Development and Evaluation of a TaqMan Real-Time PCR Assay for Fusarium oxysporum f. sp. spinaciae.

Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the United States suitable for commercial spinach seed production. A TaqMan real-time polymerase chain reaction (PCR) assay was developed for rapid identification and quantification of the pathogen, based on sequencing the intergenic spacer (IGS) region of rDNA of isolates of the pathogen. A guanine single-nucleotide polymorphism (G SNP) was detected in the IGS sequences of 36 geographically diverse isolates of F. oxysporum f. sp. spinaciae but not in the sequences of 64 isolates representing other formae speciales and 33 isolates representing other fungal species or genera. The SNP was used to develop a probe for a real-time PCR assay. The real-time PCR assay detected F. oxysporum f. sp. spinaciae at 3-14,056 CFU/g of soil in 82 soil samples collected over 3 years from naturally infested spinach seed production sites in western Washington, although a reliable detection limit of the assay was determined to be 11 CFU/g of soil. A significant (P < 0.05), positive correlation between enumeration of F. oxysporum on Komada's agar and quantification of the pathogen using the TaqMan assay was observed in a comparison of 82 soil samples. Correlations between pathogen DNA levels, Fusarium wilt severity ratings, and spinach biomass were significantly positive for one set of naturally infested soils but not between pathogen DNA levels, wilt incidence ratings, and spinach biomass for other soil samples, suggesting that soilborne pathogen population is not the sole determinant of spinach Fusarium wilt incidence or severity. The presence of the G SNP detected in one isolate of each of F. oxysporum ff. spp. lageneriae, lilii, melongenae, and raphani and reaction of the real-time PCR assay with 16 of 22 nonpathogenic isolates of F. oxysporum associated with spinach plants or soil in which spinach had been grown potentially limits the application of this assay. Nonetheless, because all isolates of F. oxysporum f. sp. spinaciae tested positive with the real-time PCR assay, the assay may provide a valuable means of screening for resistance to Fusarium wilt by quantifying development of the pathogen in spinach plants inoculated with the pathogen.

First Report of Root Rot Caused by Rhizoctonia solani AG-10 on Canola in Washington State.

Canola (Brassica napus L) production has gained renewed interest in Washington State over the past few years, primarily for the purpose of producing biofuel. Plants were observed to be showing symptoms of Rhizoctonia root rot and postemergence damping-off. In many cases, this was due to Rhizoctonia solani AG-2-1, which was previously documented (4). However, additional plants were occasionally observed that were stunted and had reduced vigor, but lacked the distinctive severe stem damage and postemergence damping-off, which are both symptoms of infection with R. solani AG-2-1. Isolates of R. solani AG-10 were collected from symptomatic plants or baited from root zone soil at various dryland production locations in eastern Washington, including sites near Colfax, Pullman, and Walla Walla. Initial identification was determined by quantitative (Q)-PCR using R. solani AG-10 specific primers (3). The identity was verified by sequencing random isolates identified by Q-PCR (GenBank Accessions Nos. JQ068147, JQ068148 and JQ068149). All sequenced isolates had 99% identity to previously reported isolates of R. solani AG-10. Six isolates were chosen to test pathogenicity on canola plants in the greenhouse. Sterilized oats were inoculated with each of six isolates of R. solani AG-10 and grown for 4 weeks. The soil was infested with ground oat inoculum (1% wt/wt) and spring canola cv. Sunrise was seeded into 3.8 × 21-cm containers. After 3 weeks of incubation at 15°C, plants were harvested and assessed. Emergence was reduced in the infested soil with 73 to 93% (average 81%) emergence compared with 100% emergence in the noninfested soil. There was no evidence of postemergence damping-off. However, all six isolates of R. solani AG-10 significantly reduced the plant height and top dry weights compared with the noninfested controls. The plant height in infested soil was 28 to 42% (average 34%) shorter and top dry weights were 37 to 70% (average 54%) lower than in noninfested soil. Roots of infected plants had a light brown discoloration along with reduced length and fewer lateral roots. Additional host plants were tested, including wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), pea (Pisum sativum L.), chickpea (Cicer arietinum L.), and lentil (Lens culinaris Medik.). There was no significant reduction in plant height or plant dry weight for any of these hosts. R. solani AG-10 was previously found to be weakly virulent on canola and other cruciferous hosts in Australia (1,2). To our knowledge, this is the first report of R. solani AG-10 causing disease on canola in Washington State. Reference: (1) R. K. Khangura et al. Plant Dis. 83:714, 1999. (2) G. C. MacNish et al. Australas. Plant Pathol. 24:252, 1995. (3) P. A. Okubara et al. Phytopathology 98:837, 2008. (4) T. C. Paulitz et al. Plant Dis. 90:829, 2006.

