Integrative Morphometric and Molecular Approach to Update the Impact and Distribution of Potato Cyst Nematodes Globodera rostochiensis and Globodera pallida (Tylenchida: Heteroderidae) in Algeria

Morphological and molecular studies were conducted to characterize the specific identity of 36 isolates of potato cyst nematodes (PCNs) recovered from soil samples collected in several potato producing areas of Algeria. Morphometric data revealed that 44% of isolates contained Globodera pallida alone, 28% contained Globodera rostochiensis alone and 28% mixtures of the two species. Morphometric values of cysts and second-stage juveniles were generally distributed with slight differences in the expected ranges for both Globodera species. Inter- and intraspecific morphometric variability in nematode isolates was noted. Molecular analysis using conventional multiplex PCR with species-specific primers and TaqMan real-time PCR confirmed the morphological identification. In addition, the distribution of both potato cyst nematode species throughout various parts of the country was investigated. In the central areas, the isolates of G. pallida alone dominate, whereas isolates of G. rostochiensis alone are more frequent in the southern areas. In the eastern regions, mixed isolates are more representative. Most isolates examined in the western areas are mixtures of the two species or G. rostochiensis alone. Comparatively, G. pallida remains the most widely distributed species in its geographic range. This study confirms the presence of two PCN species, G. pallida and G. rostochiensis, in Algeria and provides additional information on their biogeographic distribution.


Introduction
Potato cyst nematodes (PCNs), Globodera rostochiensis [1,2] and Globodera pallida [3], are damaging to potato (Solanum tuberosum L.) in various countries [4]. These parasites constitute the second group of the 10 main plant-parasitic nematodes of scientific and economic importance [5], causing annual losses estimated at 9% of world potato production [6]. Due to their harmful potential, both species are classified as quarantine organisms and were added to the European and Mediterranean Plant Protection Organization (EPPO) A2 list in 1975 [7]. These nematode species originated in the Andean region of South America [8] and from there they have spread to different parts of the world, mainly by soils adhering to potato tubers from infested fields. PCN have been reported throughout Europe, South America and parts of Asia, North America, Oceania and Africa where potatoes are grown [7]. However, new detections of Globodera sp. continue to be reported [9][10][11][12][13][14][15].

Morphometric Data
Morphometric investigation of cysts and J2s allowed a first specific identification of the different isolates of potato cyst nematodes. Globodera pallida and G. rostochiensis species were present separately or as a mixture in the various fields prospected.

Globodera Pallida
Globodera pallida species was noted in 16 fields represented by 9 to 22, 26 and 29 isolates (Tables 1 and 2). The morphometric of the specimens analyzed was comparable overall to that of G. pallida mentioned by [40] and [47]. The measurements and the mean values calculated were distributed with slight variations in the ranges proposed for this species. The majority of isolates showed a greater upper limit of the fenestra-anus distance than that defined for G. pallida (67 μm). The average values of the vulva diameter of the samples except for 9, 11 and 20 isolates were superior to the maximum value of G. pallida (21 μm). Most of the extreme values of the vulva diameter lay outside the measurement range for G. pallida (18-21 μm). The maximum stylet lengths for 9 and 17 isolates were respectively 27.2 and 27.6 μm, slightly exceeding the values reported for G. pallida (26 μm). Second-stage juveniles (J2): They were vermiform, and tap at the tail with a hyaline part; ventro-lateral overlapping of esophageal glands over intestine ( Figure 1D). The head was rounded and slightly offset with prominent cephalic sclerotization. The mouth had an apparent stylet developed with pointed basal knobs in Globodera pallida ( Figure 1E) and rounded in G. rostochiensis ( Figure 1F).

Morphometric Data
Morphometric investigation of cysts and J2s allowed a first specific identification of the different isolates of potato cyst nematodes. Globodera pallida and G. rostochiensis species were present separately or as a mixture in the various fields prospected.

Globodera pallida
Globodera pallida species was noted in 16 fields represented by 9 to 22, 26 and 29 isolates (Tables 1 and 2). The morphometric of the specimens analyzed was comparable overall to that of G. pallida mentioned by [40,47]. The measurements and the mean values calculated were distributed with slight variations in the ranges proposed for this species. The majority of isolates showed a greater upper limit of the fenestra-anus distance than that defined for G. pallida (67 µm). The average values of the vulva diameter of the samples except for 9, 11 and 20 isolates were superior to the maximum value of G. pallida (21 µm). Most of the extreme values of the vulva diameter lay outside the measurement range for G. pallida (18-21 µm). The maximum stylet lengths for 9 and 17 isolates were respectively 27.2 and 27.6 µm, slightly exceeding the values reported for G. pallida (26 µm).

