10.6092/issn.2531-7342/15373
Under natural conditions in Spain, the fruiting bodies of Pleurotus eryngii var. eryngii, a basidomycete of great economic and gastronomic importance, appears at the base of the stem of (mainly) Eryngium campestre, surrounded by the plant’s dried-out basal leaves. Traditionally collected in the wild of Mediterranean areas, this fungus is now cultivated all over the world for its culinary and even medicinal properties. However, controversy exists regarding its lifestyle. Some authors indicate it is a saprotroph, while others suggest it is a weak or even virulent parasite that causes male sterility in E. campestre. The present work aims to clarify whether P. eryngii behaves as a saprotroph, a weak or virulent parasite to E. campestre. The soil beside the roots of naturally growing E. campestre plants was inoculated with a commercial strain of P. eryngii, and the trophic mode of the fungus recorded. Soil and root samples were subjected to molecular analysis to confirm the absence/presence of the fungus. The death of the plants and the molecular analysis showed P. eryngii is a facultative necrotrophic parasite of E. campestre in natural conditions and a virulent parasite in greenhouse.
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The king oyster mushroom <em>Pleurotus eryngii</em> behaves as a necrotrophic pathogen of <em>Eryngium campestre</em>
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Ital J Mycol
José Luis Manjón
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Department of Life Sciences (Botany area), University of Alcalá, 28871-Alcalá de Henares, Madrid, Spain
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Zervakis G, Venturella G, Papadopoulou K (2001) Genetic polymorphism and taxonomic infrastructure of the Pleurotus eryngii species-complex as determined by RAPD analysis, isozyme profiles and ecomorphological characters. Microbiology 147:3183–3194. https://doi.org/10.1099/00221287-147-11-3183
The king oyster mushroom <em>Pleurotus eryngii</em> behaves as a necrotrophic pathogen of <em>Eryngium campestre</em>
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                IJM - Italian Journal of Mycology
                ISSN 2531-7342 - Vol. 52 (2023): 22-31
                Journal homepage: https://italianmycology.unibo.it/

Research article

       The king oyster mushroom Pleurotus eryngii behaves as a
            necrotrophic pathogen of Eryngium campestre

                                  Juan R. Carlavilla, José L. Manjón
Department of Life Sciences (Botany area), University of Alcalá, 28871-Alcalá de Henares, Madrid, Spain

Corresponding author e-mail: josel.manjon@uah.es



Article info
Received 16/08/2022; accepted 28/12/2022
https://doi.org/10.6092/issn.2531-7342/15373

Abstract
Under natural conditions in Spain, the fruiting bodies of Pleurotus eryngii var. eryngii, a basidomycete of
great economic and gastronomic importance, appears at the base of the stem of (mainly) Eryngium campestre,
surrounded by the plant’s dried-out basal leaves. Traditionally collected in the wild of Mediterranean areas,
this fungus is now cultivated all over the world for its culinary and even medicinal properties. However,
controversy exists regarding its lifestyle. Some authors indicate it is a saprotroph, while others suggest it is
a weak or even virulent parasite that causes male sterility in E. campestre. The present work aims to clarify
whether P. eryngii behaves as a saprotroph, a weak or virulent parasite to E. campestre. The soil beside the
roots of naturally growing E. campestre plants was inoculated with a commercial strain of P. eryngii, and the
trophic mode of the fungus recorded. Soil and root samples were subjected to molecular analysis to confirm
the absence/presence of the fungus. The death of the plants and the molecular analysis showed P. eryngii is a
facultative necrotrophic parasite of E. campestre in natural conditions and a virulent parasite in greenhouse.

Keywords
Apiaceae, basidiomycetes, trophic mode, molecular analysis



Introduction

Pleurotus eryngii (DC.) Quél. – known as the king oyster mushroom in the USA and UK, “seta
de cardo” in Spain, “pleurote du panicaut” in France, “fungo cardoncello” in Italy, and “Braune
Kräuter-Seitling” in Germany – grows in association with different members of the family Apiaceae
(Umbelliferae) when humidity and temperature conditions are favourable. In Spain, P. eryngii var.
eryngii (DC.) Quél. is commonly seen in association with Eryngium campestre, a plant found across
Central and Western Europe, North Africa, the Middle East and the Caucasus, as well as in North
America and Australia, where it is an introduced species. Between spring and summer, E. campestre
is encountered in flat, arid areas, along roadsides, in meadows and on uncultivated land, from sea
level up to 1,900 m asl. As perennial hemicryptophyte, its aerial part usually detaches after fruit
formation, facilitating the dispersal of the seeds as it is blown around by the wind. However, the
storage root remains anchored in the ground where, at its top, the buds are protected by leaf bases,
from which new aerial parts grow in the following year.

Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and maintained by ABIS-AlmaDL. © 2023 The Authors. This work is licensed under CC BY 4.0
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       The relationship between P. eryngii and E. campestre and other umbellifers is controversial.
Some authors indicate the fungus to be a saprotroph, while others suggest it is a weak or even virulent
parasite that causes male sterility in E. campestre. For example, Hilber (1982) described P. eryngii
as a parasite of the roots of members of the family Apiaceae and demonstrated its pathogenicity – at
least in the laboratory – through mycelium inoculations of the roots of different wild and cultivated
umbellifers. Urbanelli et al. (2002) found P. eryngii and P. eryngii var. ferulae (Lanzi) Sacc. to be
weak parasites, although they indicate the former to be largely saprophytic and the latter to be a
pathogen. Zervakis and Balis (1996) described P. eryngii as a weak parasite affecting the roots of
umbellifers in the northern hemisphere. Johansson et al. (1999) studied the presence of laccases
in putatively pathogenic P. eryngii, as did Punelli et al. (2009). Serino et al. (2013) proposed that
the species shows facultative biotrophic growth. Following laboratory-based inoculations into E.
campestre, Lanau-Galceran (2014) described it as a parasite of the roots of members of the family
Apiaceae. Similarly, Cailleux and Joly (1987) indicated P. eryngii to be a parasite of the roots of E.
campestre and to be associated over time with “cinéique épidémiologique des ronds de sorcières”,
“fairy rings” or “false mycorrhizal rings”. Joly et al. (1990) found P. eryngii to cause male sterility
in host plants and proposed a model in which the introduction of P. eryngii to a population of E.
campestre leads to the systematic destruction of susceptible plants. The present work aims to clarify
whether P. eryngii var. eryngii behaves as a saprotroph, a weak or virulent parasite to E. campestre
under natural conditions, in greenhouse and in vitro culture.

Materials and Methods
Area of study
The location chosen to carry out the field experiments is the External Campus of the University of
Alcala, on the outskirts of the city of Alcala de Henares (Madrid, Spain) between two alluvial terraces
of the Henares River. On these terraces, there are meadows and pastures from the degradation of
the original vegetation of the area. Nitrophilous plants abound (graminoids and hemicryptophyte
vegetation), among them a high abundance of E. campestre and Foeniculum vulgare. The first terrace
produces P. eryngii. In contrast, the second terrace, which is located 10 meters above and is less
humid, is characterized by the absence of P. eryngii and it is considered a P. eryngii-free field plot.

Pleurotus eryngii strain
Three strains were used in this study. Two strains were isolated from commercial basidiocarps bought
in Mercamadrid (Madrid, Spain) and labelled as “R. Álvarez (León)” and “Laumount”; another strain
was isolated from the fruiting body harvested in the study area and labelled as “wild strain”. In 1998,
in vitro cultures were performed in malt extract agar and maintained in culture on the same substrate
until the experiments were carried out. The mycelium for inoculation was cultured and expanded
as grain spawn on sterilized (120 oC, 90 min) hydrated barley grain following previously described
methods (Pérez et al., 1996; Manjón et al., 2004).

Field experiments
To examine the trophic mode of P. eryngii in E. campestre, 30 plants growing naturally in the P.
eryngii-free field plot were selected, and their positions marked with a numbered metal tag during
March 2005 (Supplementary Fig. S1). Fifteen of these plants, growing in one area of this plot, were
chosen for inoculation (Supplementary Fig. S2) with P. eryngii (test plants); the remaining 15, in
another area of the field plot, were not inoculated (control plants). A 20 cm deep hole was dug next
to all 30 plants using a sterilized auger. The holes next to the test plants were inoculated with the
grain spawn colonized by the isolated strain of P. eryngii (R. Álvarez, León) before being refilled

