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Journal of Invertebrate Pathology 197 (2023) 107891
Available online 27 January 2023
0022-2011/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Genetic variability of Metarhizium isolates from the Ticino Valley Natural
Park (Northern Italy) as a possible microbiological resource for the
management of Popillia japonica
Gian Paolo Barzanti
a
,
*
, Jürg Enkerli
b
, Claudia Benvenuti
a
, Agostino Strangi
a
,
Giuseppe Mazza
a
, Giulia Torrini
a
, Stefania Simoncini
a
, Francesco Paoli
a
, Leonardo Marianelli
a
a
CREA Research Centre for Plant Protection and Certication, 50125 Florence, Italy
b
AGROSCOPE Institute for Sustainability Sciences ISS, Molecular Ecology, 8046 Zürich, Switzerland
ARTICLE INFO
Keywords:
Invasive alien species
Microbiological control
Japanese beetle
Metarhizium natural occurrence
Habitat
Genotype
ABSTRACT
The natural occurrence of entomopathogenic fungi (EPF) was investigated along the Ticino River (Ticino River
Natural Park, Novara Province, Piedmont, Italy), at the center of the area of the rst settlement of the invasive
alien pest Popillia japonica. Using Zimmermann’s “Galleria bait method”, EPF were successfully isolated from 83
out of 155 soil samples from different habitats (perennial, cultivated, or uncultivated meadows, woodlands, and
riverbanks). Sequencing of the 5’ end of the Translation Elongation Factor 1 alfa (5’-TEF) region allowed the
assignment of 94% of the isolates to Metarhizium spp., while 8% and 7% were assigned to Beauveria spp. and
Paecilomyces spp., respectively. Four Metarhizium species were identied: Metarhizium robertsii was the most
common one (61.5% of the isolates), followed by M. brunneum (24.4%), M. lepidiotae (9%), and M. guizhouense
(5.1%). Microsatellite marker analysis of the Metarhizium isolates revealed the presence of 27 different geno-
types, i.e., 10 genotypes among M. robertsii, 8 among M. brunneum, 5 among M. lepidiotae, and 4 among
M. guizhouense. Metarhizium brunneum appeared to be associated with woodlands and more acid soils, while the
other species showed no clear association with a particular habitat. Laboratory virulence tests against P. japonica
3rd instar larvae allowed the identication of one M. robertsii isolate that showed efcacy as high as 80.3%. The
importance of this kind of study in the frame of eco-friendly microbiological control is discussed.
1. Introduction
Popillia japonica Newman (Coleoptera: Scarabeidae) (Pj) is a pest
native to Japan and far eastern Russia (EPPO Global Database, http
s://gd.eppo.int/taxon/POPIJA) with a wide host range (more than
300 plant species, Potter and Held, 2002). In 2014, it was detected in
Italy in the Ticino Valley Natural Park, a natural environment located
between the Lombardy and Piedmont regions (Pavesi, 2014). Since this
rst report, which represents the rst interception in mainland Europe,
the beetle quickly spread into the new territory, damaging important
crops such as corn, grapevines, plum trees, apple trees, and soya (EPPO,
2014; Marianelli et al., 2017; Santoiemma et al., 2021), and crossing the
border to Switzerland in 2017 (EPPO, 2017). To date, Pj in mainland
Europe is conrmed only in Northern Italy (Piedmont, Lombardy, Aosta
Valley, and Emilia-Romagna Regions) and in Southern Switzerland
(Canton Ticino) (EPPO Global Database, https://gd.eppo.int/tax
on/POPIJA).
Popillia japonica has a one-year life cycle that includes 3 larval in-
stars: rst-instar larvae can be found in the soil from the end of June, the
second instar from mid-July onwards, and the third instar can be found
starting from early August. Larval abundance seems to be related to less
acidic soils, especially with sandy-skeletal particles (Simonetto et al.,
2022). Pj larvae feed on plant roots and organic matter all summer long
and overwinter as third instar. During the following March-April, Pj
larvae start feeding again, pupate (starting from mid-May), and after
2–3 weeks adults start emerging and feeding on leaves, owers, and
fruits of their numerous host plants (Marianelli et al., 2017). In contrast
to North America (Althoff and Rice, 2022), in Northern Italy, Pj adults
show a longer oviposition period (from May to September), with the
ight peaks in July (Marianelli et al., 2017). Females mate upon emer-
gence and lay their eggs into the rst layer of the soil (up to 7.5 cm below
the soil surface; Potter and Held, 2002).
