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Interaction with the
entomopathogenic fungus
Beauveria bassiana influences
tomato phenome and
promotes resistance to
Botrytis cinerea infection
Assunta Russo
1,2
, Jana Barbro Winkler
3
, Andrea Ghirardo
3
,
Maurilia M. Monti
2
, Susanna Pollastri
2
, Michelina Ruocco
2
,
Jörg-Peter Schnitzler
3
*and Francesco Loreto
2,4
*
1
University of Naples Federico II, Department of Agricultural Sciences, Portici, Italy,
2
National
Research Council of Italy, Institute for Sustainable Plant Protection (CNR-IPSP), Portici, Italy,
3
Helmholtz Zentrum München, Research Unit Environmental Simulation, Neuherberg, Germany,
4
Department of Biology, University of Naples Federico II, Naples, Italy
Plants are central to complex networks of multitrophic interactions. Increasing
evidence suggests that beneficial microorganisms (BMs) may be used as plant
biostimulants and pest biocontrol agents. We investigated whether tomato
(Solanum lycopersicum) plants are thoroughly colonized by the endophytic
and entomopathogenic fungus Beauveria bassiana, and how such colonization
affects physiological parameters and the phenotype of plants grown under
unstressed conditions or exposed to the pathogenic fungus Botrytis cinerea.
As a positive control, a strain of the well-known biocontrol agent and growth
inducer Trichoderma afroharzianum was used. As multitrophic interactions are
often driven by (or have consequences on) volatile organic compounds (VOCs)
released by plants constitutively or after induction by abiotic or biotic stresses,
VOC emissions were also studied. Both B. bassiana and T. afroharzianum
induced a significant but transient (one to two-day-long) reduction of stomatal
conductance, which may indicate rapid activation of defensive (rejection)
responses, but also limited photosynthesis. At later stages, our results
demonstrated a successful and complete plant colonization by B. bassiana,
which induced higher photosynthesis and lower respiration rates, improved
growth of roots, stems, leaves, earlier flowering, higher number of fruits and
yield in tomato plants. Beauveria bassiana also helped tomato plants fight B.
cinerea, whose symptoms in leaves were almost entirely relieved with respect to
control plants. Less VOCs were emitted when plants were colonized by B.
bassiana or infected by B. cinerea, alone or in combination, suggesting no
activation of VOC-dependent defensive mechanisms in response to both fungi.
KEYWORDS
beneficial microorganisms, photosynthesis, plant pathogens, plant phenotyping,
volatile organic compounds
Frontiers in Plant Science frontiersin.org01
OPEN ACCESS
EDITED BY
Gianfranco Romanazzi,
Marche Polytechnic University, Italy
REVIEWED BY
Alessandro Vitale,
University of Catania, Italy
Elsherbiny A. Elsherbiny,
Mansoura University, Egypt
*CORRESPONDENCE
Francesco Loreto
francesco.loreto@unina.it
Jörg-Peter Schnitzler
joergpeter.schnitzler@helmholtz-
munich.de
RECEIVED 08 October 2023
ACCEPTED 29 November 2023
PUBLISHED 19 December 2023
CITATION
Russo A, Winkler JB, Ghirardo A, Monti MM,
Pollastri S, Ruocco M, Schnitzler J-P and
Loreto F (2023) Interaction with the
entomopathogenic fungus Beauveria
bassiana influences tomato phenome and
promotes resistance to
Botrytis cinerea infection.
Front. Plant Sci. 14:1309747.
doi: 10.3389/fpls.2023.1309747
COPYRIGHT
© 2023 Russo, Winkler, Ghirardo, Monti,
Pollastri, Ruocco, Schnitzler and Loreto. This
is an open-access article distributed under
the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 19 December 2023
DOI 10.3389/fpls.2023.1309747
1 Introduction
The use of beneficial microorganisms (BMs) has been promoted in
recent years as a novel strategy to ensure food safety and security of
agricultural products while reducing the application of pesticides and
chemical fertilizers and pursuing agroecology principles (Parnell et al.,
2016). In this context, plant protection by endophytic fungi may be also
considered, establishing a mutually beneficial symbiotic relationship
with the host plant and being exploited as an alternative source of
secondary metabolites (Bamisile et al., 2018;Sinno et al., 2020;Wen
et al., 2022). The entomopathogenic fungus, Beauveria bassiana (Bals.)
Vuill. (Ascomycota: Hypocreales) can endophytically colonize tissues
of many plants. Colonization was successfully demonstrated by using
different inoculation methods such as seed coating, soil watering, root
dipping, and foliar spraying (Tefera and Vidal, 2009). Beauveria
bassiana is well-known for its endophytic potential in the biocontrol
of insect herbivores (Vega, 2018), and its mechanism of action as an
entomopathogen has been extensively studied (Ortiz-Urquiza and
Keyhani, 2013;Pedrini et al., 2013;Wang et al., 2021). More
recently, B. bassiana was proposed as a dual-purpose microbial
control organism against both insect pests and plant pathogens
(Allegrucci et al., 2017;Saranraj and Jayaparakash, 2017;Wei et al.,
2020). In addition, tomato plants colonized by B. bassiana apparently
show improved nutrient root uptake, perhaps via enhanced activity of
phytohormones or growth regulators (Gonzalez-Guzmanetal.,2022).
There are several bioformulates based on B. bassiana that are
already commercially available, and interest on the practical use of
this and other BMs is spurring further research (Felizatti et al.,
2021). However, despite some efforts (Macuphe et al., 2021), the
effect of B. bassiana on plant physiology and improvement of plant
resistance to pathogens has just recently started to be investigated
(Proietti et al., 2023).
To evaluate whether endophytic colonization by B. bassiana
strain ATCC 74040 (Naturalis, CBC Europe s.r.l.,Biogard division,
Grassobbio, Italy) affects the plant phenome, we used tomato
(Solanum lycopersicum) plants. In particular, we studied whether B.
bassiana colonization a) expands quickly across plant vegetative and
reproductive organs; b) is quickly sensed by plants, causing the onset
of defensive responses; c) has a biostimulant effect inducing long-
term changes in the plant phenotype; d) improves plant resistance to
Botrytis cinerea (the gray mold), a destructive fungal pathogen of a
wide range offruits, vegetable and ornamental crops, and considered
a“high-risk”necrotrophic pathogen characterized by short life cycle,
high reproduction, and large genetic variation (Poveda et al., 2020).
Our results prompt for a rapid and complete plant colonization of B.
bassiana that is first recognized as a foreign invader, and then rapidly
elicits plant growth and protection against the pathogen B. cinerea.