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with part of the same extracted soil. An approximate volume of 7 × 7 × 14 cm of grain spawn
colonized by the fungal mycelium of P. eryngii (Supplementary Fig. S3) was used to inoculate each
plant (Supplementary Fig. S4). The holes near the control plants received no such inoculum or any
additional substrate; the extracted soil was simply replaced to simulate stress conditions (possibility
of breakage of lateral thin roots) to the inoculated plants. All plants were monitored through to the
following spring for signs of fungal infection, including the production of fruiting bodies, and the
appearance of symptoms such as chlorosis, root degradation and lack of regrowth of the aerial parts
after the appearance of fruit bodies.
       The experiment was replicated in the spring of 2013, involving 20 E. campestre plants – 10 test
plants and 10 controls – and again in 2014, involving five test plants that served as control plants in
the 2013 round, plus five again used as controls. Fruiting bodies of the inoculated strain and of the
wild strains were harvested in terrace 1.
       An ITS molecular analysis (see below) of soil and roots samples was carried out to confirm
the absence/presence of P. eryngii before and after the inoculation treatments. In addition, in order
to confirm that the inoculated plants were effectively infected with the inoculated strains and not by
a wild strain, subregion 2 of the intergenic spacer (IGS2), (comparing DNA sequences and by RFLP
analysis), the most polymorphic region of P. eryngii ribosomal DNA, was examined (Martin et al.,
1999; Zhang et al., 2006) (see below).

Pleurotus eryngii mycelial development in soil
The mycelial distribution of P. eryngii around E. campestre plants and its involvement in formations
like “fairy rings” was examined in natural populations of E. campestre in 2015-2016 in the P. eryngii
field plot of the External Campus of the University of Alcala. Soil samples (250 g; n = 38) were taken
using a sterilized auger (Supplementary Fig. S5) at a depth of 20 cm from inside and outside of a
random “fairy ring” during the spring of 2015, at flowering and when the soil was damp. All samples
were stored separately, transported to the laboratory, and dried at 30 ºC overnight before storing at
-20 ºC until ITS analysis.

Greenhouse experiments
Adult plant inoculation
Taproots from adult plants of E. campestre in vegetative standstill were collected under natural
conditions in March 2018 (Supplementary Fig. S6). The terminal root apex was paraffin-embedded,
and the taproots were kept at 4 ºC until the next day, when they were planted in raised pots with legs
(2,200 ml capacity) to avoid potential contamination (Supplementary Fig. S7). The substrate used
was sterilized soil (autoclaved at 121 ºC for 60 min) from the P. eryngii-free field plot. The taproots
were cultivated for one month, until the plants generated new well-developed basal leaves. Then 21
plants were inoculated with P. eryngii “Laumount” strain during April 2018. The equal number of
uninoculated taproots was chosen as control plants. The pots were placed in the greenhouse of the
University of Alcalá.

Seedling inoculation
Seeds were collected from random specimens of E. campestre from P. eryngii-free field plot between
April and June 2018. These seeds were observed under a magnifying glass to discard those that
presented deterioration or incomplete development. For in vitro culture, the seeds were surface-
sterilized, soaked for 5 min in an aqueous solution of 0.5% sodium hypochlorite, and then rinsed
three times with sterile distilled water and air-dried.



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      Eryngium campestre seeds were grown in containers (26 × 26 × 8 cm). These seeds were sown
semi-concentrically with sterilized substrate from the P. eryngii free field plot and cultivated under
natural conditions. Subsequently, they were inoculated with the wild strain of P. eryngii. Similarly, a
container with control seeds was prepared (Supplementary Fig. S8).

In vitro experiments
Twenty E. campestre seeds were transferred into 20 mL test tubes, where they were grown under
aseptic conditions in MS liquid medium (Murashige-Skoog, 1962) and vermiculite. These seeds were
grown in controlled conditions until cotyledon development. Once this development was achieved,
the seedlings were transferred into a Petri dish with MS agar. Pleurotus eryngii mycelium of the strain
was placed in the middle of the Petri dish, placing the E. campestre seedling at one end and a drop
of sterile distilled water at the other end as a negative control (Supplementary Fig. S9). All seedlings
were monitored for 15 days for signs of fungal infection, including symptoms such as chlorosis, root
degradation and a lack of regrowth of the aerial parts.

