Communication

Assessing the Endophytic Potential of a Commercially

Available Entomopathogenic Beauveria bassiana Strain in
Various Citrus Rootstocks
Marco Arnoldi 1 , Emily B. Duren 2 , Pasco B. Avery 2 and Lorenzo Rossi 1, *

1 Horticultural Sciences Department, Institute of Food and Agricultural Sciences, Indian River Research and
Education Center, University of Florida, Fort Pierce, FL 34945, USA
2 Entomology and Nematology Department, Institute of Food and Agricultural Sciences, Indian River Research
and Education Center, University of Florida, Fort Pierce, FL 34945, USA
* Correspondence: [email protected]; Tel.: +1-(772)-577-7341

Abstract: The citrus industry is challenged by numerous arthropods, yet extensive research has
not been conducted to determine the potential use of entomopathogenic fungi as endophytes in
pest management strategies. Two inoculation methods (i.e., soil drench and foliar spray) using a
suspension of Beauveria bassiana (strain PPRI 5339 contained in Velifer® ) containing 4 × 107 conidia
mL−1 in 0.01% Tween 80 were conducted on three commercially available citrus rootstocks (i.e.,
‘US-942’, ‘US-812’, ‘Swingle’). Seedlings were grown under greenhouse-controlled conditions over a
7-week observation period. Similarly, a third inoculation method (seed soaking) was conducted using
seeds from the same three rootstocks. The fungus was re-isolated post-inoculation from ‘US-942’ and
‘US-812’ in the foliar spray and seed soaking treatments. In addition, the fungus was recovered from
root tissue in the foliar-sprayed seedlings, suggesting possible systemic movement from leaves to
Citation: Arnoldi, M.; Duren, E.B.; roots. The fungus was not recovered from soil-drench-treated seedlings, nor from any of the ‘Swingle’
Avery, P.B.; Rossi, L. Assessing the cultivars. This study assessed the potential of B. bassiana to endophytically colonize certain citrus
Endophytic Potential of a rootstocks in planta.
Commercially Available
Entomopathogenic Beauveria bassiana Keywords: citrus; endophyte; entomopathogenic fungi; rootstocks
Strain in Various Citrus Rootstocks.
Appl. Microbiol. 2022, 2, 561–571.
https://doi.org/10.3390/
applmicrobiol2030044 1. Introduction
Academic Editor: Katarzyna Entomopathogenic fungi (EPF) are types of fungi that are parasitic to arthropods [1].
Turnau Fungal propagules in contact with the arthropods cuticle first germinate, and then form an
Received: 19 July 2022
appressorium, followed by a penetration peg. The fungus produces chitinase and other
Accepted: 9 August 2022
enzymes to break down the cuticle. This leads to continued hyphal growth through the
Published: 11 August 2022
cuticle until it reaches the hemocoel, where it produces hyphal bodies (or blastospores),
eventually forming a mycelial mass and killing the arthropod [2,3]. Some species release
Publisher’s Note: MDPI stays neutral
mycotoxins, such as beauvericin, that can injure or kill the insect before mycosis fully
with regard to jurisdictional claims in
takes place [3]. When conditions are favorable, the fungus forms conidiophores on the
published maps and institutional affil-
surface of the arthropod’s body, which release spores for new infections [4,5]. Commercially
iations.
available EPF-based biopesticides are being used as an alternative to synthetic chemical
insecticides, since they can have negative health, environmental, ecological, and economic
consequences [4,6]. EPF-based biopesticides, however, have some drawbacks as well, e.g.,
Copyright: © 2022 by the authors.
decreased efficacy when exposed to ultraviolet light and low percent relative humidity,
Licensee MDPI, Basel, Switzerland. non-compatible interactions when mixed with other insecticides during field application,
This article is an open access article and relatively short-term persistence [7]. To circumvent these issues, an alternative ap-
distributed under the terms and proach utilizing EPF as endophytes has received increased research attention over the past
conditions of the Creative Commons decade [8].
Attribution (CC BY) license (https:// Fungal endophytes are microorganisms that live within plant tissues (in planta); they
creativecommons.org/licenses/by/ are common, diverse, symbiotic, and potentially mutualistic, especially among ento-
4.0/). mopathogenic species [9]. It is believed that most plant species in natural ecosystems

Appl. Microbiol. 2022, 2, 561–571. https://doi.org/10.3390/applmicrobiol2030044 https://www.mdpi.com/journal/applmicrobiol

