagnostic feature of Ulocladium, namely, conidia essentially obovoid in shape. However, A. cheiranthi and E. indefessa also clustered within this group with high bootstrap support but did not posses this diagnostic feature. This paraphyletic clade resolved basal to the core Alternaria clade with high bootstrap support, unlike previous studies in which its position was imbedded within the primary Alternaria clade. Thus, the status of the genus as an independent lineage and a unique taxon is strongly supported. As previously reported, U. alternariae and U. oudemansii, which posses the key conidium characteristics of Ulocladium, clustered as a separate clade sister to the core Ulocladium clade. Further studies are necessary to determine if these taxa represent an independent lineage or share a common ancestor with other Ulocladium species. Obovoid conidia were poorly represented in the isolate of U. lanuginosum that was included in these analyses (the only U. lanuginosum isolate currently available), and the isolate resolved as A. radicina based upon all three loci sequenced. Based upon these data and the origin of the isolate, which was originally deposited as A. malvae, a reassessment of its identity is supported.

Mycologia, 2003

DNA sequences from rDNA and proteincoding regions were determined for six Embellisia and two Nimb... more DNA sequences from rDNA and proteincoding regions were determined for six Embellisia and two Nimbya spp. and were compared to those from Alternaria, Ulocladium and Stemphylium spp. Sequences determined included rDNA from the nuclear internal transcribed-spacer region (ITS1/5.8S/ ITS2) and the mitochondrial small-subunit (mt SSU) and a portion of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene. Phylogenetic analyses were performed on each dataset separately and then combined for total evidence analysis using methods of maximum parsimony and maximum likelihood. Results revealed that Embellisia and Nimbya clustered within a large monophyletic Alternaria-Nimbya-Embellisia-Ulocladium clade with Stemphylium as the sister taxon. Members of the infectoria species-group were the most basal group in this large polygeneric clade. Embellisia and Nimbya were sister taxa of the remaining Alternaria and Ulocladium spp. and were related more closely to Alternaria than was Stemphylium. Four Embellisia spp. formed a monophyletic clade. However, E. allii clustered with the two Nimbya spp. and E. indefessa clustered with Alternaria and Ulocladium spp., revealing that Embellisia, as currently circumscribed, is polyphyletic. Potential revisions of taxonomy for all genera are discussed.

Mycologia, 2008

The phylogenetic relationship between Alternaria radicina and A. carotiincultae was reexamined ba... more The phylogenetic relationship between Alternaria radicina and A. carotiincultae was reexamined based on morphology, sequence analysis of rDNA (ITS and mitochondrial small subunit [mtSSU]), protein coding genes (actin [ACT], btubulin, chitin synthase [CHS], translation elongation factor [EF-1a], Alternaria allergen a1 [Alt a1], and glyceraldehyde-3-phosphate dehydrogenase [gpd]), and RAPD and ISSR analysis of total genomic DNA. Although some morphological characters overlapped to a degree, with A. radicina isolates expressing moderate variation and A. carotiincultae isolates being highly uniform, A. carotiincultae could be differentiated from A. radicina based on significantly greater growth rate on potato dextrose agar (PDA) or acidified PDA (APDA) and average number of transverse septa per conidium. Sequence of rDNA and two protein coding genes, ACT and CHS, were invariant between species. However polymorphism with the EF-1a, b-tubulin, and Alt a1 gene strictly separated the population of A. radicina and A. carotiincultae as distinct lineages, as did RAPD and ISSR analysis. The polymorphic gpd gene did not strictly separate the two species. However isolates of A. radicina encompassed several haplotypes, one of which was the exclusive haplotype possessed by A. carotiincultae isolates, suggesting evidence of incomplete lineage sorting. The results suggest that A. carotiincultae is closely related to A. radicina but is a recently divergent and distinct lineage, which supports its status as a separate species.

