Isolating, characterizing, and utilizing Trichoderma asperellum to antagonize Neurospora spp. causing scab disease on King mandarin fruits (Citrus sinensis)
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Abstract
The research aimed to (i) isolate, select, and assess the pathogenic potential of fungi that pose a risk of causing scab disease on King mandarin fruits (Citrus sinensis), and (ii) evaluate the antagonistic potential of Trichoderma spp. against Neurospora spp., , which cause scab disease on King mandarin fruits under in vitro conditions. The isolation process identified four fungal strains from ten King mandarin fruits showing scab symptoms, collected from ten orchards in Vung Liem District, Vinh Long Province. The three most virulent scab-causing fungal strains were KMS-01, KMS-02, and KMS-04, with growth diameters of 7.60–7.63 cm after 72 hours of culture (HoC). Additionally, the antagonistic ability of 50 Trichoderma spp. strains against Neurospora spp. ranged from 49.8% to 87.6% at 72 HoC. Among these, three Trichoderma spp. strains—T-SP03, T-SP26, and T-SP41—exhibited high antagonistic efficiency (86.1%–87.7%) against all three scab-causing strains. Based on the ITS region, the pathogenic fungal strains were identified as Neurospora intermedia KMS-01, N. intermedia KMS-02, and N. crassa KMS-04, while the Trichoderma spp. strains were identified as Trichoderma asperellum T-SP03, T-SP26, and T-SP41 with 100% similarity. T. asperellum shows potential as a biological control agent against Neurospora spp., the causative agents of scab disease in King mandarin.
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References
Ahmed S, Chowdhury AN, Dey AK, Moniruzzaman M, & Kowser A. 2022. Isolation and identification of rhizosphere soil fungi from papaya (Carica papaya L.) and eggplant (Solanum melongena L.) at BCSIR campus in Rajshahi, Bangladesh. IJSRP. 12(4): 21–26. https://doi.org/10.29322/IJSRP.12.04.2022.p12404
Alam MM. 2006. Management of Scab and Die-Back of Citrus (Citrus limon L.) through Bioagent, Plant Extracts and Fungicides. Master Thesis. Sher-e-Bangla Agricultural University, Dhaka.
Alam MS, Hossain A, Hossain MI, Islam MZ, Bazzaz MM, Naznin S, Kabir MH, Hossain MA, Miah MJ, & Akter H. 2020. Maximum congenial period for outbreak of die–back and scab diseases in citrus and their management under sub–tropical region. Thai J. Agric. Sci. 53(1): 32–52.
Awad NE, Kassem HA, Hamed MA, El-Feky AM, Elnaggar MAA, Mahmoud K, & Ali MA. 2018. Isolation and characterization of the bioactive metabolites from the soil derived fungus Trichoderma viride. Mycology. 9(1): 70–80. https://doi.org/10.1080/21501203.2017.1423126
Barahoei N, Alaei Shahvali Anar H, Riseh S, & Sedaghati E. 2023. Evaluation of the antagonistic effect of Trichoderma species on Phytophthora citrophthora, the causal agent of citrus root and crown rot. Iranian J. Plant Prot. Sci. 53(2): 295–311. https://doi.org/10.22059/ijpps.2023.352568.1007015
Bell DK, Wells HD, & Markham CR. 1982. In vitro antagonism of Trichoderma species against six fungal plant pathogens. Phytopathology.72(4): 379–382. https://doi.org/10.1094/Phyto-72-379
Burgess LW, Knight TE, Tesoriero L, & Phan HT. 2008. Diagnostic Manual for Plant Diseases in Vietnam. pp. 129–210. ACIAR Monograph No 129. Canberra.
