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.

The beet cyst nematode (Heterodera schachtii): an ancient threat to sugar beet crops in Central Europe has become an invisible actor.

The beet cyst nematode (BCN) was one of the first discovered plant parasitic nematodes. Heterodera schachtii was observed in 1859 in Halle in Central Germany by the botanist Herman Schacht and described later by Adolf Schmidt in 1871, who named this cyst nematode species after its original discoverer. Partly due to the lack of knowledge about the effect of sugar beet monocultures on the population build-up of BCN, this nematode had a devastating impact on sugar production in 1876 that led to the shutdown of 24 sugar factories in Germany. This chapter discusses the economic importance, distribution, host range, damage symptoms, biology and life cycle, interactions with other nematodes and pathogens, recommended integrated management, and management optimization of H. schachtii. Future research requirements and future developments are also mentioned.

Molecular insights into the compatible and incompatible interactions between sugar beet and the beet cyst nematode

Background Sugar beet (Beta vulgaris subsp. vulgaris) is an economically important crop that provides nearly one third of the global sugar production. The beet cyst nematode (BCN), Heterodera schachtii, causes major yield losses in sugar beet and other crops worldwide. The most effective and economic approach to control this nematode is growing tolerant or resistant cultivars. To identify candidate genes involved in susceptibility and resistance, the transcriptome of sugar beet and BCN in compatible and incompatible interactions at two time points was studied using mRNA-seq. Results In the susceptible cultivar, most defense-related genes were induced at 4 dai while suppressed at 10 dai but in the resistant cultivar Nemakill, induction of genes involved in the plant defense response was observed at both time points. In the compatible interaction, alterations in phytohormone-related genes were detected. The effect of exogenous application of Methyl Jasmonate and ET-generator ethephon on susceptible plants was therefore investigated and the results revealed significant reduction in plant susceptibility. Genes putatively involved in the resistance of Nemakill were identified, such as genes involved in phenylpropanoid pathway and genes encoding CYSTM domain-containing proteins, F-box proteins, chitinase, galactono-1,4-lactone dehydrogenase and CASP-like protein. Also, the transcriptome of the BCN was analyzed in infected root samples and several novel potential nematode effector genes were found. Conclusions Our data provides detailed insights into the plant and nematode transcriptional changes occurring during compatible and incompatible interactions between sugar beet and BCN. Many important genes playing potential roles in susceptibility or resistance of sugar beet against BCN, as well as some BCN effectors with a potential role as avr proteins were identified. In addition, our findings indicate the effective role of jasmonate and ethylene in enhancing sugar beet defense response against BCN. This research provides new molecular insights into the plant-nematode interactions that can be used to design novel management strategies against BCN.

Cyst Nematode Infection Elicits Alteration in the Level of Reactive Nitrogen Species, Protein S-Nitrosylation and Nitration, and Nitrosoglutathione Reductase in Arabidopsis thaliana Roots

Reactive nitrogen species (RNS) are redox molecules important for plant defense against pathogens. The aim of the study was to determine whether the infection by the beet cyst nematode Heterodera schachtii disrupts RNS balance in Arabidopsis thaliana roots. For this purpose, measurements of nitric oxide (NO), peroxynitrite (ONOO−), protein S-nitrosylation and nitration, and nitrosoglutathione reductase (GSNOR) in A. thaliana roots from 1 day to 15 days post-inoculation (dpi) were performed. The cyst nematode infection caused generation of NO and ONOO− in the infected roots. These changes were accompanied by an expansion of S-nitrosylated and nitrated proteins. The enzyme activity of GSNOR was decreased at 3 and 15 dpi and increased at 7 dpi in infected roots, whereas the GSNOR1 transcript level was enhanced over the entire examination period. The protein content of GSNOR was increased in infected roots at 3 dpi and 7 dpi, but at 15 dpi, did not differ between uninfected and infected roots. The protein of GSNOR was detected in plastids, mitochondria, cytoplasm, as well as endoplasmic reticulum and cytoplasmic membranes. We postulate that RNS metabolism plays an important role in plant defense against the beet cyst nematode and helps the fine-tuning of the infected plants to stress sparked by phytoparasitic nematodes.

Effect of Winter Oilseed Rape Cropping on the Development of the Sugar Beet Cyst Nematode, Heterodera schachtii, and Control of Volunteer Plants as a Trap Crop Method

The integration of oilseed rape (OSR) into sugar beet rotation systems is restricted due to the very good host status of OSR for the beet cyst nematode (BCN) Heterodera schachtii. In contrast to sugar beet, the cultivation of winter OSR covers a longer period, but at a lower soil temperature regime. Thus, presumably one or two generations of BCN may develop during the cultivation of winter OSR, resulting in moderate multiplication rates of 1–2 in the present study. This multiplication rate was year-dependent, but not affected by different sowing times. For the first time, the present study identified volunteer OSR emerging in high densities post-harvest as a major risk for a high multiplication of BCN at optimum temperatures. The emergence of BCN females with offspring was observed very early, resulting in a significant population increase before 350-degree days (>8 °C) in inoculation experiments and in field investigations. Conducting treatment trials with glyphosate to control volunteer OSR in micro-plots and field experiments confirmed effective suppression of BCN reproduction when growth of volunteer OSR was interrupted at 250–350-degree days. Thus, data gained from BCN reproduction studies under controlled and field conditions provided a unique basis for the development of a trap crop method. The degree day model has been successfully implemented as part of an open access management tool.