Appl. Microbiol. 2022, 2 562

contain fungal endophytes, with woody species having hundreds or even thousands of
species [9,10]. While most are merely symbiotic, EPF can provide the plant with distinct
benefits, particularly increased resistance to arthropods. The artificial inoculation of plants
with entomopathogenic fungi has shown to be useful in controlling arthropod pests, pro-
moting plant growth, and increasing tolerance to plant pathogens, in part by releasing
secondary metabolites [11–15]. Additionally, arthropods that encounter fungal propagules
present within the plant may suffer harm, or even death. However, some evidence shows
that the presence of fungal conidia within the plant is uncommon. This lends additional
support to the hypothesis that antibiosis occurs via secondary metabolites—which act as a
deterrent rather than by fungal propagules [16].
Crops have been successfully endophytically colonized using a variety of EPF and
inoculation methods. The use of EPF as endophytes for insect pest control was pioneered
by Lewis and Bing [17], who were able to suppress European corn borer, Ostrinia nubilalis
Hübner (Lepidoptera: Crambidae), populations in corn using Beauveria bassiana (Bals.-
Criv.) Vuill. (Ascomycota: Hypocreales). This fungal species has been the most used in
studies involving inoculation and colonization of crops [8] such as cotton, tomato, common
bean, strawberry, coffee, sweet pepper, corn, sorghum, and potato, among others [18–26].
Successful colonization has been achieved using various inoculation methods, such as
foliar sprays, seed soaking, and soil drenching. Inoculation methods may be differentially
effective depending on the plant species, and may also influence the localization of the
fungus. For example, a foliar spray may be more effective for the inoculation of leaves,
while a soil drench may be more effective for roots [13,15,27]. Biotic and abiotic factors,
such as soil microorganisms or temperature, and other variables can influence the fungal
endophytic colonization of a crop. In addition, inoculation methods, growth medium,
fungal species, conidial concentration, crop age, and crop cultivar have all been reported to
influence colonization effectiveness [13].
The citrus industry in Florida faces challenges from multiple arthropod pests, with
the most important being the Asian citrus psyllid (Diaphorina citri Kuwayama; Hemiptera:
Liviidae), the main vector that spreads the pathogen associated with citrus greening (Huan-
glongbing, HLB) [28,29]. Other common pests include aphids, whiteflies, scales, weevils,
and mites [30]. The need to manage arthropod pests in citrus agroecosystems, has led to a
dependency on frequent applications of synthetic chemical insecticides, which has resulted
in arthropods developing resistance and secondary pest resurgence [28,31,32]. Additionally,
these intensive chemical applications have caused a decrease in the biodiversity and abun-
dance of beneficial insects in agroecosystems [32]. Therefore, more management strategies
that are sustainable and economically viable need to be researched [33].
Although there is increasing interest in the applications of EPF as endophytes, the
number of studies incorporating them into citrus is marginal. Given the numerous arthro-
pod pests and pathogens that challenge the citrus industry, it would be beneficial to increase
the knowledge about inoculating citrus plants with these fungi. The applications of EPF
can be used in a sustainable and integrated pest management strategy. Our objective in
this study was to determine the potential endophytic colonization of plant tissues with B.
bassiana in different citrus rootstocks, using different inoculation methods. This information
would be an important first step in determining the potential application and efficacy of
endophytic fungal entomopathogens in the citrus industry, principally for the control of
damaging arthropod pests.

2. Materials and Methods

2.1. Fungal Strain and Conidial Stock Suspension
Beauveria bassiana strain PPRI 5339, contained in the EPF-based biopesticide Velifer®
ES (BASF SE, Florham Park, NJ, USA), was used for this study. To prepare the conidial stock
suspension, 1 mL of Velifer was diluted in 100 mL of sterile deionized H2 O and stirred with
a magnetic stir bar for 30 min. The conidial concentration of the dilution was determined to
be 2 × 106 conidia mL−1 by using a Neubauer Improved C-Chip hemacytometer (INCYTO
Appl. Microbiol. 2022, 2 563

Co., Ltd., Cheonan, Chungnam, Korea). Then, 100 µL of the dilution was pipetted onto
potato dextrose agar (PDA) plates amended with streptomycin sulfate and chloramphenicol
and spread with a sterilized bent-glass rod (according to Avery et al. [34]). The top cover
was replaced; plates were then sealed with Parafilm “M”® (Bemis Inc., Neenah, WI, USA)
and placed in a growth chamber at 25 ◦ C with a 14 h photophase for 2 weeks. This
process was repeated weekly to ensure an adequate supply of conidia for inoculation
methods and trial repetitions. Conidial viability was determined to be greater than 95% for
all experiments.
After 2 weeks in the growth chamber, the plates were removed, and the conidia were
harvested. This was achieved by flooding each PDA plate with 15 mL of 0.01% Tween
80 solution, and the agar surface was gently scraped with a flame-sterilized, bent-glass
rod (Millipore-Sigma, Burlington, MA, USA) to release the conidia. Each plate suspension
was then carefully poured through four layers of sterile cheesecloth into individual 200 mL
Erlenmeyer flasks to filter out hyphal fragments from the conidia. The concentration of
this suspension was adjusted to 4 × 107 conidia mL−1 . This conidial stock suspension
was used in each of the inoculation methods, which included foliar spray, soil drench, and
seed soaking.

