Keywords
Agaricales, cocoa, Marasmiaceae, tropical phytopathogens
This article is included in the From genes to genomes: Investigating the population species boundary in non-model Fungi collection.
The thread blight disease (TBD) of cacao (Theobroma cacao) in the department of Amazonas, Peru was recently reported to be caused by Marasmius tenuissimus (sect. Neosessiles). This same species is known to be the main causal agent of TBD in West Africa. However, some morphological characteristics, such as the presence of rhizomorphs, the almost exclusively white color, and pileus sizes less than 5 mm, among others, differ to the description of M. tenuissimus. Therefore, we aimed to conduct a taxonomic revision of the cacao-TBD causal agent in Peru, by using thorough micro and macro morphological, phylogenetic, and nuclear and mitochondrial genomic approaches. We showed that the causal agent of TBD of cacao in Amazonas, Peru, belongs to a new species, Marasmius infestans sp. nov. This study enriches our knowledge of species in the sect. Neosessiles, and strongly suggests that the M. tenuissimus species complex is highly diverse.
Agaricales, cocoa, Marasmiaceae, tropical phytopathogens
This new version of the manuscript differs from the original version in several aspects. First, it contains a map where specimens where collected. The description of the new species is much more detailed, including examination of micro structures with Melzer´s reagent, and important statistical measurements of basidiospores, such as quotients (length by width), arithmetic mean and standard deviation of dimensions, which were not reported in the previous version. Also, new phylogenetic analyses have been included with ITS, LSU, and ITS-LSU concatenated datasets. Additionally, since this study is a taxonomic study, we have included a paragraph about the current classification of the genus Marasmius in the introduction. In the discussion, we added a comparison of the main differences of the new species with phenetically similar taxa, which was missing in the original version.
See the authors' detailed response to the review by Nopparat Wannathes
See the authors' detailed response to the review by Atik Retnowati
Marasmius (Marasmiaceae, Agaricales, Agaricomycetes, Basidiomycota) is a hyperdiverse genus of fungi with 1,560 legitimate species names registered in MycoBank, as of Jan. 14th, 2024.1 It has been traditionally classified into twelve sections, based on morphological characteristics.2 Thus far, molecular re-examination proved that sections Globulares, Marasmius, Sicci, Leveilleani and Neosessiles belong to Marasmius sensu stricto.3,4 Other sections from the traditional classification, such as Androsacei, Alliacei, Epiphylli, and Hygrometrici, have been accommodated outside Marasmius s.s., while sections Fusicystides, Inaequales, and Scotophysini have not been yet molecularly studied.5–8
The great majority of species of Marasmius are decomposers of leaves and twigs in natural ecosystems, but some species can be pathogenic in agricultural settings, such as the cacao (Theobroma cacao) agroecosystem.9–11 Cacao can get infected by several species of Marasmius, including M. crinis-equi (sect. Marasmius) and some species within the section Neosessiles, causing thread-blight disease (TBD).12 The sect. Neosessiles is a paraphyletic group mainly characterized by the pleurotoid habit of growth and the absence or rudimentarity of the stipe.2,13 Among the TBD-causing Neosessiles species, M. tenuissimus seems to be the most frequent in West Africa,11 and in native Awajun and Wampis communities from Northern Peru.14 Marasmius tenuissimus is characterized by pilei between 7 and 22 mm in diameter, with rusty brown, light grayish fusco or greyish orange color; and basidiospore dimensions of about 9-10 × 4-6 μm.3,9 Even though, nuclear rDNA sequence similarities over 99% point to M. tenuissimus reference strains,3 the morphological characteristics of specimens from West Africa and Northern Peru do not quite match the original description.10,11 Pileus sizes less than 5 mm, the white color, and the presence of rhizomorphs, as in the West African and Peruvian specimens, were never reported before for M. tenuissimus,10,11 suggesting they may not be of that species. Moreover, the mitochondrial genome of six strains and the genome of one strain of M. tenuissimus from West Africa have been recently published,12,15 which opens up the door to perform mitochondrial and nuclear genomic comparisons between the Neosessiles TBD agents from both continents. Therefore, in this study, we aimed to conduct a taxonomic and phylogenetic revision of the status of the cacao TBD-causal agent in Northern Peru, including nuclear and mitogenomic evidence.
