Detection and characterization of phytoplasmas in some cucurbits (Cucurbitaceae) and bindweed (Convolvulaceae) in Hatay Province of Turkey

Öz

Aims: During a disease survey conducted in 2019, typical symptoms including yellowing of the leaves, stunting, shortening of internodes, proliferation of auxiliary shoots (witches'-broom), the bunchy appearance of growth at the ends of stems (rosetting), virescence of flowers or sterility, phyllody, small and deformed leaves and fasciation were observed on suspected cucurbit plants growing in Hatay province. Therefore, this study was carried out to determine the phytoplasma infection(s) on cucurbit crops and bindweed in Hatay province of Turkey.

Methods and Results: In total; 4 pumpkins (winter squash: Cucurbita moschata Duchesne), 12 summer squash (Cucurbita pepo L.) and 2 bindweed (Convolvulus arvensis L.) samples were collected from suspected plants in cucurbit fields in Hatay province. Some of the plants (2 pumpkins, 10 summer squash and 2 bindweeds) exhibited symptoms related to phytoplasmas. Total nucleic acid was extracted by CTAB method. PCR amplification of 16S rDNA with phytoplasma specific primer pairs; F1/R0 followed by R16F2n/R2 confirmed the phytoplasma presence. Expected size amplicons of ~1.2 kb were obtained from infected samples from symptomatic cucurbits (12/12) and bindweed (1/2), but not from asymptomatic cucurbit plants (0/4). The sequences of the 4 symptomatic samples were deposited in NCBI GenBank (MT163353, MT163393, MT163396, and MT163469).

Conclusions: After sequencing and phylogenetic analyses it was revealed that the squash isolates had 99% sequence identity with “Candidatus phytoplasma trifolii” (16SrVI) and the bindweed isolate had 99% sequence identity with “Candidatus phytoplasma solani” (16SrXII).

Significance and Impact of the Study: There is a lack of knowledge on the status of phytoplasma infections and natural hosts in cucurbits in Turkey. According to our knowledge, this is the first report of a member of 16SrVI group, Candidatus phytoplasma trifolii associated with phytoplasma infection in winter squash-pumpkin (C. moschata) and summer squash (C. pepo) in Turkey.

Anahtar Kelimeler

• Ca. phytoplasma solani
• Ca. phytoplasma trifolii
• Phytoplasma
• Summer squash (Cucurbita pepo)
• Turkey.

Hatay İli Bazı Kabak (Cucurbitaceae) ve Tarlasarmaşığı'nda (Convolvuluceae) Fitoplazmaların Belirlenmesi ve Karakterizasyonu

Öz

Amaç: 2019’da bir hastalık surveyinde, şüpheli kabak bitkilerinde yaprakların sararması, bodurluk, boğum aralarının kısalması, çoklu sürgün oluşumu, tepe sürgünlerinde çalı görünümü, çiçeklerin yeşillenmesi ve kısırlık, fillodi, küçük ve şekli bozulmuş yapraklar ve gövde yassılaşması gibi fitoplazmalara özgü simptomlar gözlenmiştir. Böylelikle, bu çalışma Hatay ili kabak bitkilerinde ve tarla sarmaşığında fitoplazma hastalıklarının araştırılması amacıyla yürütülmüştür.

Yöntem ve Bulgular: Hatay ili kabak tarlalarından şüpheli toplam 4 kış kabağı (Cucurbita moschata), 12 yaz kabağı (Cucurbita pepo) ve 2 tarla sarmaşığı (Convolvulus arvensis) örnekleri toplanmıştır. Bitkilerden bazıları (2 kış kabağı, 10 yaz kabağı ve 2 tarla sarmaşığı) fitoplazmalara özgü simptom sergilemiştir. Toplam nükleik asit izolasyonu, CTAB metoduna göre yapılmıştır. Direct ve Nested PCR çalışmalarında sırasıyla F1/R0 ve R16F2n/R2 evrensel primer çiftleri kullanılmıştır. Simptom sergileyen kabak bitkilerinde (12/12) ve tarla sarmaşığında (1/2) beklenen ~1.2 kb büyüklüğünde bant elde edilirken simptomsuz kabaklarda elde edilememiştir. Simptom gösteren örneklerden 4 tanesi NCBI Genbankası’na (MT163353, MT163393, MT163396 ve MT163469) yüklenmiştir.

