Colmán et al. IMA Fungus (2021) 12:1 https://doi.org/10.1186/s43008-020-00052-w IMA Fungus RESEARCH Open Access A fungus-eat-fungus world: Digitopodium, with particular reference to mycoparasites of the coffee leaf rust, Hemileia vastatrix Adans A. Colmán1, Harry C. Evans1,2, Sara S. Salcedo-Sarmiento1, Uwe Braun3, Kifle Belachew-Bekele4 and Robert W. Barreto1* Abstract Digitopodium hemileiae was described originally in 1930 as Cladosporium hemileiae; growing as a mycoparasite of the coffee leaf rust (CLR), Hemileia vastatrix, in a sample of diseased leaves of Coffea canephora collected in the Democratic Republic of Congo. No cultures from this material exist. More recently, the type material was re- examined and, based on morphological features, considered to be incorrectly placed in Cladosporium. The new genus Digitopodium was erected to accommodate this species. Interest in fungal antagonists of H. vastarix,as potential biocontrol agents of CLR, led to comprehensive surveys for mycoparasites, both in the African centre of origin of the rust, as well as in its South American exotic range. Among the rust specimens from Ethiopia, one was found to be colonized by a fungus congeneric with, and similar to, D. hemileiae. Pure cultures obtained from the Ethiopian material enabled a molecular study and for its phylogenetic position to be elucidated, based on DNA sequence data from the ITS and LSU regions. Molecular data showed that two members of the recently erected genus Hyalocladosporiella (Herpotrichiellaceae: Chaetothyriales) are congeneric with Digitopodium from Ethiopia and morphologically similar to both D. hemileiae and the two Ethiopian isolates. These isolates were found to be morphologically and genetically identical to H. tectonae, described previously from Brazil. Thus, species of Hyalocladosporiella are re-allocated to Digitopodium here; including D. tectonae, and a novel species, D. canescens, recently found in Brazil growing as a mycoparasite of Puccinia thaliae. The potential use of D. hemileiae and D. tectonae for classical biological control of CLR is discussed. Keywords: Classical biological control, Ethiopia, Fungicolous fungi, Herpotrichiellaceae, Hyalocladosporiella, New taxa`, Phylogenetics INTRODUCTION growers in Central America (Avelino et al. 2015, Talhin- Hemileia vastatrix is the most important pathogen of has et al. 2017) and have prompted mass migrations – coffee plants worldwide, causing coffee leaf rust (CLR) refugee caravans – to Mexico and the USA (Ward et al. (Zambolim 2016, Talhinhas et al., 2017). The economic 2017). and social crisis provoked by CLR outbreaks of the past Efforts in mitigating the impact of CLR have included are well documented (Avelino et al. 2015, McCook & a pioneering initiative towards the development of a Vandermeer 2015). Since 2012, disastrous outbreaks of classical biological control management strategy, based CLR have been destroying the livelihoods of the coffee on the use of fungal natural enemies from the native range of coffee and Hemileia vastatrix in Africa. A num- ber of mycoparasitic fungi of CLR have been reported * Correspondence: [email protected] 1Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, MG previously (Carrion & Rico-Gray 2002, James et al. 36570-900, Brazil 2016). However, the latter records are all from the Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Colmán et al. IMA Fungus (2021) 12:1 Page 2 of 11 Americas, where coffee and H. vastatrix are exotic spe- obtained directly from rust pustules were mounted in cies. Such mycoparasites are interpreted, therefore, as lactoglycerol or lactofuchsin and the microscope slides generalists that have jumped from other fungal hosts were examined under a light microscope, Olympus BX and did not co-evolve as specialized parasites of the CLR 53 (Olympus, Melville, NY, USA), connected to an fungus. Only two mycoparasites have been reported ex- Olympus Q-color 5 camera (Olympus, Center Valley, clusively from the centre of origin of cultivated Coffea in PA, USA). Conidial morphology was based on shape, Africa, namely: Digitopodium hemileiae (Steyaert 1930, colour, and presence or absence of septation. Biometric Heuchert et al. 2005) and Paranectriella hemileiae (Piro- data were generated from the observation of at least 30 zynski 1977). In order for any classical biocontrol agent structures. to be introduced against its target in an exotic situation, DNA was extracted from single spore isolates cultivated it is critical to have its taxonomy fully elucidated (Scott on potato dextrose liquid medium at 25 °C for 5 d. Total 1995). This publication deals