Phylogeny and nucleotide variation
The alignment resulted in 2,649 characters, which included gaps in which the ITS locus contributed 580 characters and CAM, RPB2 and TUB2 contributed 559, 978 and 532, respectively. The best model adopted by PAUP*4.0 for BI data for all loci was GTR+I+G, while for the ML analyses the best fit model was TIM2e+I+G4 for ITS and TNe+I+G4 for CAM, TUB2 and rpb2. The ML and BI analyses revealed that all isolates in the present study were grouped in the ser. Erubescentia species. The isolates INPA-AP10 and INPA-AP10a clustered with P. labradorum, while isolates INPA-AP07 and INPA-AP25 formed a sister group with P. rubidurum, with 87 bootstrap support in the ML analysis and 0.51 of PP in the BI analysis (Fig. 1).
The nucleotide variation between closely related species for INPA-AP07 and INPA-AP25 showed a total of 32 nucleotide differences (ND) in relation to P. rubidurum (22 = CAM, 7 = TUB2, 3 = ITS); 39 ND in relation to P. guttulosum (25 = CAM, 12 = TUB2, 2= ITS); 38 ND in relation to P. pimiteouiense (29 = CAM, 8 = TUB2, 1 = ITS) and 74 ND in relation to P. menorum (46 = CAM, 16 = TUB2, 12 = ITS). Furthermore, INPA-AP07 and INPA-AP25 showed 15 single nucleotide polymorphisms (SNPs) (9 = CAM, 5 = TUB2, 1 = ITS) (Fig. 2). These data, together with the morphological analysis, indicate that INPA-07 and INPA-AP25 represent a new fungal species, named here as Penicillium amapaense.
Taxonomy
Penicillium amapaense I. J. S. Silva; T. F. Sousa and G. F. Silva, sp. nov. (Mycobank: MB842234); Figure 3; Genus: Penicillium, section: Exilicaulis, Series: Erubescentia.
Etymology: Refers to the Amapá state, Brazil, which is the location where the type species was isolated.
Type: BRAZIL. Amapa, sediments of Amazon River, I. J. S. Silva, T. F. Sousa and G. F. Silva, holotype: INPA-AP25. Sequences are deposited in GenBank under accession numbers: ITS = OL764382, CAM= OL782584, TUB2= OL782590.
Culture characteristics: Colonies grown at 25°C for 7 days on PDA medium have an average diameter of 14 mm. The edges have white aerial mycelium (ISCC-NBS 263) with a yellowish gray color in the center (ISCC-NBS 93), velvety, regular edges, presence of abundant exudate and bright yellow soluble pigment (ISCC-NBS 83), the reverse has a slightly grooved colony center, with bright yellow coloration (ISCC-NBS 82) and bright yellow edges. YES: 17 mm diameter; front: white aerial mycelium (ISCC-NBS 263), velvety with high center, highly grooved, irregular edges, absence of exudate and soluble pigment; reverse: highly grooved, dark brown (ISCC-NBS 56), edges bright greenish yellow (ISCC-NBS 97). MEA: 15 mm diameter; front: aerial mycelium white on the edges and yellowish gray on the center, velvety, smooth, regular edges, presence of bright greenish yellow exudate, absence of soluble pigment; reverse: slightly grooved and strong yellowish brown (ISCC-NBS 74) in the center and bright yellow around the edges. OA: 14 mm diameter; front: aerial mycelium white (color code) and cottony and elevated in the center of the colony, hyaline and regular borders, presence of colorless exudate and absence of soluble pigment; reverse: smooth, light yellow color (ISCC-NBS 86). CYA: 12 mm diameter; front: white, velvety mycelium, raised center and slightly grooved, irregular edges, absence of exudate, presence of soluble strong orange pigment (ISCC-NBS 50); reverse: smooth, strong yellowish brown center (Fig. 3a, 3b, 3c). In addition to the conditions described above, the strain was analyzed at temperatures of 30°C and 37°C, and showed the same characteristics mentioned above, with the exception of the size of the colony, which presented sizes of 15 mm and 5 mm, respectively.
Micromorphology: The conidiophores have stipes that measure 9.6-20.6 µm (M = 16.4 µm) and are strictly monoverticillate and vesiculate, containing solitary phialides (Fig. 3f) or 3-7 phialides per conidiophore that measure 2 – 8.5 µm (M = 5.8 µm) \(\times\) 2.0 – 4.2 µm (M = 2.8 µm) (Fig. 3i). The conidia have a spiny ellipsoid shape, 1.3 – 2.0 (M = 1.5) \(\times\) 1.1 – 11.8 (M = 1.3) (Fig. 3d).
