Abstract
Hemileia vastatrix, causal agent of coffee leaf rust (CLR), is an aggressive pathogen of coffee plants worldwide. Conventional fungicides play a major role in the suppression of this disease, but a recent shift toward eco-friendly farming practices has occurred and additional novel, effective, and sustainable strategies for CLR control are needed. Naturally occurring fungal antagonists could be well-positioned to meet this demand, but these fungi need to be isolated and tested for efficacy to identify organisms with potential. In this study, a survey of fungi associated with CLR lesions in four districts of Hawai‘i Island, HI, USA (Kona, Ka‘ū, Hāmākua, and Hilo) was conducted. Coffee leaves infected with CLR were collected from 22 locations and over 600 lesions were plated on ½ APDA and CTC 4T media. DNA was extracted from purified isolates and the internal transcribed spacer region (ITS) was sequenced and analyzed by BLASTn. In total, 194 isolates comprising 50 taxa were recovered. Several of the genera are known antagonists of CLR or other plant pathogens, including Simplicillium, Akanthomyces, Cladosporium, Fusarium, and Clonostachys. The wide diversity of fungi associated with CLR lesions provide a wealth of possibilities for identifying potential CLR antagonists that could serve as a valuable tool for coffee farmers as part of an integrated pest management plan.
Avoid common mistakes on your manuscript.
Introduction
Coffee leaf rust (CLR) is caused by an obligate parasitic fungus (Hemileia vastatrix) that infects the leaves of coffee (Coffea sp.) and causes considerable economic losses for farmers in all coffee growing regions [1]. In late 2020, coffee leaf rust was detected in Hawai‘i, which was the last coffee-growing region free of CLR in the world [2]. Since then, CLR has been identified on the six major islands where coffee is grown, including Hawai‘i Island, Maui, O‘ahu, Lāna‘i, Moloka‘i, and Kaua‘i [3, 4]. The origin of the outbreak is unknown; however, isolates recovered locally have been identified as Race XXIV [3] and are most genetically similar to genotypes found in Central America and Jamaica [4]. Due to the recent nature of this detection and Hawai‘i being the primary coffee producing state in the United States, there are few fungicides registered for use that farmers can employ to combat the disease and most of them are contact fungicides or prophylactics [5]. Additionally, applying fungicides to target CLR is a significant economic burden for farmers, which has been estimated as high as 11% of profits [6]. Importation of resistant coffee varieties, one of the best methods for controlling CLR as part of an IPM strategy, is restricted by regulations that limit the continental United States as the origin of the shipment followed by a one-year quarantine [4]. These logistical and financial challenges make it difficult for coffee farmers in Hawai‘i to control CLR.
Microbial antagonists of H. vastatrix could serve as a valuable tool in a farmer’s arsenal for controlling CLR [7]. Fungal endophytes are of particular interest when surveying for antagonists [8], which can secrete toxic metabolites capable of inhibiting their host pathogens [9, 10]. Additional antagonistic actions include parasitism, competition, and antibiosis. Historically, studies on mycoparasites of CLR have focused on the “white-halo fungi” originally believed to be from the genus Lecanicillium [11, 12]. This group of fungi has undergone taxonomic revision, which has separated many of these fungi into different genera including Akanthomyces and Simplicillium [13, 14]. This finding has allowed for more comprehensive analyses of these fungi as predators of CLR [15,16,17,18]. Fungi in other genera have also been discovered to be antagonistic to CLR [19,20,21], suggesting that broad surveys for potential antagonists of CLR will be fruitful.
Biological control is often a key component of organic farming systems but can be an equally important part of an integrated pest management strategy used in general coffee cropping systems. Due to the limited repertoire of effective CLR control measures in Hawai‘i, locally isolated antagonistic fungi have the potential to be an organic supplement to current control recommendations. Thus, the aim of this study was to survey coffee farms on Hawai‘i Island, HI, USA, one of the main coffee producing islands in the state, for fungi associated with CLR lesions and urediniospores to identify potential CLR-specific antagonists.
