Biofilm formation and response in Exserohilum turcicum

doi:10.21203/rs.3.rs-5178972/v1

Bacteria are well known and studied for biofilm formation in varying environments. There are however limited studies that have characterised biofilm formation in plant pathogenic fungi. The aim of this study was to assess the biofilm-forming capacity of Exserohilum turcicum, the causal pathogen of Norther leaf blight, under varying environmental conditions, including growth media, temperature, and pH. Biofilm traits namely, metabolic activity, biomass, and extracellular matrix (ECM) production, were analysed on 16 strains under various pH and temperature conditions at two-time points (3 and 7 days). All strains studied formed self-produced gelatinous matrix at an optimum temperature of 25 °C and a pH of 10 at both time points. To further assess the capacity of E. turcicumto form biofilms, two (best and least) biofilm-forming strains among the 16 were subjected to heat treatment at 45 °C and analyzed using scanning electron microscopy (SEM), confocal laser microscopy (CLSM), transmission electron microscopy (TEM). Both strains exhibited increased amounts of ECM when heat-treated compared to the non-heat-treated biofilm. Additionally, the hyphal cell wall thickened under heat treatments. The ECM, being a hallmark of biofilm formation, is often produced by microbial biofilms in response to stressful environments. Therefore, our findings demonstrated that E. turcicum produces biofilms as a survival mechanism, particularly under specific environmental conditions, which supports its persistence and survival in the field.

Biofilm

Extracellular Matrix (ECM)

Exserohilum turcicum

Fungus

northern leaf blight

northern corn leaf blight

Microorganisms that thrive in hostile environments demonstrate a variety of adaptive strategies, most notably biofilm formation (Fröls, 2013; Yin et al., 2019). A multitude of bacteria and fungi can shift between planktonic and multicellular biofilm communities, of which approximately 80% exist in the environment as biofilms (Donlan, 2002; Ramage et al., 2012). Fungal biofilms can significantly amplify the resistance to environmental factors (Manganyi et al., 2015). Therefore, studying biofilm formation is essential for understanding the behavior and structural changes of plant pathogenic fungi thriving under hostile conditions.

Several microbes have evolved mechanisms of forming and encapsulating themselves, either singly or in communities, within outer coating structures known as biofilms. These biofilms facilitate microbes to stick to one another or to surfaces (Motaung et al., 2020; Penesyan et al., 2021). The material that encases cells and binds them together is known as the extracellular matrix (ECM) (López et al., 2010), a hallmark of biofilm formation by fungal pathogens (Costa-Orlandi et al., 2017; Liu et al., 2022; Mitchell et al., 2016a, 2016b; Zarnowski et al., 2021). This matrix is composed mainly of a polysaccharide biopolymer along with other components such as proteins, nucleic acids, lipids, and exopolysaccharides (Branda et al., 2005; Motaung et al., 2020). In particular, exopolysaccharides can vary significantly based on growth requirements, nutrient composition, (López et al., 2010), temperature, and pH. Biofilm formation offers cells the ability to withstand difficult conditions and the possibility of stretching out and finding new locations to inhabit (Hall-Stoodley et al., 2004).

Formation of biofilms is implicated in having negative consequences to healthcare, biotechnological, and agricultural industries. This is partly due to the limited understanding of the mechanisms underlying biofilm adaptation to different microenvironments (Cámara et al., 2022; Mishra et al., 2020). As biofilms form, they can interact with surfaces and result in the corrosion, deposition, and microbiological degradation of materials. This can be a challenge to the healthcare sector, as biofilms can be difficult to remove from medical equipment such as dentures, endotracheal tubes, orthopedic prostheses, cardiac pacemakers, catheters, and heart valves. Their ability to persist is due to antifungal resistance of the three-dimensional (3D) biofilm structure which in turn escalates biofilm-related clinical infections, thereby increase the cost of treatment (Bryers, 2008; Coad et al., 2016; Desai et al., 2014; Donlan, 2001; Wi & Patel, 2018). The negative outcomes of biofilm formation in biotechnological environments can include biofouling, corrosion, and a decrease in the quality of drinking water (Mishra et al., 2020). Similarly, the same challenges may occur in agriculture when the necessary machinery and tools are contaminated with harmful fungal biofilms, making it a challenge to remove them. Biofilm colonization on farming equipment has the potential to contaminate unaffected fields with unique biofilm-generated propagules, some of which may have acquired resistance to fungicides (Motaung et al., 2020; Peremore, 2022b; Shay et al., 2022). In agro-based industries namely aquatic, meat, dairy, and food processing plants, biofilm formation contaminates food and food sources, which poses a threat to food safety (Toushik et al., 2022).