Specimens were obtained during an expedition into the Imaza District (4°47′09.4″S 78°16′51.6″W) in the department of Amazonas, Peru (Figure 1), during August–September 2022 (Table 1). Collections were performed under the authorization N° AUT-IFL-2021-052 granted by the Peruvian National Forestry and Wildlife Service Agency - SERFOR. Morphological studies were conducted in the Plant Health Laboratory of the National University Toribio Rodriguez de Mendoza de Amazonas, Peru (UNTRM-A). The macroscopic characterization was made from fresh pilei. We described the color, shape, and size using a stereomicroscope SMZ18 (NIKON, Tokyo, Japan). The microscopic description was made from fresh and dried material. First, a tiny piece of dried material was carefully sectioned and placed onto a microscope slide. We applied 95% ethanol to the sample for 30 seconds, and then a drop of distilled water was added. The slides were dried out and 5% KOH was applied. Additionally, ethanol-washed sections were mounted on Melzer’s reagent as in.3 We placed a cover slip onto the sample and observed it under an OLYMPUS DP74 (Tokyo, Japan) microscope. Dimensions of microstructures were reported with the ranges between the 5th and 95th percentile, with extreme values in parentheses, as in.16 Additionally, for basidiospores, the arithmetic mean ± standard deviation (xm) and the quotient of the length by the width, expressed as range (Q) and arithmetic mean (Qm), were calculated. The dimensions of basidia and basidiospores were measured with at least thirty individual structures, i.e., n ≥ 30. Finally, pure cultures were obtained from rhizomorph tissues by the hyphal tip technique. The holotype specimen (TAIM04) was deposited in the KUELAP herbarium of UNTRM-A under voucher number KUELAP-2940.
Section | Species | Specimen ID | Origin | Accession number | ||||
---|---|---|---|---|---|---|---|---|
ITS | LSU | RPB1 | EF1α | COX1 | ||||
Neosessiles | Marasmius infestans | TAIM 04* | Peru | OR359411 | OR364495 | OR420729 | OR420730 | − |
Marasmius infestans | AFHP-31 | Peru | OM720123 | OM720135 | KAK1231915§ | KAK1236186§ | OQ343345¥ | |
Marasmius infestans | AFHP-101 | Peru | OR359410 | OR364494 | – | – | − | |
Marasmius neosessiliformis nom. prov. | Buyck 97.615 | Madagascar | KX149007 | − | − | − | − | |
Marasmius tenuissimus | AKD 304/2015 | India | MF189066 | − | − | − | − | |
Marasmius tenuissimus | NW199 | Thailand | EU935569 | − | − | − | − | |
Marasmius tenuissimus | NW192 | Thailand | EU935568 | − | − | − | − | |
Marasmius tenuissimus | SCAU111 | China | MF061773 | − | − | − | − | |
Marasmius sp. 1 | RAK 339 | Cameroon | MN930548 | − | − | − | − | |
Marasmius sp. 1 | GHA07 | Ghana | MN794171 | − | − | − | UEX92801 | |
Marasmius sp. 1 | GHA74 | Ghana | MN794173 | − | − | − | UEX92859 | |
Marasmius sp. 1 | MS2 | Ghana | MN794183 | MN794074 | − | − | UEX92814 | |
Marasmius sp. 1 | GHA37 | Ghana | MN794147 | − | KAJ8088874 | KAJ8084062 | UEX92895 | |
Marasmius sp. 2 | GHA64 | Ghana | MN794166 | − | − | − | UEX92879 | |
Marasmius sp. 3 | GHA63 | Ghana | MN794165 | MN794071 | − | − | UEX92917 | |
Marasmius sp. 4 | GHA79 | Ghana | MN794177 | MN794073 | − | − | UEX92836 | |
Marasmius sp. 5 | C2/33 | Brazil | KM246277 | KM246082 | − | − | − | |
Marasmius sp. 5 | C2/06 | Brazil | KM246261 | KM246066 | − | − | − | |
Marasmius griseoroseus var. diminutus | JO390* | Brazil | JX424044 | KF742003 | − | − | − | |
Marasmius conchiformis var. lenipileatus | JO287* | Brazil | JX424042 | KF742001 | − | − | − | |
Marasmius conchiformis var. dispar | JO290* | Brazil | JX424039 | KF742002 | − | − | − | |