Mycologia, 2009

Small-spored Alternaria species are a taxonomically challenging group of fungi with few morpholog... more Small-spored Alternaria species are a taxonomically challenging group of fungi with few morphological or molecular characters that allow unambiguous discrimination among taxa. The protein-coding genes most commonly employed in fungal systematics are invariant among these taxa, so noncoding, anonymous regions of the genome were developed to assess evolutionary relationships among these organisms. Nineteen sequence-characterized amplified regions (SCAR) were screened for phylogenetic utility by comparing sequences among reference isolates of small-spored Alternaria species. Five of nineteen loci were consistently amplifiable and had sufficient phylogenetic signal. Phylogenetic analyses were performed with 150 small-spored Alternaria isolates using sequence data from an endopolygalacturonase gene and two anonymous loci. Associations among phylogenetic lineage, morphological classification, geography and host were evaluated for use as practical taxonomic characters. Samples included isolates from citrus in Florida, pistachio in California, desert plants in Arizona, walnuts in France/Italy and apples in South Africa. No associations were found between host or geographic associations and phylogenetic lineage, indicating that these characters were not useful for cladistic classification of small-spored Alternaria. Similarly strict congruence between morphology and phylogenetic lineage was not found among isolates grouped morphologically with A. alternata or A. tenuissima. In contrast 34 isolates grouped morphologically with A. arborescens fell into discrete clades for all datasets. Although 5-9 well supported clades were evident among isolates, it is currently unclear if these clades should be considered phylogenetic species or emerging evolutionary line-ages within the phylogenetically defined alternata species-group.

Mycologia, 2002

Alternaria radicina, A. carotiincultae, and A. petroselini are closely related pathogens of umbel... more Alternaria radicina, A. carotiincultae, and A. petroselini are closely related pathogens of umbelliferous crops. Relationships among these fungi were determined based on growth rate, spore morphology, cultural characteristics, toxin production, and host range. Random amplified polymorphic DNA (RAPD) analysis of these species, other species of Alternaria, and closely related fungi was also performed. A. petroselini was readily differentiated from A. radicina and A. carotiincultae on the basis of spore morphology, production of microsclerotia, host range, and RAPD analysis. Alternaria radicina and A. carotiincultae were considerably more similar to each other than to A. petroselini, but could be differentiated on the basis of growth rate, spore morphology, colony morphology, and, to a limited extent, RAPD analysis. When grown on media having a high nutritional content, A. radicina produced a diffusible yellow pigment and crystals of the fungal metabolite radicinin. In contrast, A. carotiincultae produced little or no radicinin. However, when A. carotiincultae was grown on the same medium amended with radicinin, growth rate and colony and conidial morphology were more similar to those of A. radicina. These results suggest that the morphological differences between A. radicina and A. carotiincultae are due, at least in part, to radicinin production, and that these fungi are conspecific. Therefore, we propose that A. carotiincultae be considered a synonym of A. radicina.

Mycologia, 2013

The systematics of Alternaria and allied genera traditionally has been based on the characteristi... more The systematics of Alternaria and allied genera traditionally has been based on the characteristics of conidia and the sporulation apparatus. This emphasis on morphology in the reconstruction of organismal relationships has resulted in taxonomic uncertainty and flux for a number of taxa in Alternaria and the related genera Stemphylium, Embellisia, Nimbya and Ulocladium. The present study used a molecular phylogenetic approach for systematic resolution and incorporated extensive taxon sampling (n 5 176 species) representing 10 genera and analyses of 10 protein-coding loci. Phylogenetic analyses based on five of these genes revealed eight distinct asexual lineages of Alternaria that cluster as the sister group to the asexual paraphyletic genus Ulocladium, while taxa with known teleomorphs currently circumscribed as Alternaria (the infectoria species-group) cluster among genera that also have representatives with known teleomorphs. This work proposes to elevate the eight well supported asexual lineages of Alternaria to the taxonomic rank of section. Evolutionary relationships among Alternaria and closely related genera are discussed.

