Corresponding author: Maria Alves Ferreira (
Academic editor: Nalin Wijayawardene
In recent decades, commercial
Sanchez-Gonzalez EI, Farias Soares TdP, Galafassi Zarpelon T, Valverde Zauza EA, Gonçalves Mafia R, Alves Ferreira M (2022) Two new species of
Currently, 130
In Brazil, a total of 35 species have been described: eleven species isolated from diseased tissues of
Brazil is one of the main producers of pulp, paper, and wood panels in the world, mainly due to the genus
In February 2020, during a disease survey conducted in three commercial plantations of
Samples were stored in paper bags and transported to the Laboratory of Forest Pathology at the Universidade Federal de Lavras. From each leaf, small segments of 1 cm2 from the transition section between healthy and diseased tissue were cut and the surface was disinfected by washing with 1% sodium hypochlorite for 1 min, with 70% ethanol for 30 s and with sterilized water three times before culture on 2% malt extract agar (MEA; malt extract 20 g·L-1, agar 20 g·L-1, yeast extract 2 g·L-1, sucrose 5 g·L-1) plates at 25 °C. After 48 h of incubation,
Total genomic DNA was extracted from fresh mycelia of single spore cultures grown on malt extract broth (MEB; malt extract 20 g·L-1, yeast extract 2 g·L-1, sucrose 5 g·L-1) for ten days at 25 °C in the dark. The protocol described by
Based on a previous study (
The PCRs were carried out in a 25 μL final volume containing molecular biology-grade water (Sigma–Aldrich, St. Louis, MO, USA) 1X PCR buffer (Promega, Madison, WI, USA), 2.5 mM MgCl2, 0.2 mM deoxyribonucleotide triphosphate (
PCR products were separated by electrophoresis at 120 V for 1 h in a 1.2% agarose gel, stained with Diamond Nucleic Acid Dye (Promega, Madison, WI, USA), and visualized using an ultraviolet light transilluminator. Successful PCR products were purified and sequenced in both directions using the same primer pairs used for amplification by Macrogen Inc. (Macrogen, Seoul, Korea). Raw sequences from each gene region were edited, consensus sequences were generated using SeqAssem software ver. 07/2008 (
The generated sequences were aligned with other sequences of closely related
Species complex | Species | Isolate representing the species‡,§ | Other isolate numbers | Host/ Substrate | Country | Genbank accession numbers | |||||
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Soil | Ecuador |
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N/A |
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Soil | Ecuador |
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N/A |
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Soil | Ecuador |
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N/A |
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Soil | Ecuador |
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N/A |
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Soil | Ecuador |
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N/A |
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Soil | Ecuador |
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N/A |
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Brazil |
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PFC2 | Brazil |
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PFC3 | Brazil |
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PFC4 | Brazil |
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PFC5 | Brazil |
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Soil | Ecuador |
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Soil | Ecuador | N/A | N/A |
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N/A | N/A |
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ATCC44730, CSF11330 | USA |
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Switzerland |
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Soil | Brazil |
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Soil | Brazil |
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Soil | Brazil | N/A |
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N/A |
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Soil | Brazil | N/A |
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N/A |
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Soil | Mexico |
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Soil | Mexico |
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Brazil |
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Brazil |
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Soil | Colombia |
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N/A |
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Soil | Colombia |
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N/A |
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Brazil | N/A |
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N/A |
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Brazil | N/A |
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N/A |
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Brazil | N/A |
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N/A |
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LPF141.1 |
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Brazil | N/A |
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N/A |
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Soil | Brazil | N/A |
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N/A |
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Brazil | N/A |
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N/A |
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Brazil | N/A |
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N/A |
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Brazil |
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PFC7 |
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Brazil |
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PFC8 |
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Brazil |
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PFC9 |
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Brazil |
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Brazil | N/A |
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N/A |
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Brazil | N/A |
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N/A |
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Brazil | N/A |
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N/A |
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Brazil |