Caserta R, Teixeira-Silva NS, Granato LM, Dorta SO, Rodrigues CM, Mitre LK, Yochikawa JTH, Fischer ER, Nascimento CA, Souza-Neto RR, Takita MA, Boscariol-Camargo RL, Machado MA, & De Souza AA. 2019. Citrus biotechnology: What has been done to improve disease resistance in such an important crop?. Biotechnol. Res. Innov. 3(1): 95–109. https://doi.org/10.1016/j.biori.2019.12.004
Choi CW, Hyun JW, Hwang RY, Park JS, & Jung KE. 2020. First Report of citrus scab on trifoliate orange (Poncirus trifoliata). Res. Plant Dis. 26(1): 57–60. https://doi.org/10.5423/RPD.2020.26.1.57
De la Cruz-Quiroz R, Roussos S, Rodríguez-Herrera R, Hernandez-Castillo D, & Aguilar CN. 2018. Growth inhibition of Colletotrichum gloeosporioides and Phytophthora capsici by native Mexican Trichoderma strains. Karbala Int. J. Mod. Sci. 4(2): 237–243. https://doi.org/10.1016/j.kijoms.2018.03.002
Elliott AJ, van Raak MMJP, Barnes AV, Field CJ, van Duijnhoven AALAM, Webb K, & van de Vossenberg BTLH. 2023. Real-time PCR detection of Elsinoë spp. on citrus. PhytoFrontiers. 3(1): 164–172. https://doi.org/10.1094/PHYTOFR-03-22-0017-FI
Esparza?Reynoso S, Ruíz?Herrera LF, Pelagio?Flores R, Macías?Rodríguez LI, Martínez?Trujillo M, López?Coria M, Sánchez-Nieto S, Herrera-Estrella A, & López?Bucio J. 2021. Trichoderma atroviride?emitted volatiles improve growth of Arabidopsis seedlings through modulation of sucrose transport and metabolism. Plant Cell Environ. 44(6): 1961–1976. https://doi.org/10.1111/pce.14014
Ferreira FV, Herrmann?Andrade AM, Calabrese CD, Bello F, Vázquez D, & Musumeci MA. 2020. Effectiveness of Trichoderma strains isolated from the rhizosphere of citrus tree to control Alternaria alternata, Colletotrichum gloeosporioides and Penicillium digitatum A21 resistant to pyrimethanil in post?harvest oranges (Citrus sinensis L.(Osbeck)). J. Appli. Microbiol. 129(3): 712–727. https://doi.org/10.1111/jam.14657
Gañán-Betancur L & Gazis R. 2023. Genome sequence resource of the avocado scab pathogen Elsinoe perseae. Microbiol. Resour. Announc. 12(6): e00190-23. https://doi.org/10.1128/mra.00190-23
Gopal K, Govindarajulu B, Ramana KTV, Kishore Kumar CS, Gopi V, Gouri Sankar T, Mukunda Lakshmi L, Naga Lakshmi T, & Sarada G. 2014. Citrus scab (Elsinoe fawcettii): A review. RRJAAS . 3(3): 49–58.
Gouit S, Chair I, Belabess Z, Legrifi I, Goura K, Tahiri A, Lazraq A, & Lahlali R. 2024. Harnessing Trichoderma spp.: A promising approach to control apple scab disease. Pathogens. 13(9): 752. https://doi.org/10.3390/pathogens13090752
Guzmán-Guzmán P, Kumar A, de Los Santos-Villalobos S, Parra-Cota FI, Orozco-Mosqueda MD, Fadiji AE, Hyder S, Babalola OO, & Santoyo G. 2023. Trichoderma species: Our best fungal allies in the biocontrol of plant diseases—A review. Plants. 12(3): 432. https://doi.org/10.3390/plants12030432
Hieu TS, Hau TV, & Bang PC. 2011. Investigation of plant descriptors of some lime cultivars (Citrus aurantifolia L.) in Cai Be district, Tien Giang province. Can Tho Univ. J. Sci. 20b: 106–116.
Huang HM & Huang HJ. 1999. The occurrence of citrus scab and its control. South China Fruits 28: 1–8.
