Optical clearing reveals fungal pathogen behavior on apple surfaces

The study, published in Applied Microscopy, demonstrates that optical clearing techniques can effectively remove pigments from apple tissues, allowing for the clear observation of the pathogenic fungus Botryosphaeria dothidea. This approach provides a new window into the pre-penetration behavior of the fungus, a phase that is typically difficult to observe in intact, pigmented plant tissues without extensive sectioning or destructive preparation.

The central challenge in observing plant-pathogen interactions lies in the optical properties of plant cells. Cell walls, fibers, and natural pigments such as chlorophyll, carotenoids, and anthocyanins absorb and scatter visible light. This creates a refractive index mismatch between cellular components, which significantly deteriorates the resolution of conventional light microscopy. As a result, observing fungal structures on the surface of intact apple fruits or twigs without removing these pigments is technically challenging. Optical clearing techniques, which render bulk specimens transparent by modifying cellular components and removing pigments, offer a solution to this problem.

Botryosphaeria dothidea is an ascomycete fungus that causes canker and dieback diseases in over 24 plant genera worldwide, including apple trees. In apples, it is the causal agent of white rot, a disease characterized by light brown, concentric ring rots on slightly sunken fruits and blisters on stems. While previous studies using scanning electron microscopy have revealed that the fungus germinates, forms appressoria, and enters microcracks on apple fruits, limited information was available regarding its behavior on host surfaces using conventional upright light microscopy. The significance of understanding these latent infections is considerable, as they suggest the pathogen may evade detection by quarantine systems.

The study aimed to evaluate a clearing solvent mixture suitable for apple tissues and to observe fungal conidial germination and appressorial formation on host surfaces. The researchers collected naturally infected apple fruits from an orchard in Suwon, Korea, and isolated B. dothidea from a fruit exhibiting typical white-rot symptoms. The fungal isolate, designated BD-3, was grown on potato dextrose agar amended with streptomycin sulfate to prevent bacterial contamination. Conidia were harvested from barley grain media and adjusted to a concentration of 1.0 × 10^5 conidia per milliliter for inoculation experiments.

For the inoculation process, mature red fruits and two-year-old twigs from ‘Fuji’ apple trees were surface sterilized and marked for point inoculation. A droplet of the conidial suspension was placed on the fruit surface and twig lenticels. The specimens were then placed in a dew chamber at 25 °C under continuous dark conditions for a wetness period of 9 hours. Following this period, the specimens were removed and processed for optical clearing to observe the fungal behavior on the host surfaces.

The optical clearing procedure involved excising epidermis and cork tissues from the inoculated fruits and twigs. These tissue samples were immersed overnight in an ethanol-chloroform solution (3:1 v/v) containing 0.15% trichloroacetic acid. This mixture was chosen to decolorize the specimens by removing pigments. After clearing, the tissues were stained with lactophenol cotton blue, a stain that specifically colors fungal structures blue by binding to chitin in the fungal cell walls. The stained specimens were then observed using conventional upright light microscopy.

The results showed that the morphology and behavior of the inoculated B. dothidea could be clearly observed in the optically cleared specimens. On the apple fruit epidermis, the fungal structures were specifically stained blue against the cleared background. The conidia, which were spindle-shaped and approximately 20 micrometers in length, exhibited either monopolar or bipolar germination. In monopolar germination, a germ tube emerged from one end of the conidium, while in bipolar germination, germ tubes emerged from both ends. Instances of bipolar orthogonal germination, where germ tubes emerged perpendicular to each other from opposite ends, were also noted.

Following germination and germ tube elongation, the conidia formed appressoria at the terminal ends of the germ tubes. These appressoria are specialized adhesion structures that facilitate penetration into the host tissue. In the two-dimensional light micrographs, the appressoria appeared round, hook-shaped, or irregular in shape. The variation in shape may reflect distinct developmental stages during appressorial formation or specific responses to environmental stimuli such as substrate composition and topography. The clear visualization of these structures on the fruit surface provided detailed insights into the pre-penetration phase of the infection.

Similar observations were made on the apple twig lenticels, which are natural openings in the periderm that facilitate gas exchange. The fungal behavior on the twigs was similar to that on the fruit surface, with bipolar germination and appressorial formation observed. However, the fungal structures on the twigs were not as densely stained blue as those on the fruits. Notably, multiple appressoria were observed on the suberized phellem cells within the twig lenticels. This finding suggests that the fungus develops adhesion structures potentially for the initiation of penetration pegs to invade the lenticels, which may serve as infection courts for the pathogen.

