Morphological, phylogenetic, and genomic evidence reveals the causal agent of thread blight disease of cacao in Peru is a new species of Marasmius in the section Neosessiles, Marasmius infestans sp. nov.

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.


Introduction
Marasmius (Marasmiaceae, Agaricales, Agaricomycetes, Basidiomycota) is a hyperdiverse genus of fungi with 1,560 legitimate species names registered in MycoBank, as of Jan. 14 th , 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. 3,40][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). 12The sect.Neosessiles is a paraphyletic group mainly characterized by the pleurotoid habit of growth and the absence or rudimentarity of the stipe. 2,13Among 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. 14Marasmius 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 Â 4-6 μm. 3,9ven 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. 10,11ileus 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.

Sampling and morphological analysis
Specimens were obtained during an expedition into the Imaza District (4°47 0 09.4 00 S 78°16 0 51.6 00 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 5 th and 95 th percentile, with extreme values in parentheses, as in. 16Additionally, for basidiospores, the arithmetic mean AE standard deviation (x m ) and the quotient of the length by the width, expressed as range (Q) and arithmetic mean (Q m ), 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.

REVISED Amendments from Version 1
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.
Any further responses from the reviewers can be found at the end of the article Phylogenetic analysis 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. 17The 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. 15In 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. 12The introns of COX1 sequences were removed as in. 180][21][22][23][24][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 MUSCLE 26 implemented in MEGA-X, 27 and concatenated with SeaView 4.7. 28e used jModelTest v2 29 to identify the most appropriate nucleotide evolution models under the Akaike information criterion.Phylogenetic analysis was performed with IQ-TREE v2 30 which implements the Maximum Likelihood algorithm, 31 in the CIPRES Science Gateway v3.3 portal. 32The phylogenetic trees were visualized and edited with FigTree. 33

Genomic analysis 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. 14A 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 Â 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 v0.36 34 and TrimGalore software, 35 respectively.Jellyfish v.2. 36was used for k-mer counting, and Genome Scope v1.0.0 37 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.

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. 66Genes were annotated with MITOS, 67 MFannot 68 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. 70Our results were compared to the ones reported for another published M. aff.tenuissimus mitogenomes. 12

Sampling and morphological analysis
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)Â (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 TBDcausal agent in Peru is a new species.

Phylogenetic analysis
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). 13,24The 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.

Nuclear and mitochondrial genomic analyses
The nuclear genome assembly of Marasmius infestans AFH-31 reveals a size of 84.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).
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.
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. 3,4,9Pilei in M. infestans can reach up to 5 mm broad, while pilei in M. tenuissimus can have diameters typically of 7-40 mm. 3,4Lamellae 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.

Discussion
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. 14However, other important characteristics, such as the micro morphological features of lamella and pileus, were not considered in reports from both, West Africa and Peru. 11,14In 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. 9,72Additionally, 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. 9,13,73The 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. 9,13However, 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. 9,13Moreover, 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. 19The 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 Â 5-6 μm) than M. infestans. 19 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). 11Besides the molecular differences, these TBD causal agents presented five rhizomorph morphotypes. 11The 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. 11If 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. 3In 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. 12,76Marasmius 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 . 76oreover, 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). 11Therefore, they also represent other Neosessiles species in need of formal description (Marasmius sp. 3 and Marasmius sp. 4, respectively).
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.There are some suggestions and comments, please look carefully at the following: Introduction: the current classifications of Marasmius should be informed.Since, this research aimed to conduct a taxonomic and phylogenetic revision of the fungi in this genus. 1.
Methods: The spore Quotient (Q) should be determined, and the statistical analysis of spore sizes should show the arithmetic mean with standard deviations.The type specimens (holotype, lectotype, or neotype) are designated to a single specimen.So please correct the data in Table 1 and Figure 3. Please correct the abbreviation of the translation elongation 2.
Results: Please make the scale bar in Figure 1 more clearly.In the Taxonomy subsection, the description that the author provided is not concordant with the specimen photo shown in Figure 1.Please correct the spacing, and number of the lamellae and clarify whether lamellae are intervernose or not.Please indicate the attachment of the stipe base and substrate.The Melzer's reaction is a taxonomic chemical reaction used in Marasmiaceae.
Please inform the results of the test in the spores and all kinds of tissue.The color of spores, cells, and hypha are required to be stated in a description.The type of pileipellis, and all kinds of trama have to be informed.In Notes, a comprehensive comparison of related taxa is required especially for the Amazonia species.