with a reappraisal of the genomic DNA was extracted from approximately 50–80 taxonomy of D. hemileiae and related taxa, based on mg of mycelium. Mycelial masses were disrupted with a newly-collected specimens obtained during surveys for L-Beader 3 (Loccus Biotecnologia, Cotia, SP, Brazil) ad- mycoparasites of H. vastatrix in Africa and of related justed to a speed of 4000 rpm, 2 cycles of 10 s each. DNA material collected in Brazil. extraction was carried using the Wizard Genomic DNA Purification Kit (Promega, Madison,WI, USA), according MATERIAL AND METHODS to the manufacturer’s recommendations. Surveys involved scientists from Ethiopia, Brazil, and the DNA PCR amplifications were performed with the pri- UK and were concentrated in areas where Coffea arab- mer pairs LR0R/LR5 (Vilgalys & Hester 1990) and ITS4/ ica still occurs in the wild or is cultivated in semi-wild ITS5 (White et al. 1990) for the partial 28S rDNA (LSU) conditions, as in Ethiopia. At each selected site, coffee and ITS/5.8 nr-DNA (ITS) regions. The polymerase plants were examined for rust pustules – with particular chain reactions (PCR) were performed using a total vol- attention to collecting rust colonies overgrown by other ume of 12 μL in reactions with mixture containing 30 μg fungi, or appearing to be abnormal (unusual colour, DNA, 0.5 μm of each primer and 1X Master mix Dream- poor sporulation). Specimens were dried in a plant press Taq DNA polymerase, as recommended by the manufac- for later processing in the laboratory (preliminary identi- turer (Thermo Fisher Scientific Baltics, Vilnius, fication and isolation). The dried samples were proc- Lithuania). The amplification was performed for LSU essed within 2 weeks of collection after transport to with an initial denaturing at 94 °C at 5 min, followed by laboratories in the UK or Brazil. Mono-conidial cultures 40 cycles of denaturation at 94 °C for 30 s, annealing at were obtained by direct isolation of the fungi by aseptic 55 °C for 30 s, extension initial at 72 °C for 30 s, and 7 transfer of fungal propagules from colonized tissue with min final extension at 72 °C. The PCR products were a sterile fine point needle onto potato dextrose-agar purified by using an ExoSAP-IT purification kit (Amer- (PDA) plates. Pure cultures were preserved temporarily sham Biosciences, Arlington Heights, IL, USA), accord- in potato carrot-agar (PCA) slants and long-term preser- ing to the manufacturer’s recommendations. Amplified vation was in silica-gel and in 10% glycerol at -80 °C, as fragments were sequenced by Macrogen (Seoul, South described in Dhingra & Sinclair (1995). Pure cultures Korea, http://www.macrogen.com). were deposited in the culture collection of the Universi- dade Federal de Viçosa (COAD) and dried specimens Phylogenetic analyses were deposited in the herbarium of the Universidade The nucleotide sequences obtained from forward and re- Federal de Viçosa (VIC). verse primers were used to obtain consensus sequences Culture characteristics were described based on col- using SeqAssem (SequentiX—Digital DNA Processing, onies formed on 2% malt extract-agar (MEA), PDA, and Klein Raden, Germany) (Hepperle, 2004). Complemen- oatmeal-agar (OA) for 7 d at 25 ± 2 °C under a 12 h light tary sequences used in the analyses were obtained from regime (light provided by two white and one near-UV GenBank (http:// www.ncbi.nlm.nih.gov) (Table 1). The lamps placed 35 cm above the plates). Colony colour ter- alignment performed using MUSCLE implemented in minology followed Rayner (1970). the MEGA 7 (Kumar et al. 2016). The aligned sequences Morphology was described based on the structures were manually corrected where needed. The consensus formed on colonized rust pustules on dried specimens, sequences were deposited in GenBank (Table 1) and complemented with observations made on slide cultures, taxonomic novelties in MycoBank (Crous et al. 2004). as described in Waller et al. (1998); colonies being Phylogenetic analyses were reconstructed by means of formed on blocks of synthetic nutrient poor-agar (SNA) methods based on an analysis of Bayesian Inference (BI) (Nirenberg, 1981) for 14 d, under the conditions men- of the combined LSU/ITS alignments using the Markov tioned above. Slide cultures and fungal structures chain Monte Carlo (MCMC) algorithm. Models of Colmán et al. IMA Fungus (2021) 12:1 Page 3 of 11 Table 1 Isolates included in the phylogenetic analyses. GenBank numbers in boldface indicate new sequences Species Isolates Substrate Genbank number LSU ITS Cladosporium adianticola CBS 582.92 Adiantum tenerum DQ008143 – Cladosporium adianticola CBS 735.87 Adiantum sp.