Efficient control of the fungal pathogens Colletotrichum gloeosporioides and Penicillium digitatum infecting citrus fruits by native soilborne Bacillus velezensis strains

Abstract

Destruction of citrus fruits by fungal pathogens during preharvest and postharvest stages can result in severe losses for the citrus industry. Antagonistic microorganisms used as biological agents to control citrus pathogens are considered alternatives to synthetic fungicides. In this study, we aimed to identify fungal pathogens causing dominant diseases on citrus fruits in a specialized citrus cultivation region of Vietnam and inspect soilborne Bacillus isolates with antifungal activity against these pathogens. Two fungal pathogens were characterized as Colletotrichum gloeosporioides and Penicillium digitatum based on morphological characteristics and ribosomal DNA internal transcribed spacer sequence analyses. Reinfection assays of orange fruits confirmed that C. gloeosporioides causes stem-end rot, and P. digitatum triggers green mold disease. By the heterologous expression of the green fluorescent protein (GFP) in C. gloeosporioides using Agrobacterium tumefaciens-mediated transformation, we could observe the fungal infection process of the citrus fruit stem-end rot caused by C. gloeosporioides for the first time. Furthermore, we isolated and selected two soilborne Bacillus strains with strong antagonistic activity for preventing the decay of citrus fruits by these pathogens. Molecular analyses of 16 S rRNA and gyrB genes showed that both isolates belong to B. velezensis. Antifungal activity assays indicated that bacterial culture suspensions could strongly inhibit C. gloeosporioides and P. digitatum, and shield orange fruits from the invasion of the pathogens. Our work provides a highly effective Bacillus-based preservative solution for combating the fungal pathogens C. gloeosporioides and P. digitatum to protect citrus fruits at the postharvest stages.

Keywords: Antifungal activity; Bacillus velezensis; Citrus fruits; Colletotrichum gloeosporioides; GFP tagging; Host colonization; Penicillium digitatum; Postharvest pathogens.

Fig. 1

Identification of the stem-end rot pathogen in citrus fruits. (A) Two fungal strains (HGC201, HGC286) resembling Colletotrichum fungi were isolated from stem-end rot orange fruits. Fungal strains were cultivated on PDA at 25 °C for 5 days for morphological analyses. (B) Molecular identification of strains HGC201 and HGC286 by ITS rDNA sequencing. The ITS sequences were compared to GenBank using the BLAST tool. The phylogenetic tree was constructed with MEGA X using the neighbor-joining method and 1000 bootstrap replications. Statistical support values at branch nodes of the tree, genetic distance scale, and accession numbers for the ITS sequences from other fungi are indicated. The ITS sequences for two C. gloeosporioides strains (HGC201 and HGC286) are deposited in GenBank with accession numbers OL739236−OL739237. (C) Reinfection of the isolated strains on citrus fruits to confirm their pathogenicity. Fungal spore suspensions (10⁶ spores/mL) were employed for the fruit infection assays. Negative control fruits (mock) were inoculated with SDW. The fruits were incubated in sterile plastic boxes for 10 days. Scale bars indicate the sizes of the images. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

GFP tagging of the fungal pathogens by ATMT for inspecting citrus fruit colonization. (A) The transformants generated from C. gloeosporioides HGC201 were confirmed for the presence of the GFP gene in their genomes by PCR and examined for the GFP expression under fluorescence microscopy. (B) Orange fruits were infected with spores of the transformants of C. gloeosporioides for 5−10 days at 25 °C. Thin slices of the infected fruits were prepared to detect the host colonization by the fungus via the GFP signal. Scale bars indicate the sizes of the images. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Antifungal activity of the Bacillus isolates. The dual-culture and agar well diffusion methods were used to inspect the antifungal activity of Bacillus isolates against two stem-end rot C. gloeosporioides strains (A) and three green mold P. digitatum strains (B). For the dual-culture method, three Bacillus strains (B14, B15, B16) were cultivated on the PDA plates in the presence of the fungal pathogens. Control plates were inoculated with only fungal strains. For the agar well diffusion method, the Bacillus strains were grown in conical flasks containing the LB medium at 30 °C for 1 day. Culture supernatants collected by centrifugation were added to agar wells on the PDA plates fully covered with fungal spores. For control plates, SDW was used instead of the culture supernatants. The plates were incubated at 25−28 °C for 5 days. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

In vivo assays of the antagonistic B. velezensis strains in protecting citrus fruits. Oranges were soaked in bacterial cultures for 30 min and air-dried at room temperature. The fruits were wounded and infected with fungal spores from (A) C. gloeosporioides HGC201 or (B) P. digitatum HGP01. The infected oranges were incubated in sterile boxes at 25 °C for 3−10 days. (C) The antifungal effects of bacterial cultures on citrus fruit infection were evaluated using the GFP-tagged strain of C. gloeosporioides HGC201 or C. gloeosporioides HGC201. The fungal strains were re-isolated from the infected fruit tissues using PDA supplemented with chloramphenicol (100 μg/mL) to eliminate bacterial cells and facilitate fungal growth. The plates were incubated at 25 °C for 5 days.