The environment including pH levels, temperature, nutrient availability, and hydrodynamics impact on biofilm formation. These conditions influence the composition and structural development of biofilms by affecting the enzymes and regulatory processes involved in biofilm maturation and dispersal (Flores-Vargas et al., 2021; Goller & Romeo, 2008). For instance, changes in nutrient composition can result in alterations within ECM produced by microbial communities and this affects the stability and protective functions of biofilms (Goller & Romeo, 2008; Mirghani et al., 2022). Furthermore, variables such as the presence of specific ions and water flow velocity can influence the 3D structure of biofilms, impacting their resilience and interaction with the surrounding environments (Balcázar et al., 2015; Flores-Vargas et al., 2021). Understanding these environmental factors is critical for understanding how biofilms grow and adapt under various conditions.

Although research on phytopathogenic biofilm formation and its effects on the microbial environment in the agricultural sector exists (Peiqian et al., 2014; Peremore, 2022b; Ratsoma et al., 2023; Shay et al., 2022; Tyzack et al., 2023), but it is not as extensive as with other sectors. Very recently, formation of biofilm within plant fungal pathogens namely; Fusarium verticilloides, Fusarium circinatum, Fusarium oxysporum f. sp. cucumerinum, Fusarium graminearum and Zymoseptoria tritici has been confirmed (Motaung et al., 2020; Peremore, 2022b; Ratsoma et al., 2024; Shay et al., 2022). From these studies, there is consensus that biofilms provide protection from host defences and extreme environments.

Exserohilum turcicum, the causal fungal pathogen of northern leaf blight (NLB), the most prevalent global foliar disease of maize in sub-Saharan Africa (Nsibo, 2019). Exserohilum turcicum survives on infected maize residues and spreads either by wind or splash under favourable weather. (Wise, 2011; Yang et al., 2017). The pathogen is implicated in causing up to 75% yield losses if not managed effectively and hence is a major threat to Africa’s food security (Berger et al., 2020). Control measures currently used to manage infections include crop rotation, deep tillage, NLB-resistant maize cultivars, and fungicides (Human et al., 2016; Kotze, 2020). Exserohilum turcicum is known to exhibit high genetic diversity in South Africa (Human et al., 2016; Nieuwoudt et al., 2018) being driven by several evolutionary forces namely; migration, sexual reproduction, and/or mutation (Haasbroek, 2014). The high genetic diversity found within the E. turcicum populations may lead to the emergence of strains that are more resistant to fungicides and other management strategies in the long run. Additionally, sexual recombination among strains can facilitate the exchange of genetic material, thus giving rise to more virulent strains in nature and adding complexity to the strategies for managing fungicide resistance (Bankole et al., 2023; Human et al., 2016; Nieuwoudt et al., 2018).

To our knowledge, no study has characterized biofilm formation in E. turcicum within a single strain or at a population level. The lack of this knowledge derails the development of more effective control strategies of the pathogen as well as the effort to understand how it behaves in nature. Therefore, the aim of this study was to screen and characterize biofilm formation in E. turcicum strains and determine the role of ECM production in response to abiotic stresses. The study explored several scientific techniques (plate and microscopic analysis (SEM, CLSM and TEM)) to screen and characterize E. turcicum biofilm formation and its environmental response at the morphological and metabolic levels.

2.1 Fungal strain sampling

Symptomatic maize leaves with NLB were collected from three provinces of South Africa: Gauteng, Mpumalanga, and Kwa-Zulu Natal. To obtain strains from the leaves, a method from (Jindal et al., 2019) and (Haasbroek et al., 2014) was used with minor modifications. Briefly, infected leaves were cut into 1 cm × 1 cm pieces and sterilized using 2% sodium hypochlorite (NaOCl) for 2 min. They were then rinsed twice with sterile water. The surface-sterilized leaves were later placed on damp filter paper in Petri dishes (60 mm) and incubated at 25 ˚C for three days in the dark to allow sporulation. Exserohilum turcicum-like conidia were picked with a sterile needle under a dissecting microscope at 90x magnification and then transferred into ¼ potato dextrose agar (¼ PDA) (Merck Group, Modderfontein GP, South Africa) in 60 mm petri dishes. The plates were incubated at 25°C in the dark for 3–4 days to obtain monoconidial strains of the Exserohilum species.

2.2. Culture maintenance

A pure culture, hereafter referred to as a strain, was selected and sub-cultured on fresh ¼ PDA (90 mm Petri dishes) supplemented with 50 mg/ml cefotaxime (Aspen, Pharmacare, Durban, South Africa). For long-term storage, pure cultures were resuspended in 30% glycerol stock and stored at -80°C at the University of Pretoria’s Cereal Pathology Group (CPR) collection. Seventeen E. turcicum strains were selected for use in this study. Culture, morphological, and molecular techniques were used to confirm that the selected strains were E. turcicum.