Frontiers in Cellular and Infection Microbiology, 2021

Alternaria alternata is a ubiquitous fungus and a major allergen associated with the development ... more Alternaria alternata is a ubiquitous fungus and a major allergen associated with the development of asthma. Inhalation of intact spores is the primary cause of human exposure to fungal allergen. However, allergen-rich cultured fungal filtrates are oftentimes used in the current models of fungal sensitization that do not fully reflect real-life exposures. Thus, establishing novel spore exposure models is imperative. In this study, we established novel fungal exposure models of both adult and neonate to live spores. We examined pathophysiological changes in the spore models as compared to the non-exposure controls and also to the conventional filtrate models. While both Alternaria filtrate- and spore-exposed adult BALB/c mice developed elevated airway hyperresponsiveness (AHR), filtrates induced a greater IgE mediated response and higher broncholavage eosinophils than spores. In contrast, the mice exposed to Alternaria spores had higher numbers of neutrophils. Both exposures induced c...

HortScience, 2010

Lettuce drop caused by Sclerotinia spp. is an economically important disease of lettuce (Lactuca ... more Lettuce drop caused by Sclerotinia spp. is an economically important disease of lettuce (Lactuca sativa L.), and cultivars with resistance to mycelial infection by Sclerotinia sclerotiorum (Lib.) de Bary and S. minor Jagger as well as to S. sclerotiorum ascospores are needed. Assessing resistance in field experiments can be complicated by fast bolting or small stature lettuce lines that may escape rather than resist the pathogens. Therefore, methods to select resistant lines from morphologically variable populations are needed. We used S. sclerotiorum and S. minor-infested field experiments, regression analysis, field experiments with artificially high plant densities, and S. sclerotiorum ascospore inoculations to identify lettuce lines with resistance to both pathogens. Three replicated experiments in S. sclerotiorum-infested fields were conducted in Yuma, AZ, and three replicated experiments in a S. minor-infested field were conducted in Salinas, CA, using diverse populations of i...

African Journal of Microbiology Research, Mar 5, 2013

Isothiocyanates (ITCs) is a group of defense related compounds synthesized by Brassicas that have... more Isothiocyanates (ITCs) is a group of defense related compounds synthesized by Brassicas that have positive effects on human health which suggest that they are environmentally friendly compounds to control fungi infections. However, the development of ITC-resistant strains of fungi is a matter of concern. The objective of the present work was to study the response of Alternaria alternata to allylisothiocyanate (AITC) and benzyl isothiocyanate (BITC) chronic exposure and the effect of the treatment on some of the inter simple sequence repeats (ISSR) regions. Five strains of A. alternata isolated from tomato and five isolated from cabbage were independently exposed in vitro to AITC and BITC. Concentrations were increased until it reached 0.08 and 0.6 mg/mL of AITC and BITC, respectively. Genomic DNA from both wild type and isogenic adapted strains to ITCs was isolated and five primers were used for ISSR amplification: (GACA) 4 :5´-GACAGACAGACAGACA-3´; M13:5´-GAGGGTGGCGGCGGTTCT-3´;(AAG) 8 :5´-AAGAAGAAGAAGAAGAAGAAGAAG-3´; (ACA) 5 : 5´-ACAACAACAACAACA-3´ and T3B: 5´-AGGTCGCGGGTTCGAATCC-3´. A lower degree of polymorphism was found to be induced by the treatment in the wild-type strains isolated from cabbage as compared with the wild-type strains isolated from tomato. It can be concluded that ITCs exposure induced random mutations in different ISSR regions of the A. alternata genome which does not lead to the development of strains with a hereditable resistant phenotype.