* Corresponding author.
E-mail address: gianpaolo.barzanti@crea.gov.it (G.P. Barzanti).
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology
journal homepage: www.elsevier.com/locate/jip
https://doi.org/10.1016/j.jip.2023.107891
Received 13 September 2022; Received in revised form 17 January 2023; Accepted 24 January 2023
Journal of Invertebrate Pathology 197 (2023) 107891
2
In the last decades, concerns regarding the use of chemicals in
agriculture reached the attention of public opinion. On one hand, public
awareness of such a problem has fostered a more conscious consumption
by the consumer (e.g., organic products), and on the other hand, pro-
moted research and application of environmentally friendly alternatives
for pest control.
Biological control approaches are crucial alternatives to pesticide use
and provide important components of sustainable agriculture. These
approaches are applied worldwide and are usually included in inte-
grated pest management strategies (Barbosa, 1998; Baker et al., 2020).
To make microorganisms applicable as Biological Control Agents
(BCAs) it is pivotal to know their natural occurrence and ecology in
respect of the environment where they are supposed to be applied or
exploited (Meyling and Eilenberg, 2007).
Several entomopathogenic fungi (EPF), in particular Metarhizium
spp. Sorok. (Hypocreales: Clavicipitaceae) and Beauveria sp. Vuill.
(Hypocreales: Cordycipitaceae) have been tested against larvae, pupae,
and adults of Pj in laboratory, semi-eld, and eld trials worldwide but
with contrasting results (Potter and Held, 2002; Ramoutar et al., 2010;
Behle et al, 2015). The treatment with a commercial product of Beau-
veria bassiana in Italy was found to be ineffective, while the trials carried
out with a commercial product based on Metarhizium sp. appeared to be
more promising and thus deserve further research (e.g., Benvenuti et al.,
2019; Mori et al., 2022).
Metarhizium spp. are ubiquitous, mostly soilborne entomopathogenic
fungi (Roberts and St. Leger, 2004; Inglis et al., 2019), and together with
Beauveria spp., are the most widely used EPF in biological control
(Schneider et al., 2012). In 2007, a review by de Faria and Wraight
(2007) showed that the list of Metarhizium-based mycoinsecticides is
almost completely covered by the species M. anisopliae (Metch.) Sorok.
According to Bischoff et al. (2009), M. anisopliae is a species complex
including at least ten different species (Kepler et al., 2014, Rehner and
Kepler, 2017, Lopes et al., 2018). Following this new classication,
M. brunneum Petch appears to be the only Metarhizium species developed
as BCA in Europe. Moreover, only two M. brunneum strains (BIPESCO
5/F52 and CB 15-III, the latter with an emergency authorization) have
been registered as active substances and used in commercial phytosa-
nitary products to date in Europe (EPPO database, EU Pesticides
Database).
Given the necessity to develop and implement an eco-friendly
approach to cope with the spread of P. japonica in the newly infested
territory, the aims of this study were to 1) assess the presence of
indigenous EPF in the Pj-infested area, and 2) molecularly characterize
the recovered isolates. Moreover, 3) the selected isolates were tested for
virulence to allow the selection of promising Metarhizium strains for
P. japonica biological control. To the best of our knowledge, no data are
available on EPF occurrence in this region. To achieve our goal, we have
sampled soils in areas recently infested by Popillia japonica along the
Ticino River (Ticino River Natural Park, Novara Province, Piedmont,
Italy) and collected EPF isolates. Since Beauveria and Paecilomyces were
isolated in low percentages (see the Results section), our study focused
only on the Metarhizium genus.
2. Materials and methods
2.1. Soil sampling and fungal isolation
A sampling campaign was performed in April-May 2017 along the
western bank of the Ticino River (Novara province, Piedmont region,
Northern Italy), at the center of the area invaded by Popillia japonica.
