MORPHOLOGICAL AND MOLECULAR IDENTIFICATION OF POTATO CYST NEMATODE POPULATIONS IN SERBIA

Quarantine species such as potato cyst nematodes Globodera rostochiensis and G. pallida are present in Serbia since 1999 and 2005, respectively. These nematodes are sibling species and their morphological identification is complex due to their morphometric overlap. The cysts from the localities of Kladnica, Šanac, Gojna Gora and Milatovići were grown on susceptible potato varieties and their morphological differences have been discussed. To avoid ambiguities in species morphological designation a duplex PCR method was chosen for a rapid and accurate species identification. The whole procedure, from DNA extraction to DNA isolation, can be performed in a single day.


INTRODUCTION
The year 2008 was designated the International Year of the Potato (FAO UN) signifying the importance of this plant in food production.The most important potato parasites are Globodera rostochiensis and G. pallida and both have quarantine status and are present in Serbia since 1999 and 2005, respectively.The species originate from the area around Lake Titicaca in Peru where the potato was grown 1200 years before Christ.
Potato cyst nematodes cause up to £300M sterling worth of damage to the potato crop in the EU each year (Ryan et al., 2000).
For these reasons an accurate identification is essential for the phytosanitary system of every country.The morphological identification of potato cyst nematodes is based on the combination of morphological and morphometric characteristics of cysts and invasive larvae (J2).Globodera rostochiensis and G. pallida are sibling species and their morphometric features can overlap among different populations, which can lead to their incorrect identification.
The aim of this study was to confirm the identity of potato cyst nematodes (PCN) by molecular methods for morphologically identified populations and to find a simple and reliable molecular method for species identification, since molecular analyses for plant parasitic nematodes have not been performed in Serbia before.

Nematodes
The cysts of Globodera from Kladnica, Šanac, Gojna Gora and Milatovići, Serbia, were grown on susceptible potato varieties in a climatized chamber at 15-25 o C for a 16 h photoperiod from 2006 to 2009.
The cyst extraction was done by elutriation with the Spears apparatus (Spears, 1968) and collected on a 150 μm sieve while invasive larvae were obtained by cutting vital cysts under dissecting microscope.

Morphological characteristics
For morphological study the cysts and larvae were fixed in formalin-glycerol fixative (Hooper, 1970), mounted on glycerol and observed with a light microscope.Morphometric characteristics were measured with an eyepiece micrometer.The cysts were air dried and gold plated for scanning electron microscopy studies.The morphological identification comprises larval stylet length and stylet knob shape, cyst vulval basin diameter, distance between vulva and anus, Granek´s ratio (the vulva -anus distance divided by vulval basin diameter) and number of cuticular ridges in perineal area (EPPO Standards, 2004).

DNA extraction
DNA was extracted with DNAzol TM (MRC, Inc.) using 10 cysts in 10 μl doubly distilled water and 100 μl DNAzol was added.The procedure was done in accordance to the manufacturer´s instructions except that all reagents were used in lower quantities, i.e.50 μl of 100% ethanol, 100 μl of 75% ethanol and 25 μl of 8mM NaOH.

DNA amplification and separation of PCR products
Amplification was done with both sets of primers used in one reaction according to Vejl et al., (2002).The PCR master mix contained reagents as in Subbotin et al., (2001).
The positive controls were PCN populations previously identified by molecular methods: for G. pallida population Javor (Radivojevic et al., 2006) and for G. rostochiensis population Ponikve (Radivojevic et al., 2001), and a negative control was without nematode DNA.The marker was a DNA Ladder mix #SMO331.Electrophoretic 1.5% agarose gel was stained with ethidium bromide and visualized under UV light.

RESULTS
Differences between the PCN populations were analyzed by measuring the larval morphological characteristics: stylet length (fig.1), body length, tail and hyaline tail length (fig.2) and cyst morphological characters: cyst size (fig.3), vulval basin diameter, the distance between the vulva and anus (fig.4), Granek´s ratio and the number of ridges in perineal area (fig.5).As for the morphometrics of cysts, the specimens from Milatovići have the highest values of Granek´s ratio, number of ridges and vulva-anus distance, which fits well with the G. rostochiensis description.The specimens from Kladnica have the lowest values of Granek´s ratio, number of ridges and vulva-anus distance and they are within the range observed for the G. pallida type population.
Regarding Granek´s ratio and the number of ridges in the perineal area, the closest to the G. pallida type population are the Gojna Gora and Šanac populations.
Duplex PCR revealed that the Kladnica, Šanac and Gojna Gora localities were G. pallida (lanes 3, 4 and 5) and Milatovići was G. rostochiensis (lane 6) (Fig. 8).The two sets of primers amplified products of the 239 bp fragment for G. pallida and the 274 bp fragment for G. rostochiensis in the ITS-1 region.

DISCUSSION
The taxonomic features of the two sibling species G. pallida and G. rostochiensis overlap among different populations.The characteristics that vary most are larval length and cyst size.The mean Granek´s ratio is the most reliable morphological characteristic for species morphological designation.Comparing the values of the different populations from Serbia to type population values resulted in the unambiguous populations to the German one.This hypothesis can be resolved by nucleotide sequencing, which will be the subject of further investigations.

Fig. 6 .
Fig. 6.Comparison of mean larval characteristics (in micrometers) from different populations

Fig. 7 .
Fig. 7. Comparison of mean cyst characteristics (in micrometers) from different populations