Caratteristiche biologiche di Punctularia atropurpurascens durante il suo sviluppo: studio mediante analisi morfologiche e molecolari
10.6092/issn.2531-7342/9574
Abbiamo riportato e descritto in precedenza la presenza del fungo saprotrofo (sub)tropicale Punctularia atropurpurascens nel Parco urbano di Villa Ada a Roma, nel Lazio. In questo studio vengono riportate ulteriori evidenze della sua presenza, due delle quali come parassita, a Roma. Punctularia atropurpurascens sebbene sia reputata una specie fungina abbastanza rara in Italia, nei siti in cui appare ha la capacità di espandersi con facilità, se i fattori climatici ed ambientali sono favorevoli. Una comparazione delle analisi morfologiche e molecolari ha dato informazioni suggestive per la biologia del fungo.  Inoltre, analisi di NMR in protone sul contenuto delle gocce rosse secrete dal fungo evidenziano la presenza di molecole relative al phlebiarubrone e di specifici lipidi che sono coinvolti in sistemi di trasduzione dei segnali cellulari.
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Amalia Ferretti
Italian Journal of Mycology
parasitic fungi
Biological characteristics of Punctularia atropurpurascens through morphological and molecular analyses during development
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Articles
2019-07-01
2019-06-21
2019-03-01
2020-01-20
39-49
Ital J Mycol
Amalia Ferretti
2019/07/01
10.6092/issn.2531-7342/9574
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University of Tartu
2531-7342
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Weiss RB (1992) The anthracyclines: will we ever find a better doxorubicin? Seminars in Oncology 19(6):670–686.
Biological characteristics of Punctularia atropurpurascens through morphological and molecular analyses during development
https://italianmycology.unibo.it/article/download/9574/9368
48
Knijn et al.                                                              Italian Journal of Mycology 48: 39-49 (2019)

DOI: https://doi.org/10.6092/issn.2531-7342/9574

Original paper

     Biological characteristics of Punctularia atropurpurascens
      through morphological and molecular analyses during
                            development
                                Arnold Knijn1, Irja Saar2, Amalia Ferretti1
1
Via Arno 36, 00198 Rome, Italy
2
Institute of Ecology and Earth Sciences, University of Tartu, Ravila 14A, 50411 Tartu, Estonia

Corresponding author e-mail: knijn_roma@hotmail.com
Received 10/04/2019; accepted 12/06/2019


Abstract
In this study, further evidences of the presence of the (sub)tropical Punctularia atropurpurascens in Rome are reported.
Besides its generally described saprotrophic behaviour, some of the new-found specimens revealed a parasitic ability of
the fungus. Moreover, although reputed a quite rare fungal species in Italy, P. atropurpurascens shows great capacity to
develop in its places of manifestation under favourable climatic and environmental conditions. A comparison of
morphological and molecular analyses of the fungus under various conditions gives suggestive information on the
underlying biology. Furthermore, proton NMR analyses of the biochemical composition of the red drops secreted by the
fungus showed the presence of lipids involved in cellular signal transduction pathways as well as phlebiarubrone-related
molecules.

Key words: phlebiarubrone; lipids; NMR analysis, Quercus spp.; ITS region; parasitic fungi