Sampling sites included habitats such as permanent meadows and un-
cultivated areas (with a variable density of Pj larvae in the samples), and
woodlands (without Pj larvae in the samples) within the Popillia
japonica-infested area, where no BCAs products had previously been
applied. Each sampling site was described by recording geographical
coordinates, agricultural/soil management, or vegetation cover
(Supplementary S1).
Soil samples were collected from a total of 155 sampling sites
distributed 30 km along the Ticino River (Novara Province, Piedmont,
Italy). At each sampling site, a plot of 25 m
2
was specied, within which
ve randomly distributed soil cores (20x25 cm and 15 cm depth) were
collected. Approximately 200 cm
3
of each soil core were combined into
a single 1 Kg bulk soil sample per sampling site, taken to the laboratory
in a cool box, and stored at room temperature until processed as in
Torrini et al. (2020). The pH of each soil sample was analyzed in the
laboratory following standard procedures (Violante, 2000).
Entomopathogenic fungi were collected from soil samples using the
“Galleria bait method” (Zimmermann, 1986). Each soil sample was
thoroughly mixed, and a sub-sample of 500 g was placed in a plastic box,
covered with a perforated lid, and marked with the sampling site
number. Five Galleria mellonella L. (Lepidoptera: Pyralidae) larvae ob-
tained from Agripetgarden s.r.l. (Italy) and ve Tenebrio molitor L.
(Coleoptera: Tenebrionidae) larvae from the same Company were
inserted in each box. Boxes were kept at room temperature for 10 days,
checked daily for dead larvae, and the soil was sprayed with distilled
water when needed to avoid drying out of the soil. After a sodium hy-
pochlorite wash (1 min) and three rinses in sterile distilled water, dead
larvae with symptoms of mycosis were individually transferred to sterile
plastic Petri dishes that were lined with sterile wet lter paper and
sealed with lab tape and incubated at room temperature until fungal
sporulation. A sterile scalpel blade was used to isolate fungi from ca-
davers and plate them on Rose Bengal Chloramphenicol Agar (RBCA,
VWR International PBI s.r.l.). Grown colonies were then transferred to
quarter-strength Sabouraud Dextrose Agar (SDA, VWR International PBI
s.r.l.) plus 0.25 % Yeast Extract (YE, Sigma-Aldrich Chemie GmbH)
(SDAY1/4, Liu et al., 2003) and incubated at 24 ◦C in the dark for two
weeks. Isolates were then morphologically identied, puried as single
spore colonies, and preserved as part of the Entomopathogenic Fungi
Collection maintained at CREA-DC, Florence, Italy.
2.2. DNA extraction, amplication, sequencing, and analysis
DNA extraction was performed from lyophilized mycelium. For this
purpose, isolates were grown on sterile cellophane sheets laid on
SDAY1/4 medium for 7–10 days at 24 ◦C in the dark. Mycelium was
collected with a sterile spatula, placed in 1.5 ml plastic microcentrifuge
tubes, and lyophilized overnight in a Modulyo (Edwards High Vacuum)
freeze-dryer. A small portion of lyophilized mycelium (50–100 mg) was
subsequently transferred to a 2 ml screw-capped plastic tube along with
about 150 mg of glass beads (1:1 mix v/v of 0.5 and 2.0 mm in diameter)
and homogenized with a Precellys 24 mechanical beater (Precellys).
Genomic DNA was subsequently extracted from disrupted mycelium
using the Qiagen Plant Mini Kit (Qiagen, Hilden, Germany), following
the manufacturer’s protocol. One hundred micro-liters of double-
distilled sterile water were used for the nal elution. Diluted DNA was
then lyophilized and conserved for subsequent analyses.
