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Australasian Plant Disease Notes

, Volume 7, Issue 1, pp 133–135 | Cite as

Identification of potato cyst nematode in Indonesia by polymerase chain reaction

  • LisnawitaEmail author
  • Supramana
  • Gede Suastika
Article
  • 802 Downloads

Abstract

Polymerase chain reaction (PCR) with specific primers was used to amplify the ITS-rDNA regions of potato cyst nematodes (PCN) in soil samples from Indonesia. Amplified DNA fragments showed that a single species, Globodera rostochiensis, with fragment size 238 bp occurred in East Java, and West Java, while mixed populations of G. rostochiensis and G. pallida with fragment size 391 bp were only found in Central Java.

Keywords

Globodera rostochiensis G. pallida 

Potato Cyst Nematode (PCN) is the most important plant parasitic nematode of potatoes (Solanum tuberosum). It has spread from Europe to tropical and subtropical regions of Asia, such as Pakistan, India, Philippines (Jatala and Bridge 1990). In March 2003, potato cyst nematodes were found in East Java, Indonesia (Indarti et al. 2004). In further investigations we have found the nematode in Central Java and West Java and now it is a damaging pathogen in potato plantations in Indonesia. To control PCN effectively, it is important to identify its species and the pathotypes. Traditionally, routine soil sampling and identification of PCN in Indonesia has been based on examination of morphological features. However, this is time-consuming and frequently unreliable because of morphological similarity of the two PCN species, Globodera rostochiensis and G. pallida. Identification by molecular techniques, for example DNA finger printing, offers a more rapid and precise identification of PCN species. Here, we report studies conducted to identify Indonesian populations of PCN by polymerase chain reaction (PCR).

A soil survey was conducted at potato plantations in 24 regions of Java Island. A total of 40 soil samples were collected from these regions. Twenty samples were found to be infested with PCN in 11 regions (Table 1). Soil samples consisting of 100 cores of 15–20 g of soil were taken in a grid pattern throughout the plantations (EPPO 1998). Each 1,500–2,000 g composite sample was collected from soil down to a depth of approximately 100 mm, placed in a plastic bag and was labelled with the producer’s name, field location, collection date, GPS coordinates, and sample number for laboratory identification. Samples were put into coolers for storage and transportation from the field and were submitted for processing to the Laboratory of Nematology, Department of Plant Protection, Faculty of Agriculture, Bogor Agricultural University, Bogor. Cysts were collected from 100 g soil sub-samples using a pair of sieves (840 and 250 mesh size) (Sheperd 1985) and then stored in Eppendorf tubes at room temperature.
Table 1

Regions in Java Island Indonesia in which cysts of Potato Cyst Nematode (PCN) were collected from soil and codes of isolates used in this study

Regions of Indonesia

Populations of PCN

Province

City and District

No. cysts/100 g soil

Isolate codes

East Java (location 1)

Tulung Rejo, Kota Batu

675

S1

East Java (location 2)

Tulung Rejo, Kota Batu

44

S2

East Java (location 3)

Tulung Rejo, Kota Batu

21

S3

Central Java

Pawuhan, Banjarnegara

400

S4

Central Java

Karangtengah, Banjarnegara

270

S5

Central Java

Patak Banteng, Wonosobo

2

S6

Central Java

Kepakisan, Banjarnegara

21

S7

West Java (location 3)

Sukamanah, Pengalengan

2

S8

West Java (location 4)

Sukamanah, Pengalengan

19

S9

West Java (location 5)

Sukamanah, Pengalengan

28

S10

West Java

Mekarwangi, Sindangkerta

17

S11

DNA was extracted using the modified method described by Subbotin et al. (2001) and Fullaondo et al. (1999). Five cysts from each region were collected randomly into an Eppendorf tube filled with 150 μl lyses buffer (125 mM KC; 25 mM Tris–HCl, pH 8.0; 3.75 mM MgCl2; 2.5 mM DTT; 1.125 % Tween 20 and 0.025 % gelatin) and 5 μl Proteinase K (600 μg/ml) (USB, UK) and were crushed using a plastic pistil. Following consecutive incubation periods at 65 °C for 1 h and 95 °C for 10 min, and centrifugation at 11,000 g for 10 min. DNA then was purified into chloroform-isoamyl alcohol (24:1) and precipitated with 3 M sodium acetate and absolute ethanol. DNA extract was resuspended into 20 μl sterile water and kept at −20 °C. Three primers used were as follows; specific primer for G. rostochiensis: ITS-1 (5′-TGT TGT ACG TGC CGT ACC TT-3′), G. pallida: ITS-1 (5′-GGT GAC TCG ACG ATT GCT GT-3′), and universal primer for PCN: 5.8S rDNA (5′-GCA GAA GGC TAG CGA TCT TC-3′) (Mulholland et al. 1996). Twenty five ng DNA template from each region was transferred into 25 μl PCR mixture containing 50 mM Tris–HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2 so that the 2 is subscript, 0.1 % Triton X-100, 0.2 mM dNTP (New England Biolabs, Hitchin, UK), 50 ng primer and 0.5 unit Taq polymerase (New England Biolabs, Hitchin, UK). PCR products were analysed using 2 % agarose gel electrophoresis in 0.5× TBE buffer and stained with ethidium bromide (Maniatis et al. 1989).

The primers successfully amplified targeted DNA fragments for each PCN region. Paired primers of ITS-1 and 5,8S rDNA produced 238 bp DNA fragment for all regions (West Java, East Java, and Central Java) (Fig. 1), while ITS-1 and 5,8S rDNA resulted in a 391 bp DNA fragment only for the Central Java isolates (Fig. 2). According to Mulholland et al. (1996) the fragments of 238 bp and 391 bp are related to G. rostochiensis and G. pallida, respectively.
Fig. 1

Amplified PCR products of cysts of potato cyst nematode collected in Indonesia using specific and universal G. rostochiensis primers (Mulholland et al. 1996). M, 100 bp ladder; 1–3 are isolates S1, S2 and S3 from East Java; 4-7 are isolates S4, S5, S6 and S7 from Central Java; 8–11 are isolates S8, S9, S10 and S11 from West Java

Fig. 2

Amplified PCR products of cysts of potato cyst nematode collected in Indonesia using specific and universal G. pallida primers (Mulholland et al. 1996). M, 100 bp ladder; 1–4 are isolates S4, S5, S6 and S7 from Central Java

The results presented here give evidence of the occurrence of G. rostochiensis and G. pallida in Indonesia. Both species are present in Central Java (Wonosobo and Banjarnegara Districts), while in East Java and West Java only single species were found. This is the first report of identification with molecular characterisation of PCN in Indonesia. This method is not only highly effective as a species diagnostic tool but also a sensitive method which can be used for taxonomic and quarantine purposes.

Notes

Acknowledgements

The authors wish to express our sincere appreciation to Prof. GA Wattimena and Prof. Meity S Sinaga (Faculty of Agriculture, Bogor Agricultural University,Indonesia) for their advice and counsel. The authors would like to thank Dr Kerrie A. Davies (The University of Adelaide) for helpful criticism of the manuscript.

References

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Copyright information

© Australasian Plant Pathology Society Inc. 2012

Authors and Affiliations

  1. 1.Department of Agroecotechnology, Faculty of AgricultureThe University of Sumatera UtaraMedanIndonesia
  2. 2.Department of Plant Protection, Faculty of AgricultureBogor Agricultural UniversityBogorIndonesia

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