Materials and methods
Sample collection
Collections of leaf samples infected with H. vastatrix spores were conducted at 22 sites in the Kona (14 sites), Ka‘ū (4 sites), Hāmākua (1 site), and Hilo (3 sites) districts of Hawai‘i Island, Hawai‘i, USA from January 2021 to January 2023 (Table 1). All sites were actively managed coffee farms except for sites 19 and 22, which were a feral coffee stand located in a riparian forest comprised of non-native tree species and a homeowner’s backyard coffee plant, respectfully. Leaves were collected by hand and placed between layers of paper towels in a resealable plastic bag. Leaves containing lesions overgrown with mycelium (a potential indication of mycoparasite infection) were prioritized for collection. In the absence of this scenario, any leaves with actively sporulating rust infections were collected. Once collections at a site were completed, the filled plastic bags were placed in a cooler with a cold pack. A sheet of polystyrene foam or cardboard was placed between the plastic bag and the cold pack to keep the leaf samples from directly contacting the cold pack.
Fungal isolation
Isolations were conducted in a biosafety cabinet rated for biosafety level 2 (Labconco, Kansas City, MO, USA) at the USDA ARS Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center (PBARC) in Hilo, HI. Coffee leaf rust pustules with urediniospores were scraped with a sterile scalpel with a #10 blade and streaked on selective agar. From January-September 2021, CTC 4T agar (PDA plus 1 g/l yeast extract supplemented with 500 mg/l chloramphenicol, 4 mg/l thiabendazole, and 250 mg/l cycloheximide) was used for isolation [22]. This agar, which is selective for entomopathogenic fungi, was initially used since many of the well-known parasites of CLR belong to the family Cordycipitaceae [15, 17]. Half-strength potato dextrose agar amended with lactic acid (9.75 g PDA, 3.75 g agar, 500 ml distilled water, 500 µl 85% lactic acid after autoclaved media has cooled) (½ APDA) and CTC 4T agar were used for isolations from December 2021 to January 2023 to broaden the scope of the survey. Cultures were incubated in plastic bins in the laboratory (~ 23 °C) for 4–7 days. The resulting fungal colonies were transferred by single or mass hyphal tips to full-strength PDA and incubated under the same conditions.
DNA extraction, amplification, sequencing, and isolate identification
Fungal isolates were identified via molecular sequence analysis. Cultures that were 7–22 days old were scraped with flocked-tip swabs (FLOQSwabs, Copan Diagnostics Murrieta, CA, USA) placed in 2 ml homogenization tubes filled with 6 mm zirconium beads. Samples were extracted using the NucleoSpin Plant II mini kit (Macherey-Nagel, Allentown, PA) using the manufacturers specifications and PL1 lysis buffer. To identify the isolates, the internal transcribed spacer (ITS) region was amplified via polymerase chain reaction using primer set ITS5/ITS4 [23] and the following cycling parameters: initial denaturation of 2 min at 95 C; followed by 40 cycles of 30 s at 95 C, 30 s at 55 C, and 30 s at 72 C; final extension of 5 min at 72 C. Successful PCR amplicons were determined by gel electrophoresis and visualized on a 1% agarose gel under UV illumination. PCR products were cleaned-up using ExoSAP-IT (Thermofisher) and prepared and submitted for bulk Sanger sequencing (Eurofins Genomics, Louisville, KY). Sequencing data was analyzed in Geneious Prime (Boston, MA).
Fungal identification was conducted via BLAST (http://www.ncbi.nlm.nih.gov/BLAST) using parameters adapted from Vega et al. [24]. The top 100 matches with a sequence identity of 97% or higher were analyzed for each sequence and isolates were identified to genera based on a consensus of the BLAST results. If the search results were mixed, the most specific taxonomic unit shared by the two most abundant matches in the top 100 closest matches was reported as the identity of the isolate. Isolates identified solely via sequence data were not identified down to the species level out of caution. Fungal isolate sequence data was deposited into GenBank (Accession Nos. OQ878295-OQ878344, see Table 2).