Multilocus genotyping (MLG) with 15 microsatellite markers of all
the collected isolates was performed based on the protocols described by
Mayerhofer et al. (2015). Microsatellite allele sizes were determined on
an ABI 3500 Series genetic analyzer using POP-7 polymer (Applied
Biosystems, Foster City, CA, USA). GenScan ROX400 (Applied Bio-
systems) was used as an internal size standard. Data were analyzed using
GenMarker V2.4.0 (SoftGenetics, State College, PA, USA) and allele sizes
were corrected according to fragment sizes of reference strains
M. brunneum ARSEF7524 and M. robertsii ARSEF7532.
MLGs were assigned to species by sequencing the 5′end of the
Translation Elongation Factor 1 alfa (5′-TEF) region of one isolate per
genotype and subsequent sequence alignment with conrmed Meta-
rhizium reference sequences. The 5′-TEF region was amplied using
primers EF1T 5′-ATGGGTAAGGARGACAAGAC-3′and EFjmetaR 5′-
TGCTCACGRGTCTGGCCATCCTT-3′and sequenced as described by
Mayerhofer et al. (2019). Sequences were deposited at GeneBank
G.P. Barzanti et al.
Journal of Invertebrate Pathology 197 (2023) 107891
3
(accession numbers from OP688426 to OP688452, Supplementary S2).
Obtained sequences were aligned with reference sequences of Meta-
rhizium spp. for species allocation. Reference sequences were down-
loaded from GenBank, and alignments were performed using Clustal-W
implemented in MEGA 11.0.9 (Kumar et al., 2018) followed by manual
editing. A Maximum likelihood phylogenetic analysis was performed
based on the Kimura 2-parameter model with default settings in MEGA
11.0.9. Bootstrap values were determined from 1000 bootstrap
iterations.
2.3. Virulence tests
A single representative of each MLG (n =27, see results section) was
assayed against 3rd instar Pj larvae collected in autumn 2019 from an
infested corneld (Novara Province, Italy) and maintained at 4 ◦C in
native soil at the CREA-DC laboratory in Florence (Italy) until their use.
Before performing the virulence test, larvae were individually trans-
ferred in plastic cups containing about 30 g of sterile soil (autoclaved
twice, 121 ◦C, 20 min) and ryegrass seeds and acclimated at 20 ◦C for 4
Fig. 1. Metarhizium species (and genotypes) isolated along the Ticino River (Piedmont region). Different colors indicate different genotypes within the four species
identied (symbols in Supplementary Table S2). A cross indicates a sampling point where Metarhizium was not detected.
G.P. Barzanti et al.
Journal of Invertebrate Pathology 197 (2023) 107891
4
days for quarantine purposes (Torrini et al., 2020). Only healthy-looking
larvae according to Koppenh¨
ofer et al. (2012) were selected for the
following tests. Sterile plastic plates (Cytoone, Starlab Int. GmbH), with
12 wells each (2.5 cm diameter, 2 cm depth) and lid, were used in
laboratory trials to test the virulence of Metarhizium MLGs from Ticino
against Pj larvae. A single larva was placed inside each well together
with approximately 4 g of sterilized eld soil inoculated with 1x10
8
dry
fungal conidia collected from colonies grown on SDAY1/4 overlayed
with a sterile cellophane sheet. Some perennial ryegrass seeds were
added to each well as food for the larva. Boxes were kept at room
temperature (20–22 ◦C) and relative humidity (about 60 %), and larval
mortality was assessed at 14 days, after which the experiment was
closed. Dead larvae were individually incubated in plastic Petri dishes
lined with sterile wet lter paper and sealed with lab tape and incubated
at room temperature until fungal outgrowth to conrm the larvae were
killed by the fungus. In 2 replicates, a total of 24 larvae were treated per
Metarhizium MLG and the experiment was repeated twice.
2.4. Statistical analysis
A chi-square test was performed to assess the correlation between
Metarhizium species and environment, while pH values were checked for
normality and homoskedacity by the Shapiro-Wilk and Bartlett tests
respectively, and then subjected to the ANOVA procedure followed by
Tukey’s HSD post-hoc test. Mortality data from the virulence test were
corrected according to Schneider-Orelli’s formula (Schneider-Orelli,
1947) to obtain efcacy values (i.e., treatment mortality corrected
considering control mortality). After the arcsine transformation, percent
mortality values were subjected to the ANOVA procedure followed by
the Tukey HSD post-hoc test to evaluate the best-performing strain. All
the analyses were performed with R statistical software version 4.1.1 (R
Core Team, 2021).
