Patricia A. Okubara

Engineering deoxynivalenol metabolism in wheat through the expression of a fungal trichothecene acetyltransferase gene

Abstract.Fusarium head blight occurs in cereals throughout the world and is especially important in humid growing regions. Fusarium head blight (FHB) has re-emerged as a major disease of wheat and barley in the U.S. and Canada since 1993. The primary causal agents of FHB, Fusarium graminearum and Fusarium culmorum, can produce deoxynivalenol (DON), a trichothecene mycotoxin that enhances disease severity and poses a health hazard to humans and monogastric animals. To reduce the effects of DON on wheat, we have introduced FsTRI101, a Fusarium sporotrichioides gene formerly known as TriR, into the regenerable cultivar Bobwhite. TRI101 encodes an enzyme that transfers an acetyl moiety to the C3 hydroxyl group of trichothecenes. Four different transgenic plants carrying the FsTRI101 gene were identified. Although expression levels varied among the four lines, all of them accumulated FsTRI101 transcripts in endosperm and glume. TRI101-encoded acetyltransferase activity was detected in endosperm extracts of a single plant that accumulated FsTRI101 mRNA. Greenhouse resistance tests indicated that the accumulation of FsTRI101-encoded acetyltransferase in this plant confers partial protection against the spread of F. graminearum in inoculated wheat heads (spikes).

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 Defense Responses to Fungal Pathogens: A Molecular Perspective

This review will focus on the molecular and genetic mechanisms underlying defense responses of roots to fungal pathogens. Soil-borne pathogens, including Phytophthora, Pythium, Fusarium, and Bipolaris, represent major sources of biotic stress in the rhizosphere and roots of plants. Molecular recognition and signaling leading to effective resistance has been demonstrated to occur between host and Phytophthora, or Pythium. The hypersensitive response and apoptotic cell death, two oxidative processes that limit biotrophic pathogens, generally act to exacerbate disease symptoms induced by necrotrophic organisms. Although pathogenesis-related proteins can be expressed in roots during pathogen challenge, salicylic acid has not been implicated in root-mediated interactions. Jasmonic acid and ethylene have been found to mediate parallel as well as synergistic pathways that confer partial tolerance to necrotrophic pathogens, as well as induced systemic resistance to root and foliar pathogens. Genomics approaches are revealing new networks of defense-signaling pathways, and have the potential of elucidating those pathways that are important in root-defense responses.

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.

Real-time polymerase chain reaction: applications to studies on soilborne pathogens

Real-time, or quantitative, polymerase chain reaction (Q-PCR), offers a rapid, sensitive, and specific method for the diagnosis of plant pathogens in soil, water, air, and plant samples. Fluorescence is used to monitor the accumulation of the PCR product after each PCR cycle. The fluorescence data are used to extrapolate the amount of target DNA present in the sample before amplification so that detection and quantification are achieved in a single assay. Detection limits are generally in the femtogram range for purified pathogen DNA, in the picogram range for pathogen DNA in plant samples, and one to several spores for soil samples. Quantification is reliable over a dynamic range of five to seven orders of magnitude of target DNA, in which differences of two fold or more in target DNA can be detected. Real-time PCR has already been recognized as a valuable tool for epidemiological studies, disease management, detection of emerging pathogens, evaluation of resistance and tolerance to disease, and phytosanitary screens. In this review, we compare four major real-time chemistries that have been used for the detection and quantification of soilborne plant pathogens and discuss the advantages and limitations of TaqMan™ and SYBR™ green I, the most widely used chemistries. Current applications of real-time PCR to the diagnosis of plant pathogens in soil and plant samples, considerations for assay development, and variations in measurements are also discussed

Detection and discrimination of Pratylenchus neglectus and P. thornei in DNA extracts from soil

A species-specific polymerase chain reaction (PCR) method was developed to detect and identify the root-lesion nematodes Pratylenchus neglectus and P. thornei from soil. A primer set was designed from Pratylenchus 28S rRNA gene sequences of the D3 expansion domain. Primer specificity was confirmed with 23 isolates of 15 nematode species and other plant-parasitic and non-plant-parasitic nematodes typically present in the soil communities, and with six fungal species commonly associated with wheat root rot. DNA obtained using a commercially available kit and a method developed in our laboratory gave comparable amplification. PCR conditions were optimized and the two species were differentiated by PCR products of 144 bp for P. neglectus and 288 bp for P. thornei. With this assay, we detected a single juvenile in 1 g of sterile, inoculated soil. Examination of 30 field soil samples revealed that this method was applicable to a range of soils naturally infested with these two pathogens in Oregon. This PCR-based method is rapid, efficient, and reliable, does not require expertise in nematode taxonomy and morphology, and could be used as a rapid diagnostic tool for commercial and research applications for disease forecasting and management.

