Simple lab recipe coaxes stubborn tree-killing fungi to release their spores

Fungal pathogens that attack orchards, vineyards and forests are notoriously difficult to study, and one of the biggest bottlenecks has been almost embarrassingly basic: getting them to make spores in the lab. Now, an international team of researchers has shown that a single, chemically defined culture medium can reliably coax dozens of species from three important fungal genera into sporulating, offering plant pathologists a standardised tool where once only unpredictable, hit-or-miss methods existed. The work, published in IMA Fungus, tested Vogel’s Minimal Medium on 190 isolates spanning the genera Diplodia, Lasiodiplodia and Neofusicoccum, and demonstrated that a protocol originally developed for a bread mould in the 1950s could unlock the reproductive machinery of some of the world’s most economically damaging woody-plant pathogens.

The fungi in question belong to the Botryosphaeriaceae, a sprawling family of Dothideomycetes that live quietly inside the tissues of trees and shrubs around the globe. Most of the time they behave as endophytes, harmless passengers in branches and trunks, but when their hosts are stressed by drought, heat or injury, they switch into opportunistic killers, causing cankers, dieback, fruit rots and the grapevine trunk diseases that cost growers millions each year. Understanding how these fungi reproduce is central to understanding how they spread and infect, because their main dispersal units, asexual spores called conidia, are produced inside flask-shaped fruiting bodies known as pycnidia. In nature these pycnidia form on dead and necrotic plant tissue, oozing masses of conidia that are carried off by rain splash, wind and insects to new hosts.

In the laboratory, however, these fungi are famously reluctant performers. On standard media such as Malt Extract Agar or Potato Dextrose Agar they simply grow as mycelium and refuse to fruit. Researchers have traditionally resorted to adding sterilised plant material, most often pine needles, but also poplar twigs and pistachio leaves, to water agar to stimulate pycnidia. The problem is that host tissue varies enormously in composition depending on species, origin and physiological state, so results are inconsistent between batches, laboratories and experiments. A breakthrough came in 2023, when researchers showed that Diplodia sapinea, a pine pathogen, sporulates prolifically and predictably on Vogel’s Minimal Medium, a defined formulation first devised in 1956 for the growth of Neurospora crassa, incubated at 25 degrees Celsius under constant white light.

The new study, led by researchers working with fungal cultures from the Instituto Canario de Investigaciones Agrarias in the Canary Islands, Spain, together with a reference collection from the University of Pretoria in South Africa, put that protocol through its most comprehensive test yet. The team used 123 freshly collected isolates from hosts ranging from Canary Island dragon trees and royal poincianas to avocado, plum and grapevine, plus 67 older Neofusicoccum strains from long-term storage. Before any sporulation assays began, every isolate was identified to species level by sequencing four genetic loci: the internal transcribed spacer region, the translation elongation factor 1-alpha gene, the beta-tubulin gene and the RNA polymerase II second-largest subunit gene, then running maximum likelihood and Bayesian phylogenetic analyses. This rigorous framework assigned the isolates to ten species among the three target genera.

The results were striking in their breadth but equally revealing in their variation. All three Diplodia species formed pycnidia, yet only Diplodia seriata reliably released conidia, doing so faster and more abundantly than its relatives; Diplodia africana and Diplodia mutila produced fruiting bodies but yielded no countable spores. Both Lasiodiplodia species, L. brasiliensis and L. theobromae, formed pycnidia and discharged conidia efficiently, reaching yields of roughly seven to nine million conidia per millilitre. Among the 26 Neofusicoccum species tested, 20 formed pycnidia and 15 released conidia. The standout performer was Neofusicoccum luteum, which averaged 1.5 times ten to the seventh conidia per millilitre, more than 600 times the highest yield reported in earlier studies using plant-substrate media. Timing also varied dramatically: pycnidia appeared within three days in D. seriata but took up to two weeks in D. africana and D. mutila, while conidia harvesting required anywhere from 14 to 35 days depending on the species.

