First report of cultivated Cretan mountain tea (Sideritis syriaca) as a host of Meloidogyne hapla and M. javanica in Crete, with some additional records on the occurrence of Meloidogyne species in Greece

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Society of Nematologists

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First report of cultivated Cretan mountain tea (Sideritis syriaca) as a host of Meloidogyne hapla and M. javanica in Crete, with some additional records on the occurrence of Meloidogyne species in Greece

Emmanuel A. Tzortzakakis / Carolina Cantalapiedra-Navarrete / Antonio Archidona-Yuste / Maria Kormpi / Juan E. Palomares-Rius / Pablo Castillo *

Keywords : Meloidogyne hispanica , Meloidogyne incognita , Corn, Soybean, Aloe

Citation Information : Journal of Nematology. Volume 51, Pages 1-4, DOI: https://doi.org/10.21307/jofnem-2019-010

License : (CC-BY-4.0)

Published Online: 16-April-2019

ARTICLE

ABSTRACT

Cultivated Cretan mountain tea or Malotira (Sideritis syriaca L.) was found to be infected by Meloidogyne hapla and Meloidogyne javanica in the island of Crete. The authors provide the first molecular characterization of M. hapla in Greece and the first report of Cretan mountain tea or Malotira as a host of Meloidogyne species worldwide. In addition, Meloidogyne hispanica was found infecting aloe (Andros island) and corn (Drama, North Greece) consisting the first reports of natural infection of these plants by M. hispanica in Europe. Furthermore, infection of corn by M. incognita and soybean by M. javanica (Drama, North Greece) are reported for the first time in Greece. Integrative taxonomical approach based on perineal pattern and EP/st ratio, as well as the region of the mitochondrial genome between the cytochrome oxidase subunit II (coxII) and 16S rRNA mitochondrial DNA (mtDNA) genes was used to differentiate Meloidogyne species.

Root-knot nematodes (RKNs), Meloidogyne spp., are recognized worldwide as the most economically important nematodes in agriculture, with a wide host range, and common in the Mediterranean area (Lamberti, 1981; Karssen and Moens, 2006). Knowledge of their occurrence in agricultural land is of vital importance for designing control measures. However, Meloidogyne spp. identification is complex, difficult and time-consuming, even for experts. In Greece, RKNs have been found in several locations and their identification was based on morphologic and morphometric characters and/or differential host tests until the middle of 1990s (Tzortzakakis et al., 2011). Within the last 20 years, the three major RKN species M. javanica (Treub, 1885; Chitwood, 1949); M. incognita (Kofoid and White, 1919; Chitwood, 1949); M. arenaria (Neal, 1889; Chitwood, 1949); and also M. ethiopica (Whitehead, 1968) and M. hispanica (Hirschmann, 1986) have been identified with molecular and/or biochemical markers (Tzortzakakis et al., 2011, 2014; Conceicao et al., 2012). In a recent publication, it was indicated that all of the reports of M. ethiopica from Europe, including the one from Greece, should actually refer to the species, M. luci (Carneiro et al., 2014; Geric Stare et al., 2017).

In spring–summer 2017, root and soil samples from five fields with RKN infestation from Heraklion Province, Island of Crete, Island of Andros and Drama province, North Greece were examined to identify the RKN species and to report the results herein. Egg masses were picked and put in water to release second-stage juveniles and males, whereas additional single egg masses were used to inoculate potted tomatoes (Solanum lycopersicum L.). In two cases of the corn (Zea mays L.) samples where mature egg masses were not found in galled roots, the original soil from the rhizosphere was used to fill pots (1,000 cm3) to which tomatoes were planted. After a 50 day growing period in a growth room, at 23–26 °C and 16 hr photoperiod, females, egg masses, second-stage juveniles and males were isolated from the tomato roots. All of the nematode stages isolated from the infected plants and the potted tomatoes were used for nematode identification.

Perineal patterns of mature females were prepared according to standard procedures (Hartman and Sasser, 1985). Briefly, root tissues were teased apart with forceps and half spear to remove adult females. The perineal pattern was trimmed and transferred to a drop of glycerin. In each sample, perineal patterns and the distance from excretory pore to the anterior end/stylet length ratio (EP/st) character were established for species identification in more than 10 mature female specimens. Perineal pattern and EP/st ratios were made using a Zeiss III compound microscope with Nomarski differential interference contrast at powers up to 1,000x magnification.