Marasmius conchiformis | JO117* | Brazil | JX424038 | KF741998 | − | − | − | |
Marasmius griseoroseus | JO465 | Brazil | KJ173479 | KJ173480 | − | − | − | |
Marasmius conchiformis | JO45 | Brazil | KF741996 | KF741997 | − | − | − | |
Sicci | Marasmius nodulocystis | DED 8278 | Madagascar | KX953741 | − | − | − | − |
Marasmius nodulocystis | DED 8269 | Madagascar | KX953740 | − | − | − | − | |
Marasmius nodulocystis | DED 8283 | Madagascar | KX953742 | − | − | − | − | |
Marasmius linderioides | JO286* | Brazil | JX424037 | KF742000 | − | − | − | |
Marasmius longisetosus | JO248* | Brazil | JX424040 | KF741999 | − | − | − | |
Marasmius siccus | LE 295985 | Russia | KF774132 | − | − | − | − | |
Marasmius siccus | LE 295984 | Russia | KF774131 | − | − | − | − | |
Marasmius siccus | VA 08.69 | Korea | FJ904992 | − | − | − | − | |
Marasmius siccus | KG 028 | Korea | FJ904985 | FJ904980 | − | − | − | |
Marasmius | Marasmius aff. curreyi | JES 135 | Madagascar | KX149008 | − | − | − | − |
Marasmius curreyi | Buyck 97.374 | Madagascar | KX148980 | − | − | − | − | |
Marasmius ruforotula | TYS438 | Malaysia | FJ431271 | − | − | − | − | |
Marasmius ruforotula | TYS369 | Malaysia | FJ431269 | − | − | − | − | |
Marasmius nigrobrunneus | TYS281 | Thailand | EU935575 | − | − | − | − | |
Marasmius nigrobrunneus | NW223 | Thailand | EU935572 | − | − | − | − | |
Marasmius nigrobrunneus | NW162 | Thailand | EU935570 | − | − | − | − | |
Marasmius gracilichorda | TYS411 | Malaysia | FJ431244 | − | − | − | − | |
Marasmius gracilichorda | TYS396 | Malaysia | FJ431242 | − | − | − | − | |
Marasmius berambutanus | TYS398 | Malaysia | FJ431227 | − | − | − | − | |
Marasmius berambutanus | TYS337 | Malaysia | FJ431225 | − | − | − | − | |
Marasmius graminum | NN005953 | Denmark | JN943595 | JN941141 | − | − | − | |
Marasmius graminum | FO 46723 | Germany | − | AF291345 | − | − | − | |
Marasmius crinis-equi | GHA76 | Ghana | MN794174 | MN794072 | − | − | UEX92959 | |
Leveilleani | Marasmius leveilleanus | NW268 | Thailand | EU935567 | − | − | − | − |
Marasmius leveilleanus | NW248 | Thailand | EU935566 | − | − | − | − | |
Marasmius aff. leveilleanus | RAK 392 | Cameroon | MN930527 | − | − | − | − |
DNA extractions were performed from pure cultures using the Wizard® genomic DNA purification kit (Catalogue number A1120; Promega, Wisconsin, USA). DNA was quantified with the BioSpectrometer® Basic (Eppendorf, New Jersey, USA), and diluted to 0.5 ng/μl for PCR reactions. The internal transcribed spacer region (ITS1-5.8S-ITS2, or simply ITS), the large subunit (LSU) rDNA gene, and partial fragments of the genes Elongation Factor 1-α (EF1α) and the largest subunit of RNA polymerase II gene (RPB1) were amplified following.17 The amplified PCR products were Sanger sequenced at MACROGEN (Seoul, South Korea). LSU, EF1α, and RPB1 sequences from the West African TBD-causing specimen GHA37 were retrieved from the recently published genome.15 In addition, we included the mitochondrial cytochrome oxidase I gene (COX1) from isolate INDES-AFHP31, which was retrieved from the mitogenome generated in this study, and from other phylogenetic-related species.12 The introns of COX1 sequences were removed as in.18 Most other sequences used in phylogenetic analyses were obtained from other relevant literature.3,4,11,13,14,19–25 We also included unpublished sequences available in NCBI from M. tenuissimus-phylogenetically related isolates C2/06 and C2/33 (accession numbers KM246261 and KM246277 for ITS, and KM246066 and KM246082 for LSU, respectively).
