Five new species of endophytic Penicillium from rubber trees in the Brazilian Amazon

Abstract

The Amazon rainforest is the world's most diverse ecosystem, full of fauna and flora. Among the trees that make up the forest are the rubber trees of the genus Hevea (H. brasiliensis and H. guianensis), which stand out for the industrial use of latex. It was previously shown that endophytic fungi colonize the leaves, stems, and roots of Hevea spp. In this study, 47 Penicillium spp. and three Talaromyces spp. isolates were analyzed using specific DNA barcodes: internal transcribed spacers region (ITS), β-tubulin (BenA), calmodulin (CaM), and the DNA-dependent RNA polymerase II second largest subunit (RPB2) genes and additionally, for species delimitation, the genealogical concordance phylogenetic species recognition (GCPSR) criteria were applied. The phylogenetic analyses placed the Penicillium isolates into four sections Lanata-Divaricata, Sclerotiora, Citrina, and Fasciculata. The morphological and molecular characteristics resulted in the discovery of five new species (P. heveae sp. nov., P. acrean sp. nov., P. aquiri sp. nov., P. amazonense sp. nov., and P. pseudomellis sp. nov.). The five new species were also compared to closely related species, with observations on morphologically distinguishing features and colony appearances. Bayesian inference and maximum likelihood analysis have supported the placement of P. heveae sp. nov. as a sister group to P. globosum; P. acrean sp. nov. and P. aquiri sp. nov. as sister groups to P. sumatrense; P. amazonense sp. nov. closely related to isolates of P. rolfsii, and P. pseudomellis sp. nov. closely related to P. mellis. The study of endophytic Penicillium species of rubber trees and the description of five new taxa of Penicillium sect. Citrina, Lanata-Divaricata, and Sclerotiora as endophytes add to the fungal biodiversity knowledge in native rubber trees. Reports of fungi in native tropical plants may reveal taxonomic novelties, potential pathogen control agents, and producers of molecular bioactive compounds of medical and agronomic interest.

Keywords: Hevea spp.; DNA barcoding; Endophyte; Taxonomy.

Fig. 1

Phylogeny based on the ITS sequences for species classified in Penicillium spp. and Talaromyces spp. Bootstrap values (≥ 70%) of the ML analyses, as well as posterior probability scores (≥ 0.95) from a BI of the same dataset, are indicated at well-supported nodes together with thickened branches. The scale bar represents the expected substitutions per site (0.03). Isolates belonging to the known species obtained in this study are in bold. Isolates of the new species described in this study are shown in bold red. Holotype labeled with an asterisk. The tree was rooted in three species of Talaromyces.“–” indicates a lack of support

Fig. 2

Phylogeny based on the CaM sequences for species classified in Penicillium spp. and Talaromyces spp. Bootstrap values (≥ 70%) of the ML analyses, as well as posterior probability scores (≥ 0.95) from a BI of the same dataset, are indicated at well-supported nodes together with thickened branches. The scale bar represents the expected substitutions per site (0.02). Isolates belonging to the known species obtained in this study are in bold. Isolates of the new species described in this study are shown in bold red. Holotype labeled with an asterisk. The tree was rooted in three species of Talaromyces.“–” indicates a lack of support

Fig. 3

Phylogeny based on the BenA sequences for species classified in Penicillium spp. and Talaromyces spp. Bootstrap values (≥ 70%) of the ML analyses, as well as posterior probability scores (≥ 0.95) from a BI of the same dataset, are indicated at well-supported nodes together with thickened branches. The scale bar represents the expected substitutions per site (0.03). Isolates belonging to the known species obtained in this study are in bold. Isolates of the new species described in this study are shown in bold red. Holotype labeled with an asterisk. The tree was rooted in three species of Talaromyces.“–” indicates a lack of support