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Soil | Brazil | N/A |
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N/A |
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Soil | Brazil | N/A |
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N/A |
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Soil | South Africa |
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China |
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Soil | Brazil | N/A |
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N/A |
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Soil | Brazil | N/A |
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N/A |
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Soil | Brazil | N/A |
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N/A |
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Brazil | N/A |
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N/A |
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Soil | Brazil | N/A |
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N/A |
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Soil | Brazil | N/A |
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N/A |
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Brazil |
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Soil | Brazil |
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LPF081 | Soil | Brazil | N/A |
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N/A |
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Soil | Brazil | N/A |
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N/A |
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Brazil |
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Colombia |
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Soil | Venezuela |
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Soil | Colombia |
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CSF11239 | Soil | Colombia |
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† New
The partition homogeneity test (
Maximum parsimony analysis was performed using PAUP 4.0b10 (
The best evolutionary model of nucleotide substitution for each gene region was selected according to the Akaike Information Criterion (
Individual and partitioned
Phylogenetically closely related species were analyzed using the Genealogical Concordance Phylogenetic Species Recognition (
The mating-type idiomorph of each
Morphological characterization of representative isolates of each
One representative isolate of each
A total of 34 isolates with the typical morphology of
Sequences from 50 isolates corresponding to 25
Alignments for each gene region and the concatenated dataset were as follows:
Tree topologies derived from the
Phylogenetic tree based on maximum likelihood analysis of concatenated
Phylogenetic analyses of the six individual gene regions showed that the five isolates from the
Single nucleotide polymorphisms unique to
Species |
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188‡ | 189 | 190 | 191 | 192 | 193 | 194 | 195 | 196 | 197 | 142 | 144 | 170 | 185 | 217 | 270 | 437 | 444 | 455 | 483 | ||
a | g | a | a | a | a | a | g | a | a | t | a | c | c | t | c | a | a | g | a | ||
t | - | - | - | - | - | - | - | - | - | a | c | t | t | c | t | g | g | a | c | ||
t | - | - | - | - | - | - | - | - | - | a | c | t | t | c | t | g | g | a | c | ||
t | - | - | - | - | - | - | - | - | - | a | c | t | t | c | t | g | g | a | c | ||
t | - | - | - | - | - | - | - | - | - | a | c | t | t | c | t | g | g | a | c | ||
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c | t | t | - | - | - | a | g | a | t | c | t | t | a | g | c | t | g | g | c | a | |
t | a | c | c | t | c | t | a | g | - | - | a | t | t | t | |||||||
t | a | c | c | t | c | t | a | g | - | - | a | t | t | t | |||||||
- | a | c | c | a | c | c | a | g | c | t | c | c | g | a | - | - | a | a | t | t | |
- | a | c | c | c | c | c | a | g | c | t | c | c | g | a | - | - | a | t | t | t |
† Only polymorphic nucleotides occurring in all the isolates are shown, not alleles that partially occur in individuals per phylogenetic group. ‡ Numerical positions of the nucleotides in the DNA sequence alignments.
Phylogenetic analyses of the individual gene regions of
Single nucleotide polymorphisms found in
Species |
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57‡ | 62 | 71 | 121 | 171 | 187 | 210 | 319 | 376 | 403 | 405 | 418 | 444 | 8 | 44 | 46 | 53 | 56 | 60 | 66 | |
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a | c | c | g | g | c | c | t |
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t | c | c | t | c | c | g | a | |
c | g | t | g | g | c | c | c | c | c | a | t | t | c | t | c | c | g | a | ||
a | c | c | g | g | c | g | t | c | c | a | t | c | t | c | c | c | g | a | ||
a | g | c | c | a | c | c | c | c | c | a | c | c | c | - | a | c | a | c | ||
t | a | c | c | g | g | a | g | t | c | t | a | t | c | c | t | c | t | g | a | |
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c | c | c | a | t | t | t | t | c | c | a | c | c | a | g | c | a | a | t | g | |
c | c | c | t | t | t | t | t | c | c | a | c | c | a | g | c | a | a | t | g | |
c | c | c | a | t | t | t | t | c | a | a | c | c | a | g | c | a | a | t | g | |
t | t | c | a | c | t | c | g | t | g | g | t | a | g | a | t | g | g | c | a | |
c | c | a | a | t | c | t | t | c | c | a | c | c | a | g | c | a | a | t | g | |
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c | t | t | c | g | c | t |
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c | g | a | t | t | c | c | c | |
c | t | t | c | g | t | t | c | g | t | t | t | c | c | c | ||||||
c | t | t | c | a | c | t | c | a | a | t | t | c | c | c | ||||||
t | t | c | t | g | c | g | t | g | a | a | c | a | t | c | ||||||
c | c | t | c | g | c | t | t | g | t | c | t | c | g | a | t | t | c | c | t | |
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c | c | c | a | a | c | a | g | t | c | c | g | c | t | t | t | - | - | |||
c | t | t | a | a | c | a | g | t | c | c | g | c | t | t | t | a | c | |||
c | t | t | a | a | c | a | g | t | c | c | g | c | t | t | t | a | c | |||
t | c | c | a | a | g | g | a | c | t | t | a | a | c | c | g | a | c | |||
c | c | c | c | g | c | a | g | t | c | c | g | c | t | t | t | a | c |
† Only polymorphic nucleotides occurring in all the isolates are shown, not alleles that partially occur in individuals per phylogenetic group. ‡ Numerical positions of the nucleotides in the DNA sequence alignments.