BMC Genomics, 2015

Background: Alternaria is considered one of the most common saprophytic fungal genera on the plan... more Background: Alternaria is considered one of the most common saprophytic fungal genera on the planet. It is comprised of many species that exhibit a necrotrophic phytopathogenic lifestyle. Several species are clinically associated with allergic respiratory disorders although rarely found to cause invasive infections in humans. Finally, Alternaria spp. are among the most well known producers of diverse fungal secondary metabolites, especially toxins. Description: We have recently sequenced and annotated the genomes of 25 Alternaria spp. including but not limited to many necrotrophic plant pathogens such as A. brassicicola (a pathogen of Brassicaceous crops like cabbage and canola) and A. solani (a major pathogen of Solanaceous plants like potato and tomato), and several saprophytes that cause allergy in human such as A. alternata isolates. These genomes were annotated and compared. Multiple genetic differences were found in the context of plant and human pathogenicity, notably the pro-inflammatory potential of A. alternata. The Alternaria genomes database was built to provide a public platform to access the whole genome sequences, genome annotations, and comparative genomics data of these species. Genome annotation and comparison were performed using a pipeline that integrated multiple computational and comparative genomics tools. Alternaria genome sequences together with their annotation and comparison data were ported to Ensembl database schemas using a self-developed tool (EnsImport). Collectively, data are currently hosted using a customized installation of the Ensembl genome browser platform. Conclusion: Recent efforts in fungal genome sequencing have facilitated the studies of the molecular basis of fungal pathogenicity as a whole system. The Alternaria genomes database provides a comprehensive resource of genomics and comparative data of an important saprophytic and plant/human pathogenic fungal genus. The database will be updated regularly with new genomes when they become available. The Alternaria genomes database is freely available for non-profit use at http://alternaria.vbi.vt.edu.

Phytopathology®, 2007

Fusarium oxysporum f. sp. lactucae, causal agent of Fusarium wilt of lettuce, is a serious pathog... more Fusarium oxysporum f. sp. lactucae, causal agent of Fusarium wilt of lettuce, is a serious pathogen recently reported in Arizona. Sequence analysis of the mitochondrial small subunit (mtSSU), translation elongation factor 1-α (EF-1α) gene, and the nuclear ribosomal DNA intergenic spacer (IGS) region was conducted to resolve relationships among f. sp. lactucae isolates, F. oxysporum isolates from other hosts, and local non-pathogenic isolates. Analysis of mtSSU sequences provided limited phylogenetic resolution and did not differentiate the lactucae isolates from 13 other F. oxysporum isolates. Analysis of EF-1α sequences resulted in moderate resolution, grouping seven formae speciales with the lactucae isolates. Analysis of the IGS region revealed numerous sequence polymorphisms among F. oxysporum formae speciales consisting of insertions, deletions, and single nucleotide transitions and substitutions. Repeat sequence analysis revealed several duplicated subrepeat units that were di...

International Journal of Speleology, 2011

Fungal communities on speleothem surfaces in Kartchner Caverns, Arizona, USA. INTRODUCTION Kartch... more Fungal communities on speleothem surfaces in Kartchner Caverns, Arizona, USA. INTRODUCTION Kartchner Caverns, located in Benson, Arizona, USA (N31° 50' 08", W110° 20' 37"), is a wet, actively forming carbonate cave in the Whetstone Mountains. First discovered in 1974, the cave was kept a closely guarded secret among the cave's discoverers in an effort to preserve the cave from anthropogenic harm. In 1988, the State of Arizona purchased the land surrounding the cave and began development of Kartchner Caverns State Park (Tufts & Tenen, 1999). Special effort was made during the development process to preserve the pristine status of the cave while making portions of it accessible to tourism. In

Plant Disease, 2008

Field experiments were conducted over 2 years in Yuma County, AZ, and Imperial County, CA, to det... more Field experiments were conducted over 2 years in Yuma County, AZ, and Imperial County, CA, to determine the efficacy of several biocontrol agents for the management of lettuce drop caused by Sclerotinia spp. Commercial formulations of Trichoderma harzianum (Plantshield, Supersivit), Gliocladium virens (Soilgard), Coniothyrium minitans (Contans), and Bacillus subtilis (Companion) were evaluated and compared with the chemical fungicide iprodione (Rovral) against Sclerotinia sclerotiorum and S. minor. A single application of biocontrol products or of Rovral did not reduce lettuce drop caused by either Sclerotinia species. However, two applications of Contans, one at planting and one at post-thinning, significantly reduced the incidence of lettuce drop caused by S. sclerotiorum and increased yield but had no effect on S. minor at both locations in both years. Two applications of other biocontrol products did not significantly reduce disease incidence despite medium to high recovery foll...