2.3 Molecular identification of fungal strains

Genomic DNA was extracted from 7 to 14 days old mycelia using the cetyltrimethylammonium bromide (CTAB) method, as described by (Haasbroek et al., 2014). DNA quantity and quality were assessed using a NanoDrop spectrophotometer (NanoDrop Technologies) and stored at -20°C in preparation for molecular identification. Mating-type diagnostics were performed to confirm that E. turcicum is the causal pathogen of NLB using species-specific mating-type primers MAT1-1 (GTCCATGGGATACGCG) and MAT1-2 (ACCGATTGCTTCG). PCR amplification was performed following an initial denaturation step at 95°C for 3 min. This was followed by 35 cycles at 95°C for 10 s, 61°C for 10 s, and 72°C for 20 s, followed by 25 cycles at 95°C for 30 s, 62°C for 45 s, and 72°C for 60 s. The procedure was concluded with a final extension step at 72°C for 20 minutes. The PCR products were subjected to electrophoresis on a 2% agarose gel at 80 V for 60 min and were observed using the GelDoc system (Bio-Rad Laboratories Ltd., South Africa). The PCR product of each strain was identified by analyzing the size of the PCR amplicon.

2.4. Screening for biofilm production

The inoculum to form biofilms was prepared as described by (Kotze et al., 2019) with slight modifications. A total of 100 µL spore suspension from each 7-day old cultures of E. turcicum (1 × 10⁵ conidia/mL) was placed at the centre of a sterile ¼ PDA to allow even growth on a petri dish. To form biofilms, 5 mL phosphate-buffered saline (PBS) (0.2 M PBS; 10 mM NaH₂PO₄, 10 mM Na₂HPO₄, 150 mM NaCl, pH 7.2) was pipetted on the surface of pure cultures of E. turcicum cultured on ¼ PDA. The plates were gently swirled to release mycelia and conidia. To ensure the swirled cell suspension was filtered through nylon filter membranes (0,22 µM, Merk Millipore) to retain conidia. Conidial cells were then adjusted to 1 × 10⁵ conidia/mL using an automated cell counter (Countess 3 FL, Thermo Fisher).

2.5. Standardization of biofilm formation in Exserohilum turcicum

2.5.1. Grow media assay

Three E. turcicum strains (CPR 726, CPR 721 and CPR 723) were randomly selected and screened for biofilm formation on full strength PDB, ½ PDB, or ¼ PDB to determine the best medium that facilitates the rapid formation of biofilms. Biofilm formation was performed as previously described (Peiqian et al., 2014; Peremore, 2022; Ratsoma et al., 2024), with slight modifications. Briefly, the conidial suspension (1 × 10⁵ conidia/mL) of each of the three strains was inoculated into 10 mL full strength PDB, ½ PDB, and ¼ PDB. Triplicate plates were then stationary incubated at 25°C for seven days in the dark and monitored daily for biofilm formation.

2.5.2 Biofilm formation at varying pH and temperature

The effects of pH on biofilm formation were evaluated using 16 E. turcicum strains (Table 1). Following the growth media assay, biofilm formation was performed using ¼ PDB in 96 polystyrene microtitrel plates (Corning® Costar® TC-Treated Multiple Well Plates) in the stationary state. The effect of different pH values: (3, 4, 5, 7, 8, and 10) on biofilm formation was assessed as previously described (Peiqian et al., 2014; Peremore, 2022a; Ratsoma et al., 2024). The pH of the solution was adjusted using HCL and NaOH. Biofilm formation was monitored at two time points, 3 days and 7 days post inoculation and the two-time points were choosen to allow for observation of any changes in the biofilm composition and structure over time as done by (Ratsoma et al., 2024). Biofilm formation was conducted in the dark at 25°C. In addition to pH, biofilm formation was assessed at 4˚C,16˚C, 25, 29, and 37°C over the course of 3 and 7 days in the dark.

To assess the impact of pH and temperature, we quantified metabolic activity using a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. For the metabolic activity of biofilms formed by all 16 E. turcicum strains, XTT was prepared according to the manufacturer’s recommended specifications and added to three- and 7-day-old biofilms. The biofilms were then incubated for 3 h at 37°C in the dark prior to measurement of cell viability using a microtiter plate reader (SpectraMax Paradigm) at 492 nm. During the XTT assay, tetrazolium salt is reduced by the pathogen’s mitochondrial dehydrogenase activity, thereby resulting in an orange formazan salt, which is indicative of cell viability (Costa-Orlandi et al., 2017). The OD measurement obtained determined how each strain formed a biofilm in terms of biofilm metabolic activity.