Generated sequences were edited and assembled with Sequencher v.5.4 (Gene Codes, USA). Once all sequences were gathered, they were aligned with MUSCLE26 implemented in MEGA-X,27 and concatenated with SeaView 4.7.28 We used jModelTest v229 to identify the most appropriate nucleotide evolution models under the Akaike information criterion. Phylogenetic analysis was performed with IQ-TREE v230 which implements the Maximum Likelihood algorithm,31 in the CIPRES Science Gateway v3.3 portal.32 The phylogenetic trees were visualized and edited with FigTree.33
DNA extraction, sequencing, and assembly
Total DNA extraction was performed using the Wizard Purification Kit (Promega Corp., Madison, Wisconsin) following the manufacturer’s instructions. For nuclear and mitogenome sequencing, we used the strain INDES-AFHP31 from Northern Peru, previously reported.14 A paired-end sequencing library was constructed using the TruSeq Nano DNA Kit, according to the manufacturer’s instructions. The library was sequenced on an Illumina NovaSeq 6000 platform in paired-end, 2 × 150 format.
The raw reads were checked by FastQC v.0.11.9. Also, quality trimming (Phred Q > 25) and remotion of adapters were conducted with Trimmomatic v0.3634 and TrimGalore software,35 respectively. Jellyfish v.2.36 was used for k-mer counting, and Genome Scope v1.0.037 for assessing genome size, repeat content, and heterozygosity rate. This analysis involved utilizing the output of Jellyfish and the count of 17-mers for k-mer analysis. Additionally, k-depth estimation was performed to identify a predominant single-peak pattern in the frequency distribution analysis of k-mers.
De novo assembly was performed with two assembly algorithms: SOAPdenovo2 v.2.04,38 and Masurca v.4.0.6.39 Next, we used QUAST v.5.2.040 to evaluate the statistics of assemblies. The assembly validation process employed two distinct methods. First, the filtered paired-end Illumina reads were realigned to identify any errors in the assembly. This was accomplished using Bowtie2 v.2.4.241 and SamTools v.1.742 software. Second, the completeness of the assembly was evaluated using the Agaricales-specific profile of the BUSCO strategy.43 To identify vector contamination, we employed VecScreen JCVI (https://github.com/tanghaibao/jcvi), which utilizes the Univecdatabase (https://ftp.ncbi.nlm.nih.gov/pub/UniVec/). We also performed a BLAST v.2.2.2644 analysis, mapping the scaffolds against the nt/nr NCBI database (found at https://www.ncbi.nlm.nih.gov/). Following this, any contaminated scaffolds and vectors were eliminated before submitting the remaining data to the NCBI database. This assembly has been deposited at DDBJ/ENA/GenBank under the accession: JANHQD01.
Nuclear genome annotation
Genome annotation was performed using Funannotate v1.8.12.45 Repetitive elements were soft-masked by TANTAN.46 Ab initio gene prediction was carried out using AUGUSTUS v.3.3.3,47 GlimmerHMM v.3.0.4,48 and SNAP49 trained with alignments of the BUSCO agaricales_odb10 dataset,46 and with GeneMark-ES v.4.6950 self-trained on the repeat-masked genome sequence. Protein evidence from the UniProt/SwissProt database51 was mapped to the genome using DIAMOND v.2.0.1552 and Exonerate v.2.4.0.53 Finally, EVidenceModeler v.1.1.154 was used to generate consensus gene models based on all the above ab initio and evidence-based gene models. The tRNA genes were identified with tRNAscan-SE v.2.0.9.55 Functional annotations were assigned by similarity to UniProtKB (2021_02),51 InterPro v89.0,56 Pfam v.34.0,57 EggNOG v.5.0,58 BUSCO (agaricales_odb10 dataset),43 dbCAN v.10.0,59 and MEROPS v.12.0.60 Phobius61 and SignalP v.5b62 were used to predict transmembrane topology and signal peptides, respectively. DeepLoc 2.063 was used to determine protein localization. Effectors were predicted by EffectorP 3.064 based on the subset of extracellular proteins. AntiSMASH v.6.065 was used to predict secondary metabolite gene clusters (SMGCs) and secondary metabolite Clusters of Orthologous Groups (smCOGs).