Introduction

Punctularia atropurpurascens (Berk. & Broome) Petch forms the genus Punctularia together with
the species P. strigosozonata (Schweinitz) P. H. B. Talbot, in the family Punctulariaceae and order
Corticiales (Larsson, 2007). Punctularia atropurpurascens is a purplish coloured, saprotrophic
fungus that grows on rotting wood, in prevalence deciduous. Its superior surface is fluffy,
membranous, waxy, with a purplish-brownish coloured central zone. The lower surface is adherent
to the growth substrate. An aqueous exudate in the form of reddish coloured droplets is often present.
The hymenium is found on the upper surface and it is distributed in a non-uniform manner. The
hyphal system is monomitic, made up of only generative hyphae, which appear branched, septate and
with clamp connections. Basidia produce four sterigma. The conidia, produced through sporogenesis,
are chained, subglobose or ellipsoid, with a violet brown colour (Martini, 2016).
       Recently, we reported the presence of P. atropurpurascens in the Villa Ada urban Park in
Rome, Latium (Knijn and Ferretti, 2018). This report referred to a specimen growing on a rotting
stump of Quercus suber L., thus showing a saprotrophic behaviour. The stump was contemporarily
colonised at its base by Meripilus giganteus (Pers.) P. Karst., Ganoderma lucidum (Curtis) P. Karst.
and Fomes fomentarius (L.) Fr. Here, we report further evidences of the presence of P.
atropurpurascens in Rome, with some specimens showing a parasitic capability, as well as findings
of a longitudinal study of this fungus.
       The morphological evolution of five specimens is described; also, microscopic analyses were
performed at different stages of the fungus’ life cycle as well as at different stages of the fungus’ age.
The biochemical content of red droplets secreted by P. atropurpurascens was investigated by NMR
analysis.
________________________________________________________________________________________________________________________
ISSN 2531-7342
Department of Agricultural and Food Sciences in partnership with the Italian Mycological Society (UMI)
The journal is hosted and maintained by ABIS-AlmaDL
© 2019 The Authors. This work is licensed under CC BY 4.0
Knijn et al.                                                                          Italian Journal of Mycology 48 (2019)




Materials and Methods

Samples
After the first basidioma described by Knijn and Ferretti (2018), two more specimens of P.
atropurpurascens were found in the same area of the Villa Ada urban Park called “Sughereta”
dominated by holm oaks and cork oaks. The hosts of these specimens were two living trees of Quercus
ilex L. already colonised by other lignicolous fungi (Fig. 1a). A fourth specimen was found outside
of the park at a distance of two kilometres on a trunk of Platanus × acerifolia (Aiton) Willd.
      In order to test P. atropurpurascens’ capacity of proliferation, a fifth specimen was obtained
by inoculating conidia into the knot of a cut branch of Q. cerris. Conidia were taken from the fungus
growing on the cork oak stump. The inoculated branch was then put in an external environment, far
from the site where conidia were collected. After a year and a half, in autumn 2018, P.
atropurpurascens had colonised the surface and the inside of the trunk and proliferated on some
leaves of Celtis australis L. that lay underneath the trunk (Fig. 1b). The specimens are itemised in
Table 1.

Table 1 - Description of the five samples of Punctularia atropurpurascens used in this study. The columns feature the
specimen codes in University of Tartu herbarium (Estonia) and ITS sequence codes in the UNITE database, the species
and status of their host, the dates of first observation (asterisks * indicate sites that had been frequently inspected also
before this date without observing the fungus) and of collection
Sample         Herbarium/UNITE codes      Host                                          Date of first    Collection date
                                                                                        observation
PA1            TU111531/UDB034596          Quercus suber                stump           19/10/2016       14/01/2018
PA2            TU114604/UDB035018          Platanus × acerifolia        stump           25/05/2018       29/05/2018
PA3            TU114603/UDB035019          Quercus ilex                 living tree     19/05/2018*      30/06/2018
PA4            TU114791/UDB039800          Quercus ilex                 living tree     17/10/2018*      01/11/2018
PA5            TU114792/UDB039785          Quercus cerris               stump           03/11/2018*      03/11/2018
                                           Celtis australis             leaves




Fig. 1 - Punctularia atropurpurascens growing on a living tree of Quercus ilex (a), on a Quercus cerris cut branch (b);
red droplets secreted by P. atropurpurascens (c); hyphae layer formed in saline solution (d)
      Moreover, several conidia were put in a suspension of sterilised saline solution (NaCl 0.9%,
EDTA 0.002%) at room temperature. After more than a month, hyphae had formed on the surface of
the suspension (Fig. 1d).

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Morphological analysis
Microscope images of the fungi were obtained with a digital camera AmScope (Irvine CA, USA)
MU500 mounted on an AmScope B490 microscope, operating at ×800 and ×2000 magnifications.
Samples were analysed dry mount and wet mount in water.
      Microscopical observations were made on fragments taken from the various specimens in the
feathery-felted state as well as a part in the crusty state and in the latter case from both the almost dry
centre and the still fluffy margins. Moreover, from the feathery-felted fungus, samples were taken
from the inside of a pore left by the exuded red droplets as well as from the external contour of the
pore. The sample in saline solution was analysed by microscopy at two intervals (after 20 days and
40 days).