Biomass of the biofilms and ECM of the strains were quantified using crystal violet (CV) and safranin, respectively. To perform the assay, 200 µl of 99% methanol was initially added to the biofilm for 15 min to fix the biofilm. The methanol was then removed, and the plates were allowed to dry for 5 min. Subsequently, 200 µl of 0.3% crystal violet (CV) solution diluted in PBS was added to each well and incubated at room temperature for 25 min. Thereafter, CV was discarded, and the microtiter plate wells were washed with PBS three times. The biofilm cultured 96 well plates were allowed to air-dry before adding 200 µl of absolute ethanol for 5 min prior to analysis at 590 nm using a microplate reader (SpectraMax Paradigm). The OD measurement obtained determined how each strain formed a biofilm in terms of biofilm biomass.

The amount of ECM in E. turcicum biofilms was measured using safranin staining (Mello et al., 2016) of three- and 7-day-old biofilms. Briefly, the biofilms were stained with 200 µl 0.1% safranin for 5 min. Safranin was then washed with PBS until it was translucent. Subsequently, 200 µl absolute ethanol was added to decolorize the extracellular matrix. Analysis was performed using a microplate reader at 530 nm (SpectraMax Paradigm). The OD measurement obtained determined how each strain formed a biofilm in terms of biofilm ECM.

2.6 Biofilm architecture and structures of CPR 1175 and CPR 383

2.6.1 Plate analysis

Among a pool of 16 Exserohilum turcicum two strains— CPR 1175 and CPR 383—were singled out to understand biofilm dynamics and architecture. These strains were identified respectively as the best and least biofilm formers based on the pH and temperature assays. The two strains were monitored daily for biofilm formation and photos captured daily for 7 days, with the last photos captured on day 30 post inoculation.

2.6.2 Ultrastructural analysis

To study biofilm architecture, strains CPR 1175 (best biofilm producer) and CPR 383 (least biofilm producer) were selected. Biofilms of CPR 1175 and CPR 383 were grown in sterile petri dishes with glass coverslips in ¼ PDB at room temperature for 3 and 7 days. The following steps were then performed:

Biofilms were fixed in a mixture of 2.5% glutaraldehyde (Merck) and formaldehyde (Merck) and washed with PBS before being treated with 1% osmium tetroxide for 1 h. The washing step was repeated twice, after which the biofilms were dehydrated using a range of ethanol concentrations (30%, 50%, 70%, 90% and 3x 100%) for 15 min each. After dehydration, 5 ml hexamethyldisilazane (HMDS): ethanol solution was applied to the biofilms for 1 h, and then the HMDS: ethanol solution was discarded and only HMDS was added. The samples were dried overnight. Glass slides were mounted onto stubs and coated with carbon for 15 min. Finally, the stubs were examined using a JEOL JSM 6490LV Scanning Electron Microscope (Gen Tech Scientific Inc., Arcade, NY, USA).

After biofilm formation, preformed biofilms were subjected to heat treatment for 1 h at 45°C in an oven (Ratsoma et al., 2024). Both best and least biofilm-forming E. turcicum strains were then subjected to transmission electron microscopy (TEM). TEM analysis of biofilms was performed as described by Lu et al. (2019). Samples containing mature biofilms were treated with 2.5% glutaraldehyde (Merck) for 3 h. Phosphate buffer was used to wash the 2.5% glutaraldehyde three times. The biofilms were post-fixed with 1% osmium tetroxide for 30 min. Phosphate buffer was used to wash off the adhered biofilms with osmium tetroxide. Afterwards, the samples were dehydrated in a series of ethanol solutions with increasing concentrations (30%, 50%, 70%, 90% and 3x 100%) for 10 min each, and the last 100% was left for 30 min. A 50:50 mixture of the epoxy resin and 100% ethanol was prepared and mixed for an hour. Part of the sample was scraped into 1.5 mL tubes, and a 50:50 mixture of epoxy resin and 100% ethanol was added without sample drying. The tubes were then gently centrifuged for 1 h. Subsequently, the mixture was removed, and 100% epoxy resin was added to the samples and gently centrifuged for 1 h. Fresh resin and sample numbers were then added. The samples were embedded in a mold and placed in an oven for 24 h for polymerization. Samples were trimmed and sectioned with a diamond knife into 90 nm pieces, which were then stained with uranyl acetate for 30 s and observed under a transmission electron microscope (S-3400N).