A PHI test using a five-locus concatenated dataset (
Results of the pairwise homoplasy index (PHI) test for
A PHI test using a four-locus concatenated dataset (
MAT1-1-1 and MAT1-2-1 genes were amplified in all isolates of each identified species, indicating that they are putatively homothallic. However, after a twelve-week mating test on
Based on phylogenetic analyses,
Morphological characteristics of two new
Species complex | Species | Conidiogenus apparatus | Stipe | Macroconidia | Vesicle | Reference | |||||
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Size (L × W)†,‡,§ | Branches | Size (L × W)†,‡,§ | Extension (L × W)†,‡,§ | Size (L × W)†,‡,§,| | Average (L × W)†,‡,§ | Septa | Diameter†,§ | Shape | |||
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40–113 × 45–129 | (–4) | 112–281 × 2–4 | 123–295 × 1.5–3 | (47–)56–66(–71) × (4–)4.8–5.9(–7) | 61 × 5 | 1(–3) | 8–12 | globoid to sphaeropedunculate | This study |
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49–78 × 63–123 | (–4) | 54–90 × 6–10 | 149–192 × 5–6 | (47–)50–58(–62) × (5–)6 | 54 × 6 | 1 | 10–12 | ovoid to ellipsoid to sphaeropedunculate |
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43–71 × 42–49 | 3 | 44–90 × 6–8 | 126–157 × 4–5 | (45–)48–54(–56) × (4–)5 | 51 × 5 | 1 | 10–12 | globoid to ovoid to sphaeropedunculate |
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70–100 × 25–70 | 4 | 100–350 × 5–6 | 100–250 × 2.5–3.5 | (40–)47–55(–60) × 4–5 | 52 × 4 | 1(–3) | 8–12 | sphaeropedunculate to ellipsoid | ||
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60–150 × 40–90 | 4 | 120–150 × 6–8 | 170–260 × 3–4 | (45–)65–80(–120) × (5–)6(–7)¶ | 70 × 6 | 1(–3) | 8–15 | globoid or ellipsoid to obpyriform |
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50–127 × 41–110 | (–3) | 135–227 × 2–4 | 151–254 × 1.5–3 | (38–)43–49(–52) × (2–)2.7–3.2(–4) | 46 × 3 | (–1) | 3–6 | ellipsoid to narrowly obpyriform | This study |
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20–60 × 35–80 | 2 | 50–110 × 4–6 | 95–130 × 2–3 | (38–)47–52(–60) × 3–5 | 49 × 4.5 | 1 | 3–7 | ellipsoid to narrowly obpyriform |
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50–80 × 50–135 | 3 | 55–155 × 5–8 | 90–172 × 2–3 | (35–)50–56(–65) × 3–5 | 53 × 4 | 1 | 3–7 | ellipsoid to narrowly obpyriform |
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27–45 × 25–40 | 2 | 50–130 × 5–7 | 100–165 × 2–4 | (45–)50–52(–55) × 3–5 | 50 × 4 | 1 | 3–5 | ellipsoid to narrowly obpyriform |
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25–65 × 25–60 | 3 | 35–100 × 4–8 | 85–190 × 3–6 | (48–)54–62(–65) × (4–)4.5–5.5(–7) | 58 × 5 | 1 | 5–9 | fusiform to ovoid to ellipsoid |
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† All measurements are in μm. ‡ L × W = length × width. § Minimum–maximum. | Measurements are presented in the format [(minimum–) (average – standard deviation) – (average + standard deviation) (–maximum)]. ¶ Measurements are presented in the format [(minimum–) (average) (–maximum)].
