Antagonistic activities of endophytic fungi isolated from pines against Diplodia sapinea (Fr.) Fuckel

Abstract

Diplodia sapinea, an endophytic fungus belonging to the Ascomycota, is commonly found on coniferous trees. While it typically exists as an endophyte, it can transform into an opportunistic pathogen under abiotic stress factors such as drought induced by climate change. The fungus enters the host through stomata on needles or via injured tissues, causing a disease known as Diplodia tip blight. This disease affects trees in various environments, including nurseries, plantation areas, natural forests, and urban trees. The prevalence of D. sapinea has significantly increased in Europe in recent years, and there is currently no established and effective control method worldwide. In response to this challenge, biological control method utilizing antagonist organisms have emerged as a promising alternative to combat Diplodia tip blight. The objective of this study is to evaluate the antagonistic activities of endophytic fungi isolated from different pine tree tissues against D. sapinea isolates obtained from Pinus halepensis and Pinus brutia under in vitro conditions. Identification of the isolates was carried out using both morphological and molecular methods. Fungal inhibition tests were conducted to assess the interaction between these isolates and D. sapinea isolates. The results of the tests revealed that 15 fungi, including Trichoderma sp. and Sydowia polyspora, demonstrated the potential to inhibit the growth of D. sapinea in vitro.

Keywords

• Antagonism
• Biological control
• Diplodia tip blight
• Fungal endophyte
• Türkiye

Project Number

Pamukkale University BAP department (Project no: 2021FEBE020)

Çam ağaçlarından izole edilen endofitik fungusların Diplodia sapinea (Fr.) Fuckel’e karşı antagonistik etkilerinin belirlenmesi

Abstract

Diplodia sapinea, Ascomycota’ya ait endofitik bir fungustur ve genellikle iğne yapraklı ağaçlarda bulunur. Bu fungus genellikle endofit olarak varlık gösterse de, iklim değişikliğinin neden olduğu kuraklık gibi abiyotik stres faktörleri altında fırsatçı bir patojene dönüşebilmektedir. Fungus, iğne yaprakların üzerindeki stomalar veya yaralı dokular aracılığıyla konukçusuna girmekte ve Diplodia sürgün yanıklığı olarak bilinen bir hastalığa yol açmaktadır. Bu hastalık, fidanlıklar, plantasyon alanları, doğal ormanlar ve kentsel ağaçlar gibi çeşitli ortamlardaki ağaçları etkilemektedir. Son yıllarda D. sapinea'nın Avrupa’da yaygınlığı önemli ölçüde artmış olup, dünya genelinde halen yerleşik ve etkili bir kontrol yöntemi bulunmamaktadır. Bu soruna yanıt olarak, antagonist organizmalar kullanılarak yapılan biyolojik mücadele yöntemi, Diplodia sürgün kurumasına karşı umut verici bir alternatif olarak ortaya çıkmaktadır. Bu çalışmanın amacı, farklı çam ağacı dokularından izole edilen endofitik fungusların, Pinus halepensis ve Pinus brutia'dan elde edilen D. sapinea izolatlarına karşı in vitro koşullarda antagonistik aktivitelerinin değerlendirilmesidir. İzolatların teşhisleri hem morfolojik hem de moleküler yöntemler kullanılarak gerçekleştirilmiştir. Bu izolatların D. sapinea izolatları ile olan etkileşimlerini değerlendirmek amacıyla fungal inhibisyon testleri yapılmıştır. Test sonuçları, Trichoderma sp. ve Sydowia polyspora da dahil olmak üzere 15 fungusun in-vitro olarak D. sapinea'nın gelişimini inhibe etme potansiyeline sahip olduğunu ortaya koymuştur.

Keywords

• Antagonizm
• Biyolojik mücadele
• Diplodia sürgün yanıklığı
• Endofitik fungus
• Türkiye

Project Number

Pamukkale University BAP department (Project no: 2021FEBE020)

Thanks

The authors are thankful to Pamukkale University BAP department (Project no: 2021FEBE020) for their financial support.