Globodera rostochiensis
A total of 10 G. rostochiensis isolates (3, 7, 23, 24, 25, 27, 28, 30, 32 and 34) (Tables 1 and 2) were identified. The morphometric features of the cysts and juveniles studied correspond to those proposed for G. rostochiensis by [40,47]. Measurements and calculated averages lay with some slight differences in the ranges of G. rostochiensis. The mean values of the vulvaanus distance and vulva diameter of isolates 27, 28 and 30 from the Saharan region (El Oued) exceed the maximum values reported for G. rostochiensis. Likewise, the upper limits values of the vulva-anus distance of all isolates, except for 25 and 32 isolates, are higher than those expected for G. rostochiensis (77 µm). The upper limit of the vulva diameter of all isolates was above 20 µm, which was not very typical for the species. The maximum stylet length for 24, 27, 28 and 30 isolates slightly exceeded that reported for G. rostochiensis (23 µm).

Mixture of Globodera pallida and G. rostochiensis
The two Globodera species were present sympatrially in 10 fields represented by 1, 2, 4, 5, 6, 8, 31, 33, 35 and 36 isolates (Tables 3 and 4). In each of the isolates, some specimens show the morphometric characteristics of G. pallida and others of G. rostochiensis. The measurements and average values were within the ranges of these both nematode species. However, the mean and extreme values of the vulva diameter of some isolates lay outside the ranges of PCN species. Similarly, the averages and maximum values of the vulva-anus distance of some isolates exceeded those proposed for G. pallida and G. rostochiensis.
In addition, isolates 2, 6 and 31 showed a dominance of G. pallida, while G. rostochiensis was frequent in isolates 1, 4, 5, 8, 33, 35 and 36. The ascending hierarchical classification carried out on 17 morphometric characters of cysts and second stage juveniles ( Figure 2) allowed separating the studied nematode isolates into five groups with different constitutive status, which further confirmed the morphometric variability of these isolates. The first two groups were respectively represented by isolates 1, 15, 5, 21, 32, 12, 17, 14, 27, 36, 2, 6, 33, 4, 26, 8, 11, 3, 31, 13 and 30 and isolates 10, 34, 20, 23, 35, 18, 19, 16, 22, 24, 25 and 29, belonging to various geographical origins and containing a single Globodera species or both species. This means that the morphometric similarity of the isolates of these nematodes was not related to the geographic origin of the isolate and the species it represented. The isolates 7, 9 and 28 were distributed distinctly between the third and fifth group.  6 0   28  9  7  29  25  24  22  16  19  18  35  23  20  34  10  30  13  31  3  11  8  26  4  33  6  2  36  27  14  17  12  32  21  5  The morphometric studies of specimens of the considered nematode isolates showed that among the cysts analyzed, 203 cysts were identified as G. pallida and 157  The morphometric studies of specimens of the considered nematode isolates showed that among the cysts analyzed, 203 cysts were identified as G. pallida and 157 cysts represented G. rostochiensis. In the case of juveniles, a total of 207 individuals were found belonging to G. pallida and 153 individuals to G. rostochiensis. Average morphometric values of the cysts and J2s of each Globodera species are regrouped in Table 5.
The morphometric values obtained on all the characters overlapped between the two PCN species. Analysis of the average values showed a difference between these both nematode species. For the perineal regions of cysts, a substantial variation was recorded for fenestra to anus distance (56.45 ± 12.17 and 73.83 ± 21.29 µm for G. pallida and G. rostochiensis, respectively). Likewise, a difference was noted between the number of cuticular ridges between fenestra and anus (12.43 ± 3.04 µm for G. pallida and 18.33 ± 4.21 µm for G. rostochiensis), the vulva diameter (18.90 ± 3.89µm for G. rostochiensis and 21.82 ± 4.76 µm for G. pallida) and the Granek's ratio (2.62 ± 0.48 and 4.00 ± 1.12 µm for G. pallida and G. rostochiensis, respectively). Regarding second stage juveniles, the results indicated that G. rostochiensis had a smaller body (432.99 ± 46.52 µm) than G. pallida (440.68 ± 28.71 µm). Additionally, the stylet showed a length of 24.07 ± 1.08 µm, with pointed basal knobs in G. pallida and a length of 22.20 ± 0.88 µm, with rounded basal knobs in G. rostochiensis. The tail was relatively short in G. rostochiensis (45.63 ± 4.78 µm) compared to G. pallida (47.46 ± 4.19 µm). Moreover, the comparison of the mean morphometric values revealed a significant difference (p < 0.05) between the isolates of both potato cyst nematode species for all characters considered on the biological material examined.
Variability between the mean morphometric values was also recorded between the isolates of each Globodera species. For G. pallida isolates, a significant difference (p < 0.05) was noted for all the characters except for vulva-anus distance (p = 0.0956), the vulva diameter (p = 0.0692) and juvenile body width in the middle (p = 0.5656). For G. rostochiensis isolates, all characters showed significance except for the Granek's ratio (p = 0.1413). These results showed great inter-and intra-specific morphometric variability.

