Development of a Species-Specific SCAR-PCR Assay for Direct Detection of Sugar Beet Cyst Nematode (Heterodera schachtii) from Infected Roots and Soil Samples

Sugar beet cyst nematode (SBCN, Heterodera schachtii) is an important nematode that causes significant yield losses of 25–50% or more in most areas of sugar beet production worldwide. Rapid and accurate identification of this species is essential to support decisions on pest management. However, the difference between H. schachtii and other Heterodera spp. based on morphology is a challenging task. In the present study, a SCAR-PCR assay was developed to identify and differentiate H. schachtii in infected root and soil samples. H. schachtii-species-specific SCAR-PCR primers OPA06-HsF and OPA06-HsR were designed from the randomly amplified polymorphic DNA (RAPD) marker amplified with random primer OPA06. The developed primers specifically amplify a 922-bp fragment from the target populations but did not amplify DNA from non-target cyst nematodes including Heterodera, Globodera, Cactodera, and other related species tested in this study. The sensitivity detection indicated that 5 × 10−4 of a single cyst, 1/320 of a single second-stage juvenile (J2), or 10 pg of genomic DNA could be detected. The assay accurately identifies the different stages of H. schachtii in sugar beet and oilseed rape roots as well as a single J2 in 10 g of soil. Finally, the SCAR-PCR assay detected H. schachtii in seven samples out of the fifteen field samples. The assay will not only be useful for differentiating H. schachtii from mixed populations of Heterodera spp. but also for effective detection of the species directly from infested samples. The assay also requires no expertise in the taxonomy and morphology of the species but serves to improve the diagnosis of H. schachtii in infested fields.

Developing a Real-time PCR Assay for Direct Identification and Quantification of Soybean Cyst Nematode, Heterodera glycines, in Soil and Its Discrimination from Sugar Beet Cyst Nematode, Heterodera schachtii

The soybean cyst nematode (SCN) Heterodera glycines continues to be a major threat to soybean production worldwide. Morphological discrimination between SCN and other nematodes of the H. schachtii sensu stricto group is not only difficult and time-consuming, but also requires high expertise in nematode taxonomy. Molecular assays were developed to differentiate SCN from sugar beet cyst nematode (SBCN) and other nematodes; and to quantify SCN directly from DNA extracts of field soils. SCN and SBCN-specific quantitative real-time PCR (qPCR) primers were designed from a nematode-secreted CLAVATA gene and used for these assays. The primers were evaluated based on specificity, efficiency, and target specificity to SCN or SBCN using DNA from 20 isolates of SCN and 32 isolates of other plant-parasitic nematodes. A standard curve relating threshold cycle and log values of nematode numbers was generated from artificially infested soils and was used to quantify SCN in naturally infested field soils. There was a high correlation between the SCN numbers estimated from naturally infested field soils by conventional methods, and the numbers quantified using the SYBR Green I-based qPCR assay. The qPCR assay is highly specific and sensitive and provides improved SCN detection sensitivity down to 1 SCN egg in 20 g of soil (10 eggs/200 g soil). This assay is useful for efficient detection and quantification of SCN directly from field soil. Species-specific conventional PCR assays were also developed each for SCN and SBCN, alongside a qPCR assay that simultaneously discriminates SCN from SBCN. These assays require no expertise in nematode taxonomy and morphology, and may serve as useful diagnostic tools in research, diagnostic labs, and extension services for SCN management. Sensitive and accurate detection and quantification of SCN are essential for recommending effective management measures against SCN. We also investigated the impact of soil texture and nematode life stage on molecular quantification of SCN.

Conserved Opposite Functions in Plant Resistance to Biotrophic and Necrotrophic Pathogens of the Immune Regulator SRFR1

Plant immunity is mediated in large part by specific interactions between a host resistance protein and a pathogen effector protein, named effector-triggered immunity (ETI). ETI needs to be tightly controlled both positively and negatively to enable normal plant growth because constitutively activated defense responses are detrimental to the host. In previous work, we reported that mutations in SUPPRESSOR OF rps4-RLD1 (SRFR1), identified in a suppressor screen, reactivated EDS1-dependent ETI to Pseudomonas syringae pv. tomato (Pto) DC3000. Besides, mutations in SRFR1 boosted defense responses to the generalist chewing insect Spodoptera exigua and the sugar beet cyst nematode Heterodera schachtii. Here, we show that mutations in SRFR1 enhance susceptibility to the fungal necrotrophs Fusarium oxysporum f. sp. lycopersici (FOL) and Botrytis cinerea in Arabidopsis. To translate knowledge obtained in AtSRFR1 research to crops, we generated SlSRFR1 alleles in tomato using a CRISPR/Cas9 system. Interestingly, slsrfr1 mutants increased expression of SA-pathway defense genes and enhanced resistance to Pto DC3000. In contrast, slsrfr1 mutants elevated susceptibility to FOL. Together, these data suggest that SRFR1 is functionally conserved in both Arabidopsis and tomato and functions antagonistically as a negative regulator to (hemi-) biotrophic pathogens and a positive regulator to necrotrophic pathogens.

Sugar beet nematode Heterodera schachtii distribution and harmfulness in Ukraine

Aim. To determine the distribution of sugar beet cyst nematode Heterodera schachtii in Ukraine, to defi ne its population density in soil, the degree of damage and the effi ciency of control measures. Methods. Field survey for soil samples, cyst extraction by fl otation and sieving method, light microscopy. Results. The examination of 12,130 ha of agricultural fi elds in six regions of Ukraine, conducted in 2010–2015, recorded the sugar beet cyst nematode to occur in fi ve of them (Kyiv, Chernihiv, Cherkasy, Khmelnytsky, and Vinnytsia), at a total area of 2,572 ha. Taking into consideration the results of previous nematological surveys, the presence of sugar beet cyst nematode has now been documented for 18 regions of Ukraine. In most regions H. schachtii was present in 10–11 % of the examined sugar and seed producing farms with nematode population densities reaching up to 142,000 eggs and juveniles in 100 cc of soil (710-fold above the economic threshold). Conclusions. The main reasons for this wide distribution of H. schachtii in Ukraine are seen in the negligence of prevention measures, unavailability of documented data on its occurrence (missing surveys), crop rotations with over 20 % of host plants, and unavailability of effi cient nematicides and domestic nematode-resistant sugar beet cultivars.