References

1. Baker, K.F., 1987. Evolving concepts of biological control of plant pathogens. Annual review of phytopathology, 25: 67–85.
2. Bamisile, B.S., Dash, C.K., Akutse, K.S., Keppanan, R., Wang, L., 2018. Fungal endophytes: beyond herbivore management. Frontiers in Microbiology, 9: 544.
3. Beram, R.C., Beram, A., Lehtıjarvi, H.T.D., 2016. Fungal endofitler ve etkileşimleri. Mehmet Akif Ersoy Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 7(2):161-166.
4. Bernicchia A., 2005. Polyporaceae s.l. Fungi Europaei 10: 808 p. Ed. Candusso, A.
5. Blumenstein, K., Bubkamp, J., Langer, G.J., Schlöber, R., Rojas, N.M.P., Terhonen, E., 2021. Sphaeropsis sapinea and associated endophytes in scots pine: interactions and effect on the host under variable water content. Frontiers in Forests and Global Change, 4: 655769.
6. Blumenstein, K., Langer, G., Bubkamp, J., Langer, E., Terhonen, E., 2020. The opportunistic pathogen Sphaeropsis sapinea is found to be one of the most abundant fungi in symptomless and diseased Scots pine in Central-Europe. Research Square, 3:48366.
7. Bosmans, L., Bruijn, I., Mot, R., Rediers, H., Lievens, B., 2016. Agar composition affects in vitro screening of biocontrol activity of antagonistic microorganisms. Journal of Microbiological Methods, 7-9:127.
8. Brodde, L., Adamson, K., Camarero, J.J., Castaño, C., Drenkhan, R., Lehtijärvi, A., Luchi, N., Miglliorini, D., Miranda, A.S., Stenlid, J., Özdağ, Ş., Oliva, J., 2019. Diplodia tip blight on its way to the north: drivers of disease emergence in northern Europe. Frontiers in Plant Science, 9:1818.

