Metabarcoding analysis identifies the phyllosphere fungal communities associated with a newly emerged leaf fall disease in rubber plants in Indonesia

Budi Setyawan; Arif Wibowo; Valérie Pujade-Renaud; Siti Subandiyah

doi:10.22146/ijbiotech.115802

Metabarcoding analysis identifies the phyllosphere fungal communities associated with a newly emerged leaf fall disease in rubber plants in Indonesia

https://doi.org/10.22146/ijbiotech.115802

Budi Setyawan^(1*), Arif Wibowo⁽²⁾, Valérie Pujade-Renaud⁽³⁾, Siti Subandiyah⁽⁴⁾

(1) The Graduate School of Biotechnology, Universitas Gadjah Mada, Jl. Teknika Utara Barek, Yogyakarta 55281, Indonesia; PT Riset Perkebunan Nusantara, Unit of Bogor Getas, Central Java 55072, Indonesia
(2) Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
(3) CIRAD, UMR AGAP Institut, F-34398 Montpellier, France; UMR AGAP Institut, Univ. Montpellier, CIRAD, INRAE, Institut Agro Montpellier, France; Université Clermont Auvergne, INRAE, PIAF, F-63000 Clermont-Ferrand, France
(4) Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
(*) Corresponding Author

Abstract

Leaf fall disease of Hevea brasiliensis in Indonesia and Southeast Asia is an emerging threat to rubber production. While tentatively associated with Pestalotiopsis, Colletotrichum, and Neopestalotiopsis species, the disease etiology remains poorly characterized. This study aimed to identify the fungal community associated with symptomatic rubber leaves using culture-independent ITS amplicon sequencing. Leaf samples representing three symptom severity levels were collected from Central Java and South Sumatra. Fungal community composition analysis revealed that geographic origin, rather than symptom severity, was the primary driver of community clustering. Phyllosticta and Colletotrichum were consistently dominant across all samples, with Phyllosticta displaying notably higher relative abundance. Phyllosticta was identified as a newly reported pathogen of H. brasiliensis. Minor proportions of Pestalotiopsis, Neopestalotiopsis, Corynespora, and Pestalotia were also detected. Multigene phylogenetic analysis (ITS and β-tubulin) revealed three major clades corresponding to Neopestalotiopsis, Pestalotiopsis, and Colletotrichum. Pathogenicity assays confirmed that representative isolates reproduced field symptoms. This first metagenomic assessment of the phyllosphere mycobiota in diseased rubber leaves identifies Phyllosticta as a critical yet overlooked component of the leaf fall disease complex, providing insights into fungal ecology and pathogenic interactions underlying this emerging production threat.

Keywords

Fungal community; ITS amplicon sequencing; Leaf fall disease; Phyllosticta; Rubber

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References

Aliya SSS, Nusaibah SA, Mahyudin MM, Yun WM, Yusop MR. 2022. Colletotrichum siamense and Pestalotiopsis jesteri as potential pathogens of new rubber leaf spot disease via detached leaf assay. J. Rubber Res. 25(3):195–212. doi:10.1007/s42464-022- 00157-4.

Araújo KS, Brito VN, Veloso TGR, de Leite TS, Alves JL, da Hora Junior BT, Moreno HLA, Pereira OL, Mizubuti ESG, de Queiroz MV. 2020. Diversity and distribution of endophytic fungi in different tissues of Hevea brasiliensis native to the Brazilian Amazon forest. Mycol. Prog. 19(10):1057–1068. doi:10.1007/s11557-020-01613-4.

Azeez AA, Esiegbuya DO, Lateef AA, Asiegbu FO. 2024. Mycobiome analysis of leaf, root, and soil of symptomatic oil palm trees (Elaeis guineensis Jacq.) affected by leaf spot disease. Front. Microbiol. 15. doi:10.3389/fmicb.2024.1422360.

Bhunjun CS, Phillips AJL, Jayawardena RS, Promputtha I, Hyde KD. 2021. Importance of molecular data to identify fungal plant pathogens and guidelines for pathogenicity testing based on Koch’s postulates. Pathogens 10(9):1096. doi:10.3390/pathogens10091096.

Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, Knight R, Mills DA, Caporaso JG. 2013. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat. Methods 10(1):57– 59. doi:10.1038/nmeth.2276.

Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F, Bai Y, Bisanz JE, Bittinger K, Brejnrod A, Brislawn CJ, Brown CT, Callahan BJ, Caraballo-Rodríguez AM, Chase J, Cope EK, Da Silva R, Diener C, Dorrestein PC, Douglas GM, Durall DM, Duvallet C, Edwardson CF, Ernst M, Estaki M, Fouquier J, Gauglitz JM, Gibbons SM, Gibson DL, Gonzalez A, Gorlick K, Guo J, Hillmann B, Holmes S, Holste H, Huttenhower C, Huttley GA, Janssen S, Jarmusch AK, Jiang L, Kaehler BD, Kang KB, Keefe CR, Keim P, Kelley ST, Knights D, Koester I, Kosciolek T, Kreps J, Langille MGI, Lee J, Ley R, Liu YX, Loftfield E, Lozupone C, Maher M, Marotz C, Martin BD, McDonald D, McIver LJ, Melnik AV, Metcalf JL, Morgan SC, Morton JT, Naimey AT, Navas-Molina JA, Nothias LF, Orchanian SB, Pearson T, Peoples SL, Petras D, Preuss ML, Pruesse E, Rasmussen LB, Rivers A, Robeson n M S, Rosenthal P, Segata N, Shaffer M, Shiffer A, Sinha R, Song SJ, Spear JR, Swafford AD, Thompson LR, Torres PJ, Trinh P, Tripathi A, Turnbaugh PJ, Ul-Hasan S, van der Hooft JJJ, Vargas F, Vázquez-Baeza Y, Vogtmann E, von Hippel M, Walters W, Wan Y, Wang M, Warren J, Weber KC, Williamson CHD, Willis AD, Xu ZZ, Zaneveld JR, Zhang Y, Zhu Q, Knight R, Caporaso JG. 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37(8):852–857. doi:10.1038/s41587-019-0209-9.

Brown AE, Soepena H. 1994. Pathogenicity of Colletotrichum acutatum and C. gloeosporioides on leaves of Hevea spp. Mycol. Res. 98(3):264–266. doi:10.1016/S0953-7562(09)80453-X.

Cho SE, Park KH, Shin K, Lee DH. 2025. Detection of Pestalotiopsis abbreviata sp. nov., the causal agent of Pestalotiopsis leaf blight on Camellia japonica based on metagenomic analysis. J. Fungi 11(8):553. doi:10.3390/jof11080553.

de Assis Costa OY, Tupinambá DD, Bergmann JC, Barreto CC, Quirino BF. 2018. Fungal diversity in oil palm leaves showing symptoms of fatal yellowing disease. PLoS One 13(1):e0191884. doi:10.1371/journal.pone.0191884.

Edgar RC. 2013. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10(10):996–998. doi:10.1038/nmeth.2604.

Edgar RC. 2022. Muscle5: High-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny. Nat. Commun. 13(1). doi:10.1038/s41467-022-34630-w.

Effendi Y, Aini N, Pambudi A, Sasaerila HY. 2020. Metagenomics analysis of soil microbial communities in plant agroforestry system rubber tree (Hevea brasiliensis)–Ganyong (Canna sp.). IOP Conf. Ser. Earth Environ. Sci. 468(1):012045. doi:10.1088/1755-1315/468/1/012045.

Effendi Y, Pambudi A, Sasaerila Y, WijiHastuti RS. 2019. Metagenomic analysis of diversity and composition of soil bacteria under intercropping system Hevea brasiliensis and Canna indica. IOP Conf. Ser. Earth Environ. Sci. 391(1):012023. doi:10.1088/1755- 1315/391/1/012023.

Febbiyanti TR, Fairuzah Z. 2020. Identifikasi penyebab kejadian luar biasa penyakit gugur daun karet di Indonesia [Identification of causes of rubber leaves outbreak in Indonesia]. J. Penelit. Karet p. 193–206. doi:10.22302/ppk.jpk.v37i2.616.

Febbiyanti TR, Kusdiana APJ, Fairuzah Z, Herlinawati E. 2018. The outbreak of Fusicoccum leaf disease in Indonesia and the potential yield loss. Palembang: Indonesian Rubber Research Institute.

Febbiyanti TR, Matsui M. 2025. Test of 8-hydroquinoline copper against Neopestalotiopsis thailandica in the greenhouse and budwood garden on rubber plant (Hevea brasiliensis). In: BIO Web Conf., volume 177. p. 08001. doi:10.1051/bioconf/202517708001.

Febbiyanti TR, Tistama R, Sarsono Y. 2021. Karakterisasi isolat Pestalotiopsis pada karet (Hevea brasiliensis) menggunakan karakter morfologi dan molekuler [Characterization of Pestalotiopsis isolate on rubber (Hevea brasiliensis) using morphological and molecular characteristics]. J. Penelit. Karet 39(2):151–162. doi:10.22302/ppk.jpk.v39i2.798.

