Endophytic Fungi Associated with Coffee Leaves in China Exhibited In Vitro Antagonism against Fungal and Bacterial Pathogens

Abstract

Coffee endophytes have been studied for almost 74 years, and several studies have demonstrated coffee-endophytic fungi with antibacterial and antifungal potential for human and plant pathogens. In this study, we isolated and identified a total of 235 strains of endophytic fungi from coffee leaf tissues collected in four coffee plantations in Pu'er city, Yunnan province, China. Molecular identification was carried out using maximum likelihood phylogenetic analysis of nuclear ribosomal internal transcribed spacer (ITS1-5.8S rDNA-ITS2) sequences, while the colonization rate and the isolation frequency were also calculated. Two pathogenic fungi (Alternaria alternata and Penicillium digitatum) and two pathogenic bacteria (Pseudomonas syringae and Salmonella enterica subsp. enterica) were used for screening the antagonistic activities of 61 strains of coffee-endophytic fungi by a dual-culture test assay while maximum likelihood phylogenetic analysis confirmed their natural classification. This is the first study of coffee-leaf-endophytic fungal diversity in China, and the results revealed that coffee-endophytic fungi from this study belong to the Ascomycota, distributed among two classes, 10 orders, and 17 families. Concurrently, endophytic fungi isolates distributed in Arthrinium, Biscogniauxia, Daldinia, Diaporthe, and Nigrospora showed strong antagonistic activities against the pathogens. For the pathogens Alternaria alternata and Pseudomonas syringae, Nigrospora XCE-7 showed the best inhibitory effects with inhibition rates of 71.76% and 61.11%, respectively. For the pathogen Penicillium digitatum, Daldinia ME-9 showed the best inhibitory effect with a 74.67% inhibition rate, while Biscogniauxia PTE-7 and Daldinia T5E-1-3 showed the best inhibitory effect with a rate of 60.42% against the pathogen Salmonella enterica subsp. enterica. Overall, our study shows the diversity of coffee endophytes in four coffee-growing areas in Pu'er city, Yunnan province, China, and their potential use as biological control agents against two fungal and two bacterial pathogens.

Keywords: Coffea; biocontrol agents; coffee-endophytic fungi; pathogenic bacteria; pathogenic fungi.

Figure 5

Illustration of an antagonist test by the dual-culture technique with two bacterial pathogens and an inhibition rate greater than or equal to 60%. The two pathogens were cocultivated with the coffee fungal endophytes on NA plates and incubated for 10 days at 28 °C. (a) Pseudomonas syringae control plate; (b,c) Coffee endophytes inhibit Pse. syringae; (d) Salmonella enterica subsp. enterica control plate; (e,f) Coffee endophytes inhibit S. enterica subsp. enterica.

Figure 6

Illustration of an antagonist test using the dual-culture technique with two fungal pathogens and an inhibition rate greater than or equal to 60%. The two pathogens were cocultivated with the coffee fungal endophytes on PDA plates and incubated for ten days at 28 °C. (a) Alternaria alternata control plate; (b–k) Coffee endophytes inhibit A. alternata; (l) Penicillium digitatum control plate; (m–y) Coffee endophytes inhibit Pen. digitatum.

Figure 1

(a) Aerial view of a coffee plantation in Pu’er city; (b) Arabica coffee trees; (c) Catimor coffee trees; (d) Yellow Bourbon coffee trees.

Figure 2

Dual-culture assay steps and the percentage inhibition formula.

Figure 3

Taxonomic distribution of the fungal taxa (n = 235) from the coffee.

Figure 4

A maximum likelihood analysis showing the phylogenetic relationships of coffee fungal endophytes. The tree has a total of 167 fungal sequences, of which 61 fungal sequences came from the present study and 106 reference sequences of close relatives came from the GenBank. Mucor circinelloides (NR126116; Mucoraceae; Mucorale; Mucoromycetes) was used as the outgroup taxon. The bootstrap support values (≥50%) are indicated at the nodes. The bold font indicates the type species from the GenBank and data presented in the red font are the isolate code and culture number of the fungal endophytes obtained from the coffee leaves. Fungal isolates are further highlighted in different colors according to the family classification. Images on the right side of the phylogeny tree are the morphotypes of each clade.