Fusarium incarnatum Causing Fusarium Wilt on Protea (Protea cynaroides L.) in Turkey

Abstract

Protea (Protea cynaroides L.) is one of the most important ornamental plants commercially cultivated in Turkey and other parts of the world. In May 2017, protea plants heavily showed wilting symptoms were obtained from the protea growing areas at Hatay province of Turkey. Brown vascular tissue in the stems of the infected plants was observed. To determine the causal agent of the disease, necrotic tissue pieces taken from diseased plants were surface-sterilized with 10% sodium hypochlorite, two times rinsed with sterile distilled water, placed on Petri dishes containing potato dextrose agar (PDA), and incubated at 24 oC for seven days. Microconidia were no septate, ovoid, hyaline, single-celled, and 9 to 12 × 1 to 3 μm. Macroconidia were slightly curved, four to six septate, and 27 to 31 × 3 to 5 μm. Based on the morphology and cultural features, the fungus was identified as F. incarnatum, which belongs to the F. incarnatum-equiseti species complex (FIESC). In addition, a pathogenicity test was made using the apple fruits. The pathogen was re-isolated from inoculated apple fruits and both cultural and morphological characteristics of the pathogen were identical. To confirm the identity of one representative isolate, the internal transcribed spacer (ITS) region including 5.8S rDNA was amplified and sequenced with primers of ITS-1 and ITS-4. Fusarium MLST, Fusarium-ID and GenBank database were used for the identification of sequence. The amplified 465 bp product has 99% nucleotide identity with the sequences of F. incarnatum-equiseti species complex of MLST types and deposited in GenBank under accession number (MH005097). According to the literature, this is the first molecular identification of F. incarnatum on protea plants in Turkey.

Keywords

• Protea
• Fusarium incarnatum
• protea
• wilting
• ITS region
• pathogen

References

1. Baxter, A.P., Van der Westhuizen, G.C.A., Eicker, A., 1983. Morphology and taxonomy of South African isolates of Colletotrichum. South African Journal of Botany, 2(4): 259-289.
2. Bruns, T.D., 2001. ITS reality. Inoculum, 52(6): 2-3.
3. Burgess Tan, Y.P., Garnas, J., Edwards, J., Scarlett, K.A., Shuttleworth, L.A., Daniel, R., Dann, E.K., Parkinson, L.E., Dinh, Q., Shivas, R.G., Jami, F., 2019. Current status of the Botryosphaeriaceae in Australia. Australasian Plant Pathology, 48(1): 35-44.
4. Catal, M., Adams, G.C., Fulbrigt, D.W., 2010. Evaluation of resistance to rhabdocline needlecast in douglas fir variety shuswap, with quantitative polymerase chain reaction. Phytopathology, 100(4): 337-344.
5. Chang, X., Dai, H., Wang, D., Zhou, H., He, W., Fu, Y., Ibrahim, F., Zhou, Y., Gong, G., Shang, J., Yang, J., Wu, X., Yong, T., Song, C., Yang, W., 2018. Identification of Fusarium species associated with soybean root rot in Sichuan Province, China. European Journal of Plant Pathology, 151(5): 563-577.
6. Crous, P.W., Denman, S., Taylor, J.E., Swart, L., Bezuidenhout, C.M., Hoffman, L., Palm, M.E., Groenewald, J.Z., 2013. Cultivation and Diseases of Proteaceae: Leucadendron, Leucospermum and Protea. CBS-KNAW Fungal Biodiversity Centre, Netherlands.
7. Crous, P.W., Denman, S., Taylor, J.E., Swart, L., Palm, M.E., 2004. Cultivation and Diseases of Proteaceae: Leucadendron, Leucospermum and Protea. CBS Biodervisity, Netherlands.
8. Crous, P.W., Summerell, B.A., Swart, L., Denman, S., Taylor, J.E., Bezuidenhout, C.M., Palm, M.E., Marincowitz, S., Groenewald, J.Z., 2011. Fungal pathogens of Proteaceae, Persoonia. Molecular Phylogeny and Evolution of Fungi, 27: 20-45.