Fonseca PLC, Badotti F, de Oliveira TFP, Fonseca A, Vaz ABM, Tomé LMR, Abrahão JS, Marques JT, Trindade GS, Chaverri P, Aguiar ERGR, Góes-Neto A. 2018. Virome analyses of Hevea brasiliensis using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus. Virol. J. 15(1):184. doi:10.1186/s12985-018-1095-3.

Fonseca PLC, Skaltsas D, da Silva FF, Kato RB, de Castro GM, García GJY, Quintanilha-Peixoto G, Mendes-Pereira T, do Carmo AO, Aguiar ERGR, de Carvalho DS, Costa-Rezende DH, Drechsler-Santos ER, Badotti F, Ferreira-Silva A, Oliveira G, Chaverri P, Vaz ABM, Góes-Neto A. 2022. An integrative view of the phyllosphere mycobiome of native rubber trees in the Brazilian Amazon. J. Fungi 8(4):373. doi:10.3390/jof8040373.

Guevara A, López M, Rivano F, Castro O. 2022. First report of secondary leaf fall in rubber trees caused by Phyllosticta capitalensis in the Eastern Plains of Colombia. New Dis. Rep. 45(2). doi:10.1002/ndr2.12096.

Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, Ciulla D, Tabbaa D, Highlander SK, Sodergren E, Methé B, Human Microbiome Consortium, Petrosino JF, Knight R, Birren BW. 2011. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res. 21(3):494–504. doi:10.1101/gr.112730.110.

Hasbunallah MD, Hermawan B, Falahuddin I. 2023. Isolasi cendawan Pestalotiopsis sp. penyebab gugur daun circular pada tanaman karet (Hevea brasiliensis) [Isolation of Pestalotiopsis sp. fungus causing circular leaf fall on rubber plants (Hevea brasiliensis)]. J. Multidisip. Kapalamada 2(3):193–200. doi:10.62668/kapalamada.v2i03.821.

Herath IHMIS, Manamgoda DS, Udayanga D. 2019. Morphological and molecular identification of fungal pathogens associated with cultivated rubber trees in Sri Lanka. In: Proc. First Natl. Symp. Sri Lanka Assoc. Mycol. Plant Pathol. (SLAMPP), Plant Health 2019. Kandy, Sri Lanka. p. 12.

Hiệu ND, Uyên NTK, Vinh NP, Trang NTT, Mai NN, Luân DN, Tuấn BT, Pha TA, Nghĩa NA. 2022. Tác nhân gây bệnh rụng lá đốm tròn trên cây cao su ở tỉnh Đồng Nai và Bình Phước [Causal agents of circular leaf spot/circular leaf fall on rubber in provinces]. p. 183–190.

Hsu SY, Xu YC, Lin YC, Chuang WY, Lin SR, Stadler M, Tangthirasunun N, Cheewangkoon R, AL-Shwaiman HA, Elgorban AM, et al. 2024. Hidden diversity of Pestalotiopsis and Neopestalotiopsis (Amphisphaeriales, Sporocadaceae) species allied with the stromata of entomopathogenic fungi in Taiwan. MycoKeys 101:275–312. doi:10.3897/mycokeys.101.113090.

Jatoi MT, Lan G, Wu Z, Sun R, Yang C, Tan Z. 2019. Comparison of soil microbial composition and diversity between mixed and monoculture rubber plantations in Hainan Province, China. Trop. Conserv. Sci. 12:1–9. doi:10.1177/1940082919876072.

Josephin A, Whulanza Y, Rahman SF, Lischer K, Surya MI, Martiansyah I, Rahman W, Hashim U. 2024. Easy extraction of Ganoderma boninense liquid sample using portable on-chip device. Indones. J. Biotechnol. 29(1):33. doi:10.22146/ijbiotech.83645.

Junaidi J, Tistama R, Atminingsih A, Fairuzah Z, Rachmawan A, Darojat MR, Andriyanto M. 2018. Fenomena gugur daun sekunder di wilayah Sumatera Utara dan pengaruhnya terhadap produksi karet [Secondary leaf fall phenomena in North Sumatera and its impact to rubber yield]. Warta Perkaretan 37(1):1–16. doi:10.22302/ppk.wp.v37i1.441.

Kandila A. 2023. Pengaruh dua media yang berbeda terhadap pertumbuhan tiga spesies jamur Pestalotiopsis spp. penyebab penyakit gugur daun karet [Effect of two different media on the growth of three species of the fungus Pestalotiopsis spp. causing rubber leaf disease]. Undergraduate thesis, Universitas Sriwijaya.