Plant Disease, 2010

Field experiments were conducted over 2 years in Yuma, AZ, and Holtville, CA, to establish the re... more Field experiments were conducted over 2 years in Yuma, AZ, and Holtville, CA, to establish the relationship between soil sclerotium density of Sclerotinia sclerotiorum and the incidence of lettuce drop on different lettuce (Lactuca sativa) types under different irrigation systems, and to determine the efficacy of the biocontrol agent Coniothyrium minitans (Contans) against S. sclerotiorum on crisphead lettuce at varied sclerotium densities under different irrigation systems. There was no significant interaction of irrigation (overhead sprinkler versus furrow) with either sclerotium density or with biocontrol treatment. Lettuce drop incidence was lowest in romaine lettuce compared with crisphead or leaf lettuce at all soil sclerotium densities. There was a significant positive correlation between the sclerotial density and the percent disease incidence. Disease incidence in plots infested with 2 sclerotia/m2of bed was not significantly higher than in control plots regardless of lettu...

Plant Disease, 2010

A nested polymerase chain reaction-based (nPCR) assay was developed and evaluated for the rapid d... more A nested polymerase chain reaction-based (nPCR) assay was developed and evaluated for the rapid detection of Fusarium oxysporum f. sp. lactucae in seed of lettuce. Three primers were designed from sequences of the intergenic spacer region of the rDNA and were used in the PCR amplifications. The first amplification employed the primer pair GYCF1 and GYCR4C and produced a product of 2,270 bp. The second amplification employed the forward primer GYCF1 and the nested primer R943 and produced a single 936-bp PCR product. The nPCR protocol developed successfully detected the target sequence in genomic DNA at 1 fg/μl. A seed assay was tested that included a 4-day incubation step in which seed were maintained under high humidity conditions to increase fungal biomass for DNA extraction. In seed lots prepared by mixing known amounts of F. oxysporum f. sp. lactucae–infested seed with noninfested seed, this assay permitted the detection of the pathogen from lots with infestation rates as low as...

Plant Disease, 2001

A pair of polymerase chain reaction (PCR) primers was developed based upon the sequence of a clon... more A pair of polymerase chain reaction (PCR) primers was developed based upon the sequence of a cloned random amplified polymorphic DNA (RAPD) fragment of Alternaria radicina, and a PCR-based seed assay was developed for the detection of A. radicina from infested carrot seed. The seed assay involved a 5-day incubation step, in which seed was maintained under high humidity conditions in order to increase fungal biomass. Seed was then incubated with lysis buffer, extracted with phenol-chloroform, and DNA was recovered using a silica matrix. PCR amplification of the target A. radicina DNA sequence was enhanced by the addition of skim milk to the PCR reaction mixture. With this PCR-based seed assay, A. radicina was detected from carrot seed lots with natural infestation rates as low as 0.3%. In seed lots prepared by mixing known amounts of A. radicina-infested seed with noninfested seed, this assay allowed for the detection of the pathogen from lots with infestation rates as low as 0.1%.