9. Brookhouser, L.W., Peterson, G.W., 1971. Infection of Austrian, Scots, and ponderosa pines by Diplodia pinea. Phytopathology, 61(4): 409-414.
10. Bußkamp, J., 2018. Schadenserhebung, Kartierung und Charakterisierung des Diplodia-Triebsterbens “der Kiefer, insbesondere des endophytischen Vorkommens in den klimasensiblen Räumen und Identifikation von den in Kiefer (Pinus sylvestris) vorkommenden Endophyten.PhD Dissertation, Universität Kassel, Fachbereich 10 Mathematik und Naturwissenschaften Fachgebiet Ökologie, Germany.
11. Bußkamp, J., Langer, G.J., Langer, E.J., 2020. Sphaeropsis sapinea and fungal endophyte diversity in twigs of Scots pine (Pinus sylvestris) in Germany. Mycological Progress, 19(9):985-999.
12. Capretti, P., Santini, A., Solheim, H., 2013. Pathogens, Significance and Distribution. In Infectious forest diseases, Ed. Gonthier, P., Nicolotti, G., 21 Branch and Tip Blights, Italy pp. 420-435.
13. Chou, I. M., 1987. Phase relations in the system NaCl-KCl-H2O. III: Solubilities of halite in vapor-saturated liquids above 445° C and redetermination of phase equilibrium properties in the system NaCl-H2O to 1000° C and 1500 bars. Geochimica et Cosmochimica Acta, 51(7):1965-1975.
14. Costa, D., Tavares, R.M., Baptista, P., Lino-Neto, T., 2020. Cork oak endophytic fungi as potential biocontrol agents against Biscogniauxia mediterranea and Diplodia corticola. Journal of Fungi, 6(4):287.
15. Dennis, C., Websterm, J., 1971. Antagonistic properties of species-groups of Trichoderma: I. Production of non-volatile antibiotics. Transactions of the British Mycological Society, 57(1):25-39.
16. Diekmann, M., Sutherland, J.R., Nowell, D.C., Morales, F.J., Allard, G., 2002. Pinus spp. Food and Agriculture Organization of the United Nations, Technical Guidelines for the Safe Movement of Germplasm, Rome.
17. Ganley, R., 2008. Density and diversity of fungal endophytes isolated from needles of Pinus radiata. Client Report, N 2925.
18. Grondona, I., Hermosa, R., Tejada, M., Gomıs, M. D., Mateos, P. F. Brıdge, P. D., Monte, E., Garcıa-Acha, I., 1997. Physiological and Biochemical Characterization of Trichoderma Harzianum, A Biological Control Agent Against Soilborne Fungal Plant Pathogens. Applied And Envıronmental Microbiology, 63 (8):3189–3198.
19. Guzmán-Guzmán, P., Kumar, A., Santos-Villalobos, S., Parra-Cota, F.I, Orozco-Mosqueda, M., Fadiji, A.E., Hyder, S., Babalola, O.O., Santoyo, G., 2023. Trichoderma Species: Our best fungal allies in the biocontrol of plant diseases - A Review. Plants, 12(3): 432
20. Harman G. E., 2006. Overview of mechanisms and uses of Trichoderma spp. Phytopathology,96 (2):190–194.
21. Kaya, A.G.A., Lehtijärvi, A., Kaya, Ö., Doğmuş-Lehtijärvi, T., 2014. First report of Diplodia pinea on Pseudotsuga menziesii in Turkey. Plant disease, 98(5): 689-689.
22. Kaya, A.G.A., Yeltekin, Ş., Lehtijärvi, T.D., L., Lehtijärvi, A., Woodward, S., 2019. Severity of Diplodia shoot blight (caused by Diplodia sapinea) was greatest on Pinus sylvestris and Pinus nigra in a plantation containing five pine species. Phytopathologia Mediterranea, 58(2):249-259.
23. Keller, N. P., Turner, G., 2012. Fungal secondary metabolism: regulation and function. Current Opinion in Microbiology, 15(5):508-515.
24. Kowalski, T., Bilański, P., 2021. Fungi detected in the previous year’s leaf petioles of Fraxinus excelsior and their antagonistic potential against Hymenoscyphus fraxineus. Forests, 12(10):1412.
25. Köhl, J., Kolnaar, R., Ravensberg, W., 2019. Mode of action of microbial biological control agents against plant diseases: relevance beyond efficacy. Frontiers in Plant Science, 10:845.
26. Küçük, Ç., Kivanç, M., 2003. Isolation of Trichoderma spp. and determination of their antifungal, biochemical and physiological features. Turkish Journal of Biology, 27(4):247-253.
27. Langer, G., Bressem, U., Habermann, M., 2011. Diplodia-Triebsterben der Kiefer und endophytischer Nachweis des Erregers Sphaeropsis sapinea. AFZ-Der Wald, 11: 28-31.
28. Mejía, L.C., Rojas, E.I., Maynard, Z., Van Bael, S., Arnold, A.E., Hebbar, P., Samuels, G., Robbins, N., Herre, E.A., 2008. Endophytic fungi as biocontrol agents of Theobroma cacao pathogens. Biological control, 46(1):4-14.
29. Mgbeahuruike, A.C., Sun, H., Fransson, P., Kasanen, R., Daniel, G., Karlsson, M., Asiegbu, F.O., 2011. Screening of Phlebiopsis gigantea isolates for traits associated with biocontrol of the conifer pathogen Heterobasidion annosum. Biological Control, 57(2):118-129.
30. Munck, I.A., Smith, D.R., Sickley, T., Stanosz, G.R., 2009. Site-related influences on cone-borne inoculum and asymptomatic persistence of Diplodia shoot blight fungi on or in mature red pines. Forest Ecology and Management, 257(3):812-819.
31. Muñoz-Adalia, E.J., Sanz-Ros, A.V., Flores-Pacheco, J.A., Hantula, J., Diez, J.J., Vainio, E.J., Fernández, M., 2017. Sydowia polyspora dominates fungal communities carried by two Tomicus species in pine plantations threatened by Fusarium circinatum. Forests, 8(4):127.
32. Oliva, J., Ridley, M., Redondo, M.A., Caballol, M., 2021. Competitive exclusion amongst endophytes determines shoot blight severity on pine. Functional Ecology, 35(1):239-254.