Kembel SW, Mueller RC. 2014. Plant traits and taxonomy drive host associations in tropical phyllosphere fungal communities. Botany 92(4):303–311. doi:10.1139/cjb-2013-0194.

Kumar S, Stecher G, Suleski M, Sanderford M, Sharma S, Tamura K. 2024. MEGA12: Molecular evolution genetic analysis version 12 for adaptive and green computing. Mol. Biol. Evol. 41(12). doi:10.1093/molbev/msae263.

Kusdiana APJ. 2021. Diagnosis penyakit gugur daun karet (Hevea brasiliensis Muell. Arg.) [Diagnosis of causal agent of new rubber leaf fall disease (Hevea brasiliensis Muell. Arg.)]. J. Penelit. Karet 38(2). doi:10.22302/ppk.jpk.v2i38.728.

Kusnedy D, Febrianto EB, Sayurandi, Dalimunthe CI. 2021. Uji resistensi klon-klon introduksi terhadap penyakit gugur daun Pestalotiopsis sp. di laboratorium [Resistance test of introduction clones to leaf fall disease Pestalotiopsis sp. in the laboratory]. J. Agro Estate 5(2):66–74. doi:10.47199/jae.v5i2.93.

Lan G, Wei Y, Zhang X, Wu Z, Ji K, Xu H, Chen B, He F. 2024. Assembly and maintenance of phyllosphere microbial diversity during rubber tree leaf senescence. Commun. Biol. 7(1):1192. doi:10.1038/s42003-024- 06907-x.

Lan G, Wu Z, Yang C, Sun R, Chen B, Zhang X. 2020. Tropical rainforest conversion into rubber plantations results in changes in soil fungal composition, but underlying mechanisms of community assembly remain unchanged. Geoderma 375:114505. doi:10.1016/j.geoderma.2020.114505.

Langfelder P, Horvath S. 2008. WGCNA: An R package for weighted correlation network analysis. BMC Bioinformatics 9(1). doi:10.1186/1471-2105-9-559.

Li B, Liu X, Cai J, Feng Y, Huang G. 2021. First report on Neopestalotiopsis aotearoa of rubber tree in China. Plant Dis. 105(4):1223. doi:10.1094/PDIS- 09-20-1930-PDN.

Longsaward R, Viboonjun U, Wen Z, Asiegbu FO. 2024. In silico analysis of secreted effectorome of the rubber tree pathogen Rigidoporus microporus highlights its potential virulence proteins. Front. Microbiol. 15. doi:10.3389/fmicb.2024.1439454.

Magoč T, Salzberg SL. 2011. FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27(21):2957–2963. doi:10.1093/bioinformatics/btr507.

Mei S, Hou S, Cui H, Feng F, Rong W. 2016. Characterization of the interaction between Oidium heveae and Arabidopsis thaliana. Mol. Plant Pathol. 17(9):1331– 1343. doi:10.1111/mpp.12363.

Musdalifah N, Mukrimin M, Paembonan SA, Sultan S, Hasanuddin H. 2025. Fungal community inhabiting Hevea brasiliensis organs and soil. IOP Conf. Ser. Earth Environ. Sci. 1471(1):012048. doi:10.1088/1755-1315/1471/1/012048.

Narayanan C. 2020. Variation in susceptibility to Phyllosticta capitalensis-associated leaf disease among inter-specific hybrids, half-sibs and high-yielding clones of para rubber tree (Hevea). Plant Pathol. Quar. 10(1):100–110. doi:10.5943/ppq/10/1/12.

Nizamani M, Zhang Q, Zhang H, Wang Y. 2023. Checklist of the fungi associated with the rubber tree (Hevea brasiliensis). Curr. Res. Environ. Appl. Mycol. 13(1):439–488. doi:10.5943/cream/13/1/17.

Norphanphoun C. 2019. Morphological and phylogenetic characterization of novel pestalotioid species associated with mangroves in Thailand. Mycosphere 10(1):531–578. doi:10.5943/mycosphere/10/1/9.

Nyaka Ngobisa AIC, Ndongo PAO, Doungous O, Ntsomboh-Ntsefong G, Njonje SW, Ehabe EE. 2017. Characterization of Pestalotiopsis microspora, causal agent of leaf blight on rubber (Hevea brasiliensis) in Cameroon. In: Proc. Int. Rubber Conf. 2017. USA. p. 436–447.

Pais A, Ristaino J, Whetten R, Xiang QY. 2024. Metagenomic study reveals hidden relationships among fungal diversity, variation of plant disease, and genetic distance in Cornus florida (Cornaceae). Front. Plant Sci. 14. doi:10.3389/fpls.2023.1282188.

