Exploring fungal pathogens to control the plant invasive Rubus niveus on Galapagos Island San Cristobal

Abstract

The Galapagos ecosystem faces threats from invasive species displacing native and endemic species. Rubus niveus (Hill raspberry) is particularly problematic invasive plant, covering approximately 30,000 hectares across the archipelago and rapidly outcompeting native vegetation. Current control methods, such as manual removal and herbicide application, have proven ineffective. This research aimed to identify endemic fungi pathogenic to R. niveus for potential population suppression. To achieve this goal, we sampled leaves, fruits, and stems of R. niveus in the agricultural areas of San Cristobal, Galapagos. Microbiome composition analysis of healthy and diseased R. niveus leaves revealed differences in fungal communities, representing a greater abundance of pathogenic genera in diseased tissue. These genera included Alternaria, Septoria, Fusarium, Colletotrichum, and Phanerochaete, representing well-known pathogens. Among 595 fungi isolated from Hill raspberry samples with lesions, 226 were tested for pathogenicity on healthy Hill raspberry leaves, resulting in five possible candidates consistently causing lesions. Further characterization through morphology and DNA analysis confirmed these candidates as Lasiodiplodia theobromae, Colletotrichum gloesporioides, Fusarium concentricum, Phanerochaete chrysosporium, and Penicillium rolfsii. Future research will explore the suitability of these fungal pathogens as biocontrol agents of invasive Hill rapberry.

Keywords: Rubus niveus; Biological control; Fungi; Galapagos; Invasive species.

Fig. 1

(a) Map showing the sites (red dots) on San Cristobal Island where samples of R. niveus with possible disease symptoms were collected (80 sites). Map created using ArcGIS Pro v2.6 (https://www.esri.com/en-us/arcgis/products/arcgis-pro/overview), with OpenStreetMap base layers. (b) R. niveus growing along the roadside of San Cristobal Island covering other plant species.

Fig. 2

Fungal diversity in healthy and diseased leaves of R. niveus. (a) Healthy and diseased leaves of R. niveus. (b) Principal coordinate analysis (PCoA) of fungal microbiome data obtained from healthy and diseased leaves based on Bray–Curtis dissimilarity. Variance is shown in parentheses. (c) Overview of the most abundant fungi (class level) in healthy and diseased leaves. (d) Number of plant pathogens identified by Fungi Functional Guild (FUNGuild). (e) Dot plots showing enriched fungal genera in diseased leaves samples compared to the healthy ones (log2-fold change).

Fig. 3

Disease symptoms found on leaves, stems and fruits of R. niveus plants on San Cristobal Island. On leaves (from left to right): leaf with anthracnose; leaf with blight symptoms; leaf with dark and dry spots, leaf with blight symptoms and chlorosis and leaf with anthracnose, chlorosis and blight symptoms. Stems with anthracnose and fruits colonized with fungus-like mycelium.

Fig. 4

(a) Diversity of fungi isolated from R. niveus growing on PDA plates. (b) Summary of number (N°) of samples collected and fungi isolated from leaves, stems and fruits of R. niveus.

Fig. 5

Symptoms caused by five fungal isolates in pathogenicity tests of Hill raspberry leaves inoculated with 5 µl of a fungal spore suspension consisting of 1 × 10⁶ conidia/ml. The pathogenicity tests were performed in a humid chamber at room temperature (25 °C) and scored for symptoms seven days after inoculation: (a) R. niveus leaf containing a drop of PDB without fungal spores was used as negative control; (b) Isolate 28.1a.1.2 showing necrotic symptoms; (c) Isolate 10.3n showing necrosis and a chlorotic halo; (d) Isolated 4.3b showing necrosis and mycelial growth; (e) Isolate 52h3.2 showing necrosis and a chlorotic halo; (f) Isolate 7.2a.1.2 showing phytotoxicity and mycelial growth; (g) Fungal inoculation model. PDB: Liquid culture medium (potato dextrose broth). Wound: wounding was performed using a sterile dissection needle.

Fig. 6

Phenotypic, microscopic, and molecular identification of five fungi isolated from diseased R. niveus leaf samples. ¹Fungal growth in vitro on PDA medium. Left half: top view; Right; bottom view. ²Microscopy by methylene blue staining and transparent adhesive tape with magnifications of × 40 and × 100. ³Morphology described by taxonomic key according to the isolate: C. gloesporioides, L. theobromae, F. concentricum, P. chrysosporium and P. rolfsii. ⁴For molecular identification, different regions were amplificated for each isolate: C. gloesporioides (GADPH, ITS), L. theobromae (ITS).; F. concentricum (ITS, TEF1a, TUB, RPB2); P. chrysosporium and P. rolfsii (ITS, RPB2) Molecular DNA identification was performed using BLAST.