Identification of Putative Coffee Rust Mycoparasites via Single-Molecule DNA Sequencing of Infected Pustules

Abstract

The interaction of crop pests with their natural enemies is a fundament to their control. Natural enemies of fungal pathogens of crops are poorly known relative to those of insect pests, despite the diversity of fungal pathogens and their economic importance. Currently, many regions across Latin America are experiencing unprecedented epidemics of coffee rust (Hemileia vastatrix). Identification of natural enemies of coffee rust could aid in developing management strategies or in pinpointing species that could be used for biocontrol. In the present study, we characterized fungal communities associated with coffee rust lesions by single-molecule DNA sequencing of fungal rRNA gene bar codes from leaf discs (Ϸ28 mm 2 ) containing rust lesions and control discs with no rust lesions. The leaf disc communities were hyperdiverse in terms of fungi, with up to 69 operational taxonomic units (putative species) per control disc, and the diversity was only slightly reduced in rust-infected discs, with up to 63 putative species. However, geography had a greater influence on the fungal community than whether the disc was infected by coffee rust. Through comparisons between control and rust-infected leaf discs, as well as taxonomic criteria, we identified 15 putative mycoparasitic fungi. These fungi are concentrated in the fungal family Cordycipitaceae and the order Tremellales. These data emphasize the complexity of diverse fungi of unknown ecological function within a leaf that might influence plant disease epidemics or lead to the development of species for biocontrol of fungal disease. Fungal biodiversity is closely linked to plant biodiversity, because a dominant number of fungal taxa are symbiotic with plants (1, 2). On one end of the symbiosis spectrum are plant pathogens, whose impacts are enormous in agricultural production, dramatically reducing yields or even eliminating production altogether, as well as in natural ecosystems, where they may facilitate plant biodiversity by Janzen-Connell effects, coevolutionary processes, or other mechanisms. At the other end of the spectrum are mutualists of plants, including beneficial mycorrhizae and endophytes that promote plant growth and facilitate stress tolerance (3, 4). Somewhere in the middle are the majority of plant-symbiotic fungi, with copious species detected within and on asymptomatic plant tissues. Most of these fungi have unclear roles with respect to plant health but comprise a massive and ubiquitous proportion of overall fungal biodiversity, especially in the lowland tropics, where every leaf sampled reveals the presence of endophytes by use of culture techniques (5, 6). Although some endophytes may have no direct effect on the host, what is becoming increasingly more appreciated is that endophytes can benefit the host through indirect actions, such as by protection from plant pests and pathogens (7,8).In natural ecosystems, cascading trophic interactions are tied to ecosystem stability and diversity (9). These trophic interactions involve numerous natural enemie...

PacBio metabarcoding of Fungi and other eukaryotes: errors, biases and perspectives

“…Only James et al . () and Schlaeppi et al . () utilized the potential of PacBio sequencing to account for the full internal transcribed spacer (ITS) barcode or ITS and rRNA large subunit (LSU) gene in the same amplicon, respectively.…”

“…Furthermore, long targets can be constructed to cover both the variable barcode and its flanking conserved regions, which may offer additional value for phylogenetic placement and allow the use of evolutionary ecological approaches in data analysis (for Bacteria: Bleidorn, 2016;Singer et al, 2016). Recently, PacBio has been used successfully in metabarcoding analysis of fungi (Cline & Zak, 2015;James et al, 2016;Kyaschenko et al, 2017), although the switch to this platform was neither emphasized nor validated in these studies. Only James et al (2016) and Schlaeppi et al (2016) utilized the potential of PacBio sequencing to account for the full internal transcribed spacer (ITS) barcode or ITS and rRNA large subunit (LSU) gene in the same amplicon, respectively.…”

“…Recently, PacBio has been used successfully in metabarcoding analysis of fungi (Cline & Zak, 2015;James et al, 2016;Kyaschenko et al, 2017), although the switch to this platform was neither emphasized nor validated in these studies. Only James et al (2016) and Schlaeppi et al (2016) utilized the potential of PacBio sequencing to account for the full internal transcribed spacer (ITS) barcode or ITS and rRNA large subunit (LSU) gene in the same amplicon, respectively.…”

Selección in vitro de micoparásitos con potencial de control biológico sobre Roya del café (Hemileia vastatrix)

“…Mahfund et al (2006) El género Simplicillium se reporta como parte de los hongos asociados a roya del café en Puerto Rico y México (James et al, 2016). Las especies de este género han sido registradas principalmente en asociación con H. vastatrix (Zare y Gams, 2001), mientras que su potencial como agente de control biológico de otras enfermedades ha sido poco estudiado.…”

“…The genus Simplicillium is reported as part of the fungi related to Coffee Leaf Rust in Puerto Rico and Mexico (James et al, 2016). The species of this genus have been registered mainly in relation with H. vastatrix (Zare and Gams, 2001), while its potential as a biological control agent of other diseases has had little studies.…”