2.2. Plant Material

Three citrus rootstocks were used in this experiment. The seedlings were grown and
obtained from the Southern Citrus Nurseries (Dundee, FL, USA). The rootstock genotypes
were US-812 (Citrus reticulata ‘Sunki’ × Poncirus trifoliata ‘Benecke’), US-942 (C. reticu-
lata ‘Sunki’ × P. trifoliata ‘Flying Dragon’), and Swingle citrumelo (C. paradisi Macfad ×
P. trifoliata (L.) Raf.). All seedlings (32 for each genotype) were 6 months old when received.
They were immediately transferred to one-gallon black plastic pots filled with washed
and dry-heated ‘All-natural Pure Play’TM sand (Sakrete of North America LLC., Charlotte,
NC, USA) and were given 12 g of Osmocote® 15-9-12 fertilizer (The Scotts Company LLC.,
Marysville, OH, USA). To accurately simulate natural soil conditions, the sand was not
sterilized. Although this may potentially lead to competition with the B. bassiana spores and
other microorganisms, it also is more reflective of field conditions. The pots were arranged
in a greenhouse using a split-plot experimental design, and the plants were given 5 weeks
to re-acclimate before commencing inoculations. During this time, they were watered twice
a day (~30 mL min−1 each time) using a drip irrigation system, and lateral shoots were
pruned as needed.

2.3. Plant Inoculations

The rootstocks in the greenhouse were inoculated with the conidial stock suspen-
sion. The conidial concentration for both foliar spray and soil drench treatments was
4 × 107 conidia mL−1 . Both the foliar spray and soil drench treatment groups consisted of
24 plants, with 12 treated and 12 control each.

2.3.1. Foliar Spray

A Nalgene® hand pump sprayer (Nalgene Nunc International, Rochester, NY, USA)
was used to apply a total of 150 mL of 0.01% Tween 80 with B. bassiana conidia
(4 × 107 conidia mL−1 ) to the treated group, while the control group received only 150 mL
of 0.01% Tween 80. Each plant received approximately 12 mL of inoculum. The pots were
covered with aluminum foil prior to spraying to prevent conidia from contaminating the
soil surface. There were 12 plants sprayed with conidia and 12 control, and the process was
repeated twice. To assess the efficacy of the foliar spray technique, leaf conidial deposition
was determined by pinning coverslips to the adaxial and abaxial surfaces of leaves prior
to spraying per rootstock. The plastic cover slips were allowed to dry for 24 h and then
removed from the leaf and pinned into a Styrofoam® lid. Each cover slip was prepared us-
ing the protocol of Avery, Kumar, Skvarch, Mannion, Powell, McKenzie, and Osborne [34]
and observed under a stereo light microscope (Leica ICC50 E, Leica, Wetzlar, Germany)
Appl. Microbiol. 2022, 2 564

with a digital camera. Images were taken at 400× magnification to count the number of
conidia per mm2 . Fiji imaging software [35] was used to provide quantification of conidia.
In addition, three leaves per tree were collected and pressed (adaxial and abaxial surfaces)
onto PDA plates amended with selective fungicide dodine (PDA–dodine), before and after
spray treatment to visually assess coverage of the spray.

2.3.2. Soil Drench

Each plant in the soil drench treatment received 35 mL of fungal inoculum
(4 × 107 conidia mL−1 ) in 0.01% Tween 80, and control plants received only 0.01% Tween 80.
The inoculum or the Tween 80 was applied to the soil surface at the base of the plant directly
around the stem using a battery-powered pipette (BrandTech, Essex, CT, USA). To assess the
efficacy of the soil drench technique using a drip irrigation system, allowing the B. bassiana
conidia to leach downward through the moist sand to the root depth, soil core samples
were taken with a modified 25 mL plastic pipette [36] prior to destructive sampling of the
plants. Two core samples from each pot were taken by pressing a modified 25 mL plastic
pipette with the tip sawed off into the moist sand to obtain a core sample. These cores
were then assessed to determine the presence and concentration in colony-forming units
(CFUs) of B. bassiana conidia at varying depths in the soil. The core sample was divided
into bottom, middle, and upper depth levels by sawing the pipette into three 4 ± 1 cm
sections. Sand from the same depth levels and rootstock genotypes were combined and
mixed in Ziploc® bags (S.C. Johnson and Son, Racine, WI, USA). Ten-gram random samples
were taken from the bags, placed in 50 mL plastic tubes (VWR, Radnor, PA, USA) with
30 mL of deionized water, and vortexed for 30 s [27]. Ten individual aliquots of 100 µL
were then removed from the supernatant, and each was spread onto separate PDA–dodine
plates using a flame-sterilized, bent-glass rod. The top covers were replaced, plates were
sealed with Parafilm, randomly arranged on a plastic cafeteria tray, and placed in the same
growth chamber in the environmental conditions stated above. Plates were observed daily
for approximately 1.5 weeks to visually observe and count CFUs of B. bassiana amongst the
other fungi.