33. Oskay, F., Lehtijärvi, A., Lehtijärvi, H.T.D., Woodward, S., 2018. First report of Diplodia sapinea on Cedrus libani in Turkey. New Disease Reports, 38: 13.
34. Oskay, F., Şimşek, Z., 2017. Çankırı (Eldivan) Karaçam Orman topraklarında saptanan bazı mikrofungusların in vitro koşullarda antagonistik etkileşimlerinin belirlenmesi. Anadolu Orman Araştırmaları Dergisi, 3(2):130-138.
35. Pan, Y., Ye, H., Lu, J., Chen, P., Zhou, X.D., Qiao, M., Yu, Z.F., 2018. Isolation and identification of Sydowia polyspora and its pathogenicity on Pinus yunnanensis in Southwestern China. Journal of Phytopathology, 166(6):386-395.
36. Parkinson, D., 1994. Filamentous fungi. Methods of Soil Analysis: Microbiological and Biochemical Properties, America.
37. Parnell, I.W., 1957. Some notes from Australia. Scottish Agriculture, 37(2): 84-87.
38. Prospero, S., Botella, L., Santini, A., Robin, C., 2021. Biological control of emerging forest diseases: How can we move from dreams to reality? Forest Ecology and Management, 496:119377.
39. Raghavendra, A.K., Newcombe, G., 2013. The contribution of foliar endophytes to quantitative resistance to Melampsora rust. New Phytologist, 197(3):909-918.
40. Rodriguez, R.J., White, J.F., Arnold, A.E., Redman, R.S., 2009. Fungal endophytes: diversity and functional roles. New Phytologist, 182(2):314-330.
41. Ryvarden, L., 1978. The Polyporaceae of North Europe. Inonotus-Tyromyces, 2: 219-507.
42. Sanz-Ros, A.V., Müller, M.M., San Martín, R., Diez, J.J., 2015. Fungal endophytic communities on twigs of fast and slow growing Scots pine (Pinus sylvestris L.) in northern Spain. Fungal Biology, 119(10):870-883.
43. Schulz, B., Boyle, C., Draeger, S., Römmert, A. K., Krohn, K., 2002. Endophytic fungi: a source of novel biologically active secondary metabolites. Mycological Research, 106(9):996-1004.
44. Silva, N.I.S., Brooks, S., Lumyong, S., Hyde, K.D., 2019. Use of endophytes as biocontrol agents. Fungal Biology Reviews, 33:133-148.
45. Soylu, S., Kurt, Ş., Soylu, E.M., 2001. Determination of important fungal disease agents on pine trees in the Kahramanmaraş regional forests. Journal of Turkish Phytopathology, 30: 79.
46. Stanosz, G.R., Blodgett, J.T., Smith, D.R., Kruger, E.L., 2001. Water stress and Sphaeropsis sapinea as a latent pathogen of P. brutia seedlings. New Phytologist, 149(3): 531-538.
47. Sümer, S., 2000. Shoot Blight Disease Caused by Sphaeropsis sapinea in Pine Stands at the South-Eastern Region, Turkey. (OGM research report)
48. Swart, W.J., Wingfield, M.J., 1991. Biology and control of Sphaeropsis sapinea on Pinus species in South Africa. Plant Disease, 75(8):761-766.
49. Tellenbach, C., Sumarah, M.W., Grünig, C.R., Miller, J.D., 2013. Inhibition of Phytophthora species by secondary metabolites produced by the dark septate endophyte Phialocephala europaea. Fungal Ecology, 6(1): 12-18.
50. Terhonen, E., Blumenstein, K., Kovalchuk, A., Asiegbu, F. O., 2019. Forest tree microbiomes and associated fungal endophytes: Functional roles and impact on forest health. Forests, 10(1): 42.
51. Terhonen, E., Kovalchuk, A., Zarsav, A., Asiegbu, F.O., 2018. Biocontrol Potential of Forest Tree Endophytes. In: Forestry Sciences (Ed: Pirttilä, A.M., Frank, C.), Endophytes of Forest Trees, Finland, pp. 283-318.
52. Thambugala, K., Daranagama, D., Phillips, A., Kannangara, S., Promputtha, I., 2020. Fungi vs. fungi in biocontrol: an overview of fungal antagonists applied against fungal plant pathogens. Frontiers in Cellular and Infection Microbiology, 10: 604923.
53. Trejo-Estrada, S.R., Sepulveda, I.R., Crawford, D.L., 1998. In vitro and in vivo antagonism of Streptomyces violaceusniger YCED9 against fungal pathogens of turfgrass. World Journal of Microbiology and Biotechnology, 14: 865-872.
54. Ünligil, H., Ertaş, A., 1993. İstanbul yakınlarındaki çam ağaçlarında Sphaeropsis sapinea (Fr.) Dylco & Sutton mantar hastalığı. Journal of the Faculty of Forestry Istanbul University, 43(1): 131-138.
55. Whipp, S. C., 1987. Protease degradation of Escherichia coli heat-stable, mouse-negative, pig-positive enterotoxin. Infection and immunity, 55(9), 2057-2060.
56. White, T.J., Bruns, T., Lee, S.J.W.T., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications, 18(1): 315-322.
57. Wilson, D. M., Mubatanhema, W., Jurjevic, Z., 2002. Biology and ecology of mycotoxigenic Aspergillus species as related to economic and health concerns. Mycotoxins and Food Safety, 504:3-17.
58. Witzell, J., Martín, J.A., 2018. Endophytes and forest health. Endophytes of Forest Trees: Biology and Applications, 86: 261-282.
59. Witzell, J., Martín, J.A., Blumenstein, K., 2014. Ecological aspects of endophyte-based biocontrol of forest diseases. Advances in Endophytic Research, 321-333.
60. Yeltekin, Ş., 2015. Kerpe araştırma ormanı konifer türlerinde Diplodia spp.’den kaynaklanan kozalak ve sürgün enfeksiyonlarının belirlenmesi. Yüksek lisans Tezi, Süleyman Demirel Üniversitesi. Fen Bilimleri Enstitüsü, Isparta.
61. Zlatković, M., Keča, N., Wingfield, M.J., Jami, F., Slippers, B., 2017. New and unexpected host associations for Diplodia sapinea in the Western Balkans. Forest Pathology, 47(3): e12328.