3. Results
3.1. Soil sampling and fungal isolation
Seventy-eight isolates of Metarhizium, seven Beauveria, and six Pae-
cilomyces were obtained from a total of 83 soil samples positive for EPF
presence (Fig. 1). Three soil samples harbored 2 different Metarhizium
species or genotypes, while in 4 cases Metarhizium and Paecilomyces and
in 1 case Metarhizium and Beauveria were isolated from the same sample.
Beauveria and Paecilomyces alone were isolated from 6 and 2 samples
respectively. Due to their low abundance, Beauveria and Paecilomyces
were excluded from further investigations in this study. Metarhizium spp.
was present in soil samples of all habitat types sampled, the only
exception was a single soil sample from cropland that yielded no EPF
isolates (Table 1, Supplementary S1).
3.2. Microsatellite marker Genotyping and species afliation
Microsatellite marker-based genotyping of the 78 Metarhizium iso-
lates revealed the presence of 27 different MLGs (Supplementary S2).
Seventeen MLGs were represented by single isolates, one MLG (no. 3)
was shared by 18, and two (MLG2, MLG13) by 10 isolates each (Sup-
plementary S3). The remaining MLGs were shared by 2 to 5 isolates
(Supplementary S3). Genotype n◦13, which was one of the most com-
mon genotypes (10 isolates), as well as genotype n◦17 (3 isolates) were
detected exclusively in soil samples from woodlands. Genotype n◦5 (2
isolates) was isolated from perennial meadow only and the remaining
multiple-occurring genotypes had a variable origin (Supplementary S3).
One single isolate per MLG was selected for species afliation. Mo-
lecular identication was performed by sequencing the 5′end of the
nuclear EF1-
α
gene and subsequent alignment of the sequences with
sequences of 22 Metarhizum reference strains. This allowed the assign-
ment of the 27 MLGs to 4 different species, namely M. robertsii J. F.
Bisch., Rehner and Humber, M. brunneum, M. lepidiotae J. F. Bisch.,
Rehner and Humber, and M. guizhouense Q. T. Chen and H. l. Guo
(Fig. 2).
Metarhizium robertsii was the most common species with 10 MLGs
(37 %) and 48 (61.5 %) isolates followed by M. brunneum, M. lepidiotae,
and M. guizhouense with 8 (30 %), 5 (19 %) and 4 (15 %) MLGs, and 19
(24.4 %), 7 (9 %), and 4 (5.1 %) isolates, respectively (Supplementary S3
and Table 1). The most common genotype (MLG 3, 18 isolates) was
identical to that of the reference strain Ma500 (ARSEF7532, MLG 3). A
single isolate had the same MLG as BIPESCO 5 strain (MLG 24), while all
four M. guizhouense isolates had unique genotypes.
Metarhizium robertsii was also the most widespread species, having
been isolated from each habitat, while M. brunneum appeared to be
associated with woodlands (
χ
2
=14.651, df =1, p-value <0.001)
(Table 1, Supplementary S3). Furthermore, M. robertsii was isolated
from soils with a wider pH range (3.7 – 6.19, mean 5.09), M. guizhouense
was isolated from soils with a narrow pH range (4.56 – 5.78, mean 5.19),
and M. brunneum was isolated from signicantly more acid soils (3.48 –
5.24, mean 4.21; F
(3, 74)
=9.4677, p-value <0.0001; Fig. 3, Supple-
mentary S3).
3.3. Virulence tests
The isolates tested for virulence showed a highly variable efcacy
against Pj larvae among and within species, with signicantly different
results (F
(27, 40)
=3.75, p-value <0.0001) ranging from ineffective to
80 % efcacy (Table 2). Metarhizium robertsii (efcacy range 80.3 % −
1.2 %) appeared to be the most effective species with one isolate (MLG
no. 2) reaching 80.3 %, one 78.1 %, and two more with over 60 % of
efcacy (Table 2). Two M. lepidiotae isolates (range 70.4 % −25.9 %)
achieved 70 % efcacy, while one M. brunneum isolate reached almost
68 %, and two more exceeded 50 % (range 67.8 % - −8.7 %). Meta-
rhizium guizhouense isolates (range 40.7 % - −3.8 %) generally exhibited
low virulence (<41 %, Table 2).