Results
Over 600 CLR lesions from more than 200 coffee leaves were sampled from Hawai‘i Island. In total, 194 isolates were recovered (Table 2). These isolates assorted into 50 distinct taxa: 44 to genera, one to family, one to order, one to subclass, and one to division. Fungi were isolated from CLR lesions at all sites. The median number of isolates recovered at a given site was 6.5, and Site 20 had the highest taxa richness (15 taxonomically unique isolates). The two most-isolated genera were Simplicillium (62 isolates) and Cladosporium (51 isolates). All Cladosporium isolates were assigned to the taxon “Cladosporium sp.” because they did not assort into distinct genotypic groups when the sequences were compared. Seventy-eight isolates were recovered on CTC 4T medium and 116 isolates were recovered on ½ APDA. All taxa were recovered on ½ APDA, while CTC medium was preferred by Cordycipitaceae, some yeasts, and some of the Cladosporium isolates.
Discussion
Many of the isolates recovered in this survey are suspected endophytes in coffee leaves and have been isolated previously in Columbia, Mexico, Puerto Rico, and Hawai‘i [24]. Interestingly, Vega et al. [24] recovered an abundance of Colletotrichum species from coffee leaves in Hawai‘i, totaling over 40% of their isolates. In the present study, only four Colletotrichum isolates were recovered (2% of the total). Simplicillium and Cladosporium were the two most isolated genera in this study. The abundance of Simplicillium in the present survey may be due to the selection of CLR infected leaves with the presence of mycoparasites and the use of CTC 4T medium solely in the first eight months of the survey. Cladosporium species were recovered in both surveys, though to a greater degree in the present survey (21 vs. 3 isolates collected by Vega et al. [24]). Additionally, some of the differences in taxa between the studies could be due to the lack of plant surface sterilization in the present study, which would exclude some surface-dwelling fungi that can also assume an endophytic lifestyle [25]. However, the lack of surface sterilization does not exclude fungi that are strictly epiphytic.
Several taxa that were recovered in the present study may potentially help mitigate CLR through various means of antagonism. Species of Simplicillium [16] and Cladosporium [26, 27], which were isolated in this study, are known to express antifungal VOCs. Several genera that were recovered are recognized as parasites of CLR including Simplicillium, Akanthomyces, Pleurodesmospora [28], and Clonostachys [29]. Also of interest are Fusarium spp., which have been shown to reduce urediniospore germination and disease severity on leaf disks, though mechanisms for antagonism are poorly understood [30]. Isolates from these genera will be prioritized for future efficacy studies against CLR.
The use of CLR-antagonistic fungi as an additional tool could be helpful in the hands of farmers, but further research is needed to confirm this hypothesis on coffee [7]. Testing the efficacy of these organisms against CLR via in vitro germination studies, greenhouse tests with coffee seedlings, and, eventually, field trials will be paramount. Developing a pipeline for conducting this type of research would greatly increase the timeliness and efficiency of producing results. Research into compatibility with host tissues at high spore concentrations will be conducted to identify any constraints in the application of certain CLR-antagonistic taxa. Future studies in Hawai‘i will also focus on continued isolation of potential CLR-antagonistic microorganisms from additional coffee-producing regions in the state.
Data Availability
All data supporting the findings of this study are available within the paper.
