Published February 6, 2026
| Version v1
Journal article
Open
Standardised sporulation methods for Diplodia, Lasiodiplodia and Neofusicoccum
Authors/Creators
- 1. Unidad de Protección Vegetal, San Cristóbal de La Laguna, Spain|Universidad de La Laguna, San Cristóbal de La Laguna, Spain
- 2. Unidad de Protección Vegetal, San Cristóbal de La Laguna, Spain|Sección de Laboratorio de Sanidad Vegetal, San Cristóbal de La Laguna, Spain
- 3. University of Pretoria, Pretoria, South Africa
Description
Members of the Botryosphaeriaceae are widespread fungal pathogens responsible for economically important diseases in woody plants. Despite the relevance of conidia production for understanding pathogen biology, infection processes, and disease epidemiology, sporulation in vitro remains unpredictable and inconsistent across species. In this study, we evaluated the efficacy of Vogel's Minimal Medium (VMM) for inducing pycnidial and conidial development, which has recently been shown to be effective for this purpose in Diplodia sapinea, in species of Diplodia (n = 3), Lasiodiplodia (n = 2), and Neofusicoccum (n = 26). For this purpose, we used 123 isolates recently collected in the Canary Islands (Spain), as well as 67 Neofusicoccum isolates from long-term storage. All isolates were identified through multilocus phylogenetic analysis. The results showed that D. africana, D. mutila, and D. seriata were able to produce pycnidia, although only D. seriata consistently released conidia. Lasiodiplodia brasiliensis and L. theobromae successfully formed pycnidia and released conidia, while 20 of the 26 tested Neofusicoccum species formed pycnidia, of which 15 released conidia. Significant variation was observed in the time required for pycnidial development and conidial release, as well as in the quantity of conidia produced, both among and within species. Reduced sucrose concentration in VMM delayed pycnidia formation and conidial release and reduced sporulation yields in Neofusicoccum species but increased sporulation in D. africana, D. seriata, L. brasiliensis, and L. theobromae. Long-term storage on Malt Yeast Agar medium at 4 °C negatively affected sporulation in some species, including N. luteum and N. stellenboschiana. Overall, VMM provides a standardised and reproducible medium for inducing sporulation in the Botryosphaeriaceae, although notable variation persists within and between species. These findings provide a methodological foundation for future studies on the biology, pathogenicity, molecular biology, and host-pathogen interactions of these fungi.
Files
imafungus_article_176189.pdf
Files
(10.0 MB)
| Name | Size | Download all |
|---|---|---|
|
md5:1de98c3aaabef0a1d96d2f5f0a83942a
|
9.6 MB | Preview Download |
|
md5:b1d9296ca6c680c5cd8a97b91e1753d2
|
357.2 kB | Preview Download |
Linked records
Additional details
References
- Agustí-Brisach C, Moral J, Felts D et al. (2019) Interaction between Diaporthe rhusicola and Neofusicoccum mediterraneum causing branch dieback and fruit blight of English Walnut in California, and the effect of pruning wounds on the infection. Plant Disease 103(6): 1196–1205. https://doi.org/10.1094/PDIS-07-18-1118-RE
- Ahimera N, Gisler S, Morgan DP et al. (2004) Effects of single-drop impactions and natural and simulated rains on the dispersal of Botryosphaeria dothidea conidia. Phytopathology 94(11): 1189–1197. https://doi.org/10.1094/PHYTO.2004.94.11.1189
- Almeida MC, Antunes D, Silva BMA et al. (2019) Early interaction of Alternaria infectoria conidia with macrophages. Mycopathologia 184: 383–392. https://doi.org/10.1007/s11046-019-00339-6