Pérez-Cobas AE, Gomez-Valero L, Buchrieser C. 2020. Metagenomic approaches in microbial ecology: an update on whole-genome and marker gene sequencing analyses. Microb. Genom. 6(8). doi:10.1099/mgen.0.000409.

Pornsuriya C, Chairin T, Thaochan N, Sunpapao A. 2020. Identification and characterization of Neopestalotiopsis fungi associated with a novel leaf fall disease of rubber trees (Hevea brasiliensis) in Thailand. J. Phytopathol. 168(7–8):416–427. doi:10.1111/jph.12906.

Senwanna C. 2021. Ascomycetes on Para rubber (Hevea brasiliensis). Mycosphere 12(1):1334–1512. doi:10.5943/mycosphere/12/1/18.

Sievers F, Higgins DG. 2017. Clustal Omega for making accurate alignments of many protein sequences. Protein Sci. 27(1):135–145. doi:10.1002/pro.3290.

Solarte F, Muñoz CG, Maharachchikumbura SSN, Álvarez E. 2018. Diversity of Neopestalotiopsis and Pestalotiopsis spp., causal agents of guava scab in Colombia. Plant Dis. 102(1):49–59. doi:10.1094/PDIS-01-17-0068-RE.

Solpot TC, Villanueva JD, Abello MP, Zuñiga JBM. 2023. First report of Neopestalotiopsis clavispora causing foliar disease of rubber (Hevea brasiliensis) in the Philippines. J. Phytopathol. 172(1). doi:10.1111/jph.13238.

Song X, Wang T, Liu M, Peng B, Niu Q, Liu J, Chen S, Chen K, Li M, Feng P, et al. 2024. From in silico to in vitro: A comprehensive guide to validating bioinformatics findings. doi:10.31219/osf.io/p6fzy.

Tan KLS, Mohamad SB. 2024. Fungal pathogen in digital age: review on current state and trend of comparative genomics studies of pathogenic fungi. Adv. Microbiol. 63(1):23–31. doi:10.2478/am-2024-0003.

Tang J, Lai LX, Du YL, Yang Q. 2021. First report of Neopestalotiopsis protearum causing seed rot on Camellia oleifera in China. Plant Dis. 105(12):4152. doi:10.1094/PDIS-12-20-2717-PDN.

Verly LB, Rosa ACR, Andrade JV, Miranda GS, Marques CFPM, Vieira GFA, Macedo MED, Perin ILB, Caprini HOG, de Macêdo AV, et al. 2025. Molecular mechanisms of virulence in phytopathogenic fungi. Seven Editora. chapter 35, p. 551–576. doi:10.56238/sevened2025.018-035.

Villanueva RAM, Chen ZJ. 2019. ggplot2: Elegant graphics for data analysis (2nd ed.). Meas. Interdiscip. Res. Perspect. 17(3):160–167. doi:10.1080/15366367.2019.1565254.

Vineeth VK, Babu S, Reshma TR, Philip S, Prem E. 2024. Exhaustive identification and characterization of Colletotrichum siamense and Colletotrichum fructicola as causative agents of circular leaf spot disease of rubber tree (Hevea brasiliensis) in India. Fungal Biol. 128(5):1907–1916. doi:10.1016/j.funbio.2024.05.009.

Wastie RL, Janardhanan PS. 1970. Pathogenicity of Colletotrichum gloeosporioides, C. dematium and C. crassipes to leaves of Hevea brasiliensis. Trans. Br. Mycol. Soc. 54(1):150–152. doi:10.1016/S0007- 1536(70)80136-X.

Wei Y, Lan G, Wu Z, Chen B, Quan F, Li M, Sun S, Du H. 2022. Phyllosphere fungal communities of rubber trees exhibited biogeographical patterns, but not bacteria. Environ. Microbiol. 24(8):3777–3790. doi:10.1111/1462-2920.15894.

Yang S, Johnson MA, Hansen MA, Bush E, Li S, Vinatzer BA. 2022. Metagenomic sequencing for detection and identification of the boxwood blight pathogen Calonectria pseudonaviculata. Sci. Rep. 12(1). doi:10.1038/s41598-022-05381-x.

Zheng J, Shi J, Wang D. 2024. Diversity of soil fungi and entomopathogenic fungi in subtropical mountain forest in southwest China. Environ. Microbiol. Rep. 16(4). doi:10.1111/1758-2229.13267.

DOI: https://doi.org/10.22146/ijbiotech.115802