Genel Yorum: Sekans ve filogenetik analizlerin ardından, kabak izolatlarının “Candidatus phytoplasma trifolii” (16SrVI), tarla sarmaşığı izolatının ise “Candidatus phytoplasma solani” (16SrXII) olduğunu göstermiştir.

Çalışmanın Önemi ve Etkisi: Türkiye'de kabakgillerde fitoplazma enfeksiyonlarının ve doğal konakçıların durumu hakkında bilgi eksikliği bulunmaktadır. Mevcut bilgilerimize göre, Türkiye’de 16SrVI grubunun bir üyesi olan Candidatus phytoplasma trifolii'nin kışlık kabaklardan bal kabağı (C. moschata) ve yazlık kabaklardan sakız kabağı (C. pepo) bitkilerinde enfeksiyon oluşturduğu ilk kez belirlenmiştir.

Anahtar Kelimeler

• Ca. phytoplasma solani
• Ca. phytoplasma trifolii
• Fitoplazma
• Kabak
• Türkiye

Kaynakça

1. Al-Subhi AM, Hogenhout SA, Al-Yahyai RA, Al-Sadi AM (2018) Detection, identification, and molecular characterization of the 16SrII-D phytoplasmas infecting vegetable and field crops in Oman. Plant Dis. 102: 576–588.
2. Balkaya A, Özbakır M, Karaağaç O (2010) Pattern of variation for seed characteristics in Turkish populations of Cucurbita moschata Duch. African Journal of Agricultural Research. 5(10): 1068-1076.
3. Bertaccini A (2007) Phytoplasmas: diversity, taxonomy and epidemiology. Frontiers in Bioscience. 12: 673-689.
4. Borines LM, Nuñez JAC, Duero ND, Sagarino-Borines R, Gerona RG (2020) Detection and molecular characterization of phytoplasma affecting vegetables in Eastern Visayas, Philippines. Annals of Tropical Research 42(2): 1-20. DOI: https://doi.org/10.32945/atr4221.2020
5. Choueiri E, Salar P, Jreijiri Fouad, Zammar SE, Masaad R, Abdul-Nour H, Bove ́ JM, Danet JL, Foissac X (2007) Occurrence and distribution of ‘Candidatus Phytoplasma trifolii’ associated with diseases of solanaceous crops in Lebanon. Eur J Plant Pathol 118: 411-416.
6. Davis, RE, Zhao Y, Wei W, Dally EL, Lee IM (2017) 'Candidatus Phytoplasma luffae', a novel taxon associated with witches' broom disease of loofah, Luffa aegyptica Mill. 2017. Int J Syst Evol Microbiol., 67(8): 3127-3133. doi: 10.1099/ijsem.0.001980.
7. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus (San Francisco, Calif.)12, 13–15.
8. FAO (2018) http://www.fao.org/faostat/en/#data/QC (verified October 10, 2020).