- Amponsah NT, Jones EE, Ridgway HJ et al. (2008) Production of Botryosphaeria species conidia using grapevine green shoots. New Zealand Plant Protection 61: 301–305. https://doi.org/10.30843/nzpp.2008.61.6840
- Amponsah NT, Jones EE, Ridgway HJ et al. (2009) Rainwater dispersal of Botryosphaeria conidia from infected grapevines. New Zealand Plant Protection 62: 228–233. https://doi.org/10.30843/nzpp.2009.62.4824
- Amponsah NT, Jones EE, Ridgway HJ et al. (2011) Identification, potential inoculum sources and pathogenicity of Botryosphaeriaceous species associated with grapevine dieback disease in New Zealand. European Journal of Plant Pathology 131: 467–482. https://doi.org/10.1007/s10658-011-9823-1
- Arya GC, Sarkar S, Manasherova E et al. (2021) The plant cuticle: An ancient guardian barrier set against long-standing rivals. Frontiers in Plant Science 12: 663165. https://doi.org/10.3389/fpls.2021.663165
- Baskarathevan J, Jaspers MV, Jones EE et al. (2009) Evaluation of different storage methods for rapid and cost-effective preservation of Botryosphaeria species. New Zealand Plant Protection 62: 234–237. https://doi.org/10.30843/nzpp.2009.62.4825
- Batista E, Lopes A, Alves A (2021) What do we know about Botryosphaeriaceae? An overview of a worldwide cured dataset. Forests 12(3): 313. https://doi.org/10.3390/f12030313
- Begoude BAD, Slippers B, Wingfield MJ et al. (2010) Botryosphaeriaceae associated with Terminalia catappa in Cameroon, South Africa and Madagascar. Mycological Progress 9: 101–123. https://doi.org/10.1007/s11557-009-0622-4
- Billones-Baaijens R, Úrbez-Torres JR, Liu M et al. (2018) Molecular methods to detect and quantify Botryosphaeriaceae inocula associated with grapevine dieback in Australia. Plant Disease 102(8): 1489–1499. https://doi.org/10.1094/PDIS-11-17-1854-RE
- Braun EJ, Howard RJ (1994) Adhesion of fungal spores and germlings to host plant surfaces. Protoplasma 181: 202–212. https://doi.org/10.1007/BF01666396
- Calhim S, Halme P, Petersen JH et al. (2018) Fungal spore diversity reflects substrate-specific deposition challenges. Scientific Reports 8: 1–9. https://doi.org/10.1038/s41598-018-23292-8
- Carbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553–556. https://doi.org/10.2307/3761358
- Carlile MJ, Watkinson SC, Gooday GW (2001) Spores, dormancy and dispersal. In: The Fungi (2nd edn). Academic Press, San Diego, 185–243. https://doi.org/10.1016/B978-012738445-0/50021-6
- Chen SF, Morgan DP, Hasey JK et al. (2014) Phylogeny, morphology, distribution, and pathogenicity of Botryosphaeriaceae and Diaporthaceae from English walnut in California. Plant Disease 98(5): 636–652. https://doi.org/10.1094/PDIS-07-13-0706-RE
- Copes WE, Hendrix FF (2004) Effect of temperature on sporulation of Botryosphaeria dothidea, B. obtusa, and B. rhodina. Plant Disease 88(3): 292–296. https://doi.org/10.1094/PDIS.2004.88.3.292
- Crous PW, Slippers B, Wingfield MJ et al. (2006) Phylogenetic lineages in the Botryosphaeriaceae. Studies in Mycology 55(1): 235–253. https://doi.org/10.3114/sim.55.1.235
- Crous PW, Wingfield MJ, Guarro J et al. (2013) Neofusicoccum ursorum sp. nov. and Neofusicoccum cryptoaustrale sp. nov. Fungal Planet description sheets 154–213. Persoonia 31: 188–296. https://doi.org/10.3767/003158513X675925
- Crous PW, Verkley GJM, Groenewald JZ et al. (2019) Fungal Biodiversity. Westerdijk Laboratory Manual Series. Westerdijk Fungal Biodiversity Institute, Utrecht, 425 pp.