G. rostochiensis
No PCR product was obtained in the negative controls without DNA template, which means the absence of contamination during the PCR reaction preparation.

TaqMan Real-Time PCR
The fluorescence of FAM (Flurescein amidites) and TET (Tetrachlorofluorescein) was obtained in the nematode DNA samples during TaqMan real-time PCR assay (Table  6). A total of 16 samples (9 to 22, 26 and 29) emitted only the fluorescence of FAM which corresponded to Globodera pallida and 10 samples yielded only the fluorescence of TET which corresponded to G. rostochiensis. Both types of fluorescence were noted in 10 samples (1, 2, 4, 5, 6, 8, 31, 33, 35 and 36), which indicated that these isolates contained the two PCN species. No fluorescence was observed in the negative controls. Therefore, the TaqMan real-time PCR results further confirmed the specific identity of the considered nematode isolates.
The mean threshold cycle (Ct) values highlighted a difference between DNA samples. For the detection of G. pallida, average Ct values varied from 14.25 to 29.19 which corresponded to DNA isolated from 17 and 29 isolates, respectively. Regarding the detection of G. rostochiensis, the mean Ct values were between 16.55 and 24.09 recorded respectively in the DNA extracted from 6 and 28 isolates.
Regarding the amplification curves in function of cycle numbers, all DNA samples showed typical amplification curves corresponding to a sigmoid shape (Figure 4). No PCR product was obtained in the negative controls without DNA template, which means the absence of contamination during the PCR reaction preparation.

TaqMan Real-Time PCR
The fluorescence of FAM (Flurescein amidites) and TET (Tetrachlorofluorescein) was obtained in the nematode DNA samples during TaqMan real-time PCR assay (Table 6). A total of 16 samples (9 to 22, 26 and 29) emitted only the fluorescence of FAM which corresponded to Globodera pallida and 10 samples yielded only the fluorescence of TET which corresponded to G. rostochiensis. Both types of fluorescence were noted in 10 samples (1, 2, 4, 5, 6, 8, 31, 33, 35 and 36), which indicated that these isolates contained the two PCN species. No fluorescence was observed in the negative controls. Therefore, the TaqMan real-time PCR results further confirmed the specific identity of the considered nematode isolates.   Regarding the amplification curves in function of cycle numbers, all DNA samples showed typical amplification curves corresponding to a sigmoid shape ( Figure 4).

Distribution of Potato Cyst Nematodes Species
The nematode species Globodera pallida and G. rostochiensis had a wide spread in the potato producing regions of Algeria ( Figure 5). Great variability in their distribution throughout the prospected range was noted. In central regions, G. pallida alone isolates dominated, while G. rostochiensis alone isolates were more frequent in southern regions. In the eastern areas, mixed isolates were more present. The majority of studied isolates in the western regions represented a mixture of the two species or G. rostochiensis alone isolates. Some areas were infested by a single Globodera species and others by both species either separately or mixed in a population. All combinations were present.
In addition, the comparison of the species distribution showed that G. pallida had a more significant extension, as it was found in 13 regions out of the 17 prospected, while G. rostochiensis was present in only 10 regions.

Distribution of Potato Cyst Nematodes Species
The nematode species Globodera pallida and G. rostochiensis had a wide spread in the potato producing regions of Algeria ( Figure 5). Great variability in their distribution throughout the prospected range was noted. In central regions, G. pallida alone isolates dominated, while G. rostochiensis alone isolates were more frequent in southern regions. In the eastern areas, mixed isolates were more present. The majority of studied isolates in the western regions represented a mixture of the two species or G. rostochiensis alone isolates. Some areas were infested by a single Globodera species and others by both species either separately or mixed in a population. All combinations were present.
In addition, the comparison of the species distribution showed that G. pallida had a more significant extension, as it was found in 13 regions out of the 17 prospected, while G. rostochiensis was present in only 10 regions.