Mitochondrial genome annotation
The mitochondrial genome was confirmed using the default Geneious Prime 2023.1 setup (Biomatters Ltd., Auckland, New Zealand) and GetOrganelle v1.7.6.1.66 Genes were annotated with MITOS,67 MFannot68 and manually confirmed with ORFfinder available in NCBI, and tRNAscan-SE 1.21,69 finally adjusted in Geneious. A physical map of the mitogenome was created with OGDRAW v 1.2.70 Our results were compared to the ones reported for another published M. aff. tenuissimus mitogenomes.12
We collected new cacao tissues infected by TBD-bearing fruiting bodies of the pathogen in Imaza province, department of Amazonas, in Northern Peru. Besides AFHP-31 from a previous study,14 and for which no basidiocarps and only pure agar culture were preserved, two additional specimens were included in this study: TAIM-04 and AFHP-101. The presence of white rhizomorphs, and white pilei no larger than 5 mm in diameter, smooth and non-intervenose are macro morphology hallmarks of the TBD- causing specimens in Amazonas, Peru (Figure 2). In terms of micromorphological features, these specimens produce ellipsoid and smooth basidiospores with dimensions of (6.2-)7.0-8.6(-8.8) × (3.7-)3.8-5.0(-5.2) μm; they also produce Siccus-type cheilocystidia and pileipellis broom cells; cheilocystidia are cylindrical slightly narrower at the base, and pileipellis broom cells are ovoid to globose shape (Figure 3). The examination of tissues in Melzer’ reagent revealed that most micro structures are inamyloid, except for caulocystidia which reacted in a strongly dextrinoid manner (Figure 4). All these morphological features provided the first point of evidence that the cacao TBD-causal agent in Peru is a new species.
We used fifty taxa (type and other reference specimens) of Maramius spp. in the Neosessiles and other closely related sections. As expected for paraphyletic groups, the phylogenetic analysis grouped sect. Neosessiles taxa in two different parts of the trees built with the ITS, LSU, ITS-LSU concatenated dataset, and six-gene-multilocus concatenated dataset (Figure 5).13,24 The Peruvian specimens were grouped together in a highly supported clades in all phylogenetic analyses (85–100% bootstrap support; Figure 5). The closest related clade was composed of the unpublished Marasmius isolates (C2/33, C2/06), putatively soybean endophytes according to their NCBI passport information. These two clades were phylogenetically distinct to the M. tenuissimus sensu stricto clade conformed by the reference strains NW192, and NW199, and other reference specimens (Figure 5). Therefore, these results, combined with the unique morphological characteristics of the Peruvian specimens, support they belong to a new species within the sect. Neosessiles, herein after called Marasmius infestans sp. nov.
West African specimens were phylogenetically distinct to both, M. tenuissimus sensu stricto, and the Peruvian-specimen clade (Figure 5). They were grouped in at least four phylogenetic clades. The informally described M. neosessiliformis nom. prov. was grouped together with isolate GHA64.
The nuclear genome assembly of Marasmius infestans AFH-31 reveals a size of 84.7 Mb, organized in 3,213 contigs (≥1,000 bp) with a GC content of 49.32% and a N50 value of 42,194 kb. We predicted 21,762 protein-coding genes and 656 tRNA genes in the nuclear genome of M. infestans strain AFHP31. Functional annotation resulted in the identification of 15,414 Pfam domains; 29,349 InterPro protein families; 31,346 Clusters of Orthologous Groups of proteins (COGs) and EggNog proteins; 606 proteases/protease inhibitors, and 679 carbohydrate-active enzymes (CAZymes). Moreover, we predicted 1,823 signal peptides and 4,010 transmembrane regions. About 8.9% of the proteome (2,152) are extracellular proteins, of which 25.6% (550) and 16.5% (356) were predicted to be apoplastic and cytoplasmic effectors, respectively. The genome contained 11 SMGCs, 18 biosynthetic enzymes, and 27 smCOGs. The draft nuclear genome of M. infestans AFHP-31 is larger in size than isolate GHA-37 genome size and, despite having fewer proteins, has more effectors (Table 2).