For molecular analyses, DNA of one female nematode of each RKN population was extracted and PCR assays were conducted as described by Castillo et al. (2003). The coxII-16S rRNA mtDNA was amplified using primers C2F3 (5′-GGTCAATGTTCAGAAATTTGTGG-3′) (Powers and Harris, 1993) and MRH106 (5′-AATTTCTAAAGACTTTTCTTAGT-3′) (Stanton et al., 1997). The reference M. arenaria, M. incognita , M. javanica and M. hapla Chitwood, 1949 populations from olive trees (Olea europaea L.) (Archidona-Yuste et al., 2018), and M. hispanica from grapevine (Vitis vinifera L.) (Castillo et al., 2009) previously identified and maintained on tomato in the greenhouse served as a positive control throughout this study. Detailed protocols of RFLP-PCR amplification for mtDNA fragment were studied as described previously by Powers and Harris (1993) and Maleita et al. (2012). HinfI and DraIII digestion of amplified products was conducted using 10 μl of PCR product, 1.2 μl of the restriction enzyme buffer (10X) and 15 units of the restriction enzyme (TaKaRa Biotech). Digestion was allowed to proceed for 3 hr at 37 °C. Restricted PCR products were separated on a 2% agarose gel. This pattern was compared with Powers and Harris (1993) and Maleita et al. (2012) and our positive controls. Amplification products with approximately 650 bp were identified as M. hapla, on the other hand, products with approximately 1,800 bp were digested with HinfI, this digestion generated two fragments of approximately 1,200 bp and 400 bp for M. incognita and finally, DraIII could not digest M. hispanica, but generated two fragments of approximately 1,000 and 800 pp for M. javanica.

Cretan mountain tea or Malotira (Sideritis syriaca L.) is an aromatic -medical perennial plant of the family Lamiaceae which is indigenous in the Island of Crete, naturally growing in rocky mountain areas, usually from 1,300 to 2,000 m above sea level and being recently cultivated in several areas of the island. A few plants indicating the symptoms of chlorosis and dryness with galls and egg masses in roots were found in a commercial crop in Heraklion Province, Crete, in March 2017. The crop was established in an area that had not been cultivated before and had been cleaned by natural vegetation, before planting Malotira and other aromatic-medical plants. The seed for the Malotira crop came from cultivated plants but the initial material for starting the first cultivation, had been collected from native plants of Crete, a couple of years ago. In August 2017, some more plants from the same area were uprooted and checked for nematode infection. The roots had small galls, typical of RKN infection (8–30 galls per gram of root) while in the surrounding soil were found juveniles of Meloidogyne at a density of 1.6 per gram of soil. Meloidogyne hapla was detected in the sample examined in March 2017. The species had been previously reported in Greece (Hirschmann et al., 1966; Koliopanos, 1980; Vovlas and Antoniou, 1987; Vlachopoulos, 1994) and Crete (Pyrowolakis, 1980) with identification based only on morphological characteristics. Herein, this study is the first report of molecular characterization of M. hapla in Greece. In other sample collected in August 2017, M. javanica was also detected. To our knowledge, Malotira is reported for the first time as the host of RKN worldwide. Thus, we could hypothesize that the nematode species are not indigenous in the area but introduced in the field through infested soil or plant material.

Aloe vera L. is a perennial plant and its cultivation has been recently expanded in Greece, mainly in lands which had been abandoned from cultivation or/and in areas with natural vegetation. Roots from cultivated aloe plants from the Island of Andros, Cyclades, with symptoms of stunting and leaf discoloration, were sent to the lab for nematode diagnosis. Longitudinal root sections were examined under a stereoscope and revealed the presence of egg masses and females of Meloidogyne deeply embedded inside the root tissue. The nematode species detected was identified as M. hispanica and it is hypothesized that the nematode was transferred through rooted propagating material. Aloe infection by M. javanica and M. incognita has been reported in Crete, Greece (Palomares-Rius et al., 2015). To our knowledge, this work consists the first report of infection of aloe by M. hispanica in Europe.