2.4. Seed Inoculation

Similar to the seedlings, a total of 264 seeds (88 from each rootstock) were used for the
experiment. Seeds were obtained from the nursery mentioned above. After being soaked
in water for 24 h, the seeds were surface sterilized by soaking in 70% ethanol for 3 min,
followed by 2% sodium hypochlorite for 3 min, and were then rinsed with sterile deionized
water three times [27]. Aliquots of 100 µL of the final rinse water were spread onto PDA
plates amended with selective fungicide (dodine and the previously mentioned antibiotics),
and top covers were replaced. They were sealed with Parafilm, randomly arranged on a
cafeteria tray, and placed into the same growth chamber in the environmental conditions
stated above. After 2 weeks, the plates were checked for fungal contamination to determine
if sterilization was successful. This process was performed in 12 batches so that samples
showing contamination could be excluded.
The seeds from each batch were then evenly spaced on a moistened paper towel in
large 150 mm sterile empty glass Pyrex® Petri dish (Corning Inc., Corning, NY, USA), top
covers were replaced, and Petri dishes were sealed with Parafilm to initiate germination.
Sealed dishes were placed in the dark at room temperature (~23 ◦ C). Three weeks after seed
germination had been initiated, each batch of seeds were soaked in a conidial suspension at
a concentration of 4 × 107 conidia mL−1 for 24 h. Soaked seeds were then sown in seedling
trays (SureRoots50® , T.O. Plastics, Inc., Clearwater, MN, USA) filled with the same sand
mentioned above. Seeds were allowed to grow and were watered as needed over the course
of 8 weeks. Afterwards, the sprouted seedlings were harvested for destructive sampling to
determine if B. bassiana was endophytic in the roots, stems, and leaves.
Appl. Microbiol. 2022, 2 565

2.5. Assessing Fungal Colonization of Plant Material

After 7 weeks, plants were harvested, destructively sampled, and root, leaf, and stem
samples were collected and surface sterilized. The bulk sterilization protocol developed
by Greenfield et al. [37] was used to process large volumes of plant samples by soaking
them in 70% ethanol for 2 min, followed by soaking them in 2% sodium hypochlorite
for 3 min. After sterilization, the plant samples were rinsed with sterile, deionized water
three times, and 100 µL of the final rinsate was spread onto PDA–dodine plates. The
top covers replaced; they were sealed with Parafilm and then placed in the same growth
chamber as above under the same environmental conditions. This process was to confirm
successful sterilization of the organs with no contamination from saprophytic fungi. Mature
leaves from the mid-section of the plant were also pressed onto PDA–dodine plates and
processed as described above to assess if B. bassiana propagules had remained on the surface
after sterilization.
Roots, mature, and newly emerged leaves (where mature leaves are the ones that
were present at the time of the fungal application and newly emerged leaves are the ones
that developed post-application) and stems were trimmed after sterilization to remove
edges where the sterilization process may have negatively affected endophytic B. bassiana.
Trimmed samples were then placed onto PDA–dodine plates and processed as described
above. Root samples (~5 cm) were divided into root tips (~1 cm) and upper roots (~4 cm
from root tip), and leaves were divided into upper (towards the apex), lower leaf (towards
the petiole), and newly emerged leaves sections. Similarly, four pieces (~1 cm) of the stems
were cut from the mid-section of the plant. The plant samples were placed and arranged
as described inside of Petri dishes, processed as described above, and put into the same
growth chamber under the environmental conditions described above. After 2 weeks in
the growth chamber, the plates were removed and observed for phenotypic fungal growth
of B. bassiana deriving from the plant samples. A pure colony of B. bassiana was used as a
template to compare against the other fungi. Similar endophyte colonies were compared
morphologically both under a stereo microscope (Leica EZ4E, Leica, Wetzlar, Germany)
(colony morphology) and under a stereo light microscope (described above) for phenotypic
morphology to confirm whether an outgrowth was indeed B. bassiana. Additionally, due to
suspected B. bassiana strain PPRI 5339 outgrowth, agar plugs containing the outgrowth were
removed from plates using a flame-sterilized cork borer, cultured on separate PDA–dodine
plates, and processed as described above for additional comparison. A count was recorded
for each occurrence of B. bassiana outgrowth from the plant samples.