Molecular analysis
Ribosomal DNA-based analysis was performed on all five specimens of the fungus in the frame of
the UNITE project (Kõljalg et al., 2013; https://unite.ut.ee/). DNA extraction, PCR amplification of
18S partial, ITS1, 5.8S, ITS2, 28S partial regions and sequencing were performed as in Voitk et al.
(2017). Sequences were aligned with the MUSCLE v3.8.425 algorithm (Edgar, 2004), in Aliview
1.25 software (Larsson, 2014) and fine-tuned manually. The Basic Local Alignment Search Tool
(BLAST 2.8.1+; Camacho et al., 2009) was used to assess sequence identity. The BLAST Ring Image
was generated with BRIG 0.95 software (Alikhan et al., 2011). For phylogenetic analyses, Bayesian
inference was applied as before (Knijn and Ferretti, 2018); phylogenetic trees were edited using
FigTree v1.4.3 (http://tree.bio.ed.ac.uk/). The final mean standard deviation of the separated
frequencies was less than 0.008.

Proton NMR analysis
The dried sample of red droplets secreted by the fungus at the stage shown in Fig. 1c and Fig. 3b was
dissolved in CDCl3 and proton 1D and 2D NMR spectra were collected. Proton NMR measurements
applying a single-pulse sequence have been performed on a Bruker (Billerica MA, USA) Avance 400
MHz WB (9.4 T) spectrometer at 27 ºC. In total, 6000 repetitions of 8k points were acquired and
FIDs were zero-filled to 32k points before transformation. A spectral width of 10 ppm was measured,
corresponding to 4006 Hz at the magnetic field of 9.4 T.

Results

Specimen findings
During the period of investigation (October 2016–March 2019), the weather in Rome has been
characterised by several dry periods, for instance from June to October 2017, as well as particularly
humid ones, for instance February-March 2018, as can be seen from Fig. 2. These climate dynamics
and the discovery of other specimens of P. atropurpurascens gave the opportunity to study this
species’ development in time under different climatic circumstances.
      The fungus was detected again in the “Sughereta” area of the Villa Ada Park in May 2018,
where a specimen was growing vertically on the trunk of a living holm oak tree (Fig. 1a) situated
about 70 meters from the trunk of the cork oak that had been previously observed. Already in October
2016, this tree was observed to be colonised at its base by M. giganteus and by Ganoderma adspersum
(Schulzer) Donk; moreover, in autumn 2018, groups of Armillaria gallica Marxm. & Romagn. were
growing on its trunk.
      A second observation of this species growing as a parasite occurred in October 2018 in the same
area, on a trunk of a holm oak tree, all around its circumference. This tree is situated in a relatively
open zone rather distant from the other colonized trees, a stream separated it from both the cork oak
and the other holm oak. Again, this tree was colonised by groups of A. gallica and other lignicolous
fungi at its base, causing the deterioration of the tree’s wood: some branches were evidently dry,

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Knijn et al.                                                                     Italian Journal of Mycology 48 (2019)




others still vegetating. In March 2019, about five months after the first appearance of P.
atropurpurascens, this tree had only dry branches.

                                   20

                                   18

                                   16

                                   14
                  Days with rain




                                   12
                                                                                                  2016
                                   10
                                                                                                  2017
                                    8                                                             2018

                                    6                                                             2019

                                    4

                                    2

                                    0
                                        1   2   3   4   5   6   7   8   9   10     11   12
                                                            Month

               Fig. 2 - Number of rain days (> 0.2 mm) per month for the period of investigation (data
               obtained from http://www.lineameteo.it/)

       Both observations can be considered as close to the first appearance of the fungus’ basidiomata
because the host trees are situated on a frequently inspected route. During the humid autumn of 2018,
P. atropurpurascens extended on the bark and inside the trunks, with its characteristic feathery-felted
morphology, exuding numerous reddish droplets (Fig. 1c).
       Interestingly, P. atropurpurascens was also found to be present outside of the Villa Ada Park.
Indeed, a specimen was observed on a trunk of Platanus × acerifolia (Aiton) Willd. along an avenue
tree-lined with platans (Viale Regina Margherita), distant as the crow flies about two kilometres from
the Park but only a few meters outside the large private Park of Villa Albani.

