Guiping Yan

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.

Distinguishing Heterodera filipjevi and H. avenae Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism and Cyst Morphology

The cereal cyst nematodes Heterodera filipjevi and H. avenae impede wheat production in the Pacific Northwest (PNW). Accurate identification of cyst nematode species and awareness of high population density in affected fields are essential for designing effective control measures. Morphological methods for differentiating these species are laborious. These species were differentiated using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS)-ribosomal (r)DNA with up to six restriction endonucleases (TaqI, HinfI, PstI, HaeIII, RsaI, and AluI). The method was validated by inspecting underbridge structures of cyst vulval cones. Grid soil sampling of an Oregon field infested by both species revealed that H. filipjevi was present at most of the infested grid sites but mixtures of H. avenae and H. filipjevi also occurred. These procedures also detected and differentiated H. filipjevi and H. avenae in soil samples from nearby fields in Oregon and H. avenae in samples from Idaho and Washington. Intraspecific polymorphism was not observed within H. filipjevi or PNW H. avenae populations based on the ITS-rDNA. However, intraspecific variation was observed between H. avenae populations occurring in the PNW and France. Methods described here will improve detection and identification efficiencies for cereal cyst nematodes in wheat fields.

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.

Effect of Foliarly Applied Spirotetramat on Reproduction of Heterodera avenae on Wheat Roots

The cereal cyst nematode, Heterodera avenae, has the potential to reduce yields of cereal crops in the Pacific Northwest. Spirotetramat (Movento) is a foliar-applied insecticide with ambimobile translocation that reduces fecundity of sucking insects which feed on roots as well as foliage. Spirotetramat (88 g/ha) was applied to foliage during 2010 in two wheat fields infested by H. avenae near St. Anthony, ID and Palouse, WA. In Idaho, two applications at 2-week intervals during late spring to plants already exhibiting swollen white females reduced the postharvest density of H. avenae eggs plus juveniles by 35% (P = 0.03) compared to the nontreated control. In Washington, a single application before white females became apparent reduced the nematode density by 78% (P = 0.01). Grain yields and test weights were not significantly affected by application of spirotetramat at either location. In addition, symptomatic plants from the Idaho field were transplanted into greenhouse pots and treated with spirotetramat. One application (110 g/ha) reduced numbers of eggs plus juveniles/plant by 78% (P = 0.02). Spirotetramat effectively reduced H. avenae populations and warrants further evaluation as a substitute for crop rotations or long fallow periods that reduce nematode population densities in infested fields.

Species-Specific PCR Assays for Differentiating Heterodera filipjevi and H. avenae

Heterodera avenae and H. filipjevi are economically important cyst nematodes that restrict production of cereal crops in the Pacific Northwest United States and elsewhere in the world. Identification of these two species is critical for recommending and implementing effective management practices. Primers were designed from the internal transcribed spacer (ITS) regions of H. avenae and H. filipjevi ribosomal DNA. The primers were highly specific when examined on target isolates but did not amplify DNA from nontarget Heterodera, Globodera, Meloidogyne, Pratylenchus, and other nematode species tested. Polymerase chain reaction (PCR) and amplification conditions were established, and H. avenae and H. filipjevi were clearly distinguished by PCR fragments of 242 and 170 bp, respectively. Robust PCR amplification was achieved with DNA extracted from a single egg or second-stage juvenile (J2) using a laboratory-made worm lysis buffer, and DNA from 0.5 egg or J2 using a commercial kit. The PCR assays were successfully employed for differentiation of H. filipjevi and H. avenae populations collected from eight locations in three Pacific Northwest states. This is the first report of a species-specific ITS PCR assay to detect and identify H. filipjevi. The assays for both species will enhance diagnosis of cereal cyst nematode species in infested fields.

Effects of Crop Rotations and Tillage on Pratylenchus spp. in the Semiarid Pacific Northwest United States

There is interest in converting rainfed cropping systems in the Pacific Northwest from a 2-year rotation of winter wheat and cultivated fallow to direct-seed (no-till) systems that include chemical fallow, spring cereals, and food legume and brassica crops. Little information is available regarding effects of these changes on plant-parasitic nematodes. Eight cropping systems in a low-precipitation region (<330 mm) were compared over 9 years. Each phase of each rotation occurred each year. The density of Pratylenchus spp. was greater in cultivated than chemical fallow, became greater with increasing frequency of host crops, and was inversely associated with precipitation (R2 = 0.92, α < 0.01). Densities after harvesting mustard, spring wheat, winter wheat, and winter pea were greater (α < 0.01) than after harvesting spring barley or spring pea. Camelina also produced low densities. Winter wheat led to a greater density of Pratylenchus neglectus and spring wheat led to a greater density of P. thornei. Density of Pratylenchus spp. was correlated (R2 = 0.88, α < 0.01) but generally higher when detected by real-time polymerase chain reaction on DNA extracts from soil than when detected by a traditional method. Selection of different Pratylenchus spp. by different wheat cultivars or growth habit must be addressed to minimize the level of nematode risk to future plantings of intolerant crops.

Influence of Nematicides and Fungicides on Spring Wheat in Fields Infested with Soilborne Pathogens

A complex of fungal soilborne pathogens and plant-parasitic nematodes reduces wheat yields in the Pacific Northwest. On several other crops in nematode-infested soils, seed treatment with abamectin (Avicta) or Bacillus firmus (Votivo) or foliar application of spirotetramat (Movento) reduced root injury and improved yield. These products, along with fungicide seed treatments and aldicarb (Temik), were evaluated in 13 spring wheat trials over 3 years. During 2011, the mean wheat yield at four locations was 419 kg/ha greater (valued at

Influence of Semiarid Cropping Systems on Root Diseases and Inoculum Density of Soilborne Pathogens

122/ha) from seed treated with fungicides and insecticide than from untreated seed, due to protection against soilborne fungal pathogens. Aldicarb increased the mean grain yield over the fungicide-plus-insecticide treatment by another 798 kg/ha (valued at

First Report of the Stubby Root Nematode Paratrichodorus allius on Potato in North Dakota

254/ha) and also reduced the density of Heterodera avenae but is not registered for use on wheat. Abamectin and B. firmus had negligible effects on grain yield and postharvest density of Pratylenchus spp. and H. avenae. Spirotetramat reduced density of H. avenae but did not improve grain yield. We conclude that management of fungal pathogens by seed protectants remains essential and that management of nematodes can be achieved through crop rotations and genetic resistance.