EFSA Panel on Plant Health (PLH), Jeger M, Bragard C, Caffier D, Candresse T, Chatzivassiliou E, Dehnen?Schmutz K, Gilioli G, Grégoire JC, Jaques Miret JA, MacLeod A, Navajas Navarro M, Niere B, Parnell S, Potting R, Rafoss T, Urek G, Van Bruggen A, Van der Werf W, West J, Winter S, Vicent A, Vloutoglou I, Bottex B, & Rossi V. 2017. Pest categorisation of Elsinoë fawcettii and E. australis. EFSA J. 15(12): e05100. https://doi.org/10.2903/j.efsa.2017.5100
Kakraliya S, Choskit D, Pandit D, & Abrol S. 2017. Effect of bio-agents, neem leaf extract and fungicides against Alternaria leaf blight of wheat (Triticum aestivum L.). Nat. Prod. Chem. Res. 5: 295. https://doi.org/10.4172/2329-6836.1000295
Khajuria YP, Akhoon BA, Kaul S, & Dhar MK. 2022. Secretomic insights into the pathophysiology of Venturia inaequalis: The causative agent of scab, a devastating apple tree disease. Pathogens. 12(1): 66. https://doi.org/10.3390/pathogens12010066
Khuong NQ, Nhien DB, Thu LTM, Trong ND, Hiep PC, Thuan VM, Quang LT, Thuc LV, & Xuan DT. 2023a. Using Trichoderma asperellum to antagonize Lasiodiplodia theobromae causing stem-end rot disease on pomelo (Citrus maxima) J. Fungi. 9(10): 981. https://doi.org/10.3390/jof9100981
Khuong NQ, Trang CTT, Xuan DT, Quang LT, Huu TN, Xuan LNT, Sakagami JI, & Thuc LV. 2023b. Evaluation of the antagonistic potential of Trichoderma spp. against Fusarium oxysporum F.28.1A. J. Plant Prot. Res. 63(1): 13–26. https://doi.org/10.24425/jppr.2023.144502
Kubiak A, Wolna-Maruwka A, Pilarska AA, Niewiadomska A, & Piotrowska-Cyplik A. 2023. Fungi of the Trichoderma genus: future perspectives of benefits in sustainable agriculture. Appl. Sci. 13(11): 6434. https://doi.org/10.3390/app13116434
Kulimushi SM, Muiru WM, & Mutitu EW. 2021. Potential of Trichoderma spp., Bacillus subtilis and Pseudomonas fluorescens in the management of early blight in tomato. Biocontrol Sci. Technol. 31(9): 912–923. https://doi.org/10.1080/09583157.2021.1900784
Kumar V, Koul B, Taak P, Yadav D, & Song M. 2023. Journey of Trichoderma from pilot scale to mass production: A review. Agriculture. 13(10): 2022. https://doi.org/10.3390/agriculture13102022
Kwon D, Kim S, Kim Y, Son M, Kim K, An D, & Kim BHS. 2015. An empirical assessment of the economic damage caused by apple Marssonina blotch and pear scab outbreaks in Korea. Sustainability. 7(12): 16588–16598. https://doi.org/10.3390/su71215836
Lakhdari W, Benyahia I, Bouhenna MM, Bendif H, Khelafi H, Bachir H, Ladjal A, Hammi H, Mouhoubi D, Khelil H, & Alomar TS. 2023. Exploration and evaluation of secondary metabolites from Trichoderma harzianum: GC-MS analysis, phytochemical profiling, antifungal and antioxidant activity assessment. Molecules. 28(13): 5025. https://doi.org/10.3390/molecules28135025
Leconte A, Tournant L, Muchembled J, Paucellier J, Héquet A, Deracinois B, Deweer C, Krier F, Deleu M, Oste S, Jacques P, & Coutte F. 2022. Assessment of lipopeptide mixtures produced by Bacillus subtilis as biocontrol products against apple scab (Venturia inaequalis). Microorganisms 10(9): 1810. https://doi.org/10.3390/microorganisms10091810
Li C, Shi W, Wu D, Tian R, Wang B, Lin R, Zhou B, & Gao Z. 2021. Biocontrol of potato common scab by Brevibacillus laterosporus BL12 is related to the reduction of pathogen and changes in soil bacterial community. Biol. Control. 153: 104496. https://doi.org/10.1016/j.biocontrol.2020.104496
Liu Z, Xu N, Pang Q, Khan RAA, Xu Q, Wu C, & Liu T. 2023. A salt-tolerant strain of Trichoderma longibrachiatum HL167 is effective in alleviating salt stress, promoting plant growth, and managing Fusarium wilt disease in Cowpea. J. Fungi. 9(3): 304. https://doi.org/10.3390/jof9030304
Liu D, Piao J, Li Y, Guan H, Hao J, & Zhou R. 2024. Transcriptome analysis reveals candidate genes for light regulation of elsinochrome biosynthesis in Elsinoë arachidis. Microorganisms. 12(5): 1027. https://doi.org/10.3390/microorganisms12051027
Lyubenova A, Rusanova M, Nikolova M, & Slavov SB. 2023. Plant extracts and Trichoderma spp: possibilities for implementation in agriculture as biopesticides. Biotechnol. Biotechnol. Equip. 37(1): 159–166. https://doi.org/10.1080/13102818.2023.2166869
Mannai S & Boughalleb-M’Hamdi N. 2023. Evaluation of Trichoderma harzianum, Bacillus subtilis and Aspergillus species efficacy in controlling Pythium ultimum associated with apple seedlings decline in nurseries and their growth promotion effect. Egypt. J. Biol. Pest Control. 33(1): 59. https://doi.org/10.1186/s41938-023-00705-z
Martanto E, Meliala C, & Pasorong N. 2015. Pengaruh konsentrasi Trichoderma sp. terhadap intensitas penyakit kudis (scab) pada tanaman ubi jalar. [The effect of Trichoderma sp. concentration on the intensity of scab disease in sweet potato plants]. In: Dono D, Yulia E, Hidayat Y, Widiantini F, Djaya L, Meliansyah R, Kurniawan W, & Puspasari LT (Eds.). Prosiding Plant Protection Day. Plant Protection Strategies to Improve the Competitiveness of Agricultural Products. pp. 23–27. Sumedang, Jawa Barat.