4. Discussion and conclusions
In our work, the Metarhizium genus was detected and isolated from
approximately 50 percent of the soil samples collected along the West-
ern Ticino riverbank. The genus was represented by four species:
M. robertsii, M. brunneum, M. lepidiotae, and M. guizouhense. To the best
of our knowledge, this is the rst time that the combination of all these
four species has been found in a single study area. According to Ques-
ada-Moraga et al. (2007), Metarhizium species predominated in soils
with pH lower than 7, as found in our study area, and the low abundance
of the other EPF (e.g., Beauveria) could be linked to the acidic soils, but
this topic deserves further investigations.
Several studies have indicated a heterogenous distribution of EPF in
natural, semi-natural, or cultivated habitats (Bidochka et al., 1998;
Meyling and Eilenberg, 2007; Quesada-Moraga et al., 2007; Garrido-
Jurado et al., 2015, Fern´
andez-Bravo et al., 2021). In particular, Meta-
rhizium species have been reported to be associated with arable land,
grassland, and forests (Bidochka et al., 2001; Meyling and Eilenberg,
2007; Schneider et al., 2012; Keyser et al., 2015; Fern´
andez-Bravo et al.,
Table 1
Metarhizium species and number of isolates obtained in each habitat (meadows,
woodlands, riverbanks, croplands). Metarhizium species: Mro =M. robertsii, Mbr
=M. brunneum, Mle =M. lepidiotae, Mgu =M. guizhouense.
No. of
sites
Mro Mbr Mle Mgu Total isolates
Meadows 72 28 1 3 3 35
Woodlands 79 18 18 4 1 41
Riverbanks 3 2 – – – 2
Croplands 1 – – – – –
Total 155 48 19 7 4 78
G.P. Barzanti et al.
Journal of Invertebrate Pathology 197 (2023) 107891
5
2021), but also with different types of plants (such as grasses, shrubs,
and trees) or crops (Fisher et al., 2011; Wyrebek et al., 2011; Kepler
et al., 2015; Steinwender et al., 2015; Cabrera-Mora et al., 2019). Some
studies also report that in some cases specic Metarhizium genotypes
have been found linked to or predominant in a particular environment
(Steinwender et al., 2014; Kepler et al., 2015; Fern´
andez-Bravo et al.,
2021).
The number of species recovered in our study was similar to that
reported by several other authors performing comparable studies
(Wyrebek et al., 2011; Steinwender et al., 2014; Garrido-Jurado et al.,
2015; Kepler et al., 2015; Keyser et al., 2015; Steinwender et al., 2015;
Hern´
andez-Domínguez and Guzm´
an-Franco, 2017). However, species
prevalence and abundance vary according to different authors (Keyser
et al., 2015; Steinwender et al., 2015; Hern´
andez-Domínguez and
Guzm´
an-Franco, 2017; Inglis et al., 2019). Metarhizium robertsii was the
most common species in our survey, as reported also by Kepler et al.
(2015) and Wyrebek et al. (2011). In our study M. brunneum was
correlated with woody environments and only a single isolate was ob-
tained from another environment, i.e., grasslands. Metarhizium robertsii
was instead present in all the environments sampled. In contrast to our
ndings, Fern´
andez-Bravo et al. (2021) reported a presence of
M. brunneum in arable land, grasslands, and forest, and M. robertsii and
Fig. 2. Maximum likelihood phylogenetic tree based on the alignment of 5
′-TEF-1
α
sequences of Metarhizium isolates representing the 27 different multilocus ge-
notypes (MLG) and 22 reference strains. Bootstrap values >70 %, calculated with 1000 replicates, are shown. The bar scale indicates 0.02 changes per nucleotide.
G.P. Barzanti et al.