Molecular analyses
Samples (100 mg) of roots, soil and mushroom tissues (taken in the region of the stipe) were collected
and preserved at -20oC until analysis. DNA extraction was carried out using the PowerSoil DNA
Isolation Kit (DNeasy) following the manufacturer’s recommendations with all the above sample types.
PCR was then performed in 25 µL volume using the IllustraTMpuReTaqReady-To-GoTM PCR Beads
Kit (GE Healthcare Life Sciences), a MultiGene Optimex Thermal Cycler (Labnet International).
Primers used in this study belonged to the ITS and IGS2 regions of ribosomal DNA.
       ITS region in soil samples was amplified using the primers ITS1F and ITS4 firstly, and in a
second PCR with PeryITSF and PeryITSR specific primers (Supplementary Table S1). Regarding the
PCR program used, the initial denaturing step involved a temperature of 94 ºC for 4 min; this was
followed by 35 cycles at 94 ºC for 30 s, Tm for 30 s and 72 ºC for 90 s, and a final extension step at
72 ºC for 10 min. IGS2 analysis in basidiocarp samples was amplified using the primers 5SRNAR and
invSR1R and the IGS1_PeF and IGS2_PeR specific primers. The PCR reaction conditions were as
follows: an initial denaturing cycle at 94 ºC for 4 min, followed by 40 cycles at 94 ºC for 1 min, Tm
for 1 min, and 72 ºC for 3 min, with a final extension step at 72 ºC for 7 min.
       For confirmation of the identity of the infecting fungal strain, the IGS2 amplicons obtained
were subjected to restriction length fragment polymorphism (RLFP) analysis by digesting them with
the restriction enzyme BtgI (New England Biolabs), following the supplier’s instructions. Amplicons
were then visualized in 1% agar gels stained with GelRed (Biotum) and purified using the UltraClean
PCR Clean-up DNA Purification Kit (MoBio Laboratories Inc). Finally, they were sequenced by
the Molecular Biology Unit of the University of Alcalá. Each DNA sequence was assembled and
edited manually as required. Sequence alignments were performed using the MUSCLE algorithm
in Mega 7.0. (Kumar et al., 2016). IGS2 sequences derived from this study were assigned to the
following GenBank accession numbers: MF498584, MF498585, MF498586, MF498587, MF498588
and MF498589.

Results

Field experiments
After setting the first experiment at the external campus of the Alcala University, the inoculated plants
became chlorotic and died after two weeks, showing a mortality rate of 100% for the inoculated plants
and 6.7% for the control plants. Fruiting bodies of P. eryngii were collected from all the inoculated

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plants during the following autumn, in November 2005. The basidiocarps were always found on the
inoculated roots (Table 1).
      In 2013 and 2014, the experiment was repeated. In 2013, the inoculated plants quickly showed
signs of chlorosis and died within 15 days. The control plants grew normally. The mortality rate was
100% for the inoculated plants and 10% for the control plants. The autumn of 2013 was hot and dry,
and the inoculated plants produced no mushrooms during that season; however, they did produce them
in the spring of 2014. Dead control plants never produced basidiocarps. In the 2014, the inoculated
plants became chlorotic at 12 days and died 15 days later. The controls remained asymptomatic.
Mortality rate in inoculated plants was 100%, and in control plants was 20%. Mushrooms were
collected from the test plants in October and November 2014 (Table 1).

      Table 1 – Results obtained during these inoculations.
      Year           Number of plants examined per year        Mortality rates
                     Inoculated                 Control        Inoculated        %      Control     %
      2005           15                         15             15                100    1           6.7
      2013           10                         10             10                100    1           10
      2014           5                          5              5                 100    1           20


      Total          30                         30             30                100    3           10

      The results of the ITS analysis of soil samples confirmed the absence of the fungus before
introducing the P. eryngii inoculum in the plants selected for the 2013 and 2014 experiments.
Subsequently, the ITS analysis detected the presence of the fungus in soil and root samples once the
plants were inoculated and reconfirmed its absence in control plants by ITS analysis.
      The IGS2 analysis results confirmed that the treated plants had all been infected by the inoculated
commercial P. eryngii strain (R. Álvarez, León). A band revealing a length of 1,400 bp was always
obtained for this commercial strain, being absent in the wild strain. Wild fruiting bodies samples
provided IGS2 lengths of 2,000-4,000 bp (Supplementary Fig. S10a). In one sample (line 8), an extra
band revealing a domain about 3,000 bp long was recorded. The RFLP profiles for the inoculated
plants produced a different profile to the wild fungal samples (Fig. S10b), with fragments about 700
and 200 bp (lines 2-6), instead of 750 (line 7), and 650, 500 and 200 bp (line 8). Finally, the IGS2
sequences from fruit bodies of inoculated plants were compared with the original inoculum. All the
sequences were 100% identical.