2 Materials and methods
2.1 Experimental protocol, plant material
and growth conditions
Experiments were performed for two years in two different research
institutes. In the first year, the impact of B. bassiana colonization on plant
physiology (primary metabolism) and phenotype was assessed at the
facilities of the National Research Council of Italy (CNR-IPSP) in Portici
(Naples, Italy). Trichoderma afroharzianum (strain T22), largely used as
biocontrol agent (BCA) and plant growth promoter (Thapa et al., 2020),
was used as a benchmark. The second year, the experiment was carried
outattheResearchUnitEnvironmental Simulation (EUS), Helmholtz
Zentrum München (HMGU, Munich, Germany), where we
concentrated on measuring the effect of B. bassiana on tomato plants
for a longer time course (up to fruiting) and also followed whole plant
phenotyping, VOC emission, and impact of B. bassiana on a subsequent
infection by B. cinerea.
Tomato seeds (Solanum lycopersicum cv San Marzano nano,
Semiortosementi, Sarno, Italy) were surface-sterilized in 1% NaOCl (v/
v) for 5 min, rinsed twice with sterile distilled water (SDW) and
germinated on Whatman sterile filter paper (Sigma-Aldrich,
Darmstadt, Germany) soaked with SDW, in the dark, at 24°C.
Germination occurred in 4-5 d. Seedlings were firstly individually
transplanted to 8 cm diameter pots (1.3 L) of non-sterile commercial
soil (Universal potting soil-Floragard Vertriebs-GmbH Oldenburg), then
potted in 13 cm diameter pots (2.16 L) and kept in growth chambers
(Italy) or a climatized greenhouse (Germany) at 25 ± 2°C, 70 ± 10% RH,
and a photoperiod of 14:10 h (light:dark), with a photosynthetic active
radiation (PAR) of around 700 mmol m
-2
s
-1
during the days. More than
200 plants were grown to conduct all following experiments.
2.2 Fungal cultures
Beauveria bassiana strain ATCC 74040 (Naturalis), B. cinerea
(isolate B05.10) and T. afroharzianum (strain T22) were cultured
on 4.5 g 100 mL
-1
Potato Dextrose Agar (PDA from Sigma-Aldrich,
St. Louis, MO, USA), maintained at 25 ± 2°C, and 14:10 h (light:
dark) photoperiod for 20 d. For conidial production, aerial conidia
from all the fungi were harvested by flooding the plate with sterile
distilled H
2
O containing 0.02% Tween 80 (Sigma-Aldrich).
Conidial suspensions were filtered with a sterile pipette tip
plugged with cotton wool and final conidial concentrations were
determined by direct count using a haemocytometer (Neubauer
hemocytometer chamber) under a microscope and adjusted to the
indicated concentration for final use.
2.3 Induction and assessment of
endophytic colonization by
Beauveria bassiana
Emerged tomato seedlings (27-d old plants) were treated by
drenching soil with 50 mL of 1 × 10
6
conidia mL
-1
of a conidial
solution of B. bassiana. Control plants were watered with the same
volume of SDW. The same treatment was repeated after a week on
35-d old plants. From this second treatment with B. bassiana,we
counted days post inoculum (dpi) for all the experiments.
To confirm B. bassiana endophytic colonization, tissue samples
were collected from leaves of 5 treated and 5 control tomato plants at 1,
2, 7, 15, 21, 35, 42, 49, 56, 63 dpi, respectively. At the fruiting time, 5
tomato fruits from treated and control plants were also harvested, and
Russo et al. 10.3389/fpls.2023.1309747
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tomato seeds were collected. Leaf samples and tomato seeds were
randomly chosen, and surface sterilized in 1% NaOCl for 3 min, after
which they were rinsed 3 times with SDW. These steps were useful to
ensure that appearance of mycelial growth was only due to B. bassiana
growing inside plant tissues. The success of the disinfection procedure
was assessed by plating three replicates of 100 µL each of the residual
rinsed water on PDA-medium plates. Leaf samples and seeds were
dried on sterile paper, leaves were cut into pieces of about 1 cm
2
each
and seeds into half; both leaves and seeds were placed on 90 mm wide
Petri plates containing PDA supplied with 1% (v/v) lactic acid to avoid
bacterial contamination. Plates were incubated at 25°C in the dark. Leaf
pieces and seeds were monitored daily to determine if there was fungal
growth emerging from the cut plant tissues (white, cottony, dense
hyphal growth of B. bassiana)(Humber, 2012). The fungal mycelia
were isolated and transferred on newplatescontainingPDA,inorder
to obtain pure cultures for their morphological identification.
2.4 Botrytis cinerea infection and
evaluation of damage in tomato plants
Four days after the second treatment in soil with B. bassiana,a
conidial solution of the pathogenic fungus B. cinerea (20 ml of 1×10
5
conidia mL
-1
) was applied as a foliar spray on each 39-d old tomato
plant. The plants were maintained in water-sprayed boxes for 4 d to
ensure high humidity favouring conidial germination. Control plants
received the same spraying treatment but with SDW. The disease
development was evaluated at 9, 10, 12, 15 dpi by calculating the ratio
between the surface area affected by the pathogen and the total leaf
area. These two areas (cm
2
) were estimated by harvesting and scanning
leaves and stems. The digital imagesobtainedwereanalysedwithan
open-source image processing software (Jud et al., 2018). All pixels of
the image representing plant leaves were calculated and interpolated
with leaf area values using a cubic spline function. The software allows
to manipulate and adjust colour threshold parameters (hue, saturation,
and brightness value), quantitatively determining the total leaf area
(hue= 17/180, saturation =15/100 and brightness value=18/100) and
the leaf area covered by sporulation or damaged, with necrotic and
chlorotic symptoms) (hue= 4/61, saturation= 11/51 and brightness
value= 7/100). Since the first stage, B. cinerea infection causes yellowing
of leaves, which is a rather unspecific symptom. Indeed, leaf necroses in
plants without B. cinerea infection (i.e. treated with B. bassiana and in
control conditions) are also reported, and were considered in our
experiment (added to background) to help differentiate unspecific
necrosis from symptoms due to pathogen infection. This experiment
was preceded by a pilot experiment to set up the best timing for disease
development detection.
2.5 Phenotyping of control and Beauveria
bassiana-colonized plants: growth
Five tomato plants for each treatment were randomly chosen,
uprooted, and dissected at root, stem and leaf level at 1, 2, 7, 15, 21,
35, 42, 49, 56, 63 dpi, to analyse plant growth data, respectively.
Roots, stems and leaves fresh weights (FW) were determined. Roots
were carefully washed under tap water to remove the soil. Dry
weights (DW) of roots, stems and leaves were obtained after drying
samples in an oven at 70°C for 72 h. Root length, stem height,
number of flowers and fruits, and tomato fruit weights were
also measured.