Corn and soybean (Glycine max (L) Merr) are common crops in the area of Ag. Athanasios, Drama, North Greece. Roots with surrounding soil of young corn plants (hybrid P1921) from two fields and soybean plants (variety 92B63) from one field indicating symptoms of severe stunting were sent to the lab for nematode diagnosis. The roots of corn had few galls with immature females of Meloidogyne. However, soybean roots were severely galled and egg masses were isolated. Meloidogyne hispanica and M. incognita were found in the two fields with corn while M. javanica was found in the field with soybean. Meloidogyne hispanica is indigenous in the area, as was also previously found in sunflower (Tzortzakakis et al., 2014). The nematode has a wide host range including corn as has been demonstrated in pot tests (Maleita et al., 2012). To our knowledge, this work consists the first report of natural infection of corn by M. hispanica in Europe and the first report of infections of corn by M. incognita and soybean by M. javanica in Greece.

Acknowledgements

The authors would like to thank J. Martín Barbarroja and G. León Ropero from IAS-CSIC for the excellent technical assistance. The first author would like to thank the agronomists Dr G. Zanakis for sending the samples of corn and soybean and Mrs O. Sifaki and Mr N. Fanourakis for assisting with the collection of Malotira samples.

References


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  3. Castillo, P., Vovlas, N., Subbotin, S. and Troccoli, A.. 2003. A new root-knot nematode, Meloidogyne baetica n. sp. (Nematoda: Heteroderidae), parasitizing wild olive in southern Spain. Phytopathology 93: 1093–1102.
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  4. Castillo, P., Vovlas, N., Troccoli, N., Liébanas, G., Palomares Rius, J. E. and Landa, B. B.. 2009. A new root-knot nematode, Meloidogyne silvestris n. sp. (Nematoda: Meloidogynidae), parasitizing European holly in Northern Spain. Plant Pathology 58: 606–619.
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    [CROSSREF]
  8. Geric Stare, B., Stranjar, P., Susic, N., Urek, G. and Sirca, S.. 2017. Reported populations of Meloidogyne ethiopica in Europe identified as Meloidogyne luci. Plant Disease 109: 1627–1632.
    [CROSSREF]
  9. Hartman, K. M. and Sasser, J. N.. 1985. Identification of Meloidogyne species on the basis of differential host test and perineal-pattern morphology in Bakker, K. R., Carter, C. C. and Sasser, J. N.. (Eds), An advanced treatise on meloidogyne. II. Methodology: North Carolina State University Graphics 69–77.Raleigh, North Carolina USA
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  11. Hirschmann, H., Paschalaki-Kourtzi, N. and Triantaphyllou, A. C.. 1966. A survey of plant-parasitic nematodes in Greece. Annales de l’Institut Phytopathologique Benaki, New Series 7: 144–156.
  12. Karssen, G. and Moens, M.. 2006. Root-knot nematodes In Perry, R. N and Moens, M.. (Eds), Plant Nematology CAB International, Wallingford: 59–90.
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  14. Koliopanos, C. N.. 1980. Contribution to the study of the root-knot nematode (Meloidogyne spp.) in Greece. Proceedings of the 2nd Research Planning Conference on Root-knot Nematodes, Meloidogyne spp., Athens: 35–39.
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  15. Lamberti, F.. 1981. Plant nematode problems in the Mediterranean region. Helminthological Abstracts – Series B 59: 145–166.
  16. Maleita, C. M. N., Curtis, R. H. C., Powers, S. J. and Abrantes, I.. 2012. Host status of cultivated plants to Meloidogyne hispanica. European Journal of Plant Pathology 133: 449–460.
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  17. Neal, J. C.. 1889. The root-knot disease of the peach, orange, and other plants in Florida, due to the work of the Anguillula. Bulletin United States Division Entomology No. 20, 31 pp.
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  18. Palomares-Rius, J. E., Castillo, P., Rapoport, H., Archidona-Yuste, A. and Tzortzakakis, E. A.. 2015. Host reaction of Aloe vera infected by Meloidogyne incognita and M. javanica in Crete Island (Greece). European Journal of Plant Pathology 142: 887–892.
    [CROSSREF]
  19. Powers, T. O. and Harris, T. S.. 1993. A Polymerase Chain Reaction method for identification of five mayor Meloidogyne species. Journal of Nematology 25: 1–6.
    [PUBMED]
  20. Pyrowolakis, E.. 1980. Distribution and control of root-knot nematodes in Crete. Proceedings of the 2nd Research Planning Conference on Root-Knot Nematodes, Meloidogyne spp., Athens: 30–34.
    [CROSSREF]
  21. Stanton, J., Hugall, A. and Moritz, C.. 1997. Nucleotide polymorphisms and an improved PCR-based mtDNA diagnostic for parthenogenetic root-knot nematodes (Meloidogyne spp.). Fundamentals and Applied Nematology 20: 261–268.
  22. Tzortzakakis, E. A., Anastasiadis, A. I., Simoglou, K. B., Cantalapiedra-Navarrete, C., Palomares-Rius, J. E. and Castillo, P.. 2014. First report of the root-knot nematode, Meloidogyne hispanica, infecting sunflower in Greece. Plant Disease 98: 703.
    [CROSSREF]
  23. Tzortzakakis, E. A., da Conceicao, I. L. P. M., dos Santos, M. C. V. and de O. Abrantes, I. M.. 2011. Root-knot nematodes (Meloidogyne spp.) in Greece. Hellenic Plant Protection Journal 4: 25–30.
  24. Vlachopoulos, E.G.. 1994. Plant protection problems caused by phytonematodes in Greece. Bulletin OEPP/EPPO 24: 413–415.
    [CROSSREF]
  25. Vovlas, N. and Antoniou, M.. 1987. Alterations induced by the root-knot nematode Meloidogyne hapla Chitwood in Actinidia roots. Annales del’ Institut Phytopathologique Benaki 15: 151–154.
  26. Whitehead, A. G.. 1968. Taxonomy of Meloidogyne (Nematoda: Heteroderidae) with descriptions of four new species. Transactions Zoology Society of London 31 263–401.
    [CROSSREF]
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REFERENCES