Fig. 4

Phylogeny based on the RPB2 sequences for species classified in Penicillium spp. and Talaromyces spp. Bootstrap values (≥ 70%) of the ML analyses, as well as posterior probability scores (≥ 0.95) from a BI of the same dataset, are indicated at well-supported nodes together with thickened branches. The scale bar represents the expected substitutions per site (0.06). Isolates belonging to the known species obtained in this study are in bold. Isolates of the new species described in this study are shown in bold red. Holotype labeled with an asterisk. The tree was rooted in three species of Talaromyces.“–” indicates a lack of support

Fig. 5

Phylogeny based on the combined CaM, BenA, and RPB2 data set for species classified in Penicillium spp. and Talaromyces spp. Bootstrap values (≥ 70%) of the ML analyses, as well as posterior probability scores (≥ 0.95) from a BI of the same dataset, are indicated at well-supported nodes together with thickened branches. The scale bar represents the expected substitutions per site (0.02). The isolates from known species obtained in this study are in bold. The new species’ isolates described in this study are in bold red. Holotype labeled with an asterisk. The Talaromyces spp. was chosen as an outgroup.“–” indicates a lack of support

Fig. 6

Penicillium acrean sp. nov. from Hevea brasiliensis (VIC47584). a – d Seven-day-old cultures, 25 °C, left to right; first row, all obverse, CYA, MEA, YES, CREA; second row CYA reverse, MEA reverse, YES reverse, CREA reverse. e – f Colony appearances: YES: seven days, 25 ºC; and MEA: seven days, 25 ºC. g – i Conidiophores and phialides (MEA). j – k Conidia. Scale bars: a – d = 20 mm; e – f = 5 mm; g – i = 20 μm; j – k = 10 μm

Fig. 7

Penicillium aquiri sp. nov. from Hevea brasiliensis (VIC47586). a – d Seven-day- old cultures, 25 °C, left to right; first row, all obverse, CYA, MEA, YES, CREA; second row CYA reverse, MEA reverse, YES reverse, CREA reverse. e – f colony appearances: YES: seven days, 25 ºC; and MEA: seven days, 25 ºC. g – i Conidiophores and phialides (MEA). j Conidia. K Cluster of conidia. Scale bars: a – d = 20 mm; e – f = 10 mm; g – i = 20 μm; j – k = 10 μm

Fig. 8

Penicillium amazonense sp. nov. from Hevea brasiliensis (VIC 49381). a – d Seven-day-old cultures, 25 °C, left to right; first row, all obverse, CYA, MEA, YES, CREA; second row CYA reverse, MEA reverse, YES reverse, CREA reverse. e – f Colony appearances: MEA: seven days, 25 ºC; and YES: seven days, 25 ºC. In MEA: g – h Stipes. i Hyphae anastomosis. j A cluster of stipes. K Conidia. l A cluster of conidia. Scale bars: a – d = 20 mm; e – f = 10 mm; g – i = 20 μm; j – l = 10 μm

Fig. 9

Penicillium heveae sp. nov. from Hevea brasiliensis (VIC47589). a – d Seven-day-old cultures, 25 °C, left to right; first row, all obverse, CYA, MEA, YES, CREA; second row CYA reverse, MEA reverse, YES reverse, CREA reverse. e – d Colony appearances: YES: seven days, 25 ºC; and MEA: seven days, 25 ºC. g – i Conidiophores and phialides (MEA). j Conidia. K Cluster of conidia. Scale bars: a – d = 20 mm; e – f = 10 mm; g – i = 20 μm; j = 10 μm; k = 5 μm

Fig. 10

Penicillium pseudomellis sp. nov. from Hevea brasiliensis (VIC49378). a – d Seven-day-old cultures, 25 °C, left to right; first row, all obverse, CYA, MEA, YES, CREA; second row CYA reverse, MEA reverse, YES reverse, CREA reverse. e – f colony appearances: MEA: seven days, 25 ºC; and YES: seven days, 25 ºC. g – i Conidiophores and phialides (MEA). j The apex of stipes and conidia. K Cluster of conidia. Scale bars: a – d = 20 mm; e – f = 10 mm; g – i = 20 μm; j – k = 10 μm