2.6. Statistical Analysis

One-way ANOVA and Tukey’s tests were used to determine differences in CFU counts
between soil layers. Significance for all tests was set as p ≤ 0.05. The R software was used
for data analysis (https://www.r-project.org/, accessed on 18 July 2022).

3. Results
3.1. Fungal Re-Isolation
None of the control plants showed signs of endophytic colonization by B. bassiana.
Overall, attempts at re-isolating endophytic B. bassiana from various sections of plant tissue
resulted in five successful re-isolations from 126 treated plants and seeds (Table 1). Of
these five samples, three occurred in the foliar spray treatment (24 treated plants) and two
in the seed soaking treatment (75 treated seeds), while no samples from the soil drench
treatment displayed endophytism by B. bassiana, regardless of rootstock genotype. Of the
three positive samples in the foliar spray group, one appeared in roots from US-942 and one
in new flush (leaves that sprouted after spraying), while the third appeared in US-812 from
a sprayed leaf sample (Figure 1). The two B. bassiana endophytic samples from the seed
soaking method were observed growing from the stem pieces of US-812 and US-942. Tissue
samples from the treated Swingle rootstocks did not yield any endophytism of B. bassiana.
 Of the three positive samples in the foliar spray group, one appeared in roots from US-
942 and one in new flush (leaves that sprouted after spraying), while the third appeared
in US-812 from a sprayed leaf sample (Figure 1). The two B. bassiana endophytic samples
from the seed soaking method were observed growing from the stem pieces of US-812
Appl. Microbiol. 2022, 2 and US-942. Tissue samples from the treated Swingle rootstocks did not yield any endo-
566
phytism of B. bassiana.

Table 1. Colonization frequency of Beauveria bassiana in different plant organs (leaves, roots, and
Table 1. Colonization frequency of Beauveria bassiana in different plant organs (leaves, roots, and stem)
stem) of three citrus rootstocks. Data were collected seven weeks after application of 4 × 107 conidia
of
mL three
−1. citrus rootstocks. Data were collected seven weeks after application of 4 × 10 conidia mL−1 .
7

Colonization
ColonizationFrequency
Frequency of DifferentPlant
of Different Plant Organs
Organs a,b a,b

Inoculation
Inoculation Newly
Newly
Rootstock
Rootstock Root Tip
Root Tip Upper
Upper RootLower
Root Lower
Leaf Leaf
UpperUpper
Leaf LeafStemStem
Method
Method Emerged
Emerged Leaf
Leaf
nN %% n
n %% n n % % n n % %n n% % n n %%
US-812
US-812 Foliar
Foliar spray
spray 00 0 0 00 00 1 1 12.512.5 0 0 0 00 00 0 0 0 00
US-812
US-812
Soil drench
Soil drench 0
0 0
0 00 0
0 0
0 0 0 0 0 0
00 00 0 0 0 0
0
US-812 Seed soak 0 0 0 0 0 0 0 0 1 4 NA NA
US-812 Seed soak 0 0 0 0 0 0 0 0 1 4 NA NA
US-942 Foliar spray 0 0 1 12.5 0 0 0 0 0 0 1 12.5
US-942 Foliar spray 0 0 1 12.5 0 0 0 0 0 0 1 12.5
US-942 Soil drench 0 0 0 0 0 0 0 0 0 0 0 0
US-942
US-942 Soil drench
Seed soak 00 0 0 00 00 0 0 0 0 0 0 0 00 10 4 0 NA 0NA
US-942
Swingle Seed spray
Foliar soak 00 0 0 00 00 0 0 0 0 0 0 0 01 04 0 NA 0 NA0
Swingle
Swingle Soil drench
Foliar spray 00 0 0 0 00 0 0 0 0 0 0 0 00 00 0 0 0 00
Swingle
Swingle Seed soak
Soil drench 00 0 0 00 00 0 0 0 0 0 0 0 00 00 0 0 NA 0NA
Swingle Seed soak
aFrequency
0 is expressed
0 0 as total
0 number
0 of re-isolations
0 0 and
0 as a percentage
0 0 (number
NA of re-isola-
NA
tions out of the total number of plants of each rootstock per inoculation method). b Sections of plant
a Frequency is expressed as total number of re-isolations and as a percentage (number of re-isolations out of
tissue from the plant organs were surface sterilized and then placed onto potato dextrose agar plates
the total number of plants of each rootstock per inoculation method). b Sections of plant tissue from the plant
amended with streptomycin, chloramphenicol, and dodine. Plates were placed in a growth chamber
organs were surface sterilized and then placed onto potato dextrose agar plates amended with streptomycin,
at 25 °C and with
chloramphenicol, and 14 h photophase.
dodine. Plates were After
placed 2inweeks, plates
a growth were
chamber removed
at 25 and14assessed
◦ C and with for fungal
h photophase. After
2outgrowth from
weeks, plates wereplant tissue
removed andsamples.
assessed for fungal outgrowth from plant tissue samples.