Genomic characters | M. infestans (INDES-AFHP31) | Marasmius sp. 1 (GHA37)15 |
---|---|---|
Genome Size | 84,695,575 | 71,059,514 |
Protein-coding genes | 21,762 | 24,991 |
Number of effectors prediction | 906 | 675 |
Number of genes with peptide signal | 1,823 | 1,900 |
The mitochondrial genome of Marasmius infestans is circular, 47,389 bp long, and contains 42 genes. It is A + T rich (72.83%) and includes 25 tRNA (trnR, trnL and trnS occur in duplicate, while trnM in triplicate), 14 ribosomal proteins, two rRNA (rnl, rns), and one orf (orf868) (Figure 6). A comparison with the mitochondrial genome of TBD-causal agents from West Africa reveals M. infestans differs to them in size, and gene number, and has lost all their introns in COX1 (Table 3).
Taxonomy:
Marasmius infestans Huamán, Ramos C. & Díaz Val., sp. nov. IF 901138 (Figures 1-2).
Etymology: ‘infestans’, in reference to the capacity to infect branches and leaves of cacao trees.
Diagnosis: Similar to Marasmius tenuissimus and M. neosessilis but M. infestans has white to white-cream pilei at young and mature stages, lamella non-intervenose, and presence of ovoid to globose pileipellis broom cells. It is also phenetically similar to M. griseoroseus but M. infestans lacks Rotalis- and Amyloflagellula-type pileipellis broom cells and pileus-trama is inamyloid.
Type: PERU: Amazonas department, Bagua province, Imaza district, Pumpu native community; -4.785944, -78.281000; leg. Tito Ramos-Carrasco; Sept. 2022. Holotype specimen TAIM-04 (voucher KUELAP-2940).
Other examined specimens: AFHP-101 and dried culture of AFHP-31 (voucher KUELAP-2251).
Pileus: (0.7-)1.1-4.0(-4.9) mm diam., convex, smooth, glabrous, dull to shiny, white to white-cream (n = 84). Context white, thin. Lamellae adnate, distant (1-5) with 0 series of lamellulae, narrow, smooth, non-intervenose, white, non-marginate. Stipe: absent or extremely rudimentary, eccentric, cylindrical, insititious not arising from rhizomorphs; white rhizomorphs frequently present. Odor not distinctive.
Basidiospores: (6.2-)7.0-8.6(-8.8) × (3.7-)3.8-5.0(-5.2) μm [xm = 7.8± 0.5 × 4.6 ± 0.4 μm, Q = 1.4-2.0, Qm = 1.7, n = 45], hyaline, inamyloid, ellipsoid, smooth, thin-walled (n = 44). Basidia: length (17.3-)17.7-21.5(-22.2) μm, thicker part (7.0-)7.3-10.1(-10.2) μm, thinner part (2.6-)3.1-6.1(-6.3) μm, hyaline, inamyloid, cylindrical to clavate (n = 30). Cheilocystidia: Siccus-type broom cells, hyaline, inamyloid; main body 17.8-20.6 × 7.3-9.9, cylindrical, slightly narrower at the base, thin-walled; apical setulae length 2.3-3.8(-4.1) μm, obtuse, thin-walled. Caulocystidia: setoid, hyaline, strongly dextrinoid, thin- to thick-walled, main body length (41.7-)46.5-85.1(-87.5) μm, thicker part (7.4-)7.6-10.5(-10.9) μm, thinner part 3.0-5.0(-5.1) μm (n = 5). Pileipellis: hymeniderm, mottled, composed of Siccus-type broom cells; main body (9.6-)9.8-12.1(-12.2) × (8.5-)8.9-16.8(-17.9) μm, ovoid to globose, light-brown to hyaline, thin- to thick-walled (n = 7). Pileus trama: interwoven, inamyloid. Lamellar trama: hyphae (2.4-)2.6-3.9(-4.0) μm diam., hyaline, inamyloid, non-gelatinous. Stipitipellis: hyphae diam 3.4-4.6 μm., hyaline, inamyloid, non-gelatinous. Clamp connections: present in all tissues.
Habit and habitat: Rhizomorphic colonizing leaf and twigs of cacao, which subsequently causes thread blight disease. Rhizoids are white; fruiting bodies develop on dead tissue. The fungus occurs on poorly managed cacao farms with excessive shade and high relative humidity, typical characteristics of the tropical rainforest.
