First report of morphological and molecular characterization of Moroccan populations of Globodera pallida

Soil samples were collected in 2019 to 2020 just before the harvest of potato in different potato-producing regions of Morocco: central western (Casablanca-Rabat), Eastern (Nador-Berkane), northern (Larache-Tangier), central (middle Atlas), south-eastern (Midelt-Errachidia), and (Agadir-Taroudant) locations of the country (Fig. 1). A total of 1,500 soil samples were collected according to the sampling protocol described by the European Plant Protection Directive (69/465/EEC-2007/33/EC) in a rectangular grid covering the entire field, with a minimum width of 5 meters and a maximum length of 20 meters between sampling points. Samples were then transported to the laboratory for analysis. The cysts were extracted by flotation and sieving then, they were visually sorted by stereomicroscope using Fenwick’s apparatus (Fenwick, 1940). The apparatus was filled to the brim with water then dried soil was sprayed through the upper 1 to 2 mm mesh sieve and then into a funnel running down into the body of the apparatus. The cysts float and are dragged by overflow into the recovery collar, under which a 250 µm sieve was placed. The water supply was maintained until the sample was exhausted and clear water was overflowed, then collected cysts were air-dried, placed in 0.5 ml tubes and stored at 4°C until use.

Figure 1:

In total, 50 Globodera cysts per region were prepared for a morphobiometric study on the basis of several characters especially color, size, and Granek’s ratio (the distance between the anus and the vulva divided by the diameter of the vulvar pelvis) and number of cuticular ridges between fenestra and anus for cysts, stylet length, tail length, length of hyaline terminal part of tail. Stylet knob shape of the second-stage juveniles J2 was also used for PCNs identification (EPPO, 2017 PM 7/40). The vulval cones are mounted in a 2% glycerin-agar medium as described by Correia and Abrantes (1997) and the collected larvae are killed under a low flame and then transferred to a drop of immersion oil or a drop of glycerol. Observations and measurements were made using an Olympus BX43 microscope. This operation was conducted with three repetitions and average values were calculated and compared to the corresponding reference data for Globodera species (EPPO, 2013).

DNA isolation was achieved as described by Ibrahim et al. (2001). Two cyst samples per region were placed in an Eppendorf tube (one cyst by tube) containing 3 × 2.8 mm steel beads and 8 × 1 mm zirconium beads with a 100 µl solution of lysis buffer (Tris HCl 10 mM pH = 8.0; EDTA 1 mM; Nonidet P40 1%; Proteinase K 100 μg/ml final buffer. After that, the cysts were crushed with a Vibro Mixer homogenizer BeadBlaster^TM 24, firstly for 40 sec at maximum frequency and incubated at 65°C for 1 hr and then at 95°C for 10 min after removal of the beads (Subbotin et al., 1999). The total genomic DNA suspension was centrifuged at 13,000 rpm for 1 min and was quantified and its purity assessed using Thermo Scientific™ NanoDrop spectrophotometers and then stored at −20°C for later use.

The multiplex PCR mixture consisted of 25 μl which contained: Taq DNA polymerase buffer 1X, primers 0.64 µM, MgCl2: 2 mM, dNTPs: 0.25 mM, Taq DNA polymerase (BIOLINE): 0.6 U/reaction, DNA: 20 to 30 ng/µl and Ultra-pure water Qsp 25 μl. The primers used in this study were ITS5, PITSp4, and PITSr3 (Bulman and Marshall, 1997; Skantar et al., 2007; White et al., 1990) and were purchased from BIONEER (Table 1).

PCR was carried out in an Applied Biosystems ^TM ProFlex Thermal Cycler. The cycling program consisted of an initial denaturation step of 2 min at 94°C followed by 35 cycles of denaturation of 30 sec at 94°C, 30 sec at 60°C, 30 sec at 72°C, and final elongation step of 7 min at 72°C. Once the DNA has been amplified, 5 µl of the PCR product was separated electrophoretically using 2% agarose gel in a 1% TAE buffer, stained with SYBR® Safe DNA Gel stain 10,000X, then migrated for 40 min at 100 mA and photographed under UV light. DNA fragment sizes were determined by comparing with the 100 bp DNA marker.

For this experiment, we used three restriction enzymes (AluI, MboI, and RasI) for DNA samples taken at random from samples of G. pallida cyst populations collected from Berkane in the East, Gharb, and Doukkala in the West of Morocco and were identified by morphology. As instructed by the manufacturer (Promega R6281), 1 µg/µl of the PCR product was digested with 10 U/µl restriction enzyme, 10 µg/µl BSA, 10X restriction buffer, and sterile deionized water for a reaction volume of 20 µl. After 4 hr incubation at room temperature at 25°C, the samples were deposited in wells of a 2% agarose gel in TAE 1% buffer stained with SYBR® Safe DNA Gel stain x10,000. The restriction fragments were separated by migration for 40 min at 100 mA and photographed under UV light.

The nematode cysts in all populations studied are spherical without cones, dark brown, and yellow in color (Fig. 2A, B). The cuticular ridges between fenestra and anus are visible (Fig. 2C). The cephalic structure of the juvenile J2 shows a reniform perioral disc laterally flanked by two amphids and bordered by the elliptical rim (Fig. 2D). These structures characteristic of G. pallida are consistent with those reported by Stone (1973).

Figure 2:

Morphometric studies were carried out on 20 cysts and 50 juveniles of PCNs from each potato-producing area found to be infested (Table 2). The measured mean values for cysts were 22.8 ± 2 µm for fenestra diameter, 36.8 ± 1.8 μm for distance from the fenestra to the anus of and 2.1 µm for width. The narrow fenestra structures of the cysts averaged 8.5 µm. The average number of cuticular wrinkles between the anus and the fenestra was 9. The Granek’s ratio averaged 2.2, J2 larvae indicated a stylet length of 22.6 µm (Fig. 2E), tail length between 59 and 67 µm (Fig. 2F) and average length of the hyaline part not exceeding 1.6 times the length of the stylet.

All the measured values for cysts and second-stage juveniles were within the range compared to reference data for G. pallida.

Amplification of the ITS regions of ribosomal DNA for six samples of the three regions by PITSp4 primers produced fragments of approximately 265 bp compared to those of G. pallida (Fig. 3A). Moreover, the tested samples targeted with PITSr3primer did not give any amplification and band of 434 bp. These results of multiplex PCR confirmed the results of morphological and morphometric identification, and have proven that all samples tested belong to the species pallida.

Figure 3:

Digestion of the PCR product with the three restrictions enzymes AluI, MboI, and RsaI resulted in identical fragments for all samples taken from different potato plots. The AluI digestion (Fig. 3B) showed that the samples from the three regions of Morocco are identical and generated five fragments of 100, 200, 500, 700, and 850 bp and we found that the RsaI digestion clearly identified the three populations from the sampling areas being that Globodera pallida and revealed three identical fragments of 500, 750, and 850 bp (Fig. 3C). The use of MboI digestion giving two bands of 500 and 700 bp clearly confirmed the identity of the three populations of PCNs even if it revealed poorly visible fragments of less than 100 bp and greater than 900 bp were not included in the analysis (Fig. 3D). These results confirmed that there is no genetic diversity detectable with RFLP technology among species of Globodera pallida in all sampled potato-producing regions in Morocco.