2.6 Phenotyping of control and Beauveria
bassiana-colonized plants: gas-exchange,
chlorophyll fluorescence, and emission of
volatile organic compounds
2.6.1 Gas-exchange and chlorophyll fluorescence
at leaf level
Measurements of gas exchange (CO
2
and H
2
O) and chlorophyll
fluorescence were conducted by enclosing fully mature leaves in an
8-cm
2
leaf cuvette surface of an Infra-Red Gas Analyzer system
[standard measuring head 3010-S of a portable system for
simultaneous analysis of gas exchanges and chlorophyll
fluorescence GFS-3000 (Heinz Walz GmbH, Effeltrich,
Germany)]. All measurements were conducted between 8:00 am
and 3:00 pm. After a dark adaptation period of at least 30 min, the
leaf was illuminated under standard conditions (PAR 1000 µmol
m
−2
s
−1
, leaf temperature 30°C, relative air humidity 50%, and CO
2
concentration set to 400 ppm, matching ambient CO
2
levels) until
stomata opened and steady state CO
2
and water vapour exchange
rates were reached. Values of net photosynthesis, aka CO
2
assimilation (Pn), transpiration (Tr), stomatal conductance to
water vapor (gH
2
O), intercellular CO
2
concentration (Ci) and
respiration in the dark (Rd) were calculated from gas-exchange
measurements (Von Caemmerer and Farquhar, 1981;Farquhar and
Sharkey, 1982). Minimum fluorescence (Fo), maximal fluorescence
in the dark-adapted leaf (Fm) or light-adapted leaf (Fm′), steady
state fluorescence in the light-adapted leaf (Fs), and minimal
fluorescence in the light-adapted leaf (Fo′) were determined, as
described previously (Maxwell and Johnson, 2000). The maximal
quantum yield of PSII was calculated as: Fv/Fm = (Fm –Fo)/Fm,
while the effective quantum yield of PSII in illuminated leaves was
calculated as: FPSII = (Fm′–Fs)/Fm′(Genty et al., 1989). The
electron transport rate was calculated by multiplying the FPSII with
the amount of PAR absorbed by PSII: ETR = (FPSII) × (PAR) ×
(0.84) × (0.5), where 0.84 and 0.5 estimate that leaves absorb 84% of
incident photons, 50% of which are absorbed by PSII, assuming that
the absorbed light is equally distributed between photosystem I and
II (Yamori et al., 2011). Non-photochemical energy quenching
(NPQ), a measure of heat dissipation of absorbed light energy,
was calculated as: NPQ = (Fm/Fm′) - 1, while Y(NPQ) which is the
fraction of PAR that is dissipated in PSII via the non-photochemical
quenching mechanisms was calculated as Y(NPQ) = F/Fm′−F/Fm
(Bilger and Björkman, 1990;Maxwell and Johnson, 2000).
2.6.2 Gas-exchange and VOCs at entire
plant level
Gas exchange and VOC measurements were conducted in the
VOC-SCREEN platform (Jud et al., 2018), installed in one of the
phytotron chambers at HMGU, where it was possible to control
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environmental parameters such as temperature, relative humidity
(RH), photosynthetically active radiation (PAR), maintaining them
similar to growth conditions (see above). The cuvettes in which the
plant pots were installed (total volume of ∼40 L) were made of
stainless steel and a cylindrical Duran glass cover clamped to the
base with inert Viton rings sealing the joint. The base of the cuvette
contained gas and irrigation tubing and electrical connections.
From the cuvette base, the supplied air was flushed into the
cuvette air space via a circular system of dozens small inlet holes
(Jud et al., 2018). Following the experimental setup of Figure 1A,
plants were incubated for 4 d to make possible B. cinerea conidial
germination before being enclosed in the cuvettes (9 dpi) and kept
under observation for 1 week. At 16 dpi, plants were removed from
cuvettes and discarded. The 24 cuvettes of the platform were
divided according to the following experimental design, as shown
in Figure 1B: 5 control (C) cuvettes hosted a potted tomato plant
each, grown without any fungal treatment; 5 cuvettes (Bb) hosted a
potted tomato plant each, where B. bassiana spores were inoculated
by soil irrigation in two treatments (as described in section 2.3); 5
cuvettes (Bc) hosted a potted tomato plant each, where the
pathogen B. cinerea was sprayed (as described earlier); 5 cuvettes
(Bb-Bc) hosted a potted tomato plant each, where B. bassiana was
inoculated in the soil and plants were subsequently sprayed with B.
cinerea; the remaining 4 cuvettes (soil) hosted one pot each
containing only soil (background cuvettes).
Absolute CO
2
and H
2
O concentrations of the inlet and the
outlet of the plant cuvettes were measured by two Infra-Red Gas
Analysers (IRGA; LI-840A, LI-COR Biosciences, Lincoln,
Nebraska) continuously during the entire experiment. For each
measurement, gas-exchange was measured every 5 min and 20 s
before switching to the next cuvette. After a 60-s purge time (to be
sure that no contamination of gases from the previously measured
cuvette occurred), gas exchange data were recorded. Calculations of
Pn and evapo-transpiration (E-Tr) were done in Matlab vers.2017a
(Mathworks Inc. Natick, Massachusetts) using the formulas given
above (Von Caemmerer and Farquhar, 1981). For Pn, data from
background cuvettes (containing only a pot of soil) were subtracted
from plant cuvette data to account for soil contribution to CO
2
exchange. For E-Tr, no such background correction was made, and
the data also included soil evaporation. In fact, it was not possible to
maintain the same soil moisture in all pots, and background
corrections would give a misleading result in terms of E-Tr. In
both cases, the rates were normalized for the leaf area of the plant
inside the cuvette, which was estimated as discussed by Hartmann
et al. (2011). Plant pictures were taken from two different angles
(nine from the front view, nine from a 45
°
angle from above) in a
photo-station equipped with a turntable in which a stepper motor
allowed to rotate the plants in front of an adequate image
background. These measurements were done before placing the
plants into the cuvettes and repeated after one week, in order to
capture plant growth (Jud et al., 2018). All pixels of the images
representing plant leaves were calculated and interpolated with leaf
area values using a cubic spline (measured, exact leaf area vs.
extracted pixels). The gas-exchange data were elaborated with the
R (Vers.4.2.0, using R studio) software.