Figure 1. Successful
Figure 1. Successful re-isolation
re-isolation ofof endophytic
endophytic entomopathogenic fungus Beauveria
entomopathogenic fungus Beauveria bassiana
bassiana (strain
(strain
PPRI 5339 contained in Velifer ®®
) from different plant organs of three citrus rootstocks (Swingle, US-
PPRI contained in Velifer ) from different plant organs of three citrus rootstocks (Swingle,
812 and US-942): (A) leaf, (B) stem, and (C) root tissue. Data were collected seven weeks
US-812 and US-942): (A) leaf, (B) stem, and (C) root tissue. Data were collected seven weeks after after appli-
cation of 4 ×of10
application 4 ×conidia
7 mL−1.mL
107 conidia Plant
−1 . sections were surface
Plant sections sterilized,
were surface placed
sterilized, on potato
placed dextrose
on potato agar
dextrose
plates amended with streptomycin, chloramphenicol, and dodine. Plates were
agar plates amended with streptomycin, chloramphenicol, and dodine. Plates were placed in a placed in a growth
chamber at 25 °C and with 14 h photophase. After 2 weeks, plates were removed and assessed for
growth chamber at 25 ◦ C and with 14 h photophase. After 2 weeks, plates were removed and
fungal outgrowth from plant samples. (A,C) are from foliar-spray-treated US-812 and US-942, re-
assessed for fungal outgrowth from plant samples. (A,C) are from foliar-spray-treated US-812 and
spectively. (B) is from the seed-soak treatment of US-942. No re-isolation of endophytic Beauveria
US-942,
bassiana respectively.
was observed(B) is from
with the seed-soak
the Swingle treatment of US-942. No re-isolation of endophytic
rootstock.
Beauveria bassiana was observed with the Swingle rootstock.

3.2. Plant Inoculation Methods

Leaf presses showed that the foliar spray effectively coated the adaxial and abaxial
sides of leaves with active conidia. Plants sprayed with conidia showed distinct fungal
colony growth within the outline after pressing leaves onto PDA–dodine plates (Figure 2).
 3.2. Plant Inoculation Methods
Leaf presses showed that the foliar spray effectively coated the adaxial and
Leaf presseswith
sides of leaves showed that
active the foliar
conidia. spray
Plants effectively
sprayed with coated
conidiathe adaxial
showed and
distinc
sides
colonyof growth
leaves with active
within the conidia. Plants
outline after sprayed
pressing with onto
leaves conidia showed distinct
PDA–dodine plates
Appl. Microbiol. 2022, 2 colony
2). growth within the outline after pressing leaves onto PDA–dodine567 plates
2).

Figure 2. Sample image of leaf presses on potato dextrose agar plates amended with strep
Figure
Figure 2.2.Sample
Sample
chloramphenicol, image
image and of presses
of leaf leaf presses
dodine. Citrus ondextrose
potato
leaves
on potato weredextrose
foliar
agar agar
sprayed
plates plates
with
amended amended
Beauveria
with with strept
bassiana
streptomycin, (Str
chloramphenicol,
chloramphenicol, and and dodine.
dodine. Citrus
Citrus leaves leaves
were were
foliar foliar
sprayed sprayed
with
5339 contained in Velifer ). Image was taken 14 days after spraying.
® with
Beauveria Beauveria
bassiana (Strain bassiana
PPRI (Stra
® ). Image
5339 contained
5339 contained in in Velifer
Velifer ®). Image was14taken
was taken 14 days
days after after spraying.
spraying.

