Isolation of endophytic bacterial isolates from healthy banana trees and determination of their in vitro antagonistic activities against crown rot disease agent Fusarium verticillioides

Abstract

Aims: Fungal diseases are one of the most important biotic factors causing serious losses in banana cultivation in field or greenhouses during cultivation, harvest, storage and transportation periods. Crown rot, caused by Fusarium verticillioides, is considered one of the most important postharvest fungal disease of banana fruits. In this study, endophytic bacterial isolates were obtained from fruits, branches and leaves of healthy banana trees and their antagonistic potentials were investigated against F. verticillioides as a biological control agent (BCA) in vitro conditions.

Methods and Results: A total of 23 putative endophytic bacterial isolates were obtained from fruits, stems and leaves of healthy banana trees. All bacterial isolates were identified by MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry) analysis. In vitro antagonistic efficiencies of 12 different bacterial isolates, from eight different species (Bacillus subtilis ssp. spizizenii (3), Bacillus amyloliquefaciens (2), Bacillus subtilis ssp. subtilis (2), Bacillus mojavensis, Enterococcus faecium, Enterobacter cloacae, Enterobacter ludwigii and Pseudomonas stutzeri), were tested on inhibiting mycelial growth by using dual culture tests. Among bacterial isolates, the highest antagonistic activity was displayed by Bacillus mojavensis BEn3 isolate which significantly inhibited the mycelial growth by 50.83%. Enterobacter cloacae BEn1, Enterobacter ludwigii BEn2, Enterobacter faecium BEn7, however, failed to inhibit the mycelial growth of fungi.

Conclusions: Significant suppression in the mycelial growth caused by endophytic bacterial isolates indicates that Bacillus mojavensis BEn3 isolate could be considered as possible BCA against crown rot disease agent.

Significance and Impact of the Study: Our findings suggest that B. mojavensis Ben3, as the most successful endophyte bacterial isolate that suppresses the growth of F. verticillioides, can be used as a promising biological control agent as an alternative to chemical control against crown rot disease.

Keywords

• Banana
• biological control
• endophytic bacteria
• crown rot
• Fusarium verticillioides

Sağlıklı muz ağaçlarından endofit bakteri izolatlarının izolasyonu ve taç çürüklüğü hastalığı etmeni Fusarium verticillioides’e karşı antagonistik etkinliklerinin in vitro koşullarda belirlenmesi

Öz

Amaç: Fungal hastalıklar tarla veya seralarda muz yetiştiriciliğinde ekim, hasat, depolama ve nakliye dönemlerinde ciddi kayıplara neden olan en önemli biyotik faktörlerden biridir. Fusarium verticillioides'in neden olduğu taç çürüklüğü, muz meyvelerinin en önemli hasat sonrası fungal hastalıklarından biri olarak kabul edilir. Bu çalışmada, sağlıklı muz ağaçlarının meyve, dal ve yapraklarından endofitik bakteri izolatları elde edilmiş ve biyolojik kontrol ajanı (BCA) olarak antagonistik potansiyelleri F. verticillioides'e karşı in vitro koşullarda araştırılmıştır.

Yöntem ve Bulgular: Sağlıklı muz ağaçlarının meyvelerinden, gövdelerinden ve yapraklarından toplam 23 adet aday endofit bakteri izolatı elde edilmiştir. Tüm bakteri izolatları MALDI-TOF MS (Matrix Assisted Laser Desorpsiyon Ionization-Time Of Flight Kütle Spektrometresi) analizi ile tanımlanmıştır. Sekiz farklı türden 12 farklı bakteri izolatının (Bacillus subtilis ssp. spizizenii (3), Bacillus amyloliquefaciens (2), Bacillus subtilis ssp. subtilis (2), Bacillus mojavensis, Enterococcus faecium, Enterobacter cloacae, Enterobacter ludwigii ve Pseudomonas stutzeri) misel büyümesinin engellenmesindeki in vitro antagonistik etkinlikleri ikili kültür testleri ile test edilmiştir. Bakteri izolatları arasında en yüksek antagonistik aktivite misel gelişimini %50.83 oranında engelleyen Bacillus mojavensis BEn3 izolatı tarafından gösterilmiştir. Bakteriyel izolatlar arasından Enterobacter cloacae BEn1, Enterobacter ludwigii BEn2, Enterobacter faecium BEn7 izolatları fungal etmenin misel gelişimini engellemede başarısız olmuştur.