VOC analysis was performed using gas chromatography mass
spectrometry (GC-MS), following established procedures (Ghirardo
et al., 2020). Samples were collected from the air exiting the cuvettes
in GC-MS glass tubes containing 40 mg of Tenax TA and 40 mg of
Carbopack X by diverting a constant air flow of 70 mL min
-1
through the tubes for 360 min, from 9:30 am to 3:30 pm. Sampling
was conducted at 3 different times after the plants were enclosed in
the VOC-SCREEN cuvettes: at 10, 12 and 15 dpi as illustrated in
Figure 1. Each cartridge contained 859.3 pmol of d-2-carene as
internal standard. Quantification was accomplished using three
calibration curves, which were generated independently in
triplicate and preparing six different concentrations of pure
standard mixtures (a-pinene, sabinene, limonene, methyl-
salicylate, bornyl acetate, ß-caryophyllene, a-humulene). Volatiles
that were not available as standards were quantified using calculated
response factors leading to a quantification uncertainty of 1-8%
(Ghirardo et al., 2020). Plant VOC emissions were corrected using
measurements of the background cuvettes. Non-isothermal Kovats
retention indices (RIs) were calculated based on chromatography
retention times of a saturated alkane mixture standard (van Den
Dool and Kratz, 1963). Limit of detection (LOD) was calculated
using two standard deviations.
2.7 Statistical analysis
Data are shown as means ± standard error of means (SEM)
and were subjected to analysis of variance (ANOVA) or Student’s
t-test performed using the R (Vers.4.2.0, using R studio) software.
To separate means within each parameter, the Tukey’stest
was performed. Statistically significant differences were tested
at p < 0.05.
3 Results
3.1 Colonization of tomato plants by
Beauveria bassiana and impact on plant
growth and photosynthetic gas exchange
3.1.1 Beauveria bassiana endophytic
colonization data
The presence of white, cottony, dense hyphal growth emerging
from different plant-treated tissues (Figure 2, left side of each plate)
was confirmed to be B. bassiana mycelium. Beauveria bassiana was
found in tomato leaves after the second root inoculation (one dpi),
and rapidly colonized all plant tissues, also being retrieved in seeds
of fruits of colonized tomato plants (Figure 2, bottom right plate). In
control plants (Figure 2, right side of each plate) no hyphal growth
of B. bassiana occurred, ensuring absence of fungal contamination
during the experiments.
3.1.2 Plant growth data
Colonization of B. bassiana led to significant (p < 0.05) increase
of plant growth in terms of root length (Figure 3A), fresh
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(Figure 3C) and dry weight (Figure 3F), from the beginning of the
experiment to 28 dpi; at this timepoint growth became similar in
control and treated plants and then B. bassiana-colonized plants
resumed growing more than controls at 63 dpi. Stem height
(Figure 3B), fresh (Figure 3D), and dry weight (Figure 3G), were
all promoted by B. bassiana-treatment with respect to controls until
35 dpi, and after 56 dpi. The same profile was followed by leaf
growth, based on either fresh (Figure 3E)anddryweight
(Figure 3H). Flowering started around 15 dpi in B. bassiana-
colonized plants (Figure 3I). This was earlier than in controls,
and late flowering was reduced in B. bassiana-colonized plants with
respect to controls. Fruiting was also different in plants with B.
bassiana, as more fruits where already set at 28 dpi (Figure 3J), and
fruit-set remained significantly higher than in controls during the
entire fruiting period. Tomato fruits from B. bassiana-colonized
plants were heavier than those from control plants (Figure 3K).
3.1.3 Gas exchange data at leaf level
During the first year experiment the impact on photosynthetic
gas exchange of B. bassiana was compared with that of the well-
known biocontrol agent and growth inducer Trichoderma
afroharzianum (Thapa et al., 2020). The maximal quantum yield
of PSII (Fv/Fm) did not change among controls and plants
inoculated with B. bassiana or T. afroharzianum, along the time-
course of the experiment (Supplementary Figure S1), respectively.
However, both B. bassiana and T. afroharzianum induced a
significant but transient (one to 2 dpi-long) reduction of net
photosynthesis (Pn) and stomatal conductance (gH
2
O)
(Figures 4A,B). At 21 and 35 dpi, B. bassiana improved Pn of
tomato plants with respect to controls (p < 0.05), but at 42 dpi Pn
and gH
2
O, were not different in all conditions. Measurements made
70 and 77 dpi rendered erratic results on the few plants still alive
(Figure 4B). Aging was faster in control plants than in plants
A
B
FIGURE 1
Experimental workflow is represented in figure (A); after sowing and potting, plants were inoculated twice with Beauveria bassiana, provided in the
irrigation water. Controls were irrigated only with water. Phenotyping of growth parameters, gas-exchange and chlorophyll fluorescence was carried out
in treated and control plants from one day post inoculum (dpi) until fruiting. For experiment in the VOC-SCREEN platform, 39d-old plants that were
previously either inoculated by B. bassiana or kept in control conditions, were infected with Botrytis cinerea and gas exchange was measured in cuvettes
until 15 dpi (B). Cuvettes in the VOC-SCREEN platform were divided according to different plant treatments: control plants (C); B. cinerea spray-infected
plants (Bc); B. bassiana-colonized plants (Bb); Plants colonized by B. bassiana and sprayed with B. cinerea (Bb-Bc); soil pots only (soil).
Russo et al. 10.3389/fpls.2023.1309747
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colonized by B. bassiana or T. afroharzianum, as shown by the
steeper regression line fitted to the data of Pn and gH
2
O
(Figures 4C,D).
Gas-exchange measurements at leaf level were repeated during
the second experiment at HMGU. Results of the first experiment
could be largely confirmed by the second experiment: B. bassiana
application induced a rapid and significant reduction of Pn and
gH
2
O compared to control plants within the first days after root
inoculation (Figures 5A,B). This reduction was visible until two
dpi, after which gas-exchange parameters of plants colonized by B.
bassiana resumed the levels observed in controls. From 28 dpi on,
however, Pn and gH
2
O became higher in the plants treated with B.
bassiana compared to controls. Also in this second experiment, Pn
and gH
2
O decreased over time both in controls and in plants treated
with B. bassiana, and aging was faster in control plants, as shown by
the different slopes of Figures 5C,D.
3.2 Impact of colonization of tomato
plants by Beauveria bassiana on the
development of Botrytis cinerea infection
Plants colonized by B. bassiana were less damaged than controls
when exposed to B. cinerea infection. The effect was barely
noticeable with a visual inspection at 9 dpi but became
increasingly evident with time (Figure 6A). By using a calculation
software, we were able to translate this visual effect into measurable
data (Figure 6B). At 15 dpi more than 40% and less than 5% of the
foliar surface area was damaged by B. cinerea in controls and B.
bassiana-colonized plants, respectively (Figure 6B). Chlorotic and
necrotic symptoms could both be seen in the RGB images of
severely infected B. cinerea leaves, but to a much lower extent
chlorotic spots also appeared on control plants and B. bassiana-
colonized plants (Figure 6B). These symptoms are not related to B.
cinerea infection, rather often revealing initial leaf ageing. These
chlorotic areas are visualized to give a complete picture of the results
but will be then subtracted to the total biomass of controls and B.
bassiana-colonized plants to correctly interpret the damage
specifically caused by B. cinerea infection. This experiment also
confirmed that plants treated with B. bassiana grew more than
control plants (Figure 6C), as the increase in total area is
comparable to the increase of leaf weight observed in Figures 3E,H.