The study highlights the effectiveness of the ethanol-chloroform solution with trichloroacetic acid in decolorizing apple tissues. This protocol, previously used for observing fungal infections in barley leaves, falls under hydrophobic-related clearing techniques due to the properties of ethanol and chloroform. The use of lactophenol cotton blue allowed for specific staining of fungal structures, although the researchers noted that iodine glycerol could be an alternative stain to avoid the biohazards associated with lactophenol. Further research is needed to assess integrated protocols for non-hazardous clearing and fungus-specific staining.

This work represents the first report of the application of optical clearing to document the pre-penetration behavior of B. dothidea on apple fruits and twigs using light microscopy. By providing a clear view of fungal structures on host surfaces, the technique offers a valuable tool for studying plant-pathogen interactions.

By elucidating the role of lenticels as potential infection courts, the study contributes to a better understanding of how pathogens invade host tissues.

In conclusion, the study successfully introduced a procedure for visualizing the pre-penetration behavior of B. dothidea on apple surfaces. The combination of optical clearing and fungal staining proved effective in revealing the detailed morphology of the fungus on host tissues.

The research was published in Applied Microscopy, Volume 55, article number 4, in May 2025. The authors utilized an isolate of Botryosphaeria dothidea designated BD-3, which was originally obtained from an apple fruit exhibiting typical white-rot symptoms. These symptoms included light brown, concentric ring rots on slightly sunken fruit tissues, with lesions extending to the core. The isolate was maintained on acidic potato dextrose agar amended with streptomycin sulfate to prevent bacterial contamination, a protocol consistent with previous methods described in the literature.

The optical clearing protocol employed in this study relies on the specific chemical properties of its components. Ethanol and chloroform were used in a 3:1 volume ratio, with trichloroacetic acid added at a concentration of 0.15% by weight per volume. According to the contemporary classification of optical clearing techniques for botanical applications, this protocol is categorized under hydrophobic-related techniques. This classification is due to the amphiphilic nature of ethanol and the hydrophobic properties of chloroform. The solution effectively decolorized the apple tissues by removing pigments that would otherwise obscure the fungal structures. The coloration of apple skin is attributed to chlorophyll and carotenoids in plastids, as well as phenolic pigments such as anthocyanins, flavonols, and proanthocyanidins in the vacuoles. The clearing process addressed these specific pigments to enhance visibility under light microscopy.

The staining agent, lactophenol cotton blue, functions by binding to chitin present in the fungal cell walls. This specific interaction allows for the blue staining of fungal structures, including hyphae and appressoria, against the cleared host tissue. The stain consists of cotton blue, also referred to as methyl blue, dissolved in a solution of lactic acid, phenol, and glycerol. While effective, the researchers noted that lactophenol poses biohazards. Consequently, iodine glycerol has been suggested as an alternative stain for characterizing filamentous fungi. The authors indicated that additional research is necessary to assess integrated protocols that combine non-hazardous clearing methods with fungus-specific staining. This highlights a current limitation in the methodology, where safety and efficacy must be balanced in future applications of these techniques.

The study also contextualizes the observed fungal behavior within the broader framework of plant pathology. Lenticels, the natural openings in the periderm of woody plants, are distinguished from the phellem by the presence of intercellular spaces. These structures facilitate gas exchange and have been suggested to serve as infection courts for various forest pathogens. Previous semi-thin sectioning studies revealed that hyphae of artificially inoculated B. dothidea entered apple twigs through lenticels, growing both intercellularly and intracellularly. The current findings of multiple appressoria on suberized phellem cells support the inference that the fungus develops adhesion structures to initiate penetration pegs. This suggests that optical clearing techniques can be applied to elucidate the role of lenticels as potential entry points for pathogens in woody plants, providing a clearer view of the pre-penetration phase that was previously difficult to capture using conventional upright light microscopy.

Subject of Research: Optical clearing of apple tissues for imaging fungal pathogen behavior

Article Title: Optical clearing of apple tissues for in vivo imaging of the pathogenic behavior of the fungus Botryosphaeria dothidea on host surfaces

Article References: Kim, K. W., & Park, E. W. (2025). Optical clearing of apple tissues for in vivo imaging of the pathogenic behavior of the fungus Botryosphaeria dothidea on host surfaces. Applied Microscopy, 55(1), Article 4. https://doi.org/10.1186/s42649-025-00110-w

Image Credits: AI Generated

DOI: 10.1186/s42649-025-00110-w

Keywords: Botryosphaeria dothidea, apple white rot, optical clearing, light microscopy, fungal appressoria, plant pathology, conidial germination, lenticels, lactophenol cotton blue, in vivo imaging, Optical, clearing