Journal of Invertebrate Pathology 197 (2023) 107891
6
M. guizouhense were present in arable land and grasslands only. In our
study, M. guizouhense isolates were mostly collected from meadows,
while the seven M. lepidiotae isolates were equally distributed between
woodlands and meadows. Taken together, Metarhizium species distri-
bution and abundance are highly variable among different regions,
habitats, and crops. Several studies have reported correlations of specic
ecological and/or human factors, such as clay content, soil pH, organic
matter, C:N ratio, or soil disturbance due to cropping activities with the
distribution and population structure of Metarhizium spp. (Quesada-
Moraga et al., 2007, Meyling and Eilenberg, 2007, Schneider et al.,
2012, Fern´
andez-Bravo et al., 2021). However, a comprehensive un-
derstanding of how such factors in the context of different habitats and
crops may drive Metarhizium abundance and diversity is still missing.
Future research performing systematic studies at local and regional
levels including different environmental conditions, crops as well as
insect hosts combined with meta-analyses may allow further steps to a
better understanding of the factors driving the population structure of
this important fungal genus.
Microsatellite marker-based typing revealed substantial genotypic
diversity among our M. robertsii isolates (10 MLGs among 48 isolates),
which was comparable to the results reported by Kepler et al. (2015) but
lower than that reported by Steinwender et al. (2015). On the other
hand, we found a higher variability among our M. brunneum isolates
compared to the variability reported by Kepler et al. (2015) and Stein-
wender et al. (2015). In accordance with Steinwender et al. (2014) and
Kepler et al. (2015), we found that few genotypes prevail in the entire
community, both for M. robertsii and M. brunneum. As shown in Sup-
plementary S3, the two prevalent M. robertsii genotypes have been iso-
lated from all the habitat types sampled and no habitat association was
detected. This suggests that other factors may dominate and affect the
abundance and prevalence of specic genotypes together with the
habitat type. Further studies are needed to clarify these aspects, which
could be very important in the perspective of the use of these Meta-
rhizium genotypes as BCAs in the future.
In our lab tests, we found a high variability between and within
species as regards the virulence of Metarhizium MLGs against 3rd instar
Popillia larvae (Table 2). Indeed, the most effective isolate resulted to
belong to an MLG of M. robertsii (MLG no. 2, isolate 17/T02), but within
the same species, we also found isolates with low efcacy (1.2 %), as
Fig. 3. Box plot illustrating the pH values of the soil samples from which iso-
lates of the four different Metarhizium species were recovered. The lower and
upper side of the boxes represent the rst and third quartile, respectively, the
line inside the box is the median, and the whiskers represent the minimum and
maximum value. Boxes indicated with the same letter are not signicantly
different. Metarhizium species: Mro =M. robertsii, Mbr =M. brunneum, Mle =
M. lepidiotae, Mgu =M. guizhouense.
Table 2
Virulence test results. Mean percent mortalities are reported, together with their Standard Error. Mean mortality values sharing the same letter are not signicantly
different. Percent efcacy has been calculated according to the Schneider-Orelli formula. The codes of the isolates used in the virulence test, the corresponding MLG
number they represent, and the number of isolates sharing the same MLG are reported.