Notes
Penicillium amapaense sp. nov. differs from Penicillium rubidurum by having a smaller stipe length and due to its ability to grow on CYA medium.
Dual-culture antagonistic activity of P. labradorum and P. amapaense isolates
Isolates INPA-AP10 (Supplementary Information SI 6) and INPA-AP10a (Supplementary information SI 7) were identified based on morphological and molecular characters as P. labradorum, and isolates INPA-AP25 (Fig. 3) and INPA-AP07 (Supplementary Information SI 5), herein characterized as P. amapaense sp. nov., were evaluated for antibiosis mechanisms against 12 phytopathogens.
In all, eleven out of the twelve phytopathogenic fungi tested presented inhibition of mycelial growth for at least one of the four Penicillium isolates obtained in this study (Fig. 4a). Among the phytopathogenic fungi tested, the isolates of P. amapaense and P. labradorum showed potential for inhibiting mycelial growth of up to 49 and 58%, respectively. Among the phytopathogenic fungi inhibited by all the Penicillium isolates, C. guaranicola presented a mycelial growth inhibition rate (IR) that ranged from 34.28 to 58.53%, Ps. gilvanii ranged from 44.10 to 58.09%, and Fusarium sp. ranged from 21.42 to 48.88%, while F. decemcellulare ranged from 32.22 to 47.77% (Fig. 4a). Some phytopathogenic fungi were not inhibited by all the Penicillium isolates; C. spathulatum and Colletotrichum sp. were not inhibited by the P. amapaense isolate INPA-AP07, C. siamense was not inhibited by the P. amapaense isolate INPA-AP25, and C. scovillei was not inhibited by the P. labradorum isolate INPA-AP10a. Conversely, N. formicarum was inhibited only by the P. labradorum isolate INPA-AP10, while none of the four Penicillium isolates showed inhibition of mycelial growth against Rhizoctonia sp. (Fig. 4a).
Analysis of secondary metabolites
The investigation of the secondary metabolites obtained from the different strains was carried out manually via the analysis of accurate mass, fragmentation profile and specific annotations in the databases used. In the process of chemical dereplication of extracts, the isolate P. labradorum INPA-AP10 stood out for producing the largest number of compounds previously identified (Fig. 5). In total, fourteen compounds were detected, which corresponded to the polyketide subclasses of the curvularins (compounds 1a, 1b, 1c, 1d and 2) and presented as accurate mass m/z 307.1190, m/z 307.1183, m/z 307.1169, m/z 307.1181, m/z 305.1497 and anthraquinones (compounds 3, 4 and 5) with m/z 299.0198, m/z 318.9999, m/z 332.9801 (Table 2).
In general, the P. labradorum strains presented a similar metabolic profile, but with the absence of anthraquinones in the extract obtained from the isolate INPA-AP10a. The specimens of the species P. amapaense (INPA-AP25 and INPA-AP07) also showed high similarity in the compounds produced by the two strains. Coincidentally, the production of anthraquinones was detected only in the P. amapaense isolate INPA-AP25, with the most significant distinction found between the compounds produced by this strain and P. amapaense INPA-AP07. Interestingly, when comparing the metabolic profiles of P. labradorum and P. amapaense species, the main metabolic difference occurred in the production of curvularins. As seen in Fig. 5, none of the isomers referring to the accurate mass of m/z 307 (1a-1d isomers) were identified in the P. amapaense strains, while the production of compound 2 was significantly observed (Fig. 5).
The analysis of the LC-MS data using the GNPS platform indicated that peak 1 is the curvularin, 5,13,15-trihydroxy-9-methyl-10-oxabicyclo[10.4.0]hexadeca-1 (12),13,15-triene-3,11-dione (Table 2). The cosine value of 0.92 was obtained when comparing the MS/MS spectra for compound 1a and for curvularin, which was indicated by the platform, thus suggesting high similarity in the fragmentation profile. This data is corroborated by the comparison of the spectra (Supplementary Information SI 8). The molecular formula C16H20O6, obtained from the accurate mass of compound 1a (Supplementary Information SI 9), is also in accordance with the curvularin indicated on the GNPS platform. Furthermore, for the remaining peaks referring to the possible curvularin isomers (1b-1d), lower values of cosine were obtained, suggesting other possible identities for them; all are found in the Supplementary Information SI 14. The query of the molecular formula C16H20O6 was performed in other databases (see Table 1) and indicated possible hits that correspond to other known curvularins. The hits obtained can be seen in Fig. 5b.
