Colonization ability of Penicillium strain SF-08 on walnut seedlings

【Objective】The antagonistic fungus Penicillium rubens SF-08 has demonstrated potential in disease resistance promotion and broad-spectrum antimicrobial activity. However, a comprehensive understanding of its colonization mechanisms within host plant tissues, particularly its spatial distribution and colonization dynamics, remains largely undefined. This study aimed to investigate these aspects in Juglans regia L. seedlings in order to provide a scientific foundation for the rational application of SF- 08 as a biological control agent.【Methods】The protoplasts of P. rubens SF-08 were prepared and subjected to polyethylene glycol (PEG)-mediated transformation using a plasmid containing a gene encoding green fluorescent protein (GFP) driven by a constitutive promoter. This genetic modification enabled stable expression of GFP within fungal cells for visualization. The transformants were rigorously screened based on stable GFP expression, consistent phenotypic characteristics (colony morphology, growth rate, and sporulation patterns) compared with the wild-type SF-08, and retained antagonistic activity against target pathogens through dual culture assays. A stable transformant, designated SF08gfpF- 2, exhibiting robust fluorescence and antagonistic activity comparable to the wild-type, was selected for subsequent microscopic studies. To visualize SF-08 colonization, the walnut seedlings were inoculated with spore suspensions of the GFP-labeled SF08gfpF-2 strain. At various time points post-inoculation, root, stem, and leaf tissues were harvested, sectioned, and prepared for microscopic examination. Laser confocal microscopy was employed to observe the spatial distribution of the SF08gfpF- 2 within the plant tissues. GFP’s inherent fluorescence facilitated direct, high-resolution visualization of fungal presence and localization. For quantitative assessment of the SF-08 colonization dynamics, the walnut seedlings were inoculated with spore suspensions of the wild- type SF- 08 strain. At predetermined time points (0, 7, 14, 21, 28 days post-inoculation), the samples of the roots, stems, and leaves were collected. The total genomic DNA was extracted from these collected plant tissues using a standardized protocol optimized for plant-fungal composites. A quantitative polymerase chain reaction (qPCR) assay was developed and optimized for the SF-08 detection. The specificity of the primers was validated against a panel of common plant pathogens and host plant DNA. The assay exhibited high specificity and an amplification efficiency exceeding 98%. A strong linear relationship (R2 ＞0.99) was established across a detection range from 1.5×103 to 1.5×109 copies per gram DNA, indicating high sensitivity and reliability. The DNA copy number of the SF-08 in each plant tissue sample was determined by comparing the cycle threshold (Ct) values to a standard curve generated from known concentrations of the SF- 08 DNA.【Results】A stable transformant, SF08gfpF-2, with strong fluorescence and antagonistic activity comparable to the wild-type strain, was successfully obtained. The laser confocal microscopy revealed that the SF08gfpF-2 could invade and colonize the root epidermis, intercellular spaces of the root cortex, and vascular tissues of the walnut seedlings. Furthermore, the fungus extended its presence to the intercellular spaces of the stems and leaves, confirming its endophytic and cross-tissue colonization capability. The established qPCR assay demonstrated high specificity and amplification efficiency (＞ 98% ), with a strong linear relationship (R2 ＞0.99) across the range of 1.5×103 to 1.5×109 copies per gram DNA, thus confirming its high sensitivity and suitability for quantitative analysis. The dynamic quantification results indicated that post-inoculation, the SF-08 initially established extensive colonization in the roots. Subsequently, the SF-08 DNA was detected with increasing abundance in the stems and leaves. A significant increase in the fungal load across all colonized tissues was particularly evident between 14 and 21 days post-inoculation, after which colonization levels either stabilized or exhibited a gradual decline. This dynamic pattern would elucidate the processes of establishment, systemic proliferation, and persistence of the SF-08 within the walnut seedlings.【Conclusion】This study successfully leveraged genetic labeling, advanced microscopy, and molecular quantification to investigate the colonization patterns of the antagonistic fungus P. rubens SF-08 in the J. regia L. seedlings. The GFP-labeled SF08gfpF- 2 transformant proved effective for visualizing fungal distribution. Through laser confocal microscopy and qPCR quantification, the SF-08 was confirmed to possess robust endophytic and systemic colonization capabilities within the roots, stems, and leaves of the walnut plants. The colonization process exhibited distinct spatiotemporal dynamics, characterized by initial extensive root colonization followed by systemic spread, with a peak in fungal biomass observed within 14 to 21 days post-inoculation. These findings would provide direct and substantial evidence for understanding the fundamental interaction mechanisms between the SF-08 and its host plant. The ability of the SF-08 to establish a systemic presence within walnut seedlings is important, as it supports its potential to exert systemic biocontrol effects against both foliar and root-associated pathogens. The elucidation of its colonization dynamics would offer a guidance for optimizing the timing and method of the SF-08 application as a biological control resource, aiming to maximize its efficacy in agricultural settings. This research would contribute to the knowledge of P. rubens SF-08 and its potential as a sustainable biopesticide for walnut cultivation and other plant systems.