First Report of a Ceratobasidium sp. Causing Root Rot on Canola in Washington State.

Rhizoctonia root rot occurs commonly on canola (Brassica napus L.) in Washington State. Recently, isolates of an additional pathogen were found to be involved in this disease complex. Isolates of an AG-I-like Ceratobasidium sp. were collected from roots and root zone soil in central Washington near Ritzville. Identity of selected isolates was verified by sequencing the internal transcribed spacer (ITS) region of the rDNA (GenBank Accession Nos. JQ247570, JQ247571, and JQ247572), with a 90 to 93% identity to AG-I. All isolates also amplified with AG-I-like specific primers (1). Six isolates were included in pathogenicity assays conducted in the greenhouse. There were five replicates of three plants for each treatment and the experiment was conducted twice. Pasteurized soil was infested with ground oat inoculum (1%) and placed into containers (3.8 × 21 cm). Infested soils were seeded with canola, chickpea (Cicer arietinum L.), lentil (Lens culinaris Medik.), pea (Pisum sativum L.), barley (Hordeum vulgare L.), or wheat (Triticum aestivum L.). After 3 weeks of incubation at 15°C, the plants were destructively harvested. The emergence of canola was consistently reduced in soil infested with a Ceratobasidium sp., with reductions of 0 to 23% (average 11%). There was no postemergence damping-off, a symptom commonly associated with AG-2-1 (2). Plant height and top dry weights were significantly reduced for canola seeded into infested soil. Heights of plants growing in infested soil was reduced by 25 to 53% (average 42%) and top dry weight was reduced by 37 to 81% (average 61%) compared with the noninfested control. The legume hosts tested in this study were also affected by this Ceratobasidium sp., but to a lesser extent. Compared with the noninfested controls, there was evidence of preemergence damping-off in chickpea (0 to 27%, average 13%) and pea plants were consistently stunted (5 to 23%, average 12%). Chickpea and pea plants grown in infested soil also had reduced top dry weights of 9 to 28% (average 17%) and 13 to 35% (average 21%), respectively. The roots of all infected hosts had a characteristic brown discoloration with tapered, rotted root tips (spear tips). There was no reduction in emergence or plant height of wheat and barley; there was inconsistent reduction in dry weight of these plants. To our knowledge, this is the first report of a Ceratobasidium sp. causing disease on canola in Washington State. References: (1) P. A. Okubara et al. Phytopathology 98:837, 2008. (2) T. C. Paulitz et al. Plant Dis. 90:829, 2006.

Optimum Timing of Preplant Applications of Glyphosate to Manage Rhizoctonia Root Rot in Barley.