  1. Archidona-Yuste, A., Cantalapiedra-Navarrete, C., Liébanas, G., Rapoport, H. F., Castillo, P and Palomares-Rius, J. E.. 2018. Diversity of root-knot nematodes of the genus Meloidogyne Göeldi, 1892 (Nematoda: Meloidogynidae) associated with olive plants and environmental cues regarding their distribution in southern Spain. Plos One 13: 1–40 doi: 10.1371/journal.pone.0198236.
    [CROSSREF]
  2. Carneiro, R. M. D. G., Correa, V. R., Almeida, M. R. A., Gomes, A. C. M.M., Mohammad Deimi, A., Castagnone-Sereno, P. and Karssen, G.. 2014. Meloidogyne luci n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitising different crops in Brazil, Chile and Iran. Nematology 16: 289–231.
    [CROSSREF]
  3. Castillo, P., Vovlas, N., Subbotin, S. and Troccoli, A.. 2003. A new root-knot nematode, Meloidogyne baetica n. sp. (Nematoda: Heteroderidae), parasitizing wild olive in southern Spain. Phytopathology 93: 1093–1102.
    [CROSSREF]
  4. Castillo, P., Vovlas, N., Troccoli, N., Liébanas, G., Palomares Rius, J. E. and Landa, B. B.. 2009. A new root-knot nematode, Meloidogyne silvestris n. sp. (Nematoda: Meloidogynidae), parasitizing European holly in Northern Spain. Plant Pathology 58: 606–619.
    [CROSSREF]
  5. Chitwood, B. G. 1949. Root-knot nematodes - Part I. A revision of the genus Meloidogyne Göeldi, 1887. Proceedings Helminthological Society of Washington 16: 90–104.
  6. Treub, M.. 1885. Onderzo ekingen ever sereh-zik suikerrect. Mededelingen PlTuin, Batavia, 1–39.
  7. Conceicao, I. L., Tzortzakakis, E. A., Gomes, P., Abrantes, I. and da Cunha, M. J.. 2012. Detection of the root-knot nematode Meloidogyne ethiopica in Greece. European Journal of Plant Pathology 134: 451–457.
    [CROSSREF]
  8. Geric Stare, B., Stranjar, P., Susic, N., Urek, G. and Sirca, S.. 2017. Reported populations of Meloidogyne ethiopica in Europe identified as Meloidogyne luci. Plant Disease 109: 1627–1632.
    [CROSSREF]
  9. Hartman, K. M. and Sasser, J. N.. 1985. Identification of Meloidogyne species on the basis of differential host test and perineal-pattern morphology in Bakker, K. R., Carter, C. C. and Sasser, J. N.. (Eds), An advanced treatise on meloidogyne. II. Methodology: North Carolina State University Graphics 69–77.Raleigh, North Carolina USA
  10. Hirschmann, H.. 1986. Meloidogyne hispanica n. sp. (Nematoda: Meloidogynidae), the 'Seville root-knot nematode. Journal of Nematology 18: 520–532.
    [PUBMED]
  11. Hirschmann, H., Paschalaki-Kourtzi, N. and Triantaphyllou, A. C.. 1966. A survey of plant-parasitic nematodes in Greece. Annales de l’Institut Phytopathologique Benaki, New Series 7: 144–156.
  12. Karssen, G. and Moens, M.. 2006. Root-knot nematodes In Perry, R. N and Moens, M.. (Eds), Plant Nematology CAB International, Wallingford: 59–90.
    [CROSSREF]