The current classification of Marasmius is now informed in the first paragraph of the introduction.
Methods: The spore Quotient (Q) should be determined, and the statistical analysis of spore sizes should show the arithmetic mean with standard deviations.The type specimens (holotype, lectotype, or neotype) are designated to a single specimen.So please correct the data in Table 1 and Figure 3. Please correct the abbreviation of the translation elongation factor 1-alpha gene.

In this new version we have calculated the Quotient values of spores along with other important statistical metrics such as the arithmetic means and standard deviations, as suggested by the reviewer.
○ We recognized the mistakes in the original submission regarding the designation of type specimens, as pointed out by the reviewer.Therefore, we have updated Table 1 and Figure 3 accordingly.

Regarding the abbreviation of the translation elongation factor 1-alpha gene, we have kept the abbreviation as "EF1α" following the nomenclature of Aime and Phillips-Mora (2005). We have also reviewed for consistency the abbreviations of other genes such as COX1 and updated the manuscript throughout.
○ Results: Please make the scale bar in Figure 1 more clearly.In the Taxonomy subsection, the description that the author provided is not concordant with the specimen photo shown in Figure 1.Please correct the spacing, and number of the lamellae and clarify whether lamellae are intervernose or not.Please indicate the attachment of the stipe base and substrate.The Melzer's reaction is a taxonomic chemical reaction used in Marasmiaceae.
Please inform the results of the test in the spores and all kinds of tissue.The color of spores, cells, and hypha are required to be stated in a description.The type of pileipellis, and all kinds of trama have to be informed.In Notes, a comprehensive comparison of related taxa is required especially for the Amazonia species.

Scale bars have been adjusted for better clarity.
○ As pointed out by the reviewer, there were several inconsistencies and lack of details in the description section.We have made a thorough revision in this regard.We corrected the lamellae characteristics as follows: "Lamellae adnate, distant (

Atik Retnowati
Herbarium Bogoriense, Cibinong, Indonesia This is a study that aims to describe a new species of Marasmius infestans from Peru.Combination molecular and morphological study is the best approach to describe these new species.Overall, the work is good, but here are some suggestions: A mycobank registration number must be provided for a valid description.

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The previous report of Marasmius from Peru must be added to the introduction.

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Map of locality where the species was collected must be added.It will help of the ordinary people to see where the exact type locality.
○ Scale bar at the Figure 2 can clearly be seen, and it probably white color scale bar would be better.Reviewer Expertise: Macro-fungal taxonomy I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Figure 5 .
Figure 5. Phylogenetic analyses of Marasmius taxa in the Neosessiles and other closely related sections conducted in this study.a) Phylogenetic analysis with the internal transcribed spacer (ITS1-5.8S-ITS2,ITS)rDNA region using the Hasegawa-Kishino-Yano nucleotide substitution model with a discrete Gamma model for the rate of heterogeneity (HKY + G). b) Phylogenetic analysis using the large subunit (LSU) rDNA region using the transition model 3 with equal frequency allowing for proportion of invariable sites (TIM3+I).c) Phylogenetic analysis with the ITS-LSU concatenated dataset using the transversion model with unequal base frequency with invariable site plus discrete Gamma model for the rate of heterogeneity (TVM + I +G).d) Phylogenetic analysis with the multi-locus dataset composed by ITS, LSU, and partial sequences of the Translation Elongation Factor 1-α (EF1α), the largest subunit of RNA polymerase II gene (RPB1), and the mitochondrial cytochrome oxidase I (COX1) genes, using the transversion model with unequal base frequency, with a discrete Gamma model for the rate of heterogeneity (TVM + G).All phylogenetic trees were midpoint-rooted and built under the maximum likelihood framework.Models of evolution were determined by jModelTest2.29Values above branches = Maximum likelihood bootstrap values.Taxa in blue are the specimens causing thread-blight disease of cacao in Amazonas, Peru, and conform a new species clade, Marasmius infestans sp.nov.Type specimens are marked with an asterisk.