Isolate code Genotype
n◦
Number of isolates Metarhizium species Mean mortality
(%) ± SE
Efcacy (%)
(Schneider-Orelli)
17/T02 2 10 M. robertsii 83.4 ±13.2 a 80.3
17/T29 6 3 M. robertsii 81.5 ±10.5 ab 78.1
17/T33 7 5 M. robertsii 71.0 ±0.0 abc 65.6
17/T03 3 18 M. robertsii 66.7 ±0.0 abc 60.5
17/T15 5 2 M. robertsii 64.5 ±27.8 abc 57.9
17/T61 9 5 M. robertsii 60.0 ±2.0 abc 52.6
17/T13 4 1 M. robertsii 41.7 ±8.3 abc 30.8
17/T79 10 1 M. robertsii 37.5 ±20.8 abc 25.9
17/T01 1 1 M. robertsii 29.2 ±12.5 abc 16.0
17/T39 8 2 M. robertsii 16.7 ±0.0 bc 1.2
17/T96 13 10 M. brunneum 72.9 ±16.0 abc 67.9
17/T85 12 1 M. brunneum 64.7 ±8.3 abc 58.1
17/T117 18 1 M. brunneum 58.3 ±0.0 abc 50.6
17/T116 17 3 M. brunneum 41.7 ±8.3 abc 30.8
17/T105 15 1 M. brunneum 33.3 ±0.0 abc 20.9
17/T21 11 1 M. brunneum 29.2 ±4.2 abc 16.0
17/T101 14 1 M. brunneum 25.0 ±0.0 abc 11.1
17/T111 16 1 M. brunneum 8.3 ±0.0 c −8.7
17/T57 21 1 M. lepidiotae 75.0 ±8.3 abc 70.4
17/T30 20 1 M. lepidiotae 75.0 ±0.0 abc 70.4
17/T07 19 3 M. lepidiotae 50.0 ±0.0 abc 40.7
17/T118 23 1 M. lepidiotae 49.5 ±8.5 abc 40.1
17/T80 22 1 M. lepidiotae 37.5 ±4.2 abc 25.9
17/T11 25 1 M. guizouhense 50.0 ±4.0 abc 40.7
17/T10 24 1 M. guizouhense 41.7 ±16.7 abc 30.8
17/T100 27 1 M. guizouhense 16.7 ±0.0 bc 1.2
17/T51 26 1 M. guizouhense 12.5 ±4.2 bc −3.8
Control 15.7 ±7.5 c
G.P. Barzanti et al.
Journal of Invertebrate Pathology 197 (2023) 107891
7
well as observed in all the other species. In accordance with our results,
Bidochka et al. (2001) have reported no consistent patterns of virulence
within the clonal groups they examined.
The high EPF biodiversity in this limited territory gave us the op-
portunity to nd an interesting candidate for the microbiological control
of P. japonica grubs in the soil. Metarhizium robertsii genotype no. 2
proved to be the best-performing EPF in our lab trials and, among the
four Metarhizium species found.
The possibility of controlling an insect pest in the soil with a natural
control agent is highly interesting, especially in the current perspective
of quickly abandoning or limiting the use of chemicals for such purposes.
To date, there are only very few Metarhizium strains available that are
commercialized as products for pest control (e.g., M. brunneum strain
CB15-III, against Agriotes spp. larvae, M. brunneum strain BIPESCO5,
against Popillia japonica, Phyllopertha horticola, Amphimallon spp., Otio-
rhynchus spp.). The adaptation and use of existing products to control
new and emerging pests allow to optimize and reduce efforts and costs,
e.g., for registration purposes. However, due to biosafety issues, the use
of indigenous EPF is preferred in the eco-friendly management of
invasive alien species (e.g., Lockwood, 1993) such as P. japonica.
Moreover, indigenous EPF are more adapted to the habitat and envi-
ronment they originate from and therefore are supposed to have the best
capability to survive and compete in that environment (Bidochka et al.,
1998; Jackson et al., 2010). This aspect is pivotal since fungal cycling
and survival are key aspects to be preserved (Bidochka et al., 2001) in
the perspective of maintaining EPF active in the environment for a
longer time. Furthermore, the use of indigenous isolates minimizes risks
for adverse effects on non-target organisms. For instance, Mayerhofer
et al. (2017) have reported that the use of an indigenous EPF strain did
not affect soil microbial communities.
In conclusion, assessing local EPF diversity and testing for efcient
new isolates as performed in this study is an important step to provide
new resources for biological control and increase the number of avail-
able control strains.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgments
The authors wish to thank Giovanni Bosio and Emanuela Giacometto
from the Phytosanitary Service of the Piedmont Region for eld support,
and the anonymous reviewer for the useful suggestions.
Funding
This work was funded by the Piedmont Region Grant/Award number
D. n. 1161-29/11/2016, and the European Union’s Horizon 2020
research and innovation programme “IPM-POPILLIA - Integrated Pest
Management of the invasive Japanese Beetle, Popillia japonica”, under
grant agreement No. 861852.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jip.2023.107891.
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