- Cruywagen EM, Slippers B, Roux J et al. (2017) Phylogenetic species recognition and hybridisation in Lasiodiplodia: A case study on species from baobabs. Fungal Biology 121(4): 420–436. https://doi.org/10.1016/j.funbio.2016.07.014
- Fisher NL (1982) Carnation Leaves as a Substrate and for Preserving Cultures of Fusarium species. Phytopathology 72: 151. https://doi.org/10.1094/phyto-77-151
- Furukawa T, Kishi K (2002) Production of perithecia of various ascomycotina on water agar medium emended with leaf pieces. Journal of Phytopathology 150: 625–628. https://doi.org/10.1046/j.1439-0434.2002.00822.x
- Garcia JF, Lawrence DP, Morales-Cruz A et al. (2021) Phylogenomics of plant-associated Botryosphaeriaceae species. Frontiers in Microbiology 12: 1–18. https://doi.org/10.3389/fmicb.2021.652802
- Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61(4): 1323–1330. https://doi.org/10.1128/aem.61.4.1323-1330.1995
- González-Domínguez E, Alves A, León M et al. (2017) Characterization of Botryosphaeriaceae species associated with diseased loquat (Eriobotrya japonica) in Spain. Plant Pathology 66(1): 77–89. https://doi.org/10.1111/ppa.12556
- Greenwald CJ, Kasuga T, Glass NL et al. (2010) Temporal and spatial regulation of gene expression during asexual development of Neurospora crassa. Genetics 186: 1217–1230. https://doi.org/10.1534/genetics.110.121780
- Hamer JE, Howard RJ, Chumley FG et al. (1988) A mechanism for surface attachment in spores of a plant pathogenic fungus. Science 239: 288–290. https://doi.org/10.1126/science.239.4837.288
- Hernández D, García-Pérez O, Perera S et al. (2023) Fungal pathogens associated with aerial symptoms of Avocado (Persea americana Mill.) in Tenerife (Canary Islands, Spain) focused on species of the family Botryosphaeriaceae. Microorganisms 11(3): 45–66. https://doi.org/10.3390/microorganisms11030585
- Huang M, Hull CM (2017) Sporulation: how to survive on planet Earth (and beyond). Current Genetics 63: 831–838. https://doi.org/10.1007/s00294-017-0694-7
- Hughes HB, Carzaniga R, Rawlings SL et al. (1999) Spore surface glycoproteins of Colletotrichum lindemuthianum are recognized by a monoclonal antibody which inhibits adhesion to polystyrene. Microbiology (Reading) 145(8): 1927–1936. https://doi.org/10.1099/13500872-145-8-1927
- Jami F, Marincowitz S, Slippers B et al. (2018) New Botryosphaeriales on native red milkwood (Mimusops caffra). Australasian Plant Pathology 47: 475–484. https://doi.org/10.1007/s13313-018-0586-4
- Jami F, Marincowitz S, Durán A et al. (2022) Botryosphaeriaceae diversity on Eucalyptus clones in different climate zones of Indonesia. Forest Pathology 52(2): 1–14. https://doi.org/10.1111/efp.12737
- Liu AR, Chen SC, Wu SY et al. (2010) Cultural studies coupled with DNA based sequence analyses and its implication on pigmentation as a phylogenetic marker in Pestalotiopsis taxonomy. Molecular Phylogenetics and Evolution 57(2): 528–535. https://doi.org/10.1016/j.ympev.2010.07.017
- Mendgen K, Deising H (1993) Infection structures of fungal plant pathogens – a cytological and physiological evaluation. New Phytologist 124(2): 193–213. https://doi.org/10.1111/j.1469-8137.1993.tb03809.x
- Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Gateway Computing Environments Workshop (GCE). 1–7: https://doi.org/10.1109/GCE.2010.5676129
- Mohali S, Slippers B, Wingfield MJ (2006) Two new Fusicoccum species from Acacia and Eucalyptus in Venezuela, based on morphology and DNA sequence data. Mycological Research 110(4): 405–413. https://doi.org/10.1016/j.mycres.2006.01.006
- Mohali SR, Slippers B, Wingfield MJ (2007) Identification of Botryosphaeriaceae from Eucalyptus, Acacia and Pinus in Venezuela. Fungal Diversity 25: 103–125.