2.6.3 Architectural analysis

The conidial cell suspensions of strains CPR 1175 and CPR 383 were added to ¼ PDB (pH 10) and incubated at room temperature for 3 and 7 days without shaking to allow the conidia to settle and adhere to the bottom of the chamber slide (Nunc Lab-Tek II Chamber Slide System; Thermo Fisher Scientific). The strains were exposed to heat treatment, as previously described (Ratsoma et al., 2024), while the non-heated biofilms served as positive controls. The medium was removed, and the slides were rinsed once with PBS to remove non-adhering cells before staining with fluorescent dyes to assess ECM secretion and metabolic activity. The dyes flooded on E. turcicum biofilms were concanavalin A-Alexa Fluor 488 conjugate (Con A) (25 µg/mL; Thermo-Fisher Scientific), calcofluor white stain (CFW) (25 µg/mL; Sigma-Aldrich), membrane (25 µg/mL; Biotium MemBrite® Fix 405/430; Thermo-Fisher Scientific), and 4′,6-diamidino-2-phenylindole (DAPI) (25 µg/mL; Thermo Fisher Scientific) to target the polymer of interest. The dye Con A stains exopolysaccharides, specifically a lectin that has an affinity for terminal α-d-glucosyl residues and α-d-mannosyl. CFW stains polysaccharides in the cell wall and matrix and specifically binds to cellulose, chitin, and glucose residues. Membrite stains lipids in the cell membrane, whereas DAPI stains nucleic acids. The biofilms were stained in the dark for 30 min with 100 µl of each stain. The slides were viewed using a ZEISS confocal laser scanning microscope 880 (CSLM), and CFW was visualized with excitation at 405 nm and emission in the range of 410–475 nm. Membrites were observed with emission at 419–481 nm, whereas DAPI emission occurred at 410–481 nm. Additionally, the dye Con A was excited at 488 nm, and its emission fell within the range of 499–561 nm using a 10x lens (University of Pretoria; Laboratory for Microscopy and Microanalysis). CLSM data were analyzed using the Fiji ImageJ program.

Reproducibility and statistical analysis

These experiments were conducted with three technical and four biological replicates. All experiments were repeated twice, and the average measurements are presented. The data were expressed as mean ± standard deviation (SD) using the GraphPad Prism 8 computer program. The data obtained from these assays were analyzed using one-way ANOVA in GraphPad Prism 8 or t-test analyses. Images were captured using a smartphone camera (Vivo V2027).

3.1 Standardization of biofilm formation in E. turcicum

Although all 16 isolated E. turcicum strains were screened for biofilm formation, we specifically focused on three randomly selected strains, CPR 726, CPR 721, and CPR 723, to closely assess their ability to form in vitro biofilms. Biofilms of these strains was characterized by dark pigmented adherent colonies that were surrounded by a layer of gelatinous slimy matrix that resembled an ECM (Fig S1 and Fig. 1). In addition, colonies formed in the broth adhered to the polystyrene plates by what appeared to be a gelatinous slimy matrix which was consistent even after repeated washes. The black pigment accumulated over time (Fig S2 and Fig. 1), with strains producing more of this pigment and the slimy material in ¼ PDB than in ½ PDB and full PDB (results not shown).

3.2 pH and temperature influence biofilm formation in Exserohilum turcicum

In all the 16 E. turcicum strains studied, biofilm response was measured based on XTT, biomass and ECM and represented by the average OD values within the respective heatmaps as indicated in Fig. 2, Fig. 3, and Fig. 4C, respectively. The OD measurement determined how each strain formed a biofilm in all three critical biofilm assays. Over a period of 3- and 7-days post inoculation, responses varied, with no unique patterns except for CPR 1174, whose XTT OD values changed during exposure to pH 4 to 10. Corresponding analyses of XTT, biomass, and ECM assays reaffirmed the difference in responses and gave confidence of what we observed through heatmaps at both time points with corresponding boxplots (Fig. 2 to Fig. 4). Different letters on top of all boxplots represent significant differences between the collections of strains and medians sharing the same letter per box and whisker were not significantly different as determined by the Tukey’s test (p-value ≤ 0.05). Furthermore, we observed through heatmaps that the overall optimum pH was 10 for E. turcicum biofilm formation for the majority of strains at both days 3 and 7, despite the strains coming from various regions with diverse environmental conditions. To confirm pH 10 as the best for E. turcicum biofilm formation in terms of XTT, biomass and ECM as depicted by heatmaps, corresponding boxplots and Tukey’s multiple comparison test analysis of a collection of SA strains at each tested pH (3, 4, 5, 7, 8, and 10), it was observed that at pH 10, biofilm indicators (metabolic activity, biomass and ECM) were significantly higher in a majority of strains at both time points: XTT (Fig. 2, A & B; Table S1-3), biomass (Fig. 3, A & B; Table S4-6), and ECM (Fig. 4, A & B; Table S7-9) assays. The median values on top of the box plots supported this finding, showing that pH 10 was the best with the highest median values for XTT at 0.84 (Fig. 2, C & D), biomass at 1.3 (Fig. 3, C & D), and ECM at 0.90 (Fig. 4, C & D). There was also a noted overall pH time effect in terms of E. turcicum biofilm response between day 3 and day 7 in all pH values.