  13. Kofoid, C. A. and White, W. A.. 1919. A new nematode infection of man. Journal of the American Medical Association 72: 567–569.
    [CROSSREF]
  14. Koliopanos, C. N.. 1980. Contribution to the study of the root-knot nematode (Meloidogyne spp.) in Greece. Proceedings of the 2nd Research Planning Conference on Root-knot Nematodes, Meloidogyne spp., Athens: 35–39.
    [CROSSREF]
  15. Lamberti, F.. 1981. Plant nematode problems in the Mediterranean region. Helminthological Abstracts – Series B 59: 145–166.
  16. Maleita, C. M. N., Curtis, R. H. C., Powers, S. J. and Abrantes, I.. 2012. Host status of cultivated plants to Meloidogyne hispanica. European Journal of Plant Pathology 133: 449–460.
    [CROSSREF]
  17. Neal, J. C.. 1889. The root-knot disease of the peach, orange, and other plants in Florida, due to the work of the Anguillula. Bulletin United States Division Entomology No. 20, 31 pp.
    [CROSSREF]
  18. Palomares-Rius, J. E., Castillo, P., Rapoport, H., Archidona-Yuste, A. and Tzortzakakis, E. A.. 2015. Host reaction of Aloe vera infected by Meloidogyne incognita and M. javanica in Crete Island (Greece). European Journal of Plant Pathology 142: 887–892.
    [CROSSREF]
  19. Powers, T. O. and Harris, T. S.. 1993. A Polymerase Chain Reaction method for identification of five mayor Meloidogyne species. Journal of Nematology 25: 1–6.
    [PUBMED]
  20. Pyrowolakis, E.. 1980. Distribution and control of root-knot nematodes in Crete. Proceedings of the 2nd Research Planning Conference on Root-Knot Nematodes, Meloidogyne spp., Athens: 30–34.
    [CROSSREF]
  21. Stanton, J., Hugall, A. and Moritz, C.. 1997. Nucleotide polymorphisms and an improved PCR-based mtDNA diagnostic for parthenogenetic root-knot nematodes (Meloidogyne spp.). Fundamentals and Applied Nematology 20: 261–268.
  22. Tzortzakakis, E. A., Anastasiadis, A. I., Simoglou, K. B., Cantalapiedra-Navarrete, C., Palomares-Rius, J. E. and Castillo, P.. 2014. First report of the root-knot nematode, Meloidogyne hispanica, infecting sunflower in Greece. Plant Disease 98: 703.
    [CROSSREF]
  23. Tzortzakakis, E. A., da Conceicao, I. L. P. M., dos Santos, M. C. V. and de O. Abrantes, I. M.. 2011. Root-knot nematodes (Meloidogyne spp.) in Greece. Hellenic Plant Protection Journal 4: 25–30.
  24. Vlachopoulos, E.G.. 1994. Plant protection problems caused by phytonematodes in Greece. Bulletin OEPP/EPPO 24: 413–415.
    [CROSSREF]
  25. Vovlas, N. and Antoniou, M.. 1987. Alterations induced by the root-knot nematode Meloidogyne hapla Chitwood in Actinidia roots. Annales del’ Institut Phytopathologique Benaki 15: 151–154.
  26. Whitehead, A. G.. 1968. Taxonomy of Meloidogyne (Nematoda: Heteroderidae) with descriptions of four new species. Transactions Zoology Society of London 31 263–401.
    [CROSSREF]

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