Rhizoctonia root rot, caused by Rhizoctonia solani AG-8 and R. oryzae, is considered one of the main deterrents for farmers to adopt reduced-tillage systems in the Pacific Northwest. Because of the wide host range of Rhizoctonia spp., herbicide application before planting to control weeds and volunteer plants is the main management strategy for this disease. To determine the effect of timing of glyphosate applications on the severity of Rhizoctonia root rot of barley, field experiments were conducted in 2007, 2008, and 2009 in a field naturally infested with a high level of both R. solani and R. oryzae. Crop volunteer plants and weeds were allowed to grow over the winter and plots were sprayed with glyphosate at 42, 28, 14, 7, and 2 days prior to planting. As the herbicide application interval increased, there were significant increases in shoot length, length of the first true leaf, and number of healthy seminal roots and a decrease in disease severity. Yield and the number of seminal roots did not show a response to herbicide application interval in most years. The activity of R. solani, as measured by toothpick bioassay and real-time polymerase chain reaction, declined over time in all treatments after planting barley. The herbicide application interval required to meet 80 and 90% of the maximum response (asymptote) for all plant and disease measurements ranged from 11 to 27 days and 13 to 37 days, respectively. These times are the minimum herbicide application intervals required to reduce disease severity in the following crop.

Soilborne Pathogens of Cereals in an Irrigated Cropping System: Effects of Tillage, Residue Management, and Crop Rotation.

An irrigated cropping systems experiment was conducted for 6 years in east-central Washington State to examine agronomic and economic alternatives to continuous annual winter wheat (Triticum aestivum) with burning and plowing, and to determine how root diseases of cereals are influenced by management practices. The continuous winter wheat treatment with burning and plowing was compared with a 3-year no-till rotation of winter wheat-spring barley (Hordeum vulgare)-winter canola (Brassica napus) and three straw management treatments: burning, straw removal, and leaving the straw stubble standing after harvest. Take-all disease and inoculum increased from years 1 to 4 in the continuous winter wheat treatment with burning and plowing, reducing plant growth compared to the no-till treatments with crop rotations. Inoculum of Rhizoctonia solani AG-8 was significantly lower in the tilled treatment compared to the no-till treatments. Inoculum concentration of Fusarium pseudograminearum was higher than that of F. culmorum, and in one of three years, the former was higher in treatments with standing stubble and mechanical straw removal compared to burned treatments. Residue management method had no effect on Rhizoctonia inoculum, but spring barley had more crown roots and tillers and greater height with stubble burning. This 6-year study showed that irrigated winter wheat can be produced in a no-till rotation without major disease losses and demonstrated how cropping practices influence the dynamics of soilborne cereal diseases and inoculum over time.

Effect of inoculum density and soil tillage on the development and severity of rhizoctonia root rot.

Rhizoctonia spp. cause substantial yield losses in direct-seeded cereal crops compared with conventional tillage. To investigate the mechanisms behind this increased disease, soils from tilled or direct-seeded fields were inoculated with Rhizoctonia spp. at population densities from 0.8 to 250 propagules per gram and planted with barley (Hordeum vulgare). The incidence and severity of disease did not differ between soils with different tillage histories. Both R. solani AG-8 and R. oryzae stunted plants at high inoculum densities, with the latter causing pre-emergence damping-off. High inoculum densities of both species stimulated early production of crown roots in barley seedlings. Intact soil cores from these same tilled and direct-seeded fields were used to evaluate the growth of Rhizoctonia spp. from colonized oat seeds. Growth of R. oryzae was not affected by previous tillage history. However, R. solani AG-8 grew more rapidly through soil from a long-term direct-seeded field compared to tilled soils. The differential response between these two experiments (mixed, homogenized soil versus intact soil) suggests that soil structure plays a major role in the proliferation of R. solani AG-8 through soils with different tillage histories.

Identification and quantification of Rhizoctonia solani and R. oryzae using real-time polymerase chain reaction.