We also observed that each E. turcicum strain responded differently in terms of XTT, biomass and ECM towards a wide range of temperature conditions (4–45 ˚C) although the overall optimum temperature was 25 ˚C for the majority of strains at 7 days (Fig. 5 to Fig. 7). The temperature of 25°C was significantly higher on day 7. Our analysis reaffirmed the observations and data presented using the corresponding boxplots for metabolic activity (Fig. 5, C & D; Table S10-12), biomass (Fig. 6, C & D; Table S13-15), and ECM (Fig. 7, C & D; Table S16-18). The exception was at day 3 at early biofilm development/maturity stagewhere the second-bestoptimum temperature was 29 ˚C, which was similarly reaffirmed with their corresponding boxplots. Day 3 was regarded as an early maturation stage by looking at the early formed dark adherent colony accumulation in while day 7 was regarded as lately matured due to an even more biofilm accumulation on day 7 (Fig. 8). Overall, among all the strains investigated, we observed that CPR 1175 could form thicker biofilms (Fig. 8A) while CPR 383 formed least biofilm (Fig. 8B) at both time points. These two strains were chosen for further analysis of their biofilm traits.

Visual and timeline analyses of biofilm structures

According to image analysis, the structure of E. turcicum biofilm (CPR 1175 and CPR 383) were formed in Petri-dishes with ¼ PDB, temperature of 25°C and pH level of 10, conditions previously deemed optimum for biofilm formation. Biofilms in both strains showed observable unique differences in terms of melanin dark color intensity, biofilm spatial and microcolony size distribution, biofilm texture, and biofilm thickness from day 3 to day 30 (Fig. 8). Morphologically, CPR 1175 biofilms were characterized by colonies with clear shiny slime around the dark (gray-black) attached colonies (Fig. 8, A). In contrast, CPR 383 biofilms were characterized by dark attached colonies, but displayed less slime material (Fig. 8, B). There were no major apparent morphological differences between days 7 and 30 in biofilms formed by either strain when visually inspecting the biofilm biomass aggregates in the Petri dishes. Additionally, adjusting the pH of the nutrient media to 10 further accelerated biofilm formation and development, occurring between days 7 and 3 according to image analysis (Fig. 8). Contrary to our initial biofilm formation with the conditions ¼ PDB and temperature of 25°C but no pH adjustment (Fig S1), we discovered that both stains in this instance demonstrated an early biofilm development on day 3 which is early biofilm development looking at the rapid biofilm formation in relation to biofilm forming conditions. In Fig S1, where only two stressors, namely temperature at 25°C and nutrient deprivation in ¼ PDA media, were present, the biofilm formed on day 7.

3.3 Ultrastructural analysis of Exserohilum turcicum biofilms

Both CPR 383 and CPR 1175 formed dense mycelial mats composed of multiple interconnected hyphae (Fig. 9). The SEM images revealed internal channels intricately woven together akin to trap-like formations (Fig. 9, white arrows). ECM consisted of a network of filaments covered with a smear of smooth gel-like slime/matrix (Fig. 9B, black arrows), appearing like an adhesive substance that securely anchors the hyphae in place. However, CPR 383 performed the least in forming the biofilms with no observable ECM surrounding the hyphal bundles after 3 days at RT (Fig. 9), which may have compromised its biofilm integrity.

3.3.2 Matrix component analysis of Exserohilum turcicum biofilm

The CLSM analyses revealed the presence of stained ECM biomolecule aggregates in both strains CPR 1175 and CPR 383 following staining with concanavalin A (conA), MemBrite, calcofluor white (CFW), and DAPI (Fig. 10A-D). As expected, the different stains used in this analysis highlighted various components of the biofilm (indicated by a-b and d-e): conA (green) highlighted β-glucan and mannans common in fungal biofilms, MemBrite (pink) revealed the hyphal membrane, CFW (aquamarine) highlighted the cell wall components (β-(1,4)-linked polysaccharides), and DAPI (blue) stained nucleic acids.

On day 3, the fluorescence signals displayed by various stains exhibited a transition from weak to strong response to heat treatment for strain CPR 383 (Fig. 10A, C). There was also an increased signal for both MemBrite and DAPI. This was supported by both 3D surface plots (Fig. 10A, C: a, d) and merged fluorescence intensity plots (Fig. 10A, C: c, f). A different response in terms of fluorescence signal intensity was observed for CPR 1175 (Fig. 10B, D). This strain was shown to form a thicker biofilm (Fig. 8–9). Interestingly, the signal intensity displayed by biofilms of this strain appeared to be strong only for the conA stain (Fig. 10B: a, d) in response to heat treatment on day 3, while for other stains (MemBrite, CFW, and DAPI), it was weak.

On day 7, the CPR 393 biofilm displayed a strong response based on MemBrite and CFW staining in the absence of heat (Fig. 10C). Only the DAPI signal decreased in the presence of heat treatment. In a 7-day biofilm of CPR 1175 (Fig. 10D), increased fluorescence signal for most stains, except DAPI, became intense when there was no heat present but became weak only for MemBrite and DAPI, while conA and CFW remained strong, when heat was applied. Taken together, our data strongly suggests differential structural changes in biofilms under heat stress and partly explains the molecular interactions within the biofilm that account for the different biofilm response in these strains.