Maturation phases and morphological changes
The basidoma of P. atropurpurascens is stably present throughout the year, although its morphology
undergoes evident transformations (Fig. 3). In the first stage of maturation (Fig. 3a), the fungus
appears as sparse, light pink, fluffy tufts coming out of the clefts of the hosts bark. With time (Fig.
3b), the tufts spread and merge, becoming showy pink-coloured, while reddish droplets exude from
pores. This stage generally occurs mid-autumn, but can also happen as late as mid-spring. Then,
depending on humidity, the morphology of the fungus can transform in two different ways. If the
weather is dry (Fig. 3g), the red droplets disappear and the fungus turns purple while its consistency
becomes compact. When the weather is more humid stage 3b persists for a long time and with the
temperatures rising, the centre of the fungus dries to a brownish crust and the colour of the droplets
tend to rust (Fig. 3c). At the next stage (Fig. 3d) the fungus turns purple/blue at the centre while
staying pink at the edges. Successively (Fig. 3e), the fungus darkens to deep purple and at the final
stage (Fig. 3f) the fungus appears as black spots on the tree’s bark. In dry seasons, the transition from
stage 3g to 3f occurs directly in late winter or early spring.



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Knijn et al.                                                                   Italian Journal of Mycology 48 (2019)




Microscopical observations
Samples of all specimens were observed by microscopy, including those obtained from the evolution
of the conidia in suspension (Fig. 4). All samples had similar microscopical as well as morphological
appearances, in accordance with Martini (2016). The basidia seem to coincide in particular with the
crust state of P. atropurpurascens. On the other hand, conidia are prevalent when the fungus has a
feathery-felted morphology, that is, at the stadium of primary hyphae with the appearance of
ramifications (dendrohyphidia).




     Fig. 3 - Morphological changes undergone during the year by the basidiomata of Punctularia
     atropurpurascens. The long cycle follows stages a-b-c-d-e-f, however under dry circumstances the fungus can
     transform from stage b into g and directly into stage f


      The fungus, at the initial phase of growth on each substrate (for example the P.
atropurpurascens put in saline solution, de novo growth), is composed of hyphae without clamp
connections, since these appear in the hyphae with the formation of the secondary mycelium
(Stephenson, 2010). The clamp connections appear after several growth cycles, sometime after the
fungus P. atropurpurascens has colonised its host.
      Massive conidia were present inside the pores produced by the secreted reddish droplets, while
they were scarce at the margins, essentially formed by primary filamentous hyphae. The conidia
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Knijn et al.                                                                       Italian Journal of Mycology 48 (2019)




appear with a reddish intracellular content, probably due to pigments like phlebiarubrone and its
derivatives. Furthermore, conidia have hyaline as well as brownish violet cell walls, in particular with
the growth of the fungus. Moreover, at this point violet-brownish-coloured granulations become
noticeable on the hyphal filaments. At the first stages of growth, lipid droplets appear inside the
conidia.




Fig. 4 - a) Hyphae from Punctularia atropurpurascens in saline solution for 20 days; no clamp connections were visible.
b) Similar patterns were present in hyphae found the first time (17/10/2018) on Quercus ilex. c) Basidia in the fungus in
the crust state, from the same tree. d) Hyphae in saline solution for 40 days; clamp connections were visible. e) P.
atropurpurascens from Quercus suber; clamp connections were present. f, g) Conidia found in saline solution after 20
days (f) and 40 days, with red droplets present internally (g). Bar = 10 µm


Genetics
All five specimens were confirmed to be P. atropurpurascens by BLAST searches and phylogenetics
(Fig. 5). After the alignment process, the dataset resulted in five sequences with a length of 938 bp.
Nucleotide diversity was observed in the ITS1 (one SNP very close to the 5.8S region), ITS2 (three
SNPs) and LSU (four insertions) regions (Table 2). The phylogram obtained through Bayesian
inference grouped the five specimen in two clades (PA1, PA2 and PA3, PA4, PA5) with a node
supported by a posterior probability of 0.997 (Fig. S1).