Rhizoctonia solani and R. oryzae are the principal causal agents of Rhizoctonia root rot in dryland cereal production systems of the Pacific Northwest. To facilitate the identification and quantification of these pathogens in agricultural samples, we developed SYBR Green I-based real-time quantitative-polymerase chain reaction (Q-PCR) assays specific to internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA of R. solani and R. oryzae. The assays were diagnostic for R. solani AG-2-1, AG-8, and AG-10, three genotypes of R. oryzae, and an AG-I-like binucleate Rhizoctonia species. Quantification was reproducible at or below a cycle threshold (Ct) of 33, or 2 to 10 fg of mycelial DNA from cultured fungi, 200 to 500 fg of pathogen DNA from root extracts, and 20 to 50 fg of pathogen DNA from soil extracts. However, pathogen DNA could be specifically detected in all types of extracts at about 100-fold below the quantification levels. Soils from Ritzville, WA, showing acute Rhizoctonia bare patch harbored 9.4 to 780 pg of R. solani AG-8 DNA per gram of soil.. Blastn, primer-template duplex stability, and phylogenetic analyses predicted that the Q-PCR assays will be diagnostic for isolates from Australia, Israel, Japan, and other countries.

Role of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. in the defense of plant roots.

Plants have evolved strategies of stimulating and supporting specific groups of antagonistic microorganisms in the rhizosphere as a defense against diseases caused by soilborne plant pathogens owing to a lack of genetic resistance to some of the most common and widespread soilborne pathogens. Some of the best examples of natural microbial defense of plant roots occur in disease suppressive soils. Soil suppressiveness against many different diseases has been described. Take-all is an important root disease of wheat, and soils become suppressive to take-all when wheat or barley is grown continuously in a field following a disease outbreak; this phenomenon is known as take-all decline (TAD). In Washington State, USA and The Netherlands, TAD results from the enrichment during monoculture of populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas fluorescens to a density of 10 (5) CFU/g of root, the threshold required to suppress the take-all pathogen, Gaeumannomyces graminis var. tritici. 2,4-DAPG-producing P. fluorescens also are enriched by monoculture of other crops such as pea and flax, and evidence is accumulating that 2,4-DAPG producers contribute to the defense of plant roots in many different agroecosystems. At this time, 22 distinct genotypes of 2,4-DAPG producers (designated A - T, PfY and PfZ) have been defined by whole-cell repetitive sequence-based (rep)-PCR analysis, restriction fragment length polymorphism (RFLP) analysis of PHLD, and phylogenetic analysis of PHLD, but the number of genotypes is expected to increase. The genotype of an isolate is predictive of its rhizosphere competence on wheat and pea. Multiple genotypes often occur in a single soil and the crop species grown modulates the outcome of the competition among these genotypes in the rhizosphere. 2,4-DAPG producers are highly effective biocontrol agents against a variety of plant diseases and ideally suited for serving as vectors for expressing other biocontrol traits in the rhizosphere.

Identification and Quantification of Pathogenic Pythium spp. from Soils in Eastern Washington Using Real-Time Polymerase Chain Reaction.

ABSTRACT Traditional methods of quantifying Pythium spp. rely on the use of selective media and dilution plating. However, high variability is inherent in this type of enumeration and counts may not be representative of the pathogenic population of Pythium spp. Variable regions of the internal transcribed spacer of the rDNA were used to design species-specific primers for detection and quantification of nine Pythium spp. from soils in eastern Washington. Primer pairs were designed for Pythium abappressorium, P. attrantheridium, P. heterothallicum, P. irregulare group I, P. irregulare group IV, P. paroecandrum, P. rostratifingens, P. sylvaticum, and P. ultimum and used with real-time polymerase chain reaction. Standard curves were generated for each of the species using SYBR Green I fluorescent dye for detection of amplification. Seventy-seven isolates of Pythium were screened to confirm specificity of each primer set. DNA was extracted from soil and standard curves were generated for P. irregulare group I, P. irregulare group IV, and P. ultimum to correlate populations of each species in the soil with quantities of DNA amplified from the same soil. Examination of raw field soils revealed results similar to those observed in previous studies. This new technique for the quantification of Pythium spp. is rapid and accurate, and will be a useful tool in the future study of these pathogenic Pythium spp.

Root Diseases of Wheat and Barley During the Transition from Conventional Tillage to Direct Seeding.