The
The mean
mean number
number (±SD)(±SD) of conidia
of conidia per mm2per mm slips
on cover on cover
2 slips
that were thattowere
pinned the pinne
The
leaveswas
leaves mean
was
45.69 number
45.69
(SD (SD (±SD)
= 107.41),
= 107.41), of conidia
andwas
and there there per mm 2 on cover slips that were pinned
was no significant
no significant difference indifference
mean number inofmean nu
leaves
conidia was 45.69
between (SD
cover = 107.41),
slips on and
adaxial andthere was
abaxial no significant
surfaces of the difference
leaves (Figure
conidia between cover slips on adaxial and abaxial surfaces of the leaves (Figure 3). in mean num
conidia between cover slips on adaxial and abaxial surfaces of the leaves (Figure 3

Figure
Figure 3. 3. Conidial
Conidial deposition
deposition of Beauveria
of Beauveria bassiana
bassiana (strain PPRI(strain PPRI 5339
5339 contained contained
in Velifer in Velifer ) o
® ) on a plastic ®

Figure
cover 3. that
tic cover
slip Conidial
slipwas
that deposition
was
acid of Beauveria
acidstained
fuchsin fuchsinandstained bassiana (strain PPRI 5339
and photographed
photographed at contained
at 400×
400× magnification. in Velifer®) o
magnification.
tic cover slip that was acid fuchsin stained and photographed at 400× magnification.
The soil cores revealed that the top layer of the soil had significantly (F = 92.30;
The soil cores revealed that the top layer of the soil had significantly (F = 92
df = 2, 82; p < 0.0005) higher quantities of CFUs compared to the bottom and middle layers
2, The
82; soil
p <(Tablecoreshigher
0.0005) revealed that theoftop layer of the soil
tohad significantly (F = 92.l
of the soil 2). There wasquantities CFUs compared
no significant difference per rootstock the bottom
between and middle
the number
2,
of 82;
the p <in(Table
soil
CFUs 0.0005)
the top2).higher
andThere quantities
middlewas of
theCFUs
soil. Incompared
noofsignificant
layers the bottom to
difference therootstock
per
layers, bottom
more CFUsand middle
between
were thela
the soil (Table
observed
of CFUs per the2).with
inplate There
top and was
rootstocks no significant
US-942
middle and US-812,
layers difference
of the compared per rootstock
to Swingle.
soil. In the bottom between
Plants morethe
in the
layers, CFn
of CFUs
control in the
groups didtop
not and
have middle layers
any observed CFUsof of
the soil. In the bottom layers, more CFU
B. bassiana.
observed per plate with rootstocks US-942 and US-812, compared to Swingle. P
observed pergroups
the control plate with rootstocks
did not have anyUS-942 andCFUs
observed US-812,
of B.compared
bassiana. to Swingle. Pl
the control groups did not have any observed CFUs of B. bassiana.
Appl. Microbiol. 2022, 2 568

Table 2. Mean number of colony-forming units (CFUs) of Beauveria bassiana (Strain PPRI
5339 contained in Velifer® ). Soil cores samples were taken from moist sandy soil after 6 weeks
post-inoculation via soil drench. Sample cores were taken from each pot and divided into three equal
depths (bottom, middle, and top), and suspensions were made. Ten samples from each suspension
were spread onto potato dextrose agar plates amended with streptomycin, chloramphenicol, and
dodine. Results are categorized by citrus rootstock. No CFUs of B. bassiana were observed in any of
the control rootstock groups.

Mean ± SE Number of CFUs per Soil Depth per Plate a,b

Rootstock
Bottom c Middle c Top c
US-942 10.0 ± 8.5 bB 3.0 ± 1.7 aA 81.0 ± 50.2 aC
US-812 6.0 ± 5.5 bB 1.0 ± 0.7 aA 225.0 ± 53.9 aC
Swingle 3.0 ± 1.6 aA 2.0 ± 1.3 aA 114.0 ± 30.7 aB
a 95% confidence interval (SE × 1.96); b CFU values in a column that are significantly different are followed by
lower case; letters and across rows by upper-case letters (Tukey’s HSD test, p < 0.05); c each depth = 4.0 ± 1.0 cm.