Mirzaeipour Z, Bazgir E, Zafari D, & Darvishnia M. Isolation and identification of Harzianum clade species of Trichoderma from Khorramabad County. Mycol. Iran. 10(2): 67–78. https://doi.org/10.22092/MI.2023.362910.1264
Mubashir SS, Khan NA, Padder BA, Bhat ZA, & Bhat SN. 2024. Morphological differentiation of Venturia species infecting different pome and stone fruits in Jammu and Kashmir, India. Indian Phytopathol. 77(2): 335–343. https://doi.org/10.1007/s42360-024-00726-0
Mulatu A, Megersa N, Teferi D, Alemu T, & Vetukuri RR. 2023. Biological management of coffee wilt disease (Fusarium xylarioides) using antagonistic Trichoderma isolates. Front. Plant Sci. 14: 1113949. https://doi.org/10.3389/fpls.2023.1113949
Nygren K, Dubey M, Zapparata A, Iqbal M, Tzelepis GD, Durling MB, Jensen DF, & Karlsson M. 2018. The mycoparasitic fungus Clonostachys rosea responds with both common and specific gene expression during interspecific interactions with fungal prey. Evol. Appl. 11(6): 931–949. https://doi.org/10.1111/eva.12609
Phal P, Soytong K, & Poeaim S. 2023. Natural product nanofibers derived from Trichoderma hamatum K01 to control citrus anthracnose caused by Colletotrichum gloeosporioides. Open Agric. 8(1): 20220193. https://doi.org/10.1515/opag-2022-0193
Pham NQ, Wingfield BD, Barnes I, Gazis R, & Wingfield MJ. 2025. Elsinoe species: The rise of scab diseases. Plant Pathol. 74(1): 39–58. https://doi.org/10.1111/ppa.14015
Porto JS, Rebouças TNH, José ARS, José ARS, Tebaldi ND, & Luz JMQ. 2022. Biocontrol of potato common scab cultivated on different soil mulch. Agronomy. 12(4): 904. https://doi.org/10.3390/agronomy12040904
Rajendiran R, Jegadeeshkumar D, Sureshkumar BT, & Nisha T. 2010. In vitro assessment of antagonistic activity of Trichoderma viride against post harvest pathogens. J. Agric. Technol. 6(1): 31–35.
Reuveni M, Gur L, Henriquez JL, Frank J, Tedford E, Cloud G, & Adaskaveg JE. 2022. A new highly effective hybrid fungicide containing difenoconazole and tea tree oil for managing scab of apple, pecan and almond trees and as a tool in resistance management. Plant Pathol. 71(8): 1774–1783. https://doi.org/10.1111/ppa.13610
Shivas R & Beasley D. 2005. Management of Plant Pathogen Collections. Australian Government Department of Agriculture, Fisheries and Forestry. Canberra.