- Obrador-Sánchez JA, Hernández-Martínez R (2020) Microscope observations of Botryosphaeriaceae spp. in the Mediterranean region. Phytopathologia Mediterranea 59(1): 119–129. https://doi.org/10.36253/phyto-11040
- Okane I, Nakagiri A, Ito T (2001) Identity of Guignardia sp. inhabiting ericaceous plants. Canadian Journal of Botany 79: 101–109. https://doi.org/10.1139/b00-136
- Oostlander AG, Brodde L, von Bargen M et al. (2023) A reliable and simple method for the production of viable pycnidiospores of the pine pathogen Diplodia sapinea and a spore-based infection assay on Scots Pine. Plant Disease 107(11): 3370–3377. https://doi.org/10.1094/PDIS-01-23-0107-RE
- Oostlander AG, Brodde L, von Bargen M et al. (2024) Protocol for the production and storage of Diplodia sapinea pycnidiospores. Protocols.io. https://doi.org/10.17504/protocols.io.kqdg3271pv25/v1
- Osorio JA, Crous CJ, de Beer ZW et al. (2016) Endophytic Botryosphaeriaceae, including five new species, associated with mangrove trees in South Africa. Fungal Biology 121(4): 361–393. https://doi.org/10.1016/j.funbio.2016.09.004
- Pavlic D, Slippers B, Coutinho TA et al. (2007) Botryosphaeriaceae occurring on native Syzygium cordatum in South Africa and their potential threat to Eucalyptus. Plant Pathology 56(4): 624–636. https://doi.org/10.1111/j.1365-3059.2007.01608.x
- Pavlic D, Slippers B, Coutinho TA et al. (2009) Multiple gene genealogies and phenotypic data reveal cryptic species of the Botryosphaeriaceae: A case study on the Neofusicoccum parvum/N. ribis complex. Molecular Phylogenetics and Evolution 51(2): 259–268. https://doi.org/10.1016/j.ympev.2008.12.017
- Phillips AJL (2016) Special issue on Botryosphaeriaceae. Mycosphere 7(7): 868–869. https://doi.org/10.5943/mycosphere/si/1b/12
- Phillips AJL, Alves A, Abdollahzadeh J et al. (2013) The Botryosphaeriaceae: Genera and species known from culture. Studies in Mycology 76(1): 51–167. https://doi.org/10.3114/sim0021
- Ronquist F, Teslenko M, van der Mark P et al. (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
- Sakalidis ML, Hardy GESJ, Burgess TI (2011) Use of the Genealogical Sorting Index (GSI) to delineate species boundaries in the Neofusicoccum parvum-Neofusicoccum ribis species complex. Molecular Phylogenetics and Evolution 60(3): 333–344. https://doi.org/10.1016/j.ympev.2011.04.026
- Sammonds J, Jaspers MV, Jones EE (2019) Influence of surface characteristics on germination and early growth of Botryosphaeriaceae species. European Journal of Plant Pathology 154: 1125–1134. https://doi.org/10.1007/s10658-019-01733-y
- Slippers B, Crous PW, Denman S et al. (2004a) Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea. Mycologia 96(1): 83–101. https://doi.org/10.1080/15572536.2005.11833000
- Slippers B, Fourie G, Crous PW et al. (2004b) Multiple gene sequences delimit Botryosphaeria australis sp. nov. from B. lutea. Mycologia 96(5): 1030–1041. https://doi.org/10.1080/15572536.2005.11832903
- Slippers B, Fourie G, Crous PW et al. (2004c) Speciation and distribution of Botryosphaeria spp. on native and introduced Eucalyptus trees in Australia and South Africa. Studies in Mycology 50: 343–358. (ISBN 90-70351-54-4)
- Slippers B, Johnson GI, Crous PW et al. (2005) Phylogenetic and morphological re-evaluation of the Botryosphaeria species causing diseases of Mangifera indica. Mycologia 97(1): 99–110. https://doi.org/10.1080/15572536.2006.11832843
- Slippers B, Wingfield MJ (2007) Botryosphaeriaceae as endophytes and latent pathogens of woody plants: diversity, ecology and impact. Fungal Biology Reviews 21(2–3): 90–106. https://doi.org/10.1016/j.fbr.2007.06.002
- Slippers B, Smit WA, Crous PW et al. (2007) Taxonomy, phylogeny and identification of Botryosphaeriaceae associated with pome and stone fruit trees in South Africa and other regions of the world. Plant Pathology 56(1): 128–139. https://doi.org/10.1111/j.1365-3059.2006.01486.x
- Slippers B, Boissin E, Phillips AJL et al. (2013) Phylogenetic lineages in the Botryosphaeriales: a systematic and evolutionary framework. Studies in Mycology 76(1): 31–49. https://doi.org/10.3114/sim0020