Genel Yorum: Endofit bakteri izolatlarının fungal etmenin misel gelişimini önemli düzeylerde baskılamış olması, Bacillus mojavensis BEN3 izolatının taç çürüklüğü etmenine karşı olası biyolojik kontrol ajanı olarak kabul edilebileceğini göstermektedir.

Çalışmanın Önemi ve Etkisi: Bulgularımız, F. verticillioides’in gelişimini baskılayan en başarılı endofit bakteri izolatı olan B. mojavensis Ben3’in taç çürüklüğü hastalığına karşı kimyasal mücadeleye alternatif ve umut verici bir biyolojik kontrol ajanı olarak kullanılabileceğini önermektedir.

Anahtar Kelimeler

• Muz
• Biyolojik mücadele
• endofit bakteri
• taç çürüklüğü
• Fusarium verticillioides

Kaynakça

1. Abbas A, Khan SU, Khan WU, Saleh TA, Khan MHU, Ullah S, Ali A, Ikram M (2019) Antagonist effects of strains of Bacillus spp. against Rhizoctonia solani for their protection against several plant diseases: Alternatives to chemical pesticides. C. R. Biologies 342: 124–135.
2. Akram W, Anjum T (2011) Use of bioagents and synthetic chemicals for induction of systemic resistance in tomato against diseases. International Research Journal of Agricultural Science and Soil Science 1: 286-292.
3. Aktan ZC, Soylu S (2020) Diyarbakır ilinde yetişen badem ağaçlarından endofit ve epifit bakteri türlerinin izolasyonu ve bitki gelişimini teşvik eden mekanizmalarının karakterizasyonu. KSÜ Tarım ve Doğa Derg. 23(3): 641-654.
4. Al-Hatmi AMS, Meis JF, de Hoog GS (2016) Fusarium: Molecular diversity and ıntrinsic drug resistance. PLoS Pathog. 12(4): e1005464.
5. Alvindia DG, Natsuaki KT (2009) Biocontrol activities of Bacillus amyloliquefaciens DGA14 isolated from banana fruit surface against banana crown rot-causing pathogens. Crop Prot. 28: 236-242.
6. Alvindia DG (2013) An integrated approach with hot water treatment and salt in the control of crown rot disease and preservation of quality in banana. Int. J. Pest Manag. 59: 271-278.
7. Anand M, Naik MK, Ramegowda G, and Devika Rani GS (2010) Biocontrol and growth promotion activity of indigenous isolates of Pseudomonas fluorescens. J. Mycopathol. Res. 48: 45-50.
8. Anonymous (2021) TUİK Bitkisel Üretim İstatistikleri. http://www.tuik.gov.tr/bitkiselapp/bitkisel.zul. Access date:01.11.2021.

