The cereal cyst nematode, Heterodera avenae, occurs in at least seven western states of the USA and reduces grain yield in localised regions and in selected crop management systems. Virulence phenotypes for H. avenae populations in North America have not been reported. Nine individual assays in six experiments were conducted to determine the reactions of barley, oat and wheat cultivars to five H. avenae populations in the Pacific Northwest (PNW) states of Idaho, Oregon and Washington. Three populations were evaluated for virulence to 23 entries of the 'International Test Assortment for Defining Cereal Cyst Nematode Pathotypes', plus selected local cultivars and entries representing a greater diversity of resistance genes. The virulence phenotype(s) for populations of H. avenae in the PNW did not correspond to any of the 11 pathotypes defined by the Test Assortment. Five PNW populations exhibited affinities with Group 2 but were not defined by pathotypes Ha12 and Ha22. Reproduction was prevented or greatly inhibited by barley carrying the Rha3 resistance gene and by most carriers of Rha2 resistance, and by selected oat cultivars with multigenic resistance. Wheat cultivars carrying the Cre1 resistance gene were highly effective in suppressing H. avenae reproduction. Current PNW wheat cultivars do not carry the Cre1 resistance gene. Crosses between Ouyen, an Australian bread wheat with Cre1 resistance, and several PNW wheat cultivars were resistant. The CreR gene also prevented H. avenae reproduction in the trial where it was tested. Intermediate levels of reproduction occurred on wheat cultivars carrying the Cre5, Cre7 and Cre8 resistance genes, each of which was considered useful for pyramiding into cultivars with Cre1 resistance. This research identified genetic resources of value in PNW cereal crop breeding programmes.
The endomigratory root-lesion nematode, Pratylenchus scribneri, is one of the major plant-parasitic nematodes infecting potato. Accurate identification and quantification of this nematode are essential to develop management strategies but microscopic observations are particularly challenging and time consuming. In this study, a SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. scribneri from field soil DNA extracts. A primer set was designed from the internal transcribed spacer (ITS) region of the P. scribneri rDNA gene. Primer specificity to the target nematode was evaluated by both in silico analysis and qPCR and no detection or non-specific amplification was observed for other non-target nematode species/communities tested in this study. Standard curves were generated using DNA extracts from autoclaved soil infested with varying nematode numbers for calibration. The curves were supported by a high correlation between the P. scribneri numbers artificially added to soil or estimated from naturally infested field soils by traditional methods, and the numbers quantified using the qPCR assay. The assay was able to detect 1 out of 128 (0.0078) equivalents of the DNA of a single nematode in 0.5 g of soil. The qPCR assay developed in this study provides a specific and sensitive detection and quantification of P. scribneri from field soils and a rapid alternative to time-consuming traditional nematode identification and enumeration.
The ectoparasitic stubby root nematode, Paratrichodorus allius, transmits tobacco rattle virus, which causes corky ringspot disease resulting in significant economic losses in the potato industry. A diagnostic method for direct quantification of P. allius from soil DNA using TaqMan probe and SYBR Green real-time PCR assays was developed to assist the potato industry in management of this important vector. Specificity of primers/probe designed from the internal transcribed spacer of ribosomal DNA of P. allius was demonstrated by in silico analysis and experimental PCR tests with no cross reactions using non-target nematode species and nematode communities. The SYBR Green method was more sensitive than the TaqMan probe method during detection using serial diluted DNA templates. Standard curves were generated from serial dilutions of DNA extracted from autoclaved soil with artificially inoculated P. allius individuals and were validated by high correlations between the numbers of target nematodes quantified by the assays and added to the soil. Moreover, the numbers of P. allius determined by the real-time PCR assays and estimated by the microscopic method in 17 field soil samples presented positive correlation relationships (). Although the quantification using TaqMan probe overestimated the target nematodes compared to using SYBR Green in eight out of ten field soil samples, results of the two methods correlated well (). This is the first report of P. allius quantification from soil DNA extracts using real-time PCR, providing a rapid and sensitive diagnostic method obviating time-consuming manual nematode extraction from soil and microscopic identification and quantification.
Plant-parasitic nematodes restrict crop growth and cause yield loss in field pea (dry edible pea). A 4-year survey of commercial pea fields was conducted in North Dakota, one of the leading producers of field pea in the USA, to investigate nematode distribution, prevalence, abundance and association with soil properties. Beginning in 2014, a total of 243 soil samples were collected from 16 counties, and soil properties of 115 samples were analysed to determine the association of nematodes with soil factors (texture, organic matter, nutrients). The plant-parasitic nematode genera, Paratylenchus (absolute frequency = 58-100%; mean density = 470-1550 (200 g soil)−1; greatest density = 7114 (200 g soil)−1) and Tylenchorhynchus (30-80%; 61-261; 1980, respectively), were the most frequent and widely distributed. Pratylenchus and Helicotylenchus were identified in one-third of the counties surveyed with mean densities ranging from 43 to 224 and 36 to 206 (200 g soil)−1, respectively. Xiphinema was found relatively frequently but at low densities. Hoplolaimus and Paratrichodorus were rarely detected at lower densities. Canonical correspondence analysis revealed that soil factors explained 19% of the total variance of nematode genera abundance. The relationship between nematode abundance and soil sand content and pH was significant, while clay, silt, organic matter and nutrients were not significantly related to nematode abundance. This is the first multi-year study investigating nematodes associated with field peas and their relationship with soil factors in a major field pea production region of the USA.