3.3 Impact of colonization by Beauveria
bassiana and of Botrytis cinerea infection
on gas-exchange and VOC emission of
whole tomato plants
3.3.1 Gas-exchange at entire plant level
The antagonistic effect of B. bassiana on B. cinerea infection was
further evaluated by analysis of gas exchange and VOC emission
from entire plants. Net photosynthesis (Pn), dark respiration (Rd),
and evapo-transpiration (E-Tr) were monitored continuously (24-h
long) from 9 dpi until 16 dpi (see Figure 1A for the complete
experimental design). Measurements on the first and the last day
(when plants were inserted into and removed from the cuvettes)
were not considered in our analysis, and only data from 10 until 15
dpi are shown in Supplementary Figure S2.
Based on results shown in Supplementary Figure S2, data were
further filtered and averaged on two periods:h 8:00-15:00 (when PAR
in cuvettes was stable around 700 mmol m
-2
s
-1
); and h 20:00-4:00
(when PAR was turned off). In these two periods Pn (in the light) and
Rd (in the dark) were relatively constant and could be integrated on
whole plant and whole day basis. Plants colonized by B. bassiana
showed significantly higher Pn and lower Rd than all other plants. On
the other hand, plants infected by B. cinerea that significantly reduced
the photosynthetic areas (Figure 6C) showed the lowest Pn (Figure 7).
FIGURE 2
Endophytic colonization by Beauveria bassiana evaluated on PDA plates on leaves at 1, 2, 7, 15, 21, 28, 35, 42, 49, 56, 63 dpi (A–K, respectively), and
on fruit seeds at 77 dpi (L). Leaves or tomato seeds of B. bassiana-colonized plants (left plate of each panel) are compared with leaves or tomato
seeds of control plants (right plate of each panel).
Russo et al. 10.3389/fpls.2023.1309747
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When examining the cumulative exchange of CO
2
by the plants
over the entire experimental period (six days), B. bassiana-
colonized plants and controls photosynthesized slightly more
than plants infected by B. cinerea and those that were infected by
B. cinerea and colonized by B. bassiana (Figure 8A). However, Rd
measurements in the night confirmed a much higher respiratory
emission of CO
2
by control plants (Figure 8B), which reduced the
total uptake of CO
2
(net photosynthesis –respiration) in
comparison to plants colonized by B. bassiana (Figure 8C).
3.3.2 VOC emissions at whole-plant level
We investigated the changes in tomato VOC emissions
consequent to B. bassiana colonization. Different classes of VOCs
were identified according to library match and Kovats's RIs.
However, the profile of emitted VOCs was similar under all
treatments. Main VOCs emitted were the monoterpenes b-
phellandrene, a-pinene, p-cymene, D-limonene; the sesquiterpene
b‐caryophyllene; the benzene analogues m-xylene and
phenylethyne (the latter tentatively identified); the ethanol ester
triethyl phosphate (possibly a contaminant); and the saturated fatty
aldehydes decanal and nonanal (Supplementary Table S1). b-
Phellandrene, and D-limonene were the two most emitted VOCs,
with b-phellandrene contributing on average to around 75% of the
total emissions in all treatments (Figure 9). Treatments with B.
bassiana and B. cinerea (alone or in combination) did not elicit the
emission of induced VOCs, rather they reduced the overall
constitutive emission of VOCs. A general but not statistically
significant reduction of VOC emission from the first to the third
sampling point (in leaves 44 to 50 d-old) was also observed,
particularly in control plants (Figure 9).
4 Discussion
Beneficial associations between plants and microorganisms
have been extensively studied (Yan et al., 2019;Adeleke and
Babalola, 2022;Dlamini et al., 2022;Russo et al., 2022) and are
often linked to plant physiological and metabolic reprogramming
that promote growth and strengthen the defense barriers (De Palma
et al., 2019). Recently, the communication mechanisms between
plants and endophytic fungi in presence of biotic and abiotic
stresses was deeply investigated (Lu et al., 2021), and some fungal
entomopathogens started to be proposed as beneficial endophytes
(Vidal and Jaber, 2015;Jaber and Enkerli, 2017). With this work we
intended to test whether the entomopathogenic fungus B. bassiana:
a) successfully colonizes the different organs of tomato; b) is
A
B
DE
FG
I
H
JK
C
FIGURE 3
Growth data in control (C, black bars) tomato plants and in plants colonized by Beauveria bassiana (Bb, grey bars). Root length (A), stem height (B),
root, stem and leaf fresh weight (C–E), respectively, and dry weight (F–H), respectively, numbers of flowers (I) and fruits (J) per plant, and fruit fresh
weight per plant (K) at 1, 2, 7, 15, 21, 28, 35, 42, 49, 56, and 63 dpi. Means ± SEM (N=5) are shown. Statistical significance of differences between C
and Bb means was assessed at each dpi by Student’s t-test, asterisks represent p < 0.05; ns = non-significant differences.
Russo et al. 10.3389/fpls.2023.1309747
Frontiers in Plant Science frontiersin.org07
perceived as a stressful or beneficial organism during different
stages of plant development with an overall positive effect on
plant phenotypes; c) defends tomato plant against the pathogenic
fungus B. cinerea.
Colonization by B. bassiana of internal plant tissues has been
observed in many crops (Vega, 2018;Mantzoukas et al., 2021;
Mantzoukas et al., 2022), including tomato (Proietti et al., 2023). As
the inoculum source was in the soil (we irrigated the plants with a
solution of spores), B. bassiana could have entered the plant via the
root system, or could have travelled out of the plant reaching stem
lenticels close to the soil surface from where invaded the plant.
Beauveria bassiana colonization was successfully verified in the
leaves that were used for gas-exchange analyses. The infection was
extremely rapid (within one dpi), and the leaves were proved to be
colonized at all tested timepoints. First vertical transmission such as
shown here was proved by Quesada-Moraga et al. (2014) in Papaver
somniferum.Weshowforthefirst time that B. bassiana
colonization reach tomato fruits and seeds, making trans-
generation transmission also possible. If from a biological control
perspective this could represent an advantage (allowing protection
to different generations), fruit colonization by B. bassiana also
makes us wonder if tomatoes of plants treated with this beneficial
microorganism can be eaten safely. Beauveria bassiana produces a
mycotoxin (beauvericin) with insecticidal activity, which is the basis
of B. bassiana entomo-pathogenicity (Al Khoury et al., 2020). When
produced by Fusarium sp. beauvericin may be toxic also to
mammals (Logrieco et al., 1998). More studies are needed to test
whether B. bassiana treatments may impair consumption
of tomatoes.