Phytopathology®, 2012

Since 1999, a disease of apple caused by an Alternaria sp. has been affecting orchards in norther... more Since 1999, a disease of apple caused by an Alternaria sp. has been affecting orchards in northern Italy resulting in necrotic spots on leaves and on fruit. Forty-four single-spored isolates were obtained from diseased plant materials to investigate the diversity of this fungus in Italy and to compare these isolates to isolates of Alternaria associated with apple disease in previous studies, including A. mali, causal agent of apple blotch. All isolates, including the reference strains, were tested for pathogenicity utilizing in vitro bioassays on detached leaf or on fruit (‘Golden Delicious’). In addition, morphological characterizations were conducted describing both the three-dimensional sporulation pattern and the colony morphology of each isolate. In order to assess the genetic diversity within the Italian Alternaria population, sequence characterization of specific loci and anonymous regions (endoPG, OPA1-3, OPA2-1, and OPA10-2) and genetic fingerprinting based on amplified fra...

Phytopathology®, 2002

Alternaria isolates were obtained from various pistachio tissues collected in five orchards in Ca... more Alternaria isolates were obtained from various pistachio tissues collected in five orchards in California. For all isolates, morphological characteristics of the colony and sporulation apparatus were determined and compared with those of representative isolates of A. alternata, A. tenuissima, A. arborescens, and A. infectoria. A selection of the pistachio isolates and the representative Alternaria isolates were evaluated for pathogenicity to pistachio. Molecular characteristics of these isolates were determined using random amplified polymorphism DNA (RAPD) analysis, polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of nuclear intergenic spacer rDNA, and sequence analysis of nuclear internal transcribed spacer (ITS) rDNA. Based on morphological characteristics, the pistachio isolates were grouped as identical or very similar to either A. alternata, A. tenuissima, A. arborescens, or A. infectoria. Isolates from the alternata, tenuissima, and arbor...

Mycological Progress, 2011

Previous phylogenetic analyses revealed that species within the genera Nimbya and Embellisia resi... more Previous phylogenetic analyses revealed that species within the genera Nimbya and Embellisia reside within a large monophyletic clade that also includes the genera Alternaria, Ulocladium, Undifilum, Sinomyces, and Crivellia with Stemphylium as the sister taxon. This study expands upon previous work by including many contemporary species of each genus and utilizes molecular and morphological characters to further examine relationships. Maximum parsimony and Bayesian analysis reveals that Nimbya is not a monophyletic genus but is split into two phylogenetically distant clades, which have different and distinct conidial morphologies. One of these clades resides completely within Alternaria. Phylogenetic analyses also reveals that Embellisia does not form a monophyletic genus but is split into four monophyletic lineages. Moreover, several species of Embellisia cluster individually with clades that are predominantly Alternaria, Ulocladium, or Stemphylium, yet these Embellisia spp. possess morphological characters that are diagnostically Embellisia. Thus, these data reveal that both Nimbya and Embellisia are polyphyletic as currently defined and taxonomic restructuring is necessary in order to resolve conflict between historical morphological and contemporary molecularbased phylogenies.

Mycological Progress, 2008

The genus Ulocladium represents phaeodictyosporic Hyphomycetes that produce conidia that are esse... more The genus Ulocladium represents phaeodictyosporic Hyphomycetes that produce conidia that are essentially obovoid in shape. Previous molecular studies that included Ulocladium and related taxa in Alternaria, Embellisia, and Stemphylium revealed a conflict between morphology and phylogeny, and Ulocladium was supported as polyphyletic with a paraphyletic core group. Moreover, the genus consistently resolved within a larger Alternaria/ Ulocladium clade, resulting in paraphyly of Alternaria and questions as to the taxonomic status of Ulocladium. In the present study, 13 Ulocladium species and three genetic loci were included for a more comprehensive systematic analysis of the genus than had previously been conducted. Total genomic DNA was extracted from representative taxa and sequences were determined for the nuclear internal transcribed spacer region, including the 5.8S rDNA gene, and the protein-coding genes glyceraldehyde-3-phosphate dehydrogenase and Alt a1. Subsequent phylogenetic analyses based on maximum parsimony and Bayesian methods included related Alternaria, Embellisia, and Stemphylium spp. Results supported previous findings of polyphyletic and paraphyletic relationships of Ulocladium among other taxa. Ten Ulocladium species clustered into a core Ulocladium clade and all taxa possessed the key diagnostic feature of Ulocladium, namely, conidia essentially obovoid in shape. However, A. cheiranthi and E. indefessa also clustered within this group with high bootstrap support but did not posses this diagnostic feature. This paraphyletic clade resolved basal to the core Alternaria clade with high bootstrap support, unlike previous studies in which its position was imbedded within the primary Alternaria clade. Thus, the status of the genus as an independent lineage and a unique taxon is strongly supported. As previously reported, U. alternariae and U. oudemansii, which posses the key conidium characteristics of Ulocladium, clustered as a separate clade sister to the core Ulocladium clade. Further studies are necessary to determine if these taxa represent an independent lineage or share a common ancestor with other Ulocladium species. Obovoid conidia were poorly represented in the isolate of U. lanuginosum that was included in these analyses (the only U. lanuginosum isolate currently available), and the isolate resolved as A. radicina based upon all three loci sequenced. Based upon these data and the origin of the isolate, which was originally deposited as A. malvae, a reassessment of its identity is supported.