9. Anthony S, Abeywickrama K, Dayananda R, Wijeratnam Shanthi W, Arambewela L (2004) Fungal pathogens associated with banana fruit in Sri Lanka and their treatment with essential oils. Mycopathologia 157: 91-97.
10. Appiah-Sarpong M, Kumah P, Olympio NS, Moses E (2013) In vitro management of crown rot disease of banana (Musa spp. AAA) 'Medium Cavendish' using extracts from Ocimum gratissimum. Alstonia boonei and Garcinia kola. Acta Hortic. 1007: 739-746.
11. Damasceno CL, Duarte EAA, dos Santos LBPR, de Oliveira TAS, de Jesus FN, de Oliveira LM, Goes-Neto A, Soares ACF (2019) Postharvest biocontrol of anthracnose in bananas by endophytic and soil rhizosphere bacteria associated with sisal (Agave sisalana) in Brazil. Biol. Control 137: 101016.
12. De Costa DM, Erabadupitiya HRUT (2005) An integrated method to control postharvest diseases of banana using a member of the Burkholderia cepacia complex. Postharvest Biol. Tec. 36: 31-39.
13. Dukare AS, Paul S, Nambi VE, Gupta RK, Singh R, Sharma K, Vishwakarma RK (2019) Exploitation of microbial antagonists for the control of postharvest diseases of fruits: a review. Crit. Rev. Food 59(9): 1498-1513.
14. Dutta D, Puzari KC, Gogoi R, Dutta P (2014) Endophytes: Exploitation as a tool in plant protection. Braz. Arch. Biol. Technol. 57: 621-629.
15. Eljounaidi K, Lee SK, Bae H (2016) Bacterial endophytes as potential biocontrol agents of vascular wilt diseases – Review and future prospects. Biol. Control 103: 62-68.
16. Faruk S, Soylu S (2021) Mersin ilinde yetiştirilen muz meyvelerinde hasat sonrasında görülen fungal hastalık etmenlerinin belirlenmesi. MKU. Tar. Bil. Derg. 26(2): 399-411.
17. Fisher NL, Burgess LW, Toussoun TA, Nelson PE (1982) Carnation leaves as a substrate and for preserving cultures of Fusarium species. Phytopathology 72: 151-153.
18. Fravel DR (2005) Commercialization and implementation of biocontrol. Annu. Rev. Phytopathol. 43: 337-359.
19. Fu G, Huang SL, Ye YF, Wu YG, Cen ZL, Lin SH (2010) Characterization of a bacterial biocontrol strain B106 and its efficacy in controlling banana leaf spot and post-harvest anthracnose diseases. Biol. Control 55(1): 1-10.
20. Gao FK, Dai CC, Liu XZ (2010) Mechanisms of fungal endophytes in plant protection against pathogens. Afric. J. Microbiol. Res. 4: 1346-1351.
21. Gunasinghe WKRN, Karunaratne AM (2009) Interactions of Colletotrichum musae and Lasiodiplodia theobromae and their biocontrol by Pantoea agglomerans and Flavobacterium sp in expression of crown rot of "Embul" banana. Biocontrol 54: 587-596.
22. Kara M, Soylu S, Kurt Ş, Soylu EM, Uysal A (2020) Determination of antagonistic traits of bacterial isolates obtained from apricot against green fruit rot disease agent Sclerotinia sclerotiorum. Acta Horticulturae 1290:135-142.
23. Kurt Ş, Soylu S, Uysal A, Soylu EM, Kara M (2020) Ceviz gövde kanseri hastalığı etmeni Botryosphaeria dothidea’nın tanılanması ve bazı fungisitlerin hastalık etmenine karşı in vitro antifungal etkinliklerinin belirlenmesi. MKU. Tar. Bil. Derg. 25(1): 46-56.
24. Lelliot RA, Stead DE (1987) Methods for the diagnosis of bacterial diseases of plants. (T.F. Preece. Editör). In: Methods in plant pathology. Vol 2. Blackwell Scientific Publications. pp. 176-177. Oxford.
25. Leslie JF, Summerell BA (2006) The Fusarium laboratory manual. Blackwell Publishing: Hoboken. Iowa. USA. 388 pp.
26. Maqbool M, Ali A, Alderson PG, Mohamed MTM, Siddiqui Y, Zahid N (2011) Postharvest application of gum arabic and essential oils for controlling antrachnose and quality of banana and papaya during cold storage. Postharvest Biol. Tec. 62: 71-76.