References
McCook S (2006) Global rust belt: Hemileia vastatrix and the ecological integration of world coffee production since 1850. J Global History 1:177–195. https://doi.org/10.1017/s1740022806000012x
Keith LM, Sugiyama LS, Brill E, Adams BL, Fukada M, Hoffman KM, Ocenar J, Kawabata A, Kong AT, McKemy JM, Olmedo-Velarde A, Melzer MJ (2022) First report of coffee leaf rust caused by Hemileia vastatrix on coffee (Coffea arabica) in Hawai‘i. Plant Dis 106:761. https://doi.org/10.1094/PDIS-05-21-1072-PDN
Keith LM, Matsumoto TK, Sugiyama LS, Fukada M, Nagai C, Pereira AP, Silva MdC, Várzea V (2023) First report of the physiological race XXIV of Hemileia vastatrix (coffee leaf rust) in Hawai‘i. Plant Dis 107:2528. https://doi.org/10.1094/PDIS-03-23-0460-PDN
Ramírez-Camejo LA, Keith LM, Matsumoto T, Sugiyama L, Fukada M, Brann M, Moffitt A, Liu J, Aime MC (2022) Coffee leaf rust (Hemileia Vastatrix) from the recent invasion into Hawaii shares a genotypic relationship with latin American populations. J Fungi 8:198. https://doi.org/10.3390/jof8020189
Kawabata AM, Nakamoto ST (2021) Spraying to suppress coffee leaf rust (Hemileia vastatrix) in Hawai‘i. Plant Diseases, PD-118, College of Tropical Agriculture and Human Resources, University of Hawai‘i at Mānoa, Honolulu, HI, USA
Aristizábal LF, Johnson MA (2022) Monitoring coffee leaf rust (Hemileia vastatrix) on commercial coffee farms in Hawaii: early insights from the first year of disease incursion. Agronomy 12:1134. https://doi.org/10.3390/agronomy12051134
Castillo NET, Acost YA, Parra-Arroyo L, Martínez-Prado MA, Rivas-Galindo VM, Iqbal HMN, Bonaccorso AD, Melchor-Martínez EM, Parra-Saldívar R (2022) Towards an eco-friendly coffee rust control: compilation of natural alternatives from a nutritional and antifungal perspective. Plants 11:2745. https://doi.org/10.3390/plants11202745
De Silva NI, Brooks S, Lumyong S, Hyde KD (2019) Use of endophytes as biocontrol agents. Fungal Biol Rev 33:133–148. https://doi.org/10.1016/j.fbr.2018.10.001
Lu L, Tibpromma S, Karunarathna SC, Jayawardena RS, Lumyong S, Xu J, Hyde KD (2022) Comprehensive review of fungi on coffee. Pathogens 11:411. https://doi.org/10.3390/pathogens11040411
Mousa WK, Raizada MN (2013) The diversity of anti-microbial secondary metabolites produced by fungal endophytes: an interdisciplinary perspective. Front Microbiol 4:65. https://doi.org/10.3389/fmicb.2013.00065
Eskes AB, Mendes MDL, Robbs CF (1991) Laboratory and field studies on parasitism of Hemileia vastatrix with Lecanicillium lecanii and V. leptobactrum Café Cacao Thé 35:275–282.
Shaw DE (1987) Verticillium lecanii a hyperparasite on the coffee rust pathogen in Papua New Guinea. Austral Plant Pathol 17:2-3
Sung GH, Hywel-Jones NL, Sung JM, Luangsa-ard JShrestha B, Spatafora JW (2007) Phylogenetic classification of Cordyceps and the clavicipitaceous fungi. Stud Mycol 57:5–59. https://doi.org/10.3114/sim.2007.57.01
Zare R, Gams W (2001) A revision of Verticillium section Prostrata. IV. The genera Lecanicillium and Simplicillium gen. nov. Nova Hedwigia 73:1–50
García-Nevárez G, Hidalgo-Jaminson E (2019) Efficacy of indigenous and commercial Simplicillium and Lecanicillium strains for controlling Hemileia vastatrix Mex J Phytopathol 37:237–250. https://doi.org/10.18781/r.mex.fit.1810-4
Gomes AM, Pinho DB, Cardeal ZL, Menezes HC, De Queiroz MVD, Pereira OL (2018) Simplicillium coffeanum, a new endophytic species from brazillian coffee plants, emitting antimicrobial volatiles. Phytotaxa 333:188–198. https://doi.org/10.11646/phytotaxa.333.2.2
Gómez-De La Cruz I, Pérez-Portilla E, Escamilla-Prado E, Martínez-Bolaños M, Carrión-Villarnovo GLL, Hernández-Leal TI (2018) Selection in vitro of mycoparasites with potential for biological control on Coffee Leaf Rust (Hemileia vastatrix). Mex J Phytopathol 36:172–183. https://doi.org/10.18781/r.mex.fit.1708-1
Mahfud MC, Mior Ahmad ZA, Meon S, Kadir J (2006) In vitro and in vivo tests for parasitism of Verticillium psalliotae Treschow on Hemileia vastatrix Berk. And Br. Malay J Microbiol 21:46-50