Mycologia, 2003

DNA sequences from rDNA and proteincoding regions were determined for six Embellisia and two Nimb... more DNA sequences from rDNA and proteincoding regions were determined for six Embellisia and two Nimbya spp. and were compared to those from Alternaria, Ulocladium and Stemphylium spp. Sequences determined included rDNA from the nuclear internal transcribed-spacer region (ITS1/5.8S/ ITS2) and the mitochondrial small-subunit (mt SSU) and a portion of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene. Phylogenetic analyses were performed on each dataset separately and then combined for total evidence analysis using methods of maximum parsimony and maximum likelihood. Results revealed that Embellisia and Nimbya clustered within a large monophyletic Alternaria-Nimbya-Embellisia-Ulocladium clade with Stemphylium as the sister taxon. Members of the infectoria species-group were the most basal group in this large polygeneric clade. Embellisia and Nimbya were sister taxa of the remaining Alternaria and Ulocladium spp. and were related more closely to Alternaria than was Stemphylium. Four Embellisia spp. formed a monophyletic clade. However, E. allii clustered with the two Nimbya spp. and E. indefessa clustered with Alternaria and Ulocladium spp., revealing that Embellisia, as currently circumscribed, is polyphyletic. Potential revisions of taxonomy for all genera are discussed.

Mycologia, 2008

The phylogenetic relationship between Alternaria radicina and A. carotiincultae was reexamined ba... more The phylogenetic relationship between Alternaria radicina and A. carotiincultae was reexamined based on morphology, sequence analysis of rDNA (ITS and mitochondrial small subunit [mtSSU]), protein coding genes (actin [ACT], btubulin, chitin synthase [CHS], translation elongation factor [EF-1a], Alternaria allergen a1 [Alt a1], and glyceraldehyde-3-phosphate dehydrogenase [gpd]), and RAPD and ISSR analysis of total genomic DNA. Although some morphological characters overlapped to a degree, with A. radicina isolates expressing moderate variation and A. carotiincultae isolates being highly uniform, A. carotiincultae could be differentiated from A. radicina based on significantly greater growth rate on potato dextrose agar (PDA) or acidified PDA (APDA) and average number of transverse septa per conidium. Sequence of rDNA and two protein coding genes, ACT and CHS, were invariant between species. However polymorphism with the EF-1a, b-tubulin, and Alt a1 gene strictly separated the population of A. radicina and A. carotiincultae as distinct lineages, as did RAPD and ISSR analysis. The polymorphic gpd gene did not strictly separate the two species. However isolates of A. radicina encompassed several haplotypes, one of which was the exclusive haplotype possessed by A. carotiincultae isolates, suggesting evidence of incomplete lineage sorting. The results suggest that A. carotiincultae is closely related to A. radicina but is a recently divergent and distinct lineage, which supports its status as a separate species.