The use of direct seeding (no-till) in place of tillage can reduce soil erosion and improve water infiltration. However, despite these improvements in soil quality, growers in the Pacific Northwest are reluctant to adopt direct seeding, partially because of fears of increased root diseases caused by Gaeumannomyces graminis var. tritici, Rhizoctonia spp., and Pythium spp. To examine the effect of the transition from conventional tillage to direct seeding, field plots were established at two locations. One site had been managed with direct seeding for 12 years, and the second had been conventionally tilled. Over 4 years, a portion of each plot was tilled or direct seeded, and planted to wheat or barley. Plants in the tilled plots had consistently more crown roots than plants in direct-seeded plots. Rhizoctonia root rot and yield did not differ between tillage types during the first 2 years of the study. However, in the third and fourth years of the transition to direct seeding, a higher incidence of Rhizoctonia root rot, increased hyphal activity of R. solani, and reduced yields were observed in direct-seeded plots. Populations of R. oryzae and Pythium spp., and incidence of take-all were the same for both management practices.

A New Method for the Quantification of Rhizoctonia solani and R. oryzae from Soil.

Rhizoctonia solani anastomosis group (AG) 8 and R. oryzae are important root pathogens on wheat and barley in the dryland production areas of the inland Pacific Northwest. R. solani AG-8 is difficult to isolate from root systems and quantify in soil because of slow growth and low population densities. However, both pathogens form extensive hyphal networks in the soil and can grow a considerable distance from a food base. A quantitative assay of active hyphae was developed, using wooden toothpicks as baits inserted into sample soils. After 2 days in soil, toothpicks were placed on a selective medium, and the numbers of colonies that grew after 24 h were counted under a dissecting microscope. R. solani and R. oryzae could be distinguished from other fungi based on hyphal morphology. This method was tested in natural soils amended with known inoculum densities of R. solani AG-8 and R. oryzae. Regressions were used to compare the inoculum density or toothpick colonization curves to a predicted curve based on the volume of the toothpicks. The slopes and y intercept of log-log transformed regressions did not differ from the predicted curves in most cases. This technique was used to assess the hyphal activity of R. solani AG-8 and R. oryzae from soil cores taken from various positions in and around Rhizoctonia bare patches at two locations. Activity of R. solani was highest in the center and inside edge of the patch, but there was no effect of patch position on R. oryzae. This simple and inexpensive technique can be used for detection and diagnosis in grower fields and to study the ecology and epidemiology of Rhizoctonia spp.

Genotypic and phenotypic diversity of phlD-containing Pseudomonas strains isolated from the rhizosphere of wheat.

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2, 4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.

Root-surface caries in rats and humans: inhibition by a non-antimicrobial property of tetracyclines.

The incidence of root caries has been found to increase as the population ages and as edentulism becomes less prevalent due to improved dental awareness and care, and as exposure of roots due to gingival recession has also increased in the elderly. The mechanism of root caries is thought to be mediated by both bacterial and mammalian proteases produced by plaque and the periodontal tissues, respectively. In the current study, a rat model of periodontal disease was used in which gnotobiotic rats were infected intra-orally with a periodontal pathogen (P. gingivalis). Infecting the rats with P. gingivalis increased the collagenase activity in the gingival tissue in association with severe alveolar bone loss. Treating P. gingivalis-infected rats with doxycycline or CMT-1 prevented the destruction of the periodontium by MMPs, thus preventing exposure of roots to subgingival bacterial plaque and host tissue collagenases and the subsequent development of root caries. In addition, a low-dose doxycycline (LDD, 20 mg bid, non-antimicrobial dose) for 3 months was used in humans predisposed to increased root caries as the result of heavy use of smokeless (chewing) tobacco, causing gingival recession, subgingival plaque accumulation with Gram-negative bacteria, increased gingival crevicular fluid flow (GCF), and elevated GCF collagenase. Daily administration of LDD in smokeless tobacco patients reduced the GCF collagenase and prevented the further development of root caries.