Plants responded fast to B. bassiana colonization. We showed
that the infection was extremely rapid. The photochemistry of
photosynthesis was clearly unaffected by B. bassiana infection, as
shown by the steady-state values of the maximal quantum yield of
PSII (Fv/Fm). However, both B. bassiana and T. afroharzianum
induced a significant but transient (1-2 day-long) reduction of
stomatal conductance and net photosynthesis. Our results suggest
the induction of a rapid activation of defensive (rejection) responses
against a foreign organism that invades plant organs. This might
have primed a plant response that should be further demonstrated
by examining activation of defensive metabolism (e.g., priming of
antioxidant metabolites) (Pollastri et al., 2021). Fast perturbations
of transcriptomes of tomatoes after T. afroharzianum root
treatment were previous detected (De Palma et al., 2019). ROS
signalling, SA responses and cell wall modifications were activated
24 h after treating the plants, whereas after 72 h an increased
transcription of ethylene and auxin signalling genes triggered
Pn (µmol m -2 s-1)
0
2
4
6
8
10
12
Tim e (d pi )
1 2 7 152135427077
gH
2
O (mm ol m
-2
s
-1
)
0
50
100
150
200
a
b b
a
b
ba
b b
a
a
c
b
a
a
aa a a
b
Time (dpi)
X Data
1 2 7 15 21 35 42 70 77
Tim e (d pi )
ba
a
a a
b
a
bb
AB
CD
a
a
a
a
a
aa
a
a
a
a
aa
a
b
cb
c
a
a
b
bb
b
b
Bb
C
y = 7,18 -0,042x; r
2
=0.24
y = 7,08 -0,028x; r
2
=0.21
C
Bb
y = 186,90 -15,09; r
2
=0.48
y = 135,35 -0,77x; r
2
=0.26
C
Bb
Ta
FIGURE 4
Net photosynthesis (Pn, A) and stomatal conductance (gH
2
O, C) of leaves of control tomato plants (C), and of plants colonized by Beauveria
bassiana (Bb), and Trichoderma afroharzianum (Ta) at 1, 2, 7, 15, 21, 35, 42, 70, 77 dpi during the first experiment in Italy. Statistical significance of
differences among the means of the different treatments was assessed over single time point, as indicated by the dashed lines, by ANOVA followed
by Tukey’s test. Means ± SEM (N=3) are shown. Different letters indicate significantly different means with p < 0.05. In (B, D), linear regression lines
for Pn (black) and gH
2
O (grey) over time (dpi) are shown for control tomato plants and plants colonized by B. bassiana. The 95% confidence interval
with respect to best fit lines is represented by the dashed lines of the same color of the regression lines.
Russo et al. 10.3389/fpls.2023.1309747
Frontiers in Plant Science frontiersin.org08
possible modifications in root architecture, and possibly also plant
growth stimulation. As discussed elsewhere (De Palma et al., 2019;
Proietti et al., 2023) a down-regulation of proteins related to defense
responses and up-regulation of proteins related to calcium
transport during early phases of B. bassiana colonization of
tomato plants was showed. The data set discussed by Proietti
et al. (2023) is delayed by 3 days with respect to our very rapid
reduction of Pn and gH
2
Oandmayreflect the following
establishment of a symbiotic relationship. For example, calcium
flux across the plasma membrane was found to be an early
signalling step when establishing symbiosis and immunity (Yuan
et al., 2017;Moscatiello et al., 2018;Jiang and Ding, 2022).
We did not notice any significant and prolonged stimulation of
leaf photosynthesis, implying that growth stimulation may not be
due to an improvement of carbon fixation on a leaf area unit.
However, slower plant aging might have been related to delayed
reduction in Pn and gH
2
O in plants colonized by B. bassiana with
respect to control plants. Moreover, a significant effect of enhanced
growth was seen when measuring net photosynthesis at whole plant
level rather than after normalizing on a leaf area basis. Finally,
respiratory losses of carbon overnight (Rd) were also reduced in
plants colonized by B. bassiana with respect to all other treatments,
which may also support better carbon availability/allocation for
growth and development. However, a direct correlation between net
photosynthesis and dark respiration (i.e. a simultaneous increase of
Pn and Rd) is more often observed (Poorter and Bongers, 2006).
The lower respiration rate found in our study may rather confirm
slower ageing and prolonged leaf life-span (Reich et al., 1998)inB.
bassiana-treated plants than in controls that do not interact with
the BM. Interestingly, our results indicate that any interaction with
fungi (either pathogenic or beneficial) has a significant inhibitory
effect on dark respiration in tomato.
In other crops, like corn, grapevine, bean, cotton, coffee, and
sorghum, B. bassiana endophytic colonization was proposed to
result in plant growth promotion (Posada et al., 2007;Tefera and
Vidal, 2009;Lopez and Sword, 2015;Ramos et al., 2017;Afandhi
et al., 2019;Russo et al., 2019;Mantzoukas et al., 2021). This was
also the case with tomato. Colonization of B. bassiana led to
statistically significant increase of all plant organs (roots, stems
and leaves). We observed that growth stimulation was stronger
early after the infection, was absent or somehow reduced in the
second month after the infection, and then again became evident on
older plants. This confirms recently published data showing a
reprogramming of the proteome toward energy production
processes sustaining growth in ageing plants (Proietti et al., 2023).
We note that both fresh and dry weights of the phenotyped plant
Pn (µmol m -2 s-1)
0
5
10
15
20 C
Bb
Tim e (d pi )
1 2 7 1521283542495663
gH2O (mmol m -2 s-1)
0
50
100
150
200
250
Time (d pi)
X Data
1 2 7 15 21 28 35 42 49 56 63
Time (dpi)
AB
CD
Bb
Cy = 18,80 -0,104x; r
2
=0.96
y = 16,22 -0,035x; r
2
=0.23
C
Bb
y = 249,85 -8,6 3; r
2
=0.96
y = 203,91 -0,4 4x; r
2
=0.27
******
**
*
******
*
ns
ns
ns
ns
ns
ns
FIGURE 5
Net photosynthesis (Pn, A) and stomatal conductance (gH
2
O, C) of leaves of control tomato plants (C), and of plants colonized by Beauveria
bassiana (Bb) at 1, 2, 7, 15, 21, 28, 35, 42, 49, 56, 63 dpi, measured during the second experiment in Germany. Statistical significance of differences
among the means of the different treatments was assessed over single time point, as indicated by the dashed lines, by ANOVA followed by Tukey’s
test. Means ± SEM (N=3) are shown. Asterisks and ns indicate significantly and non-significantly different means with p < 0.05, respectively. In (B, D),
linear regression lines for Pn (black) and gH
2
O (grey) over time (dpi) are shown for control tomato plants and plants colonized by B. bassiana. The
95% confidence interval with respect to best fit lines is represented by the dashed lines of the same color of the regression lines.