The molecular formula C16H22N2O4, suggested for the accurate mass m/z 305.1490 (2) (Supplementary Information SI 10), was consulted in the databases used and no hit was found, thus suggesting that it is a metabolite that has not yet been reported in the literature. Although there are two nitrogen units in the molecule, there is evidence that this compound has a biosynthetic origin that is related to the class of curvularins, due to the similarity in retention time and the observed hydrogen deficiency index (see Table 1) and, mainly, to the MS/MS spectrum when compared to that of compound 1a (Supplementary Information SI 11).
Regarding the anthraquinones observed in the extracts of P. labradorum INPA-AP10 and P. amapaense INPA-AP07, data processing on the GNPS platform and database consultation allowed the identification of emodic acid for compound 3, 5-chloro-ω-hydroxyemodin for compound 4 and 2-chloroemodic acid for compound 5. In all cases, the molecular formulas were obtained with low values of deviation from the respective exact masses (see Table 2), as well as the observed isotopic profile for 4 and 5 (Supplementary Information SI 12 and SI 13), which corroborated in confirming the identity of the compounds (Figs. 5a and 5b).
Figure 5a Penicillium labradorum and Penicillium amapaense extracted ion chromatograms of m/z 307.1181; m/z 305.1501; m/z 299.0191; m/z 319.0009, m/z 333.9880, with deviation of ± 0.005 u obtained for the INPA-AP25, INPA-AP07, INPA-AP10 and INPA-10a strains. Peaks comprising substances that could not be annotated were referred to as (*) unknown compounds. b Chemical structures of annotated polyketides: curvularin (1a), emodic acid (3), 5-chloro-ω-hydroxyemodin (4) and 2-chloroemodic acid (5).
Table 2
Chemical annotation of the LC-MS analysis of Penicillium labradorum and Penicillium amapaense sp. nov.
Peak ID
|
rt (min)
|
Accurate mass (m/z)
|
Exact mass
|
Molecular formula
|
Deviation (ppm)
|
IDH
|
Putative hit
|
1a
|
7.5
|
307.1190
|
307.1181
|
C16H20O6
|
2.6
|
7
|
Curvularin
|
1b
|
8.5
|
307.1183
|
307.1181
|
C16H20O6
|
0.6
|
7
|
(1a) isomer
|
1c
|
9.5
|
307.1169
|
307.1181
|
C16H20O6
|
3.9
|
7
|
(1a) isomer
|
1d
|
10.6
|
307.1181
|
307.1181
|
C16H20O6
|
-0.1
|
7
|
(1a) isomer
|
2
|
7.5
|
305.1490
|
305.1501
|
C16H22O4N2
|
-3.6
|
7
|
no hit
|
3
|
10.1
|
299.0198
|
299.0191
|
C15H8O7
|
-0.3
|
12
|
Emodic acid
|
4
|
10.7
|
318.9999
|
319.0009
|
C15H9O6Cl
|
-3.1
|
11
|
5-Chloro-ω-hydroxyemodin
|
5
|
11.3
|
332.9801
|
333.9880
|
C15H7O7Cl
|
-0.3
|
12
|
2-Chloroemodic acid
|
Enzymatic activity
The production of amylase, cellulase and siderophores were detected in all isolates of P. amapaense and P. labradorum. Only in Penicillium amapaense (INPA-AP07 and INPA-AP25) was it able to solubilize phosphate. No enzymatic activity was observed for chitinase, lipase and protease for all isolates analyzed (Table 3, Fig. 6).
Table 3
Enzymatic activity and in vitro physiological traits for plant growth promotion of Penicillium amapaense sp. nov. and Penicillium labradorum.
|
P. amapaense INPA-AP07
|
P. amapaense INPA-AP25
|
P. labradourum INPA-AP10
|
P. labradrourum
INPA-AP10a
|
Amylase
|
+
|
+
|
+
|
+
|
Cellulase
|
+
|
+
|
+
|
+
|
Chitinase
|
-
|
-
|
-
|
-
|
Lipase
|
-
|
-
|
-
|
-
|
Protease
|
-
|
-
|
-
|
-
|
Siderophore
|
+
|
+
|
+
|
+
|
Phosphate solubilzation
|
+
|
+
|
-
|
-
|
+ Halo formation, - No halo formation. |