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

Chen Jiang,Huan Peng,Kemei Li,Yingdong Zhang,Guangkuo Li,Deliang Peng,Ke Yao,Sulaiman Abdulsalam,Wenkun Huang,Haifeng Gao,Lingan Kong

doi:10.3390/life11121358

Abstract

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.

Highlights

This study aimed to develop a sensitive and specific sequence characterized amplified regions (SCAR)-PCR assay based on randomly amplified polymorphic DNA (RAPD) fragments for the direct detection of H. schachtii from infected plant root and soil samples
The Blastn results indicated that this sequence was similar to H. schachtii genomic sequences (GenBank accession No JAHGVF010000211.1) and H. glycines genomic DNA sequences (VAPQ01000257.1) with 99.24% and 86.35% identity, respectively
Microscopic observations show that H. schachtii becomes parasitic during the following growth stages: J2 at third stage (J3) and adult females at 3, 9, and 15 days after inoculation (DAI), respectively. These diagnostic observations reported in this study indicate that SBCN could be detected at different growth stages in sugar beet and oilseed rape roots

Summary

Introduction

Sugar beet cyst nematode (SBCN, Heterodera schachtii, Schmidt, 1871) is an economically important plant-parasitic nematode that limits the production of sugar beet across the world [1,2]. It was first identified in Germany in 1859 by Schachti as a plant pathogen when it caused yield reduction in sugar beet cultivation. The nematode has a wide host range, infecting over 218 plant species belonging to 95 genera and 23 different 4.0/). Plant families [5,6]. It was reported to cause yield losses of 25–50% or more in sugar beet and other crops grown [7]

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