9. Gundersen DE, Lee IM (1996) Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathol. Mediterr 35: 144-151.
10. Güller A, Usta M (2020) Stolbur and Clover Proliferation Phytoplasma Infections in Tomato from Bingöl province, Turkey. Türk Tarım ve Doğa Bilimleri Dergisi, 7(4): 855-866.
11. Hogenhout SA, Musić MS (2010) Phytoplasma Genomics, from Sequencing to Comparative and Functional Genomics- What Have We Learnt? Page: 19-36. Ed. Phyllis G. Weintraub and Phil Jones, PHYTOPLASMAS Genomes, Plant Hosts and Vectors. CABI, London, UK.
12. Kastalyeva TB, Bogoutdinov DZ, Bottner-Parker KD, Girsova NV, Lee I (2016) Diverse phytoplasmas associated with diseases in various crops in Russia - pathogens and vectors. Sel’skokhozyaistvennaya Biologiya (Agricultural Biology), 51: 367-375. doi: 10.15389/agrobiology.2016.3.367.
13. Khan AJ, Botti S, Subhi AMA, Zaidi MA, Altosaar I, Alma A, Bertaccini A (2003) Molecular Characterization of the 16S RNA gene of phytoplasma detected in two leafhopper species associated with alfalfa plants infected with witches’ broom in Oman. Phytopathol. Mediterr 42 (3): 257-267.
14. Khasa E, Gopala Taloh A, Prabha T, Madhupriyal, Rao GP (2016) Molecular characterization of phytoplasmas of ‘Clover proliferation’ group associated with three ornamental plant species in India. Biotech 6 (2): 237.
15. Kumar S, Singh V, Lakhanpaul S (2010) First report of ‘Candidatus Phytoplasma asteris’ (16SrI) associated with little leaf of cotton and luffa in India. Australas. Plant Dis. 5 (1): 117-119. 10.1071/DN10043
16. Kumari S, Nagendran K, Rai AB, Singh B, Rao GP, Bertaccini A (2019) Global Status of Phytoplasma Diseases in Vegetable Crops. Frontiers in Microbiology, 10: 1349.
17. Lee IM, Gundersen DE, Hammond RW, Davis RE (1994) Use of mycoplasmalike organism (MLO) group-specific oligonucleotide primers for nested-PCR assays to detect mixed- MLO infections in a single host plant. Phytopathology, 84 (6): 559-566.
18. Lee IM, Gundersen-Rindal DE, Davis RE, Bartoszyk, IM (1998) Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. Int. J. Syst. Bacteriol. 48: 1153-1169.
19. Martini M, Delić D, Liefting L, Montano H (2018) Phytopsma infecting vegetable, Pulse and Oil crops. In: Phytoplasmas: Plant Pathogenic Bacteria – I.
20. Montano HG, Brioso PST, Pimentel JP, Figueiredo DV, Cunha Junior JO (2006) Cucurbita moschata, new phyto-plasma host in Brazil. Journal of Plant Pathology, 88: 226.
21. Montano HG, Paulo STB, Roberta CP, Joao PP (2007). Sicana odorifera (Cucurbitaceae) a new phytoplasma host. Bull. Insectology. 60: 287-288.
22. NangKyuKyu W, YoungHwan K, HeeYoung, J (2014) Bitter gourd little leaf disease associated to 'Candidatus Phytoplasma asteris'. Tropical Plant Pathology, 39(1): 82-88.
23. Omar AF, Foissac X (2012) Occurrence and incidence of phytoplasmas of the 16SrII-D subgroup on solanaceous and cucurbit crops in Egypt. European Journal of Plant Pathology, 133: 353-360.
24. Paris HS, Brown RN (2005) The genes of pumpkin and squash. HortScience, 40(6): 1620-1630.
25. Rao GP, Gopala GS, Rao A (2017) First report of a ’Candidatus Phytoplasma asteris’-related strain (16SrI-B subgroup) associated with witches’ broom disease in Cucurbita pepo in India. New Dis. Rep. 35: 33. doi: 10.5197/j.2044-0588.2017.035.033
26. Robinson RW, Decker-Walters DS (1997) Cucurbits. New York Cab. International. p. 226 (Crop Production Science in Horticulture).