           Table 2 - Single nucleotide polymorphisms (SNPs) in the genetic sequences of the P.
           atropurpurascens samples. Columns report obtained sequence length, BLAST identity and gaps
           between sample PA1 and the other four samples. “–” a gap existed in the reported locus, “·” no data
           Region                                                  ITS1 ITS2               LSU
           Position (bp)                                           204    477 516 529 842 894 900 928
           Sample    Accession No      Length    Ident    Gaps
                                       (bp)
           PA1       UDB034596         915       1        0        A      A    T      C    –     –   –    ·
           PA2       UDB035018         937       0.995    0.003    G      A    C      C    G     A   A    G
           PA3       UDB035019         930       0.992    0.003    G      G    C      T    G     A   A    –
           PA4       UDB039800         600       0.993    0        G      G    C      T    ·     ·   ·    ·
           PA5       UDB039785         938       0.993    0.003    A      G    C      T    G     A   A    G




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Knijn et al.                                                                        Italian Journal of Mycology 48 (2019)




Magnetic Resonance Spectroscopy
In order to confirm the presence of phlebiarubrone in the droplets secreted by the fungus, liquid was
collected in November 2018 when fluffy tufts appeared (Fig. 3b) and 1H NMR analysis was
performed. The results show that signals from terphenylquinone (Anke et al., 1986), although of low
intensity, are present in the aromatic region of the spectra (Fig. 6). Other resonances in the spectrum
are due to acyl chains from lipids and to sterols; moreover, well visible signals arising from inositol
can be observed, probably partly –O–phosphorylated (Gutierrez et al., 2007). Signals from double
bonds are absent, indicating that the lipid acyl chains are saturated, as can be found in lipids forming
the category of glycosyl inositol phosphoryl ceramides (GIPCs), while the sterol is related to
ergosterol (Dupont et al., 2012).




Fig. 5 - Phylogenetic tree with posterior probability values obtained through Bayesian inference based upon alignment of
the nuclear ribosomal ITS sequences reported in Table S1, identified by the associated species, accession number and
place of collection. The samples described in the article are indicated by an asterisk. Bayesian posterior probabilities are
reported on each branch. The final mean standard deviation of the separated frequencies was 0.0075


Discussion

Although in Italy only few reports from Sicily are available on the (sub)tropical species P.
atropurpurascens (Mondello, 2017), the results prove that, given favourable climatic and
environmental conditions, this fungus has the ability to proliferate easily in its sites of appearance. In
fact, P. atropurpurascens is present in the Villa Ada urban Park (Rome) and in a platan tree-lined
avenue, two kilometres distant from the other specimens. Notably, the fungus showed parasitic as
well as saprotrophic behaviour. In the “Sughereta” area of Villa Ada, both holm oak trees infected by
P. atropurpurascens were alive but previously hit by the colonisation of other parasitic and
saprotrophic fungi, while no healthy trees in the same area were affected. Evident presence of P.
atropurpurascens basidiomata was found on a living holm oak where in previous years a flourishing
cluster of M. giganteus had been noted at the base of its trunk. It is conceivable that when P.
atropurpurascens behaves as a parasite, its development is successive to the aggression of the tree by
other fungal parasites, thus previously weakened by its colonisation (Schwarze et al., 2000). No
reports in literature were found on a possible synergism between different parasitic fungal genera
with P. atropurpurascens.
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Knijn et al.                                                                    Italian Journal of Mycology 48 (2019)




      Remarkably, it was found that the cyclic morphological transformations of the fungus can
follow two distinct pathways in function of conditions of humidity, which seems an adapting strategy
to withstand drought stress. It would be interesting to see if this duality also occurs in the (sub)tropical
ambient where the fungus usually grows, however no data was found regarding this particular aspect.
Petch (1916) did mention two quite different forms of P. atropurpurascens depending on humidity,
describing the phases 3b and 3c in Fig. 3. It is striking that the morphological changes occurring in
P. atropurpurascens during its life cycle are more significant than the macroscopic differences with
the similar fungus Hypochnella violacea Auersw. Ex J. Schrot., alluding to a biological significance
of these transformations.