Discussion
The morphological and morphometric data acquired on Algerian isolates of potato cyst nematodes are comparable to those provided by [40] and [47]. However, some slight differences are noted in the value amplitudes and the calculated means. This dissimilar-

Discussion
The morphological and morphometric data acquired on Algerian isolates of potato cyst nematodes are comparable to those provided by [40,47]. However, some slight differences are noted in the value amplitudes and the calculated means. This dissimilarity is due to the intra-specific variability for certain extreme values and remains very comparable with those reported by different authors in various geographic areas [26,31,[33][34][35]. In various cases, obtained morphometric data has not been very conclusive in the differentiation between the nematode species G. pallida and G. rostochiensis and it is in this context that molecular analysis has been conducted for confirmation.
Conventional multiplex PCR assays with species-specific primers (ITS5/PITSp4 + PITSr3) and TaqMan real-time PCR produced consistent results and confirmation of the identity of the cyst nematode isolates associated with potato. Despite the consideration of the molecular tool for species characterization, the morphological approach remains basic in taxonomy.
The two Globodera species identified are found separately or mixed in the various fields surveyed. Examples of cases have been reported in the Ain Defla area by [18]. In this regard, only G. pallida or G. rostochiensis alone isolates in different regions (Algiers, Boumerdes, Blida, Tipaza, Béchar and El Oued) were identified by [19]. Similar investigations carried out in various other countries have shown the presence of alone isolates and mixed isolates of the PCN species [13,[48][49][50][51][52], which corroborates with these results. In addition, according to [48], G. pallida and G. rostochiensis rarely occurred as separate species, but more often as a mixture species in the same field.
Globodera pallida alone isolates are present in 44% of the fields prospected, while G. rostochiensis alone isolates and mixed isolates are present in the rest of the fields with equal proportions (28%), showing the dominance of the G. pallida species in potato fields. This may result from the control of populations of G. rostochiensis through the intensive cultivation of resistant potato cultivars as is occurring in other countries like the Netherlands [53], United Kingdom [54][55][56] and Portugal [52] since almost all current potato cultivars are resistant or tolerant to this species [34], unlike with G. pallida species where the number of resistant varieties is limited [26,34,57,58].
The presence of mixed populations in fields represents a more worrying threat than pure populations for potato cultivation, not only because of yield losses, but also because of their extremely difficult management, especially when using resistant potato varieties as an alternative control method, since no cultivar is resistant to both Globodera species [59]. Repeated use of cultivars resistant to G. rostochiensis may favour the multiplication of G. pallida in mixed populations, which is the case in Ile de Ré [60] and in The Netherlands [61]. The consequence of the fusion is the possible reinforcement of a cross hybridization between these two nematode species that might result in a generation with new genotypes [34]. However, the crossing between these two species probably results in non-fertile hybrids [62].
Analysis of the data shows a wide geographical distribution of Globodera species in the regions prospected and with certain dominance. The majority of the isolates present in the central regions belong to the G. pallida species, while G. rostochiensis isolates are more frequent in the southern areas. In eastern regions, the two PCN species are often present in mixed isolates. Most of the samples identified in the western regions are mixed or G. rostochiensis alone. On this subject, it was reported by [19] that G. pallida mainly occupies northern Algeria, while G. rostochiensis occurs mainly in southern regions. The study conducted by [63] on the distribution of potato cyst nematode species in South America showed that north of 15.6 • S, only the G. pallida species is noted, but south of this latitude, most of the examined populations belong to the species G. rostochiensis or to both common species. Based on these data, the distribution of both PCN species is related to latitude, especially the influence of day length. The predominance of G. rostochiensis in some regions in Tunisia is determined by temperature [27]. In this regard, according to [64] G. rostochiensis is more competitive than G. pallida at an average temperature of 24 • C and conversely at an average temperature of 9.5 • C. Recently, it was shown by [65,66] that the optimum temperature for reproduction of G. rostochiensis is higher than that of G. pallida. The distribution of Globodera species depends on a set of environmental variables that govern the nematode populations in their range.
The widespread distribution of nematodes of the genus Globodera in the potato-producing regions may be due to the environmental conditions favorable to the development of these pests. Various reports have shown that temperature is a factor limiting the development of these nematode species. In addition, the type of soil plays a considerable role in the development of these nematodes. It was noted that light and porous soils favor nematodes [67]. Added to this are the invoices linked to the host plant, particularly the varieties of potato cultivated (Table 7); Spunta and Desiree, which are preferential hosts for G. pallida and G. rostochiensis, are indicate by various authors [68][69][70][71][72][73]. In addition, human activities allow more ground movement and are a factor favouring the spread of nematode cysts.
Further PCN surveys should cover other areas with attention to the seed multiplication plots. In addition to the essential species identification, investigations into the detection of pathotypes within each species deserve to be conducted. Faced with the lack of information on varietal resistance, tests of susceptibility to nematodes must be carried out for a better knowledge of resistant varieties in order to better manage these harmful pests.
The control of PCN should be based on a combination of practices, such as the use of natural crop production methods like the resistant cultivars, rotations, and biological control agents. The starting point to guide decisions is to know about the presence and spread of both Globodera species. Thus, this study is a valuable contribution to have a more complete and updated picture of the distribution of PCN in Algeria.