Colmán AA, Evans HC, Salcedo-Sarmiento SS, Braun U, Belachew-Bekele K, Barreto RW (2021) A fungus-eat-fungus world: Digitopodium, with particular reference to mycoparasites of the coffee leaf rust, Hemileia vastatrix IMA Fungus 12:1. https://doi.org/10.1186/s43008-020-00052-w
Rodríguez MCH, Evans HC, de Abreu LM, de Macedo DM, Ndacnou MK, Bekele KB, Barreto RW (2021) New species and records of Trichoderma isolated as mycoparasites and endophytes from cultivated and wild coffee in Africa. Sci Rep 11:1-30. https://doi.org/10.1038/s41598-021-84111-1
Salcedo-Sarmiento S, Aucique-Pérez CE, Silveira PR, Colmán AA, Silva AL, Corrêa Mansur PS, Rodrigues FA, Evans HC, Barreto RW (2021) Elucidating the interactions between the rust Hemileia vastatrix and a calonectria mycoparasite and the coffee plant. iScience 24:102352. https://doi.org/10.1016/j.isci.2021.102352
Fernandes ÉKK, Keyser CA, Rangel DEN, Foster RN, Roberts DW (2010) CTC medium: a novel dodine-free selective medium for isolating entomopathogenic fungi, especially Metarhizium acridum, from soil. Biol Control 54:197–205. https://doi.org/10.1016/j.biocontrol.2010.05.009
White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, Inc, New York, pp 315-322
Vega FE, Simpkins A, Aime MC, Posada F, Peterson SW, Rehner SA, Infante F, Castillo A, Arnold AE (2010) Fungal endophyte diversity in coffee plants from Colombia, Hawai‘i, Mexico, and Puerto Rico. Fungal Ecol 3:122–138. https://doi.org/10.1016/j.funeco.2009.07.002
Busby PE, Ridout M, Newcombe G (2016) Fungal endophytes: modifiers of plant disease. Plant Mol Biol 90:645–655. https://doi.org/10.1007/s11103-015-0412-0
Paul D, Park KS (2013) Identification of volatiles produced by Cladosporium cladosporioides CL-1, a fungal biocontrol agent that promotes plant growth. Sensors 13:13969–13977. https://doi.org/10.3390/s131013969
Salvatore MM, Andolfi A, Nicoletti R (2021) The genus Cladosporium: a rich source of diverse and bioactive natural compounds. Molecules 26:3959. https://doi.org/10.3390/molecules26133959
Bekele KB (2022) Potential biocontrol agents diversity for coffee leaf rust Hemileia vastatrix from Southwestern Ethiopia. Am J Life Sci 10:45–52. https://doi.org/10.11648/j.ajls.20221003.13
Kapeua-Ndacnou M, de Abreu LM, de Macedo DM, da Nóbrega TF, Pereira CM, Evans HC, Barreto RW (2023) Assessing the biocontrol potential of Clonostachys species isolated as endophytes from Coffea species and as mycoparasites of Hemileia rusts of coffee in Africa. J Fungi 9:248. https://doi.org/10.3390/jof9020248
Belachew K, Adugna G, Garedew W (2021) Biocontrol potential of indigenous antagonist fungal species for the coffee leaf rust Hemileia vastatrix in Ethiopia. Res J Plant Pathol 4:4
Acknowledgements
We would like to thank MaryAnn Villalun, Dr. Melissa Johnson, Colby Maeda, Jared Nishimoto, and Karma Kissinger for their support in the lab and field. This research was supported by the U.S. Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) Specialty Crop Research Initiative (SCRI) (2021–07760). We would also like to acknowledge the funding and support from the National Plant Disease Recovery System (NPDRS) of the USDA-Office of National Programs through Dr. Timothy Widmer. Mention of trademark, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U.S. Dept. of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing financial or non-financial interests.
Additional information
Responsible Editor: Admir Giachini
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
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/.
About this article
Cite this article
Luiz, B.C., Sugiyama, L.S., Brill, E. et al. Survey of potential fungal antagonists of Coffee Leaf Rust (Hemileia vastatrix) on Coffea arabica in Hawai‘i, USA. Braz J Microbiol (2024). https://doi.org/10.1007/s42770-024-01304-2
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s42770-024-01304-2