The term “
Sexual morph unknown. Macroconidiophores consisted of a stipe, a suite of penicillate arrangements of fertile branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, (112–)135–207(–281) × (2–)2.6–3.5(–4) μm; stipe extension septate, straight to flexuous, (123–)147–220(–295) μm long, (1.5–)1.9–2.4(–3) μm wide at the apical septum, terminating in a globose to sphaeropedunculate vesicle, (8–)8.5–10.5(–12) μm diam; lateral stipe extensions (90° to the axis) also present. Conidiogenous apparatus was (40–)56–88(–113) μm long, (45–)67–107(–129) μm wide; primary branches aseptate or 1-septate, (15.7–)18.4–25.9(–30.6) × (3.3–)4–6(–6.5) μm; secondary branches aseptate, (12.7–)14.3–19.6(–22.1) × (3–)3.5–5(–6) μm; tertiary branches aseptate, (9.9–)11.6–15.3(–17.9) × (2.8–)3.6–5.3(–6.4) μm; additional branches (–4), aseptate, (10.3–)11–13.2(–14) × (3–)3.2–4.4(–5) μm; each terminal branch produced 2–4 phialides; phialides doliiform to reniform, hyaline, aseptate, (8–)9.1–11.8(–14) × (2–)2.7–4.1(–6) μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia were cylindrical, rounded at both ends, straight, (47–)56–66(–71) × (4–)4.8–5.9(–7) μm (av. = 61 × 5 μm), (1–3) septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colorless slime. Megaconidia and microconidia were not observed.
Colonies formed abundant white aerial mycelium on MEA at 25 °C after seven days, with irregular margins and moderate sporulation. The surface had white to buff outer margins, and sienna to amber in reverse with abundant chlamydospores throughout the medium, forming microsclerotia. The optimal growth temperature was 23.8 °C, with no growth at 5 °C; after seven days, colonies at 10 °C, 15 °C, 20 °C, 25 °C, and 30 °C reached 7 mm, 23 mm, 38.3 mm, 36.1 mm, and 31.8 mm, respectively.
Leaves of
Northeast Brazil.
Brazil,• Pará state, Paragominas microregion; From infected leaves of
The term “
Sexual morph unknown. Macroconidiophores consisted of a stipe, a suite of penicillate arrangements of fertile branches, a stipe extension, and a terminal vesicle; stipe septate, hyaline, smooth, (135–)151–198(–227) × (2–)2.6–3.4(–4) μm; stipe extension septate, straight to flexuous, (151–)169–220(–254) μm long, (1.5–)1.9–2.7(–3) μm wide at the apical septum, terminating in an ellipsoidal to narrowly obpyriform vesicle (3–)3.1–4.6(–6) μm diam. Conidiogenous apparatus was (50–)66–100(–127) μm long, (41–)62–89(–110) μm wide; primary branches aseptate, (14.6–)19–24.8(–28.5) × (2.5–)3.2–4(–4.5) μm; secondary branches aseptate, (12.1–)13.5–18.2(–24.2) × (2.3–)2.8–3.7(–4) μm; tertiary branches aseptate, (10.1–)11–15(–18.1) × (1.9–)2.3–3.2(–4.1) μm; each terminal branch producing 2–4 phialides; phialides doliiform to reniform, hyaline, aseptate, (8–)9.1–13(–15) × (2–)2.7–3.3(–4) μm, apex with minute periclinal thickening and inconspicuous collarette. Macroconidia were cylindrical, rounded at both ends, straight, (38–)43–49(–52) × (2–)2.7–3.2(–4) μm (av. = 46 × 3 μm), (–1) septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colorless slime. Megaconidia and microconidia were not observed.
Colonies formed moderate aerial mycelium on MEA at 25 °C after seven days, with moderate sporulation. The surface had white to buff outer margins, and sepia to umber in reverse with abundant chlamydospores throughout the medium, forming microsclerotia. The optimal growth temperature was 25 °C, with no growth at 5 °C; after seven days, colonies at 10 °C, 15 °C, 20 °C, 25 °C, and 30 °C reached 10.1 mm, 25.5 mm, 29.1 mm, 44.5 mm, and 40.6 mm, respectively.
Leaves of
Northeast Brazil.
Brazil,• Maranhão state, Cidelândia municipality;
The conidia suspensions of the representative isolates of
Pathogenicity tests on leaves of
Two new species of
Pathogenicity tests showed that
In this study, we described two new
We thank Heitor S. Dallapiccola and Francisco J. A. Gomes for their help during sample collection. We thank the laboratories of Molecular Biology, Plant Virology, and Nematology at Universidade Federal de Lavras (
Figures S1–S6
Phylogenetic trees (pdf file)