3.3.3 Cell wall analysis of Exserohilum turcicum biofilm

According to the TEM results, CPR 383 biofilms exhibited an increase in certain components, notably the ECM and electron-dense/dark regions around the cut hyphae sections within the biofilm in response to heat (Fig. 11A). Similarly, CPR 1175 displayed an enhanced response to heat treatment, as shown by the thickened layer of both the cell wall and ECM (Fig. 11B), surpassing the thickness observed in CPR 383. Additionally, the TEM analysis aligns with the CLSM results obtained through the conA stain, whereby the thicker cell walls under heat treatments (Fig. 11B, D) (heat treatment) correlates directly with the increased Con A signal intensity under heat treatments (Fig. 10) (heat treatment) in CLSM results of both strains. Furthermore, both strains displayed a dark layer between the cell wall and ECM layers which became thinner in the presence of heat treatment for CPR 383 (Fig. 11A) or completely disappeared for CPR 1175 (Fig. 11B). The less faint ECM under heat treatment showed increased darkness as the melanin layer diminishes under heat treatment (Fig. 11).

The fungal pathogen E. turcicum threatens maize crops globally, especially in humid areas, due to its resistance to fungicides and its ability to persist in crop residues and soil (Jakhar et al., 2017; Navarro et al., 2021). In this study, we characterized biofilm formation and structure using in vitro conditions in an effort to demonstrate the potential of this pathogen to uphold this life form in its natural habitat. We believe that this lifestyle can contribute to E. turcicum’s persistence and resistance to abiotic factors including fungicides (Liu et al., 2022; Villa et al., 2017). Combined with the climatic factors on its pathogenicity and interaction with other species in the field, biofilm formation can complicate disease management (Doohan et al., 2003; Navarro et al., 2021; Ogolla et al., 2019). Therefore, more research is required to understand the role of biofilms in E. turcicum strains and develop effective control strategies.

This is the first study to critically analyse and demonstrate in vitro biofilm formation and response by E. turcicum, contrasting with the previous research that looked primarily on its growth on solid media/agar (Abdulaziz et al., 2017; Anwer et al., 2022; Bankole et al., 2023; Yamaguchi & Mutsunobu, 2010). In vitro, E. turcicum biofilm formation occurs optimally at pH 10 and 25°C and matures between three and seven days. During this period, biofilm formation was characterized by attachment, microcolony formation, maturation, and dispersion. These biofilm developmental stages have previously been demonstrated in several plant-associated filamentous biofilms (Motaung et al., 2020). Furthermore, E. turcicum biofilms exhibited a unique patterned round dark-black attached cohesive micro-colonies in liquid media, which seemed to differ from strain to strain. This was found to be surrounded by a matrix typical of other matrixes observed in other fungi (e.g., Ratsoma et al., 2024).

What was interesting was the level of matrix produced seemingly corresponding with the dark pigment of the colonies over time, which seemed to be less prioritized during heat exposure. This suggests that pigmentation, potentially melanin, may be augmented in a biofilm. We hypothesize that pigments associated with E. turcicum biofilms play a multifaceted role in enhancing the survival and pathogenicity of the fungus. TEM analysis suggests that melanin in E. turcicum is likely a part of the biofilm architecture and is affected during response to heat stress. For instance, when the biofilm was subjected to heat treatment, melanin decreased, while the ECM and cell wall layer thickened. Therefore, the interaction between melanin, ECM, and the hyphal cell wall may contribute to the overall E. turcicum biofilm response to abiotic stress. Melanin was represented by a dark layer observed between the cell wall and ECM, and this has been reported by other studies (Breitenbach et al., 2022).

Generally, fungi-produced metabolites such as melanin are known to play key roles in the thermal protection against harmful ultraviolet (UV) radiation and the host immune system (Belozerskaya et al., 2017; Dadachova et al., 2007; Frank et al., 2020; Horikoshi et al., 2010; Wang et al., 2022). It is possible that field strains of E. turcicum may adapt a biofilm lifestyle, which will add to the overall resilience of the biofilm and persistence in agricultural settings. For instance, the ability of biofilms in some of the strains we studied to withstand extreme pH and temperature may partly be influenced by the collective accumulation of melanin and matrix. This can enable the fungus to flourish in its natural habitat.