9. Crous, P.W., Summerell, B.A., Taylor, J.E., Bullock, S., 2000. Fungi occurring on Proteaceae in Australia: selected foliicolous species. Australasian Plant Pathology, 29(4): 267-278.
10. Denman, S., Crous, P.W., Groenewald, J.Z., Slippers, B., Wingfield, B.D., Wingfield, M.J., 2003. Circumscription of botryosphaeria species associated with proteaceae based on morphology and DNA sequence data. Mycologia, 95(2): 294-307.
11. Du, X.H., Zhao, Q., Yang, Z.L., Hansen, K., Taşkın, H., Büyükalaca, S., Dewsury, D., Moncalvo, J.M., Douhan, G.W., Robert, V.A.R.G., Crous, P.W., Rehner, S.A., Rooney, A.P., Sink, S., O’Donnell, K., 2012. How well do ITS rDNA sequences differentiate species of true morels (Morchella). Mycologia, 104(6): 1351-1368.
12. Dunne, C., 2004. Control of sudden death in cultivated proteas from the Southwest of Western Australia. PhD Thesis, Murdoch University, Australia.
13. Falk, S.P., Parbery, D.G., 1995. Armillaria luteobubalina population structure in horticultural plantings in Victoria, Australia. Mycological Research, 99(2): 216-220.
14. Forsberg, L., 1993. Protea Diseases and Their Control. Department of Primary Industries, Queesland, Australian.
15. Gazis, R., Rehner, S., Chaverri, P., 2011. Species delimitation in fungal endophyte diversity studies and its implications in ecological and biogeographic inferences. Molecular Ecology, 20(14): 3001-3013.
16. Geiser, D.M., Gasco, M.M.J., M., Kang, S., Makalowska, I., Veeraraghavan, N., Ward, T.J., Zhang, N., Kuldau, G.A., O'Donnell, K., 2004. Fusarium-ID v. 1.0: a DNA sequence database for identifying Fusarium. European Journal of Plant Pathology, 110(5): 473-479.
17. Geiser, D.M., Lewis-Ivey, M.L., Hakiza, G., Juba, J.H., Miller, S.A., 2005. Gibberella xylarioides (Anamorph: Fusarium xylarioides), a causative agent of coffee wilt disease in Africa, is a previously unrecognized member of the G. fujikuroi species complex. Mycologia, 97(1): 191-201.
18. Guan, Y.M., Deng, J.C., Ma, Y.Y., Li, Y., Zhang, Y.Y., 2020. Seed-associated fungal diversity and the molecular identification of Fusarium with potential threat to ginseng (Panax ginseng) in China. Plant Disease, 104(2): 330-339.
19. Jedidi, I., Soldevilla, C., Lahouar, A., Marín, P., González-Jaén, M.T., Said, S., 2018. Mycoflora isolation and molecular characterization of Aspergillus and Fusarium species in Tunisian cereals. Saudi Journal of Biological Sciences, 25(5): 868-874.
20. Kintega, K.R., Zida, P.E., Tarpaga, V.W., Sankara, P., Sereme, P., 2020. Identification of Fusarium species associated with onion (Allium cepa) plants in field in Burkina Faso. Advances in Bioscience and Biotechnology, 11(3): 94-110.
21. Knox-Davies, P.S., 1981. Comments on fungus disease of plants indigenious to the South-Western Cape. Veld & Flora, 67: 88-91.
22. Knox-Davies, P.S., Van-Wyk, P.S., Marasas, W.F.O., 1987. Diseases of protea, leucospermum and leucadendron recorded in South Africa. Phytophylactica, 19(3): 327-338.
23. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7): 1870-1874.
24. Leslie, J.F., Summerell, B.A., 2006. The Fusarium Laboratory Manual. Blackwell Publishing, UK.
25. Lubbe, C.M., Denman, S., Cannon, P.F., Groenewald, J.Z., Lamprecht, S.C., Crous, P.W., 2004. Characterization of colletotrichum species associated with diseases of proteaceae. Mycologia, 96(6): 1268-1279.
26. Migliorini, D., Messal, M., Santini, A., Ramos, A.P., Talhinhas, P., Wingfield, M.J., Burgess, T., 2021. Metabarcoding reveals southern hemisphere fungal endophytes within wood of cultivated Proteaceae in Portugal. European Journal of Plant Pathology, 160(1): 173-184.
27. Minati, M.H., Mohammed-Ameen, M.K., 2019. Novel report on six Fusarium species associated with head blight and crown rot of wheat in Basra province, Iraq. Bulletin of the National Research Centre, 43(1): 1-15.
28. Moumni, M., Allagui, M.B., Mancini, V., Murolo, S., Tarchoun, N., Romanazzi, G., 2020. Morphological and molecular identification of seedborne fungi in squash (Cucurbita maxima, Cucurbita moschata). Plant Disease, 104(5): 1335-1350.