Geographic distribution Imaza province, Amazonas department, Peru.
Notes: Marasmius infestans differs from M. tenuissimus in the color and size of the pilei, and lamellae appearance. Marasmius infestans pilei are white to white-cream, while M. tenuissimus is between light grayish fuscous to rusty brown when fresh,9 or greyish or pale orange to golden brown, light brown or orangish white.3,4,9 Pilei in M. infestans can reach up to 5 mm broad, while pilei in M. tenuissimus can have diameters typically of 7–40 mm.3,4 Lamellae in M. infestans lacks reticulation while in M. tenuissimus lamellae is heavily reticulate. Additionally, West African species within the M. tenuissimus species complex can produce pure-white to brown-colored rhizomorphs,11 while M. infestans produces pure-white rhizomorphs. At the genomic level, M. infestans is 13,636,061 pb larger than M. aff. tenuissimus GHA37. Marasmius infestans also has 3,229 fewer genes, and 231 more effectors (Table 2). Moreover, M. infestans differs from M. neosessilis and its varieties (M. neosessilis var. neosessilis and M. neosessilis var. montepiensis) in that these taxa can have various pilei colors such as apricot, salmon-flesh, and gray.9,71 Also, M. neosessilis and the other phenetically similar species M. griseoroseus have pseudoamyloid (dextrinoid) pileus-trama, while pileus-trama of M. infestans is inamyloid.
The causal agent of cacao TBD had been previously analyzed based on nuclear rDNA comparison, macro morphology of the fruiting body and rhizomorphic structures, and mycelial culture.14 However, other important characteristics, such as the micro morphological features of lamella and pileus, were not considered in reports from both, West Africa and Peru.11,14 In this study, we present morphological, phylogenetic, and genomic evidence that the species of Marasmius causing TBD in the Amazonian areas of Northern Peru is a new species in the sect. Neosessiles, namely M. infestans.
In this study we revealed morphological differences with M. tenuissimus s.s. in color and size of pilei, lamellae appearance, and growth habit. Marasmius infestans differs from M. tenuissimus mainly because of their smaller basidiocarps. With respect to color, M. infestans can be easily distinguished by its white to white-cream pilei. Also, M. tenuissimus has never been reported to colonize the leaves and stems of plants with abundant white rhizomorphs as M. infestans.9,72 Additionally, other relates species with pleurotoid-habit of growth such as M. neosessilis, M. griseoroseus, M. conchiformis, and M. jasingensis also differ in the color of pilei, which can go from gray and pale orange to brown and reddish-brown at maturity.9,13,73 The most macro morphological similar species to M. infestans is M. griseoroseus: pilei are most frequently no more than 5 mm broad, and lamellae is adnate and distant, just as M. infestans. Marasmius griseoroseus can also have white to white-cream pilei.9,13 However, pilei in M. griseoroseus can be pale orange as well. Also, several micro morphological characteristics, such as the presence of both Rotalis- and Amyloflagellula-type pileipellis broom cells, and some degree of dextrinoidity in pileus trama are marked differences with M. infestans.9,13 Moreover, the informally described species M. neosessiliformis nom. prov. is one of the few very closely related taxa for which there is ITS sequence data available in GenBank.19 The provisional description of this species pointed to several shared characteristics with M. infestans, such as the size and the non-intervenose nature of pilei, and the presence of Siccus-type broom cells in the pileipellis. However, it differs in that M. neosessiliformis nom. prov. has much larger basidiospores (10–11 × 5–6 μm) than M. infestans.19
In West Africa, four different species were reported to cause TBD of cacao: M. crinis-equi, M. aff. tenuissimus, M. scandens, and Paramarasmius palmivorus (reported as Marasmius palmivorus).11 Besides the molecular differences, these TBD causal agents presented five rhizomorph morphotypes.11 The morphotype A of rhizomorphs is characterized by abundant thin, black, “horsehair”-type rhizomorphs, and was only found on M. crinis-equi. The morphotype B is characterized by its brown coloration; and the type C, by its intense white color. Both morphotypes B and C, were found on M. aff. tennuisimus. The morphotype D is characterized by a faint cream or dull white rhizomorph, with the presence of smooth or cream-pruinose pilei, with a diameter up to 8 mm; and the morphotype E is characterized by its aggregation of shiny or silky white hyphae and white or pale yellow basidiocarps, with smooth and convex pilei, with diameter from 10 to 50 mm, observed on P. palmivorus.11 If we followed this classification in our study, we find M. infestans has rhizomorphs of morphotype C. Unfortunately, in the West African study, no fruiting bodies from this type of rhizomorphs were reported, so we cannot make a macro morphological comparison.