Russo et al. 10.3389/fpls.2023.1309747
Frontiers in Plant Science frontiersin.org09
parts increased, suggesting that the effect was not simply limited to
an improved water content of the plants.
This is to our knowledge the first experiment that has followed
the interaction between tomato and B. bassiana along the entire
plant life. The infection of B. bassiana produced an unexpected
anticipation of flowering, and a positive effect on tomato fruit-set.
Not only more fruits were set during the entire fruiting period, but
fruits were also significantly bigger in B. bassiana-treated plants
than in controls. This result confirms that B. bassiana may be used
as a growth stimulator of tomato plants, as suggested earlier in a
different experiment on the basis of biochemical responses (Proietti
et al., 2023). Normally, BMs promote plant growth either by directly
facilitating nutrient uptake (as biofertilizers) or by modulating
(stimulating) plant hormone levels (Russo et al., 2022). Improved
nutrient uptake is frequent in the case of soil BMs that do not act as
endophytes (Nasslahsen et al., 2022), although it was reported that
B. bassiana might enhance nutrient availability, particularly soluble
phosphate (Barra-Bucarei et al., 2019a). Endophytization is more
likely to activate plant hormones, especially those involved in plant
growth and development (Waadt et al., 2022). In particular, the role
of gibberellins (GAs) in the development and maintenance of plant-
beneficial microbe symbioses is an emerging area of research
(McGuiness et al., 2019). A significant up-regulation of growth-
related hormones like GA precursors and their active forms was
observed in B. bassiana-treated tomato plants, together with an
increased biosynthesis of hormones related to defense such as
benzoic acid and jasmonate (Proietti et al., 2023).
Entomopathogenic fungi control pests (Mantzoukas et al., 2015;
Mantzoukas and Lagogiannis, 2019;Bava et al., 2022). However,
several studies have demonstrated that endophytic fungi can also
protect host plants against pathogens (Ownley et al., 2008;Barra-
Bucarei et al., 2019b) and herbivores (Arnold et al., 2003;Ownley
et al., 2004;Card et al., 2016;Jensen et al., 2020). Recent results have
suggested an antifungal activity of B. bassiana against B. cinerea
(Barra-Bucarei et al., 2020;Sinno et al., 2021;Proietti et al., 2023).
Botrytis cinerea is a necrotrophic fungus. It first produces toxic
compounds that cause cell death, and then the fungus feeds on the
dead tissue, causing typical necrotic lesions (Williamson et al.,
2007). We were able to monitor B. bassiana protection of tomato
plants against B. cinerea during a long period (until the symptoms
of the pathogen made it impossible to continue with the analysis),
and to visually see its protective effect on the entire plant. Beauveria
bassiana reduced B. cinerea symptoms in leaves almost entirely. As
previously described (Ownley et al., 2004;Ownley et al., 2008), the
endophyte might have an indirect effect if it moves through the
vascular system until it reaches the leaf tissues, and competes for
space and food with the pathogen, reducing its colonizing ability; or
it could directly parasitize the pathogen (mycoparasitism),
weakening its pathogenic potential. Beauveria bassiana may also
have a role in reducing the oxidative potential, which often is a
factor inducing B. cinerea infection (Govrin and Levine, 2000;
Kuzniak and Skłodowska, 2004). A reduction of ROS and of lipid
peroxidation indicators was observed in leaves colonized by B.
bassiana and infected by B. cinerea, with respect to plants only
infected with B. cinerea (Proietti et al., 2023). However, the
mechanism underlying improved plant protection against B.
cinerea in plants colonized by B. bassiana should be further
elucidated with dedicated experiments.
We further examined VOCs to assess whether colonization by
the entomopathogen B. bassiana aids plants fight B. cinerea
A
B
C
FIGURE 6
Evaluation of Botrytis cinerea infection in plants without (Bc) or with Beauveria bassiana colonization (Bb-Bc) at 9, 10, 12, 15 dpi (see Figure 1A for
experimental design), as compared to controls (C) and to plants treated with B. bassiana (Bb). In (A), the digital RGB images of scanned green leaves
of Bc (lower plates) and Bb-Bc plants (upper plates) are shown on a white background. The black background images were obtained with the leaf
area calculator software, to better identify the damage caused by B. cinerea infection. In (B), the percentage of leaf area showing symptoms with
respect to the total area of plants (C) is shown. In (B) the symptoms attributed to C and Bb plants (below the dashed line) are not related to damage
caused by B. cinerea infection, rather representing chlorotic spots due to aging or other non-pathological causes. In both (B, C), statistical
significance of differences among the means of the four treatments was assessed at each dpi by ANOVA followed by Tukey’s test. Means ± SEM
(N=5) are shown, and different letters indicate statistically different means with p < 0.05.
Russo et al. 10.3389/fpls.2023.1309747
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infection through fungi-induced plant VOC emissions or
potentially via direct emissions of volatiles from B. bassiana.
Unexpectedly, we did not observe any enhanced or induced
emissions in the bouquet of volatiles from tomato plants
colonized by B. bassiana. VOCs can mediate plant-plant
communication (Rosenkranz et al., 2021) and BMs can also alter
plant VOC profile by interaction (Russo et al., 2022). While plant
VOCs hold promise as a natural and eco-friendly solution to defend
plants from biotic stresses (Brilli et al., 2019), their effectiveness
remain uncertain. In the case of grapevine, B. bassiana elicited VOC
emissions, although this induction did not result in improved insect
resistance (Moloinyane and Nchu, 2019). Tomato plants primarily
store terpenes in glandular trichomes of leaves and stems (Catola
et al., 2018), which are filled during the early stage of leaf
development. The glandular VOCs are either released in large
amounts upon rupture of the cuticle (stress inductions) or slowly
evaporate out of glands (constitutive releases). Burst of volatiles are
commonly observed in plant species that possess specialized storage
structures such as secretory cavities, resin ducts and glandular
trichomes when attacked by insects or because of generic
mechanical stresses (Loreto et al., 2000;Kang et al., 2010) and
rupture of glandular trichomes can induce the expression of
defense-related genes in tomato plants (Peiffer et al., 2009).
Contrary to expectations, we did not detect a burst of volatile
emissions suggesting that the growth of the fungi was not sufficient
to damage the tomato glands, which would have stimulated
glandular-dependent defense responses. We neither detected
stress-induced emissions, at least in the time frame we sampled (4
days after B. cinerea leaf infection and 10 days after root inoculation
with B. bassiana). In contrast, constitutive plant VOC emissions
significantly decreased in B. bassiana-colonized leaves, as well as in
leaves treated with B. cinerea and both fungi together. Decrease of
A
B
FIGURE 7
Net photosynthesis (Pn, A) and dark respiration (Rd, B) from whole
plants of tomato in the VOC-SCREEN platform. Control plants (C)
are compared with plants treated with Beauveria bassiana (Bb),
Botrytis cinerea (Bc), and B. bassiana and B. cinerea (Bb-Bc). Values
are represented for each treatment as an average of all days (A) or
nights (B) of experiment. Statistical significance of differences
among the four treatments was assessed over the entire
experimental period by one-way ANOVA followed by Tukey’s test.