29. Nayyar, B.G., Woodward, S., Mur, L.A., Akram, A., Arshad, M., Naqvi, S.S., Akhund, S., 2018. Identification and pathogenicity of Fusarium species associated with sesame (Sesamum indicum L.) seeds from the Punjab, Pakistan. Physiological and Molecular Plant Pathology, 102: 128-135.
30. Nilsson, R.H., Abarenkov, K., Veldre, V., Nylinder, S., De-Wit, P., Brosché, S., Alfredsson, J.F., Ryberg, M., Kristiansson, E., 2010. An open source chimera checker for the fungal ITS region. Molecular Ecology Resources, 10(6): 1076-1081.
31. O'Donnell, K., 2000. Molecular phylogeny of the Nectria haematococca-Fusarium solani species complex. Mycologia, 92(5): 919-938.
32. O’Donnell, K., Gueidan, C., Sink, S., Johnston, P.R., Crous, P.W., Glenn, A., Riley, R., Zitomer, N.C., Colyer, P., Waalwijk, C., Van Der Lee, T., 2009. A two-locus DNA sequence database for typing plant and human pathogens within the Fusarium oxysporum species complex. Fungal Genetics and Biology, 46(12): 936-948.
33. O’Donnell, K., Humber, R.A., Geiser, D.M., Kang, S., Park, B., Robert, V.A., Crous, P.W., Johnston, P.R., Aoki, T., Rooney, A.P., Rehner, S.A., 2012. Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via Fusarium-ID and Fusarium MLST. Mycologia, 104(2): 427-445.
34. O'Donnell, K., Sutton, D.A., Rinaldi, M.G., Sarver, B.A., Balajee, S.A., Schroers, H.J., Summerbell, R.C., Robert, V.A., Crous, P.W., Zhang, N., Aoki, T., 2010. Internet-accessible DNA sequence database for identifying fusaria from human and animal infections. Journal of Clinical Microbiology, 48(10): 3708-3718.
35. Park, B., Park, J., Cheong, K.C., Choi, J., Jung, K., Kim, D, Lee, Y.H., Ward, T.J., O'Donnell, K., Geiser, D.M., Kang, S., 2010. Cyber infrastructure for Fusarium: three integrated platforms supporting strain identification, phylogenetics, comparative genomics and knowledge sharing. Nucleic Acids Research, 39(1): 640-646.
36. Saitou, N., Nei, M., 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4): 406-425.
37. Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge, J.L., Levesque, C.A., Chen, W., 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences, 109(16): 6241-6246.
38. Shan, L., Abdul-Haseeb, H., Zhang, J., Zhang, D., Jeffers, D.P., Dai, X., Guo, W., 2019. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of toxigenic Fusarium temperatum in maize stalks and kernels. International Journal of Food Microbiology, 291: 72-78.
39. Soren, K.R., Gangwar, P., Chaudhary, R.G., Datta, S., 2015. Population distribution and genetic relatedness in Indian Fusarium udum isolates based on ribosomal internal transcribe spacer and elongation factor. Journal of Environmental Biology, 36(5): 1063-1070.
40. Srivastava, S., Kadooka, C., Uchida, J.Y., 2018. Fusarium species as pathogen on orchids. Microbiological Research, 207: 188-195.
41. Summerell, B.A., 2019. Resolving fusarium: current status of the genus. Annual Review of Phytopathology, 57: 323-339.
42. Summerell, B.A., Laurence, M.H., Liew, E.C.Y., Leslie, J.F., 2010. Biogeography and phylogeography of Fusarium: a review. Fungal Diversity, 44(1): 3-13.
43. Summerell, B.A., Salleh, B., Leslie, J.F., 2003. A utilitarian approach to Fusarium identification. Plant Disease, 87(2): 117-128.
44. Swart, L., Denman, S., Lamprecht, S.C., Crous, P.W., 1999. Fusarium wilt: a new disease of cultivated protea in Southern Africa. Australasian Plant Pathology, 28(2): 156-161.
45. Taylor, J.E., Crous, P.W., 2000. Fungi occurring on Proteaceae. New anamorphs for Teratosphaeria, Mycosphaerella and Lembosia, and other fungi associated with leaf spots and cankers of Proteaceous hosts. Mycological Research, 104(5): 618-636.
46. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. In PCR Protocols a Guide to Methods and Applications.
47. Zarrin, M., Ganj, F., Faramarzi, S., 2016. Analysis of the rDNA internal transcribed spacer region of the Fusarium species by polymerase chain reaction-restriction fragment length polymorphism. Biomedical Reports, 4(4): 471-474.
48. Zhu, Z., Zheng, L., Pan, L., Hsiang, T., Huang, J., 2014. Identification and characterization of Fusarium species associated with wilt of Eleocharis dulcis (Chinese water chestnut) in China. Plant Disease, 98(7): 977-987.