Figure 6 .
Figure 6.Circular map of Marasmius infestans sp.nov.mitochondrial genome.The genes inside and outside the circle are transcribed in clockwise and counterclockwise directions, respectively.Genes belonging to different functional groups are shown in different colors.The dark gray area in the inner circle denotes GC content.

©
2023 Retnowati A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Description: Lamellae adnate with 1-5 series of lamellulae.It should be "Lamellae adnate, distant (1-5) with 0 series of lamellulae".○Isthe work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesCompeting Interests: No competing interests were disclosed.

Table 2 .
Comparison of nuclear genomes with the phylogenetic close specimen from West Africa, isolate GHA37.

Table 3 .
12mparison our M. infestans with mitochondrial genomes available in GenBank.12 This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Faculty of Science and Technology, Pibulsongkram Rajabhat University, Phitsanulok, Phitsanulok, ThailandThe manuscript entitled "Morphological, phylogenetic, and genomic evidence reveals the causal agent of thread blight disease of cacao in Peru is a new species of Marasmius in the section Neosessiles, Marasmius infestans sp.nov."described a new species of Marasmius from Peru.This manuscript is interesting, with new data and nice photographs and drawings.However, some points need to be considered.This research reported a new species of Marasmius, which is a taxonomic report with some molecular evidence.So a clear morphological description of a new species is needed.The key characteristics of Marasmius species have to be informed, such as Melzer's reagent reactions of spores, and the trama tissues.A comparison of a new species with phenetically similar taxa is needed, especially for the species reported from the related geographical area --the Amazon rainforest, not scope to the Marasmius species that caused the thread blight disease (TBD) only.A comprehensive dataset for phylogenetics analyses is required, ITS sequences of related species from the Amazon rainforest (Oliveira et al 2014--the sixth reference of this manuscript) have to be included.The phylogenetic analyses of a single gene of ITS and LSU and a combined gene of ITS and LSU are required at least as supplemented data.Due to the dataset used in this study being mainly based on both genes, Only a new species and a single specimen of undescribed Marasmius sp. have other genes.

3 .
Discussion: Since a new species reported in this study caused the TBD.The comparison of new species and other TBD causal species is valuable data.However, a species is described based on the sexual stage and the basidiocarp is mainly characterized.Other characters like the rhizomorph or culture morphology could be additional data.So please keep in mind that the TBD causal strains from West Africa are identified without the morphological characteristics of basidiocarp.The author cites that ITS did not provide sufficient support for resolving species in the section Neosessiles refers to the work of Wannathes et al 2009.I disagree with this information, please check this carefully.According to the number of available sequences in GenBank and the recent data on the barcode gaps in ITS of Marasmius (Wilson et al., 2023 1 ), it would be better if the author could investigate the phylogenetic analysis on combined sequences of ITS and LSU.

microstructures were examined with Melzer's reagent, finding that only Caulocystidia were strongly dextrinoid, while all other structures and tissues were inamyloid. We have included a new figure to show these findings.
○The

color of spores, cells, and other tissues was included in the description (hyaline, except for the Siccus-type broom cells that in some cases were light brown) ○ The type of pileipellis is hymeniderm, and other characteristics of trama tissues were added.
○We

have added a discussion comparing the new species with related taxa, with emphasis on Amazonian species from Oliveira et al. (2014).
Since a new species reported in this study caused the TBD.The comparison of new species and other TBD causal species is valuable data.However, a species is described based on the sexual stage and the basidiocarp is mainly characterized.Other characters like the rhizomorph or culture morphology could be additional data.So please keep in mind that the TBD causal strains from West Africa are identified without the morphological characteristics of basidiocarp.The author cites that ITS did not provide sufficient support for resolving species in the section Neosessiles refers to the work of Wannathes et al 2009.I disagree with this information, please check this carefully.According to the number of available sequences in GenBank and the recent data on the barcode gaps in ITS of Marasmius(Wilson et al., 20231), it would be better if the author could investigate the phylogenetic analysis on combined sequences of ITS and LSU.We ○Discussion:

have included a paragraph in which we have added a comparison of the new species with other related basidiocarp-forming species in the Neosessiles section. We agree with the reviewer, so we have clarified the statement in which we indicated that ITS did not provide sufficient phylogenetic resolution power within the Neosessiles. We have also added new phylogenies with ITS alone, LSU alone, and concatenated, to provide extra evidence for the new species. Competing Interests:
No competing interests were disclosed.