27. McInroy JA, Kloepper JW (1995) Population dynamics endophytic bacteria of in field-grown sweet corn and cotton. Can. J. Microbiol. 141: 895-901.
28. Rabiey M, Hailey LE, Roy SR, Grenz K, Al-Zadjali MAS, Barrett GA, Jackson RW (2019) Endophytes vs tree pathogens and pests: can they be used as biological control agents to improve tree health? Eur. J. Plant Pathol. 155: 711-729.
29. Nunes CA (2012) Biological control of postharvest diseases of fruit. European J. Plant Pathol. 133(1)(Special Issue: SI): 181-196.
30. Ons L, Bylemans D, Thevissen K, Cammue BPA (2020) Combining biocontrol agents with chemical fungicides for integrated plant fungal disease control. Microorganisms 8(12): 1930.
31. Pınar H, Türkay C, Denli N, Ünlü M, Bircan M (2011) Türkiye’de muz üretim potansiyeli. GAP VI. Tarım Kongresi 09-12/05/2011 Şanlıurfa.
32. Ranasinghe L, Jayawardena B, Abeywickrama K (2002) Fungicidal activity of essential oils of Cinnamomum zeylanicum (L.) and Syzygium aromaticum (L.) Merr et L.M. Perry against crown rot and antrachnose pathogens isolated from banana. Lett. Appl. Microbiol. 35: 208-211.
33. Sangeetha G, Thangavelu R, Rani SU, Muthukumar A, Udayakumar R (2010) Induction of systemic resistance by mixtures of antagonist bacteria for the management of crown rot complex on banana. Acta Physiol. Plant. 32(6): 1177-1187.
34. Santoyo G, Moreno-Hagelsieb G, Orozco-Mosqueda MC, Glick BR (2016) Plant growth-promoting bacterial endophytes. Microbiol. Res. 183: 92-99.
35. Schaad NW (2001) Initial identification of common genera. (N.W. Schaad. J.B. Jones. W. Chun. Editör). In: Laboratory guide for identification of plant pathogenic bacteria. 3rd edn. The American Phytopathological Society. St. Paul. MN. St Paul. pp. 1–15. USA.
36. Snowdon AL (2010) A colour atlas of postharvest diseases and disorders of fruits and vegetables. Volume 1: General Introduction and Fruits. Wolfe Scientific. Spain. 302 pp.2.
37. Soylu EM, Soylu S, Kara M, Kurt Ş (2020) Sebzelerde sorun olan önemli bitki fungal hastalık etmenlerine karşı vermikomposttan izole edilen mikrobiyomların in vitro antagonistik etkilerinin belirlenmesi. KSU J. Agric. Nat. 23: 7-18.
38. Stein T (2005) Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol. Microbiol. 56(4): 845-857.
39. Tan S, Dong Y, Liao H, Huang J, Song S, Xu Y, Shen Q (2012) Antagonistic bacterium Bacillus amyloliquefaciens induces resistance and controls the bacterial wilt of tomato. Pest Manag. Sci. 69: 1245-1252.
40. Tariq M, Khan A, Asif M, Khan F, Ansari T, Shariq M, Siddiqui MA (2020) Biological control: a sustainable and practical approach for plant disease management. Acta Agric. Scand. B Soil Plant Sci. 70(6): 507-524.
41. Umana-Rojas G, Garcia J (2011) Efficacy of plant extracts on growth reduction of Colletotrichum musae and Fusarium proliferatum causal agents of crown rot of bananas. Acta Hortic. 906: 205-210.
42. Williamson SM, Guzman M, Marin DH, Anas O, Jin X, Sutton TB (2008) Evaluation of Pseudomonas syringae strain ESC-11 for biocontrol of crown rot and antrachnose of banana. Biol. Control 46: 279-286.
43. Wilson CL, Wisniewski M, Droby S, Chalutz E (2011) Historical perspective on biological control of postharvest diseases - past, present, and future. Acta Hortic. 905: 23-28.
44. Zhang S, Reddy MS, Kloepper JW (2004) Tobacco growth enhancement and blue mold disease protection by rhizobacteria: Relationship between plant growth promotion and systemic disease protection by PGPR strain 90-166. Plant and Soil 262: 277-288.
45. Zhang H, Mahunu GK, Castoria R, Apaliya MT Yang Q (2017) Argumentation of biocontrol agents with physical methods against postharvest diseases of fruits and vegetables. Trends Food Sci. Technol. 69: 36-45.