Molecular and morphological characterization of Globodera rostochiensis (Wollenweber, 1923) Skarbilovich, 1959 from Egypt

The golden potato cyst nematode, Globodera rostochiensis (Wollenweber, 1923) Skarbilovich (1959) is a damaging soilborne quarantine pest of Solanum tuberosum (potato) and other solanaceous crops worldwide. In spring of 2021 a survey was conducted in area of Abo El Matamer, Bahera governorates in Egypt. Soil samples were taken in zigzag pattern throughout 65 acres of potato cultivated land and processed in Nematology lab, Fayoum University, Egypt. In June 2021, two hundred soil samples were collected from nearby areas to evaluate the distribution of this potato cyst nematode in other cultivated land located in area of first infection but fortunately the golden potato cyst nematode was not detected from neighboring locations. The nematode species was identified by both morphological and molecular means as Globodera rostochiensis. To our knowledge this is the first molecular and morphological characterization of G. rostochiensis from Egypt.


Introduction Introduction Introduction Introduction
Golden nematode, Globodera rostochiensis, is one of the three potato cyst nematodes (PCN) which are economically important pests of potato and have a worldwide distribution (Gartner et al., 2021). Globodera pallida and G. rostochiensis can cause potato crop losses of approximately 9% worldwide (Turner and Subbotin, 2013). Cyst nematodes are one of the most important pest groups of economically important crop plants in Egypt (Ibrahim et al., 2017). A survey conducted over a period of four years in three governorates of Alexandria, El Behera and Sohag by Ibrahim et al. (2017), found and identified G. rostochiensis along with other cyst nematodes. The detection of the golden cyst nematode G. rostochiensis on potato in El-Nobarria, El-Behera governorate in northern Egypt is very important as this nematode species has been considered as a serious and a potential pest on potato and other solanaceous vegetable crops (Ibrahim et al., 2017). However, that study did not provide any morphological or molecular characterization of G. rostochiensis. In the present study, the surveyed area included the Abo El Matamer and the neighboring areas located in Behera governorate.

Materials and Methods Materials and Methods Materials and Methods Materials and Methods
A nematode survey was conducted between April and May of 2021 in the Behera governorate. Two hundred samples (250 g/each) were collected randomly in a zigzag pattern from about 65-acre field cultivated with potato. The samples were processed by the Nematology lab, Fayoum University, and the cysts were extracted from the soil samples using centrifuge floatation techniques (Ayoub, 1980). Juveniles were fixed in 3% formaldehyde and processed to glycerin by the formalin glycerin method (Hooper, 1970;Golden, 1990). Females and some cysts were typically removed from roots after fixation for 12 hours in 3% formaldehyde solution. Photomicrographs of the specimens were made with a Nikon Eclipse Ni compound microscope using a Nikon DS-Ri2 camera. Measurements were made with an ocular micrometer on a Leica WILD MPS48 Leitz DMRB compound microscope. All measurements are in micrometers unless otherwise stated. Living nematode juveniles (J2) recovered from the cysts were examined morphologically and molecularly for species identification and by using key to species by (Golden, 1986;Subbotin et al., 2010). DNA was extracted from three specimens using the worm smash and proteinase K protocols as described by Skantar et al. (2020). Mitochondrial cytochrome oxidase I (COI) was amplified with primers Het-Cox1F (5'-TAGTTGATCGTAATTTTAATGG -3') and Het-Cox1R (5'-CCTAAAACATAATGAAAATGWGC -3') as described in the study of Subbotin et al. (2017). The following thermal profile was used for COI gene amplification: 4 min at 94 °C, followed by 40 cycles of 1 min at 94 °C, 1 min at 45 °C and 1 min 30s at 72 °C, with a final extension at 72 °C for 10 min. PCR amplicons of 550 bp were cleaned with the Monarch DNA Gel Extraction Kit (NEB, Ipswitch, MA) and sequenced directly with the same primers at Genewiz, Inc. The newly generated DNA sequence was submitted to GenBank under accession number MZ570875.

Results and Discussion and Discussion and Discussion and Discussion
Measurements Cysts (n = 5) were light brown in color, spherical to subspherical shape with a protruding neck, circumfenestrate. Anal area prominent. Vulval region intact or fenestrated with a single circumfenestrate opening. Vulval bridge and under bridge present. No bullae. Cyst wall patterns were ridge-like to wavy lines.
Cysts body length without neck ranged from 600-795 μm with a mean of 669 μm. The fenestra diameter range was between 16-20μm, with a mean 18.2μm. Distance from edge of fenestra to anal area was 70.0-95.0 μm with a mean of 79.0 μm. Number of cuticular ridges between anus and fenestra edge were 17-21, with a mean of 19.5 and Granek's ratio was 3.5 to 5.4, with a mean of 4.6. Measurements of second-stage juveniles (J2) from Egypt (n = 15) included length of body tapering at both ends with more so in the posterior region (range = 463-500 μm, mean = 463 μm), stylet short, well developed (20.0-21.5 μm, 20.7μm) with rounded basal knobs, lateral field with four lines. Tail (43.0-56.0 μm, 50.6 μm), tapering to bluntly rounded terminus and hyaline tail terminus measures (16.0-32.0 μm, 23.2 μm). Morphometrics of J2's including shapes of the tail, tail terminus, stylet knobs and cyst morphometrics and morphology were consistent with G. rostochiensis as given in description and or re-descriptions (Golden and Ellington, 1972;Mulvey and Golden, 1983;Subbotin et al., 2010).
Morphological characters used for identification included cyst shape, characteristics of cyst terminal cone including nature of fenestration, cyst wall pattern, anal-vulval distance, number of cuticular ridges between anus and vulva, and Granek's ratio. The second-stage juvenile morphologies critical for identification were the following: body and stylet length, shape of stylet knobs, shape and length of tail and hyaline tail terminus, and number of refractive bodies in the hyaline part of tail. The morphology of cysts and second-stage juveniles and molecular analyses established the identity of the species as the golden cyst nematode Globodera rostochiensis (Wollenweber 1923) Skarbilovich, 1959 (Figure 1). Diagnosis as G. rostochiensis was clearly confirmed by molecular means as well.

Molecular analysis
DNA sequence (accession number MZ570875) was obtained for three amplicons representing three individual juveniles and aligned using Sequencher v. 5.4.6. All were found to be identical. A BlastN search of available sequences in GenBank revealed identity to COI sequences from several populations of Globodera rostochiensis, including MT240262 from Indonesia, MN095979 from Germany, MN095978 from Bolivia, MN095977 from Russia, and several others.

Conclusions Conclusions Conclusions Conclusions
Based on the strength of mitochondrial COI to differentiate species of Globodera, it is confirmed that the Egypt population is G. rostochiensis.