Detection and Quantification of Pratylenchus thornei in DNA Extracted from Soil Using Real-Time PCR

The root-lesion nematode Pratylenchus thornei is one of the most important pests restricting productivity of wheat in the Pacific Northwest (PNW). It is laborious and difficult to use microscopy to count and identify the nematodes in soils. A SYBR Green I-based real-time polymerase chain reaction (PCR) assay was developed to detect and quantify this species from DNA extracts of soil. A primer set, designed from the internal transcribed spacer region (ITS1) of rDNA, was highly specific to P. thornei and did not amplify DNA from 27 isolates of other Pratylenchus spp., other nematodes, and six fungal species present in PNW wheat fields. A standard curve relating threshold cycle and log values of nematode number was generated from artificially infested soils. The standard curve was supported by a high correlation between the numbers of P. thornei added to soil and the numbers quantified using real-time PCR. Examination of 15 PNW dryland field soils and 20 greenhouse samples revealed significant positive correlations between the numbers determined by real-time PCR and by the Whitehead tray and microscopic method. Real-time PCR is a rapid, sensitive alternative to time-consuming nematode extractions, microscopic identification, and counting of P. thornei from field and greenhouse soils.

Developing a Real-Time PCR Assay for Detection and Quantification of Pratylenchus neglectus in Soil

Pratylenchus neglectus is one of the most widespread and economically important nematodes that invades plant roots and restricts wheat productivity in the Pacific Northwest. It is challenging to quantify P. neglectus using microscopic methods for studies that require large-scale sampling, such as assessment of rotation crops, wheat cultivars, and other management practices. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. neglectus from DNA extracts of soil. The primers, designed from the internal transcribed spacer region of rDNA, showed high specificity with a single melt curve peak to DNA from eight isolates of P. neglectus but did not amplify DNA from 28 isolates of other plant-parasitic and non-plant-parasitic nematodes. A standard curve (R2 = 0.96; P < 0.001) was generated by amplifying DNA extracted from soil to which nematodes were added. The soil standard curve was validated using sterilized soil inoculated with lower numbers of P. neglectus. A significant positive relationship (R2 = 0.66; P < 0.001) was observed for nematode numbers quantified from 15 field soils using qPCR and the Whitehead tray and microscopic method but the qPCR generally tended to provide higher estimates. Real-time PCR potentially provides a useful platform for efficient detection and quantification of P. neglectus directly from field soils.

Molecular and genetic aspects of controlling the soilborne necrotrophic pathogens Rhizoctonia and Pythium

The soilborne necrotrophic pathogens Rhizoctonia and Pythium infect a wide range of crops in the US and worldwide. These pathogens pose challenges to growers because the diseases they cause are not adequately controlled by fungicides, rotation or, for many hosts, natural genetic resistance. Although a combination of management practices are likely to be required for control of Rhizoctonia and Pythium, genetic resistance remains a key missing component. This review discusses the recent deployment of introduced genes and genome-based information for control of Rhizoctonia, with emphasis on three pathosystems: Rhizoctonia solani AG8 and wheat, R. solani AG1-IA and rice, and R. solani AG3 or AG4 and potato. Molecular mechanisms underlying disease suppression will be addressed, if appropriate. Although less is known about genes and factors suppressive to Pythium, pathogen genomics and biological control studies are providing useful leads to effectors and antifungal factors. Prospects for resistance to Rhizoctonia and Pythium spp. will continue to improve with growing knowledge of pathogenicity strategies, host defense gene action relative to the pathogen infection process, and the role of environmental factors on pathogen-host interactions.

An In vitro Study of Bio-Control and Plant Growth Promotion Potential of Salicaceae Endophytes

Microbial communities in the endosphere of Salicaceae plants, poplar (Populus trichocarpa) and willow (Salix sitchensis), have been demonstrated to be important for plant growth promotion, protection from biotic and abiotic stresses, and degradation of toxic compounds. Our study aimed to investigate bio-control activities of Salicaceae endophytes against various soil borne plant pathogens including Rhizoctonia solani AG-8, Fusarium culmorum, Gaeumannomyces graminis var. tritici, and Pythium ultimum. Additionally, different plant growth promoting traits such as biological nitrogen fixation (BNF), indole-3-acetic acid (IAA) biosynthesis, phosphate solubilization, and siderophore production were assessed in all bio-control positive strains. Burkholderia, Rahnella, Pseudomonas, and Curtobacterium were major endophyte genera that showed bio-control activities in the in-vitro assays. The bio-control activities of Burkholderia strains were stronger across all tested plant pathogens as compared to other stains. Genomes of sequenced Burkholderia strains WP40 and WP42 were surveyed to identify the putative genes involved in the bio-control activities. The ocf and hcnABC gene clusters responsible for biosynthesis of the anti-fungal metabolites, occidiofungin and hydrogen cyanide, are present in the genomes of WP40 and WP42. Nearly all endophyte strains showing the bio-control activities produced IAA, solubilized tricalcium phosphate, and synthesized siderophores in the culture medium. Moreover, some strains reduced acetylene into ethylene in the acetylene reduction assay, a common assay used for BNF. Salicaceae endophytes could be useful for bio-control of various plant pathogens, and plant growth promotion possibly through the mechanisms of BNF, IAA production, and nutrient acquisition.