Means ± SEM (N=5) are presented, and statistically different means
(p < 0.05) are shown with different letters.
A
B
C
FIGURE 8
Cumulative CO
2
capture by photosynthesis (A), release by
respiration (B) and net capture by photosynthesis-respiration (C) in
whole plant cuvettes (N = 5 per treatment) are shown summing up
parameters measured each day during a 6-day long period, starting
at 9 dpi (as shown in the text). Control plants C are compared with
plants treated with Beauveria bassiana (Bb), Botrytis cinerea (Bc), and
B. bassiana and B. cinerea (Bb-Bc). Max SEM among the 5 cuvettes
per treatment and per day was always less than 3% of the reported
data. Statistical significance of differences among the four
treatments was assessed on the cumulated data (last data point) by
one-way ANOVA followed by Tukey’s test. Statistically different
means (p < 0.05) are shown with different letters.
Russo et al. 10.3389/fpls.2023.1309747
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plant VOC emissions after interaction with BMs was observed in
mycorrhized beans (Babikova et al., 2014). One possible
explanation for the reduction of the total VOC emissions could
be a depletion of the terpene pool resulting from the interaction
with the fungi.
Overall, we interpret our VOC results as an indication that
improved protection against B. cinerea by B. bassiana does not
involve VOC signalling. Nevertheless, our measurements showed a
relevant decrease (up to 80% in early phases of colonization/
infestation) of b-phellandrene and D-limonene, the two major
contributors (<90%) commonly found in the tomato volatile
bouquet (Jansen et al., 2009). Notably, b-phellandrene is known
to attract natural enemies (Colazza et al., 2004;Rasman et al., 2005),
hence the observed decrease in emission capacities upon B. bassiana
colonization could have significant ecological consequences in
tritrophic interactions.
The reduced total VOC emission might have been caused by the
fact that in plants with long-term endophytization by B. bassiana
less stress-signalling compounds are induced, or that emitted plant
VOCs are absorbed by the endophytic fungus before emission.
Further studies are needed to explain this result. Reduced VOC
emission might also contribute to save carbon and energy for
sustained growth and development of plants colonized by
B. bassiana.
It should also be noted that unfortunately we did not measure
VOCs when B. bassiana was temporarily rejected by plants, as
indicated by the reduction of net photosynthesis and stomatal
conductance (1-2 dpi) in both of our experiments. This
stimulation might have been associated to a temporary rise of
VOCs that might serve to prime defenses (Pollastri et al., 2021).
A rapid induction of monoterpenes was indeed reported in tomato
plants infected by B. cinerea (Jansen et al., 2009). This should be
assessed with future experiments, as it might be essential to fully
understand the impact of B. bassiana in tomato leaves, especially if
combined with detection of ROS and defensive metabolites in
colonized leaves (Stamelou et al., 2021).
5 Conclusions
Drawing on our results, we suggest that tomato plants in the
beginning perceive treatments with BMs (such as B. bassiana and T.
afroharzianum)asan‘infection’, translated into a significant but
short-term transient reduction in stomatal conductance and net
photosynthesis, and a possible and transient priming of defensive
metabolites. Thereafter, B. bassiana appears to establish itself as an
endophyte in tomatoes, stimulating plant growth and productivity.
Perhaps even more interestingly, B. bassiana seems to control the
infection of the widespread pathogen B. cinerea, largely reducing
the negative symptoms at foliar level. VOC emissions did not
explain how B. bassiana controlled the pathogen, but VOC
reduction might be interpreted as mirroring an improved plant
health status. These findings expand the possible use of B. bassiana
from being employed as an entomopathogen to a general and
promising use as a plant growth promoter and defender. Further
studies should focus on the mechanisms driving first negative
(lower photosynthesis and stomatal conductance) and then
positive (higher photosynthesis and growth, lower respiration)
plant responses to B. bassiana, and should also enquire whether
such responses are widespread and durable in other crops and in
natural vegetation.
Data availability statement
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
Author contributions
AR: Investigation, Data curation, Methodology, Writing –
original draft. JW: Data curation, Investigation, Methodology,
Writing –review & editing. AG: Data curation, Investigation,
Methodology, Writing –review & editing, Validation. SP: Data
curation, Investigation, Methodology, Writing –review & editing.
Time (dpi)
E-Phellandrene (pmol m
-2
s
-1
)
0
20
40
60
80
100
C
Bb
Bc
Bb-Bc
10 12 15
Time (dpi)
D - Limonene (pmol m
-2
s
-1
)
0
100
200
300
400
10 12 15
a
a
a
bb
ab
ab
b
ab
b
b
b
aa
a
b
bb
b
b
b
ab
bb
A
B
FIGURE 9
Comparison of the main two VOCs (D-limonene (A) and b-
phellandrene (B)) emitted by control tomato plants (C), and by
plants treated with Beauveria bassiana (Bb), Botrytis cinerea (Bc), and
both B. bassiana and B. cinerea (Bb-Bc) at 10, 12 and 15 dpi.
Statistical significance of differences among the means of the four
treatments was assessed at each dpi by one-way ANOVA followed
by Tukey’s test. Means ± SEM (N=5) are shown, and statistically
significant means (p < 0.05) are separated with different letters.
Russo et al. 10.3389/fpls.2023.1309747
Frontiers in Plant Science frontiersin.org12
MM: Data curation, Investigation, Methodology, Writing –review &
editing. MR: Data curation, Investigation, Methodology, Writing –
review & editing. JS: Writing –review & editing, Conceptualization,
Validation, Visualization. FL: Conceptualization, Validation,
Visualization, Writing –review & editing, Funding acquisition,
Investigation, Resources, Supervision.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This work was
supported by the Projects PRIN 2017 “Plant multitROphic interactions
for bioinspired Strategies of PEst ConTrol (PROSPECT)”funded by
the Italian Ministry of University and Research.
Acknowledgments
We would like to thank Ulrich Junghans and Georg Gerl for
their help for the greenhouse experiments in Munich, Baris Weber
for his technical assistance for GC-MS experiments in Munich, Ina
Zimmer and Petra Seibel for their chemical and biological assistance
in Munich, Dr Liberata Gualtieri and Francesca Mele for their help
with measurements in Naples.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
The author(s) declared that they were an editorial board
member of Frontiers, at the time of submission. This had no
impact on the peer review process and the final decision.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1309747/
full#supplementary-material
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