   Fig. 6 - One-dimensional 1H NMR spectrum in deuterated chloroform of a sample taken from dried red droplets
   exuded by Punctularia atropurpurascens. Signals arising from lipids’ acyl chains are present (about 0.9 and 1.3
   ppm), as well as from sterols (about 0.8, 1.1, 1.6 and 3.6 ppm) and inositol compounds (from 3.4 to 4.4 ppm). The
   aromatic region (high ppm) is shown as an insert, amplified by four to evidence signals from phlebiarubrone (7.5
   and 7.7 ppm)

       All samples of the various specimens showed a similar appearance at a morphological and
microscopical level. Our observations put in evidence that the morphological variations occurring
during growth cycle are not correlated with the host species, and neither with its saprotrophic or
parasitic behaviour. In fact, the morphological transformations are induced by climate dynamics. On
the other hand, the microscopical changes are related to growth cycle and age of the fungus.
       Complex biochemical pathways underlie variations in fungal gene expression, also in relation
with the specific nature and composition of its growth substrate (Skyba et al., 2016). It is reported
that in Fomitopsis pinicola (Sw.) P. Karst. gene expression is regulated at different stages of growth
in function of the genotype of the colonised substrate, regardless of its provenience (conifers or
hardwoods) (Baojun et al., 2018). The genes that are regulated by substrate composition, encode for

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Knijn et al.                                                                   Italian Journal of Mycology 48 (2019)




enzymes with known or potential function in rot decay, some of them are involved in redox reactions
with benzoquinones such as phlebiarubrone and its derivatives (Suzuki et al. 2006; Baojun et al.,
2018).
       Another question is what biochemical role phlebiarubrone and its derivatives play during
fungus’ development. It is reported that phlebiarubrone produced by P. atropurpurascens (Anke et
al., 1986; Gill and Steglich, 1987), as well as by other macromycetes (Järvinen et al., 2016), has
effects similar to inhibitors of DNA transcription, translation and replication in bacteria (Järvinen et
al., 2016). Phlebiarubrone is essentially a diphenylbenzoquinone or terphenylquinone, like some
DNA intercalating molecules (Di Marco et al., 1969; Hollstein, 1974; Weiss, 1992). Moreover,
phlebiarubrone shows weak antibacterial and cytotoxic activity (Calì et al., 2003).
       Proton NMR analyses performed on the reddish droplets secreted by P. atropurpurascens when
basidiomata appeared as fluffy tufts (Fig. 3b), underline that the molecular content is not casual. In
fact, the droplets are composed of derivatives of phlebiarubrone together with lipid molecules, most
probably structured as micelles. The lipids are sterols related to ergosterol and inositol containing
lipids, the major part with saturated acyl chains, as found in lipids forming the category of glycosyl
inositol phosphoryl ceramides (GIPCs) (Guan et al., 2009; Burè et al., 2014). Both kinds of molecules
are known to be involved in various signals transduction mechanisms through the cell membrane in
fungi and plants (Rodrigues et al., 2000; Gutierrez et al., 2007; Alvarez-Vasquez et al., 2011;
Gronnier et al., 2016) matching those in mammalian cells (Ferretti et al., 2003). Moreover, it was
shown that the ergosterol biosynthetic pathway increases survival in fungi during the drying-wetting
cycling periods (Dupont et al., 2012). The cycling periods are also undergone by P. atropurpurascens,
being persistent throughout the year on the colonised host (Knijn and Ferretti, 2018). These processes
could be related to the cyclic morphological transformations through two distinct pathways in
function of the climatic conditions that have been described.
       Overall, these findings suggest that the molecules secreted by P. atropurpurascens are not
merely the guttation of an ensemble of aqueous substances, but indicate a biological role exists for
the secretion of this kind of droplets and their components by the fungus. Therefore, P.
atropurpurascens can be proposed as a model for signalling molecules’ studies on how its compounds
could modulate the growth and survival of fungi and conidia (Brown and London, 2000; Guan et al.,
2009).

Acknowledgments
We would like to thank Dr. Sveva Grande and Dr. Alessandra Palma from the Istituto Superiore di Sanità for performing
1
 H NMR measurements. Irja Saar was supported by the Estonian Research Council (IUT20-30), the European Regional
Development Fund (Centre of Excellence EcolChange).


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