Shanmugam G, Jeon J, & Hyun JW. 2020. Draft genome sequences of Elsinoë fawcettii and Elsinoë australis causing scab diseases on citrus. MPMI. 33(2): 135–137. https://doi.org/10.1094/MPMI-06-19-0169-A
Sharma N, Khanna K, Kaur R, Jasrotia S, Parihar RD, Khajuria A, Tikoria R, Kour S, Kumar D, Bhardwaj R, & Ohri P. 2024. Soil microbiota and mechanisms of plant parasitic nematode suppression. In: Chaudhary KK, Meghvansi MK, & Siddiqui S (Eds.). Sustainable Management of Nematodes in Agriculture, Vol. 2: Role of Microbes-Assisted Strategies. Sustainability in Plant and Crop Protection. Vol 19. pp. 49–87 Springer, Cham. https://doi.org/10.1007/978-3-031-52557-5_3
Shuang M, Wang Y, Teng W, & Jin G. 2022. Isolation and identification of an endophytic bacteria Bacillus sp. K-9 exhibiting biocontrol activity against potato common scab. Archi. Microbiol. 204(8): 483. https://doi.org/10.1007/s00203-022-02989-5
Siddiquee TA, Islam MR, Aminuzzaman FM, Faruq AN, & Islam MM. 2011. Efficacy of foliar spray with seven fungicides and a botanical to control scab (Elsinoe fawcettii) and dieback (Colletotrichum gloeosporioides) diseases of lemon. Agric. 9(1–2): 99–105. https://doi.org/10.3329/agric.v9i1-2.9484
Silva RN, Monteiro VN, Steindorff AS, Gomes EV, Noronha EF, & Ulhoa CJ. 2019. Trichoderma/pathogen/plant interaction in pre-harvest food security. Fungal Biol. 123(8): 565–583. https://doi.org/10.1016/j.funbio.2019.06.010
Singh D, Kapur, SP, & Singh K. 2000. Management of citrus scab caused by Elsinoe fawcettii. Indian Phytopathol. 53(4): 461–467.
Takeuchi Y, Nishio S, Terakami S, Imai A, Shirasawa K, & Takada N. 2023. Genetic mapping of the pear scab resistance gene Vnlf using a pseudo-BC3 population derived from Japanese pear cultivars and European pear ‘La France’. Sci. Hortic. 321: 112260. https://doi.org/10.1016/j.scienta.2023.112260
Tam HNT, Tuoi NTK, & Toan HT. 2021. Isolation and identification of fungi on the peels of Da Xanh and Nam Roi pomelo growing in the Mekong Delta. CTU Journal of Science. 57: 108–117. https://doi.org/10.22144/ctu.jsi.2021.012
Turner BC. 1987. Two ecotypes of Neurospora intermedia. Mycologia. 79(3): 425–432. https://doi.org/10.1080/00275514.1987.12025400
Arasu MV, Vijayaraghavan P, Al?Dhabi NA, Choi KC, & Moovendhan M. 2023. Biocontrol of Trichoderma gamsii induces soil suppressive and growth?promoting impacts and rot disease?protecting activities. J. Basic Microbiol. 63(7): 801–813. https://doi.org/10.1002/jobm.202300016
White TJ, Bruns T, Lee S, & Taylor JW. 1990. Amplifcation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, & White TJ (Eds.). PCR Protocols: A Guide to Methods and Applications. pp. 315–322. Academic Press Inc. New York.
Win TT, Bo B, Malec P, Khan S, & Fu P. 2021. Newly isolated strain of Trichoderma asperellum from disease suppressive soil is a potential bio-control agent to suppress Fusarium soil borne fungal phytopathogens. J. Plant Pathol. 103: 549–561. https://doi.org/10.1007/s42161-021-00780-x
Won K, Choi ED, Kim K, Jung HW, Shin IS, Hong S, Segonzac C, & Kim YJ. 2023. An alternative method to evaluate resistance to pear scab (Venturia nashicola). Plant Pathol. J. 39(2): 228–233. https://doi.org/10.5423%2FPPJ.NT.01.2023.0006
Woo SL, Hermosa R, Lorito M, & Monte E. 2023. Trichoderma: A multipurpose, plant-beneficial microorganism for eco-sustainable agriculture. Nat. Rev. Microbiol. 21(5): 312–326. https://doi.org/10.1038/s41579-022-00819-5
Ye X, Xu C, Xie TT, Zhang Y, Zhao Y, Xia C, Li Z, Huang Y, Fan J, Cao H, Zhang Z, & Cui Z. 2023. Myxobacterial outer membrane ?-1,6-glucanase induced the cell death of Fusarium oxysporum by destroying the cell wall integrity. Appl. Environ. Microbiol. 89(1): e01236–22. https://doi.org/10.1128/aem.01236-22