An epilogue to evaluating the impact of P.L. 99-252 on decreasing smokeless tobacco use.

In a previous paper, "Evaluating the Impact of P.L. 99-252 on Decreasing Smokeless Tobacco Use," the context of this law and the theoretical framework for an evaluation plan for measuring its impact were described. In this paper, the methodology and selected findings from this project as well as their implications are discussed. This discussion includes the identification of the six indicators considered to be the most relevant, valid, reliable, accessible, and practical for measuring the impact of this law on decreasing smokeless tobacco use, as well as a report on the feasibility analysis of three of these indicators. Pilot data on two indicators--pounds of smokeless tobacco sold and incidence rates of tobacco-induced leukoplakia--are presented and analyzed.

P.L. 99-252--implications for dentists and their clinical practice.

This paper is one of a series attempting to determine the impact of Public Law 99-252, the Comprehensive Smokeless Tobacco Health Education Act of 1986, on decreasing smokeless tobacco use. Potential indicators are discussed that could be used by the dental profession to determine the efficacy of the profession's involvement in existing and future prevention and cessation efforts relative to this law. In this review, six major areas are considered for measuring declines in smokeless tobacco use relative to the role of the dental health practitioner. These areas encompass the following: (1) the dental profession's knowledge of smokeless tobacco health hazards, (2) dental practitioners' involvement in measuring changes in use patterns, (3) the influence dentists have with their patients and community in decreasing smokeless tobacco use, (4) the general attitude and intention to quit on the part of dental patients, (5) active governmental involvement efforts, and (6) health effects of smokeless tobacco that might be used for identifying decreasing smokeless tobacco use. Within each of these six broad categories, more specific indicators for measuring the impact of P.L. 99-252 relative to the dental profession were considered.

An interpretative review of smokeless tobacco research in the United States: Part II.

This is the second part of a two-part series reviewing the published literature of smokeless tobacco in the United States. The article explores smokeless tobacco as a pharmacologically addicting substance, educational interventions designed to prevent use or help users quit, and outlines areas of future research.

Morphologic and immunohistochemical evidence of human papillomavirus capsid antigen in smokeless tobacco keratoses from juveniles and adults.

Seventy-seven tissue samples from patients with either degree I, II, or III smokeless tobacco keratoses were examined. The tissues were evaluated for the presence of human papillomavirus (HPV) antigen by immunocytochemical staining. Capsid antigen could be identified in sixteen cases (20.78%). This investigation further supports the concept that smokeless tobacco-associated leukoplakias can be correlated with HPV infection.

Proposed definition of a smokeless tobacco user based on "potential" nicotine consumption.

This study examined and operationally defined "light," "moderate," and "heavy" users of smokeless tobacco according to the potential amounts of nicotine consumed per week from chewing tobacco and/or snuff products. Fifty adult male smokeless tobacco users ranging in ages from 18-85 were tested to determine nicotine content of smokeless tobacco products consumed. The smokeless tobacco products were blindly analyzed by gas chromatography to determine nicotine content. Mean nicotine yield per week was calculated by multiplying the number of cans and/or pouches used per week and placed on a normal probability distribution. The mean nicotine yielded 238 mg/wk and the categories emerged as "light" less than or equal to 87 mg/wk, "moderate" 88-388 mg/wk and "heavy" users greater than or equal to 389 mg/wk. Differences regarding type of user (light, moderate, heavy) were crossed tabbed with educational levels, years of smokeless tobacco use, age the habit was initiated, perception of harm and whether habit forming, and reason for using smokeless tobacco.

Screening for smokeless tobacco-associated lesions: recommendations for the dental practitioner.

The increasing use of smokeless tobacco and the accumulating list of associated health hazards including cancer and cardiovascular effects should be of concern to the dentist. This study documents both concern from patients' perceptions and clinical examinations of their smokeless tobacco-associated lesions. Implications for the dentists' practice among adolescents and young adults are discussed.