We further observed that while biofilm formation can be intrinsic in other strains, it was inducible in some. We narrowed this response to two strains, CPR 383 and CPR 1175, and characterized the ECM via SEM and CLSM. SEM revealed minimally produced ECM in CPR 383 while CLSM confirmed this low production in ECM, consistent with CPR 383's poor biofilm formation capacity. In contrast, CPR 1175 produced more ECM which wrapped around the hyphae and creating complex channels. Similar arrangement has been observed in biofilms formed by fungi such as F. verticillioides (Peremore, 2022), and such channels may act as conduits that facilitate diffusion of water and essential nutrients from the environment and within the biofilm (Costa-Orlandi et al., 2017; Fanning & Mitchell, 2012; Liu et al., 2022; Martinez & Fries, 2010). This can enable direct access of cells buried deep within the biofilm structure to have access to nutrients and water. Therefore, intrinsic biofilm forming field strains, such as CPR 1175, are likely forming dense biofilm structures with sophisticated channels due to high ECM production in order to retain water and nutrients. These channels can also enhance the structural integrity of the biofilm by virtue of their networks, and thus greatly influence the shape and mechanical response of the biofilm, as well as serve as a barrier to antifungals.

In conclusion, E. turcicum biofilm may promote the pathogen’s response to a wide range of field conditions by enhancing its survival, resistance to environmental stress, and persistence in agricultural settings. However, biofilm formation strength seems to be dependent on a specific strain, with formation in some strains characterized by dense biofilms with complex channels and likely heightened resilience to abiotic stressors. Our work highlights the need to take into account complex cell community structures when developing antifungal therapies against E. turcicum.

Author contributions

Wisely D. Kola: Investigation; Writing; review and editing; conseptualization: methodology: analysis

Thabiso E. Motaung: Project administration; supervision; review and editing: conseptualisation; methodology; analysis

David L. Nsibo: Project administration; co-supervision; review and editing; conseptualization; methodology; analysis

Acknowlegements

The work was funded by the The South African National Department of Science and Innovation-NRF under the Thuthuka funding instrument (Grant no. 129580) and the Centres of Excellence programme and South African Research Chairs Initiative (Grant No. 98353).

Conflict of interest statement

There was no conflict of interest

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Table 1: Origin and characterization of Exserohilum turcicum strains from maize-infected leaves in South Africa.

CPR #	Strains	Provinces	Region	Year	MT	Host	Morphological identification
CPR 403	KZN 126	KZN	Baynesfield	2013	MAT1-2	Maize	Exserohilum turcicum
CPR 406	KZN 129	KZN	Baynesfield	2013	MAT1-2	Maize	Exserohilum turcicum
CPR 383	KZN 105	KZN	Baynesfield	2013	MAT1-1	Maize	Exserohilum turcicum
CPR 404	KZN 127	KZN	Baynesfield	2013	MAT1-2	Maize	Exserohilum turcicum
CPR 721	Bay 689	KZN	Baynesfield	2020	N/A	Maize	Exserohilum turcicum
CPR 726	Bay 665	KZN	Baynesfield	2020	N/A	Maize	Exserohilum turcicum
CPR 722	Bay 651	KZN	Hopewell	2020	N/A	Maize	Exserohilum turcicum
CPR 723	Bay 670	KZN	Baynesfield	2018	N/A	Maize	Exserohilum turcicum
CPR 728	HW 580	KZN	Hopewell	2020	N/A	Maize	Exserohilum turcicum
CPR 1174	EC 202	EC	Mthingwevu	2020	N/A	Maize	Exserohilum turcicum
CPR 1175	EC 203	EC	Mthingwevu	2020	N/A	Maize	Exserohilum turcicum
CPR 1176	EC 212	EC	Matatiele	2021	N/A	Maize	Exserohilum turcicum
CPR 1177	EC 213	EC	Matatiele	2021	N/A	Maize	Exserohilum turcicum
CPR 413	MP 137	MP	Machdodorp	2013	MAT1-2	Maize	Exserohilum turcicum
CPR 414	MP 138	MP	Machdodorp	2013	MAT1-1	Maize	Exserohilum turcicum
CPR 416	MP 140	MP	Machdodorp	2013	MAT1-2	Maize	Exserohilum turcicum
CPR 423	MP 149	MP	Machdodorp	2013	MAT1-1	Maize	Exserohilum turcicum

KZN – Kwa-Zulu Natal, MP – Mpumalanga, EC – Eastern Cape, CPR #–Cereal Pathology Research group strain number, N/A –Not genetically identified, MT –Mating types, MP- Morphological identification.

The authors declare no competing interests.

FigS1.jpg
Figure S1: Biofilm formation by Exserohilum turcicum strains CPR 726 (A), CPR 721 (B), and CPR 723 (C) in 90 mm Petri dishes containing full-strength, ½-strength, and ¼-strength potato dextrose broth (PDB) over time. The strains were incubated at 25 °C in the dark until mature biofilm structures developed. The biofilms exhibit changes in color as they mature.
Kolaetal.2024SupplementoryTables.docx

Biofilm formation and response in Exserohilum turcicum

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Abstract

Figures

1. Introduction

2. Material and methods