Mycologia, 2009

Small-spored Alternaria species are a taxonomically challenging group of fungi with few morpholog... more Small-spored Alternaria species are a taxonomically challenging group of fungi with few morphological or molecular characters that allow unambiguous discrimination among taxa. The protein-coding genes most commonly employed in fungal systematics are invariant among these taxa, so noncoding, anonymous regions of the genome were developed to assess evolutionary relationships among these organisms. Nineteen sequence-characterized amplified regions (SCAR) were screened for phylogenetic utility by comparing sequences among reference isolates of small-spored Alternaria species. Five of nineteen loci were consistently amplifiable and had sufficient phylogenetic signal. Phylogenetic analyses were performed with 150 small-spored Alternaria isolates using sequence data from an endopolygalacturonase gene and two anonymous loci. Associations among phylogenetic lineage, morphological classification, geography and host were evaluated for use as practical taxonomic characters. Samples included isolates from citrus in Florida, pistachio in California, desert plants in Arizona, walnuts in France/Italy and apples in South Africa. No associations were found between host or geographic associations and phylogenetic lineage, indicating that these characters were not useful for cladistic classification of small-spored Alternaria. Similarly strict congruence between morphology and phylogenetic lineage was not found among isolates grouped morphologically with A. alternata or A. tenuissima. In contrast 34 isolates grouped morphologically with A. arborescens fell into discrete clades for all datasets. Although 5-9 well supported clades were evident among isolates, it is currently unclear if these clades should be considered phylogenetic species or emerging evolutionary line-ages within the phylogenetically defined alternata species-group.

Mycologia, 2002

Alternaria radicina, A. carotiincultae, and A. petroselini are closely related pathogens of umbel... more Alternaria radicina, A. carotiincultae, and A. petroselini are closely related pathogens of umbelliferous crops. Relationships among these fungi were determined based on growth rate, spore morphology, cultural characteristics, toxin production, and host range. Random amplified polymorphic DNA (RAPD) analysis of these species, other species of Alternaria, and closely related fungi was also performed. A. petroselini was readily differentiated from A. radicina and A. carotiincultae on the basis of spore morphology, production of microsclerotia, host range, and RAPD analysis. Alternaria radicina and A. carotiincultae were considerably more similar to each other than to A. petroselini, but could be differentiated on the basis of growth rate, spore morphology, colony morphology, and, to a limited extent, RAPD analysis. When grown on media having a high nutritional content, A. radicina produced a diffusible yellow pigment and crystals of the fungal metabolite radicinin. In contrast, A. carotiincultae produced little or no radicinin. However, when A. carotiincultae was grown on the same medium amended with radicinin, growth rate and colony and conidial morphology were more similar to those of A. radicina. These results suggest that the morphological differences between A. radicina and A. carotiincultae are due, at least in part, to radicinin production, and that these fungi are conspecific. Therefore, we propose that A. carotiincultae be considered a synonym of A. radicina.

Mycologia, 2013

The systematics of Alternaria and allied genera traditionally has been based on the characteristi... more The systematics of Alternaria and allied genera traditionally has been based on the characteristics of conidia and the sporulation apparatus. This emphasis on morphology in the reconstruction of organismal relationships has resulted in taxonomic uncertainty and flux for a number of taxa in Alternaria and the related genera Stemphylium, Embellisia, Nimbya and Ulocladium. The present study used a molecular phylogenetic approach for systematic resolution and incorporated extensive taxon sampling (n 5 176 species) representing 10 genera and analyses of 10 protein-coding loci. Phylogenetic analyses based on five of these genes revealed eight distinct asexual lineages of Alternaria that cluster as the sister group to the asexual paraphyletic genus Ulocladium, while taxa with known teleomorphs currently circumscribed as Alternaria (the infectoria species-group) cluster among genera that also have representatives with known teleomorphs. This work proposes to elevate the eight well supported asexual lineages of Alternaria to the taxonomic rank of section. Evolutionary relationships among Alternaria and closely related genera are discussed.