27. Salehi MT, Siampour M, Esmaeilzadeh-Hosseini SA, Bertaccini A (2015) Characterization and vector identification of phytoplasmas associated with cucumber and squash phyllody in Iran. Bulletin of Insectology, 68(2): 311-319.
28. Sarı N, Tan A, Yanmaz R, Yetişir H, Balkaya A, Solmaz I, Aykas L (2008) General Status of Cucurbit Genetic Resources in Turkey. Cucurbitaceae 2008. Proceedings of the IXth EUCARPIA meeting on genetics and breeding of Cucurbitaceae (Pitrat M.ed.) INRA. Avignon, France, 21-32s.
29. Seemüller E, Marcone C, Lauer U, Ragozzino A, Goschl M (1998) Current status of molecular classification of the phytoplasmas. Journal of Plant Pathology, 80: 3–26.
30. Sertkaya G, Martini M, Musetti R, Osler R (2007) Detection and molecular characterization of phytoplasmas infecting sesame and solanaceous crops in Turkey. Bull. Insec. 60 (2): 141-142.
31. Sertkaya G, Yüksel SD (2018) Virus and Phytoplasma Diseases of Edible gourd (Lagenaria siceraria L.) in Hatay Province of Turkey. I. International Agricultural Science Congress, Van Congress 2018 (09-12 May 2018 Van, Turkey): 633.
32. Streten C, Conde B, Herrington M, Moulden J, Gibb K (2005) Candidatus Phytoplasma australiense is associated with pumpkin yellow leaf curl disease in Queensland, Western Australia and the Northern Territory. Australasian Plant Pathology, 34: 103–105.
33. Tripathi S, Thorat V, Verma R, Shouche Y, Yadav A (2017) First Report of ‘Candidatus Phytoplasma asteris’ (subgroup 16SrIX) associated with bottle gourd virescence and phyllody disease in India. Plant Disease (Disease Notes), 101(11): 1949. DOI: 10.1094/PDIS-03-17-0368-PDN
34. TUIK (2019) https://data.tuik.gov.tr/Bulten/Index?p=Bitkisel-Uretim-Istatistikleri-2019-30685 (verified June 16, 2021).
35. Ulubaş-Serçe Ç, Yılmaz S (2020) First report of ‘Candidatus Phytoplasma trifolii’ (16SrVI group) infecting cabbage (Brassica oleracea) in Turkey. Journal of Plant Pathology, 102: 553.
36. Usta M, Güller A, Sipahioğlu HM (2017) Detection and Characterization of two Phytoplasma lineages on Cucumber (Cucumis sativus L.) with Same Symptomatology based on Virtual RFLP and Nucleotide Sequence Analysis of 16S rDNA. YYÜ TAR BİL DERG (YYU J AGR SCI) 27(3): 299-308.
37. Weintraub PG, Beanland L (2006) Insect vectors of phytoplasmas. Annual Review of Entomology 51(1): 91-111.
38. Whitaker TW, Robinson RW (1986) Squash breeding. In: Bassett M.J. (Ed.). Breeding Vegetable. Crops. Westport, Connecticut: Avi, (209-242), 584.
39. Wu T, Zhou J, Zhang Y, Cao J (2007). Characterization and inheritance of a bush-type in tropical pumpkin (Cucurbita moschata Duchesne). Scientia Horticulturae., 114(1):1-4.
40. Yılmaz S, Çağlar BK, Djelouah K (2019) Molecular characterization of phytoplasma diseases of pepper in Turkey. Journal of Phytopathology, 167(9): 479–483.
41. Zhao Y, Wei W, Lee IM, Shao J, Suo X, Davis RE (2009) Construction of an interactive online phytoplasma classification tool, iPhyClassifier and its application in analysis of the peach X-disease phytoplasma group (16SrIII). International Journal Systematic and Evolutionary Microbiology, 59: 2582-2593.
42. Zibadoost S, Rastgou M, Asghari S, Tazehkand SA (2016) Detection and molecular identification of ‘Candidatusphytoplasma trifoli’ infecting some cultivated crops and vegetables in West Azarbaijan province. Australas. Plant Dis. Notes. 11: 3.