Pleurotus eryngii mycelial development in soil
To confirm the “fairy ring” formation of P. eryngii origin, the distribution of its mycelium in the
soil was investigated. Figure 1a shows the distribution of seven E. campestre plants that would be
involved in a “fairy ring”, while the results of the soil samples that were positive and negative for the
presence of P. eryngii appear in Figure 1b. Positive soil samples were detected within the circle and
around E. campestre plants, but their distribution was not homogeneous; a bias was seen towards the
center and right of the circle (the positive samples recorded at the three sampling points outside the
top left of the ring probably represent the front of a different mycelium). During the spring of 2016,
it was detected that plants number 5 and 6 died due to P. eryngii.




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 Fig. 1 – a) Distribution of seven E. campestre forming a “fairy ring”. b) Results of P. eryngii soil samples in an E.
 campestre-involving fairy ring. Red = positive; White = negative.

Greenhose experiments
Adult plant inoculation
To confirm the results obtained in natural conditions additional experiments were carried out on adult
plants in greenhouse. Most of the plants inoculated with P. eryngii died (85.7% mortality), in a time
interval between 1-2 months. The symptoms (chlorosis) appeared 15 days after inoculation, at the
same time as the beginning of the fructifications (Supplementary Fig. S11). The secondary and tertiary
roots generated from the taproot were completely invaded (Supplementary Fig. S12) by secondary
(dikaryotic) white mycelium (Fig. 2a) of the inoculated strain. The absorbent roots had clear signs
of infection and cortical cell penetration (Fig. 2b) and the accumulation of coloured gums (Fig. 2c).
In addition, taproots from inoculated plants also showed the accumulation of coloured gums (Fig. 2d
and Supplementary Fig. S13) and clear signs of fungal colonization and necrosis (Supplementary Fig.
S14). These signs were absent in the control plants.

Seedling inoculation
One month after inoculation with P. eryngii, the seedlings showed 42% mortality, regardless of the
distance from the inoculation point (Fig. 3a) with the control (Fig. 3b).

In vitro trials
The 30% of the E. campestre seeds inoculated with P. eryngii in sterile conditions showed signs of
chlorosis 15 days after inoculation. However, none of the inoculated plants died during this experiment.
Signs of root infection by the mycelium were observed in symptomatic seedlings (Supplementary
Fig. S15). Chlorosis signs were absent in the control plants.

Discussion

The present results indicate that the tested strains of P. eryngii var. eryngii are necrotrophic fungal
pathogens of E. campestre. In fact, in field conditions, the inoculated plants soon became chlorotic and
died, giving rise to mushrooms at the base of the stem during the following autumn and spring when
conditions were favourable. The molecular analyses confirmed that these symptoms were caused by
the inoculated commercial strain. The analysis of the IGS2 sDNA region (i.e., IGS2 sequences and
RFLP-IGS2 profiles) confirms that these plants had been infected only by the inoculated commercial
strain and none had been infected by any wild strain. These results highlight the usefulness of IGS2


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     Fig. 2 – a) Proliferation of secondary dikaryotic mycelium (staining in Congo red) in the vicinity of the taproot
     of an adult plant of Eryngium campestre. b) Dikaryotic fungal hyphae penetration on the cortical cells absorbent
     root of an adult Eryngium campestre inoculated plant (staining in cotton blue). c) Proliferation of coloured
     gums in a longitudinal section of an absorbent root (staining in cotton blue) from an adult Eryngium campestre
     inoculated. d) Start of gum formation in a cross section of an adult Eryngium campestre taproot inoculated
     plant.




Fig. 3 – a) 75-day-old seedlings grown in a container where a large reduction in seedling density is observed one month
after being inoculated with Pleurotus eryngii, compared to the control. b) Eryngium campestre control container with a
high seedling density, compared to inoculated seedlings, just after being inoculated for the first time at 75 days of age.