In the Marasmius genus, ITS has been the main locus for phylogenetic studies of species in sect. Neosessiles.3 In this study, we found M. infestans is closely related species to M. tennuisimus s. s., both species forming individual clades with high bootstrap support (>85%) in all phylogenetic analyses conducted. We also found that the M. tenuissimus is a species complex that has at least five other species that will need proper description, including the previously reported yet not formally described M. neosessiliformis nom. prov.,19 provided the discovery of corresponding basidiocarps. One of these species (Marasmius sp. 5) corresponds to the endophytic strains C2/33 and C2/06 from soybean in Brazil forming a distinct phylogenetic clade, with 97% bootstrap support and closely related to M. infestans and M. tennuisimus. Additionally, TBD-causal strains from West Africa form separate and well-supported clades. Isolate GHA64 groups together with the informally described species M. neosessiliformis nom. prov. specimen Buyck 97.615, suggesting it may be another member of this species requiring formal description.
On top of morphological and phylogenetic evidence that M. infestans distinguishes from other species within the sect. Neosessiles, in this study we present its nuclear and mitochondrial genome sequences. The nuclear genome is about 13 Mb longer than M. aff. tennuisimus GHA37. Marasmius infestans also has 231 more effectors than M. aff. tennuisimus GHA37. Pathogenic effectors are secreted by pathogenic fungi during infection and play an important role in silencing plant defenses response,64,74,75 which may help M. infestans during cacao infection. Moreover, mitochondrial genomes are known for different sizes and rearrangements despite their similar gene function.12,76 Marasmius infestans mitogenome has 47,389 bp and has undergone the loss of introns in the COX1 gene, as opposed to M. cf. tenuissimus causing TBD in Africa (Table 3). The gain and loss of introns are common in fungal mitogenomes and are related to their evolution .76 Moreover, we found that M. cf. tennuisimus specimens GHA74, GHA37, MS2, and GHA07, which group together in the phylogenetic analyses with the six-gene-multilocus dataset, have very similar mitochondrial genome sizes, ranging from 44,399 to 44,859 bp, supporting they all belong to another species (Marasmius sp. 1; Figure 5). Additionally, specimens GHA63, and GHA79, which also formed individual specific lineages, have different mitochondrial genome sizes (51,210 and 48,952 bp, respectively).11 Therefore, they also represent other Neosessiles species in need of formal description (Marasmius sp. 3 and Marasmius sp. 4, respectively).
BioProject: The Genome Shotgun project of specimen AFHP-31. Accession number PRJNA860982; https://identifiers.org/NCBI/bioproject:PRJNA860982
The NCBI accession number of the version of the assembled genome of specimen AFHP-31 described in this paper is JANHQD010000000.
Nucleotide: The mitochondrial genome assembly. Accession number OQ343345; https://www.ncbi.nlm.nih.gov/nuccore/OQ343345.1/
SRA: The raw reads for the nuclear and mitochondrial genome under accession number SRR20354643. https://identifiers.org/insdc.sra:SRR20354643
Sequences obtained through the Sanger method used are deposited at NCBI through accession numbers OR359411, OR364495, OR420729 and OR420730 for specimen TAIM-04; OM720123 and OM720135 for specimen AFHP-31; and OR359410 and OR364494 for specimen AFHP-101.
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Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Taxonomy and systematics on agaric
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Macro-fungal taxonomy
Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Partly
Are all the source data underlying the results available to ensure full reproducibility?
No
Are the conclusions drawn adequately supported by the results?
Partly
References
1. Wilson AW, Eberhardt U, Nguyen N, Noffsinger CR, et al.: Does One Size Fit All? Variations in the DNA Barcode Gaps of Macrofungal Genera.J Fungi (Basel). 2023; 9 (8). PubMed Abstract | Publisher Full TextCompeting Interests: No competing interests were disclosed.
Reviewer Expertise: Taxonomy and systematics on agaric
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Macro-fungal taxonomy
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