- Slippers B, Crous PW, Jami F et al. (2017) Diversity in the Botryosphaeriales: Looking back, looking forward. Fungal Biology 121(4): 307–321. https://doi.org/10.1016/j.funbio.2017.02.002
- Slippers B, Ramabulana E, Coetzee MPA (2024) Botryosphaeriaceae partially overlap on asymptomatic and symptomatic tissues of Anacardiaceae in agroecosystems and conservation areas in northern South Africa. Fungal Systematics and Evolution 13(1): 131–142. https://doi.org/10.3114/fuse.2024.13.07
- Smith DH, Fergus CL (1971) Effect of light intensity on sporulation of Botryosphaeria ribis. Mycopathologia et Mycologia applicata 45: 311–315. https://doi.org/10.1007/BF02051978
- Smith H, Wingfield MJ, Crous PW et al. (1996) Sphaeropsis sapinea and Botryosphaeria dothidea endophytic in Pinus spp. and Eucalyptus spp. in South Africa. South African Journal of Botany 62(2): 86–88. https://doi.org/10.1016/S0254-6299(15)30596-2
- Smith H, Crous PW, Wingfield MJ et al. (2001) Botryosphaeria eucalyptorum sp. nov., a new species in the B. dothidea -complex on Eucalyptus in South Africa. Mycologia 93(2): 277–285. https://doi.org/10.1080/00275514.2001.12063159
- Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9): 1312–1313. https://doi.org/10.1093/bioinformatics/btu033
- Sutton TB, Arauz LF (1991) Influence of temperature and moisture on germination of ascospores and conidia of Botryosphaeria dothidea. Plant Disease 75(11): 1146–1149. https://doi.org/10.1094/PD-75-1146
- Tariq A, Naz F, Rauf CA et al. (2015) Long term and least expensive preservation methods for various fungal cultures. Pakistan Journal of Phytopathology 27(2): 147–151. http://pjp.pakps.com/index.php/PJP/article/view/192
- Úrbez-Torres JR, Gubler WD (2009) Pathogenicity of Botryosphaeriaceae species isolated from grapevine cankers in California. Plant Disease 93(6): 584–592. https://doi.org/10.1094/PDIS-93-6-0584
- Úrbez-Torres JR, Bruez E, Hurtado J et al. (2010a) Effect of temperature on conidial germination of Botryosphaeriaceae species infecting grapevines. Plant Disease 94(12): 1476–1484. https://doi.org/10.1094/PDIS-06-10-0423
- Úrbez-Torres JR, Battany M, Bettiga LJ et al. (2010b) Botryosphaeriaceae species spore-trapping studies in California vineyards. Plant Disease 94(6): 717–724. https://doi.org/10.1094/PDIS-94-6-0717
- Úrbez-Torres JR (2011) The status of Botryosphaeriaceae species infecting grapevines. Phytopathologia Mediterranea 50(4): 5–45. https://doi.org/10.14601/Phytopathol_Mediterr-9316
- Vogel HJ (1956) A convenient growth medium for Neurospora crassa. Microbial Genetics Bulletin 13: 42–47.
- Vieira RFBS, Brasil SOS, Fonseca WL et al. (2021) Sporulation methodology for fungi of the Botryosphaeriaceae family. Summa Phytopathologica 47(1): 62–64. https://doi.org/10.1590/0100-5405/231724
- Vu D, Groenewald M, de Vries M et al. (2019) Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation. Studies in Mycology 92(1): 135–154. https://doi.org/10.1016/j.simyco.2018.05.001
- Wang M, Kriticos DJ, Ota N et al. (2021) A general trait-based modelling framework for revealing patterns of airborne fungal dispersal threats to agriculture and native flora. New Phytologist 232(3): 1506–1518. https://doi.org/10.1111/nph.17659
- West JS (2014) Plant pathogen dispersal. Encyclopedia of Life Sciences, 1–7. https://doi.org/10.1002/9780470015902.a0021272
- White TJ, Bruns T, Lee S et al. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols. A Guide to Methods and Applications, 315–322. https://doi.org/10.1016/b978-0-12-372180-8.50042-1
- Yang T, Groenewald JZ, Cheewangkoon R et al. (2017) Families, genera, and species of Botryosphaeriales. Fungal Biology 121(4): 322–346. https://doi.org/10.1016/j.funbio.2016.11.001
- Zhang W, Groenewald JZ, Lombard L et al. (2021) Evaluating species in Botryosphaeriales. Persoonia: Molecular Phylogeny and Evolution of Fungi 46: 63–115. https://doi.org/10.3767/persoonia.2021.46.03