The team then probed the chemistry behind the process by systematically varying the sucrose concentration in the medium, testing levels from the standard 1.5 percent down to zero. The pattern that emerged underscored how deeply carbon availability regulates fungal reproduction, but not in a uniform way. Most Neofusicoccum species, along with both Lasiodiplodia species, sporulated best at 1 to 1.5 percent sucrose, and deprivation delayed pycnidia formation or suppressed conidial release altogether. Yet the exceptions were instructive: D. seriata produced its greatest yields at much lower sugar levels of 0.25 to 0.5 percent, and D. africana, remarkably, released quantifiable conidia only in the complete absence of sucrose. These findings suggest sucrose acts not merely as a nutrient but as a regulatory cue in reproductive development, and that tailoring the recipe to individual species could dramatically improve results.

The comparison with long-stored strains delivered a cautionary lesson for culture collections worldwide. Isolates of 26 Neofusicoccum species had been maintained on Malt Yeast Agar slants at 4 degrees Celsius since their original collection dates, some stretching back to the 1980s. When transferred to Vogel’s Minimal Medium, the oldest isolates, particularly those collected in the 1980s, consistently failed to produce either pycnidia or conidia, while more recently isolated strains sporulated far more readily. Strikingly, neither geographic origin nor host species predicted sporulation success; only culture age correlated with reproductive decline. Species such as N. luteum and N. stellenboschiana, which had sporulated robustly as fresh Canary Island isolates, failed to sporulate after years in storage. The message is clear: long-term preservation and, likely, repeated subculturing can quietly strip fungal strains of their capacity to reproduce, even when they remain alive and growing.

The practical advantages of the new protocol extend beyond yield numbers. Because the medium is chemically defined, every component can be manipulated precisely, enabling researchers to dissect exactly which nutrients and signals control sporulation, something impossible with pine needles of unknowable composition. Conidia released in sticky cirri can be harvested simply by flooding plates with a dilute Tween 20 solution, gently agitating, centrifuging and resuspending the pellet in glycerol for storage at minus 80 degrees Celsius, with no need to crush pycnidia manually. Yields from several species exceeded those of the original Diplodia sapinea study, with N. eucalypticola from the stored collection producing up to 12.4 million conidia per millilitre. The protocol also proved applicable to a second fungal genus, Oblongocollomyces, in parallel work, hinting at even wider utility.

For plant pathologists, the implications reach into infection biology, epidemiology and disease forecasting. Reliable, standardised spore production is the foundation for inoculating seedlings in resistance trials, testing fungicides, developing genetic transformation systems and modelling how disease spreads through orchards and forests under different climates. Until now, comparisons across studies were undermined by the unpredictability of plant-substrate methods; a shared, reproducible medium changes the calculus entirely. The researchers caution that substantial interspecific and even isolate-to-isolate variation persists, and that unanswered questions remain, including whether alternative light regimes could further boost yields and how best to preserve strains without eroding their reproductive competence. Vogel’s Minimal Medium, they conclude, offers not a universal key but a common, well-characterised baseline, and in a field long plagued by inconsistency, that baseline may prove transformative for research on some of agriculture’s most persistent fungal enemies.

Subject of Research: Standardised induction of sporulation in Diplodia, Lasiodiplodia and Neofusicoccum using Vogel's Minimal Medium

Article Title: Standardised sporulation methods for Diplodia , Lasiodiplodia and Neofusicoccum

Article References: Hernández-Hernández, D., Siverio de la Rosa, F., Grobler, C., & Slippers, B. (2026). Standardised sporulation methods for Diplodia, Lasiodiplodia and Neofusicoccum. IMA Fungus, 17, Article e176189. https://doi.org/10.3897/imafungus.17.176189

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.176189

Keywords: Botryosphaeriaceae, Diplodia, Lasiodiplodia, Neofusicoccum, sporulation, Vogel's Minimal Medium, conidia, pycnidia, plant pathology, fungal pathogens, culture media, woody plant diseases