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polymorphism in the identification of strains belonging to members of the genus Pleurotus (Zhang et
al., 2006). Most of the susceptible inoculated plants died in less than two weeks before their flowers
could form. These findings corroborate Joly et al. (1990) hypothesis, since the adult inoculated plants
died before flowering.
       Detection and distribution of P. eryngii in soil samples, as well as the death of E. campestre
plants, seem to confirm mycelial outgrowth in soil, where it would grow as a saprophytic fungus
decomposing organic matter. However, once the mycelium reaches the roots of a E. campestre plant,
the fungus will penetrate and infect them, as indicated by experiments carried out with inoculated
plants. Within the root tissues, the fungus will obtain the ideal nutritional conditions to complete
fruiting body development, as suggested by the collections of mushrooms on root remains in Zervakis
et al. (2001) and Lewinsohn et al. (2002). From this infected plant, P. eryngii mycelium would grow
as a saprophytic fungus again. The distribution and abundance of E. campestre plants could lead
to formations similar to “fairy rings” over time. Although, the fairy rings are not well defined, the
mycelium was found in a large area within a bare circle of E. campestre plants.
       There are more than 50 species of basidiomycete fungi which cause fairy rings; some species
grow saprotrophically on decaying organic matter, while other species also have a parasitic phase on
plant roots (Terashima et al., 2004; Nelson, 2008; Bonanomi et al., 2011). Our study would support
the hypothesis of Cailleux and Joly (1987), which stated that P. eryngii. is be associated over time
with “fairy rings” or “false mycorrhizal rings”. Furthermore, these formations would fall within the
definition of “fairy ring” described by Kirk et al. (2011), who stated that fairy rings of fungal origin
are also seen as bare circles of vegetation, as occurs under trees colonized by the black truffles Tuber
melanosporum Vittad. The non-homogeneous distribution of P. eryngii-positive soil in the present
ring might due to the more compact and stony soil in the left side of the sampled area, which may
have hindered fungal development, or the distribution/abundance of E. campestre plants.
       Due to the necrotrophic plant pathogen lifestyle of P. eryngii, it remains alive in the field,
especially in the dry, steppe-like conditions of central Spain as saprothroph. The dikaryotic mycelium
of P. eryngii, after having infected the host plants, takes refuge in the deep main roots of the killed
plants until the mycelium begins to proliferate in the autumn when the rains rehydrate the soil. This
growth might be encouraged by synergetic bacteria or their secondary metabolites (Clausen, 1996;
Kim et al., 2008). The P. eryngii inoculation of E. campestre seedlings demonstrates a preference of
the fungus for older tissue, compared to young tissue as shown by the highest mortality rate in the
old inoculated plants. Differences in pathogen preferences by tissue age have been mentioned by
Okubara and Paulitz (2003).
       Although the high virulence of P. eryngii in our experiments can be explained by the wounds that
are caused during the inoculation process which have facilitated host infection, the results obtained
in this study seem to support the pathogenicity of P. eryngii. This confirms Hilber’s conclusions
(1982), who described P. eryngii as a parasite of the roots of members of the Apiaceace family (such
as E. campestre, Ferula communis, etc.). For this reason, the Spanish administration should take the
pertinent measures to avoid the sale of P. eryngii mycelium to inoculate adult E. campestre in the
field, as certain companies indicate on their websites, to produce field basidiocarps. These practices
with phytopathogenic fungi in natural conditions may generate environmental problems.
       In conclusion, P. eryngii var. eryngii would appear to be a necrotrophic pathogen of E.
campestre, and thus perhaps of other umbellifers. It infects its hosts as a virulent pathogen, later
surviving saprotrophically on the roots after the aerial parts of the plant become detached, where the
fungus fructifies from the taproots. However, to confirm its trophic mode and its formation of “fairy
rings”, additional research will be necessary, extending the study to different strains and sites.
Acknowledgments

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This work was partially funded by the University of Alcalá via project UAH GC2016-003. The
authors thank Gustavo Díaz and Jaime Diez for their help with fieldwork, and Adrian Burton and
Bruno Parra for editing assistance.

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Carlavilla, Manjón                                              Italian Journal of Mycology 52 (2023): 22-31

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