4. Discussion
In this study, a strain of the entomopathogenic fungi B. bassiana was applied to three
different citrus rootstocks using three inoculation methods. This was carried out to deter-
mine if this strain could become endophytic in citrus, to what degree or frequency it was
successful, as well as which inoculation methods and rootstock cultivar had the highest
occurrence of fungal endophytism by B. bassiana. In doing so, this study explored the poten-
tial application of using fungal entomopathogens as endophytes in citrus agroecosystems
and nursery production for the purpose of biological arthropod pest control and other
potential benefits, such as increased disease resistance.
We found that this strain of B. bassiana had a low occurrence or incidence rate in
becoming endophytic in these citrus rootstocks tested using different inoculation methods,
but nonetheless still demonstrated potential colonization in planta under optimal environ-
mental conditions. No plants in the soil drench group had re-isolated B. bassiana, while
three plants in the foliar spray group and two plants in the seed soaking group displayed
fungal endophytism in our tissue samples. Within the foliar spray group, B. bassiana was
re-isolated from root and leaf tissue samples, as well as newly emerged leaves, demonstrat-
ing systemic movement of the fungal endophyte. A similar study involving B. bassiana
foliar-spray-inoculated lemon seedlings reported a colonization percentage of tissue sam-
ples as high as 60% and 43% for leaves, and 36% and 24% for stems at 7 and 80 days
post-inoculation, respectively [38]. In cotton plants treated by soaking seeds in a conidial
suspension, B. bassiana was reisolated from 33 to 66% of sampled plants at different growth
stages [39]. Using a soil drench inoculation on cassava plants, Greenfield et al. [40] reported
that 84% of treated plants were colonized at 7 to 9 days after inoculation, which dropped
to 40% at 47 to 49 days after inoculation. Numerous other studies using B. bassiana and
other entomopathogenic fungi to inoculate a variety of crop species have reported high per-
centages of colonization of plants by these fungi, along with a general trend of decreasing
endophytism over time [13].
There are multiple plausible reasons as to why a greater amount of endophytism by
B. bassiana was not observed in the citrus rootstocks used in this study. These include (but
are not limited to) potting substrate, fungal strain, plant species, plant age and health,
competition with other endophytes, and surface sterilization protocols [13,20,41]. The
rootstocks were already 7 months old when inoculated with B. bassiana, and re-isolations
were only carried out 7 weeks after inoculations. Bamisile et al. [42] showed that lemon
seedlings that were 6 months old were less effectively colonized than seedlings that were
3 months old. Furthermore, while B. bassiana has been shown to persist in some woody
plant species for over 6 months [22,43], this fungal endophyte has also been observed to
be less effective over time, which can be influenced by plant species and fungal strain
interactions, among other factors [40].
Appl. Microbiol. 2022, 2 569

The potting medium used can also influence endophytic colonization success, with
non-sterile soil showing lower colonization success of B. bassiana compared to sterile soil
and vermiculite [25]. The potting medium used in this study was not sterilized; therefore,
the sandy soil may have contained antagonistic microorganisms that would have competed
with the endophytism of B. bassiana. Parsa et al. [20], found that the potting substrate
used has a strong influence on the outcome of potential endophytic colonization following
seed soaking of the common bean, and predictable colonization rates are unlikely under
natural microbial soil conditions. There is high variability in the colonization success rates
of the numerous EEPF applied to different crop species. This suggests that the interaction
between plant cultivars and EPF plays a major role in determining the efficacy of employing
the EPF as endophytes [44].
Our results align with findings from similar studies in which inoculation method
and plant species/genotype influenced the success of endophytic colonization, and the
migration of B. bassiana to areas outside of the inoculated region [45]. However, this
movement was observed in un-grafted rootstocks. Whether B. bassiana is able to move
internally past the grafting union of a citrus rootstock and scion has not been tested. The
pathway for internal movement of the endophyte could be through air spaces between
parenchyma cells or through the plants’ vascular tissue [46].
No fungal growth was detected from any plants within the soil drench treatment,
which may be due to the conidia not leaching through the soil deeply enough to reach
the roots zone in significant quantities. Soil cores taken from the pots showed that the top
3–5 cm of soil contained significantly higher amounts of B. bassiana conidia compared to
the middle and bottom layers of the soil. This may have resulted in an insufficient number
of conidia reaching the roots of the plant to successfully colonize them. Meanwhile, three
plants in the foliar spray treatment had B. bassiana re-isolated from samples. Additionally,
within those foliar-spray-treated plants, the fungus was recovered from both stem and root
tissue, demonstrating that it is possible for the endophyte to move systemically within the
plant. Furthermore, B. bassiana was only re-isolated from US-812 and US-942 rootstocks
and not from Swingle rootstocks. A clear reason for this is not known; it may be by chance
or due to interactions with this strain of B. bassiana and the Swingle rootstock.

5. Conclusions
As an endophyte, the entomopathogen B. bassiana shows promise not only as a sustain-
able means for arthropod pest control, but also in providing plants with other benefits such
as increased nutrient uptake, disease resistance, plant growth, and general plant health.
This study shows that inoculating citrus rootstocks with B. bassiana is possible using certain
inoculation methods, but more research is needed to increase endophytic colonization rates
and determine their benefits. Factors such as environmental conditions and the age of
trees might prove challenging when it comes to implementing the usage of endophytic
B. bassiana in commercial citrus operations. Using B. bassiana as an endophyte may be more
applicable in a nursery production setting, and future research may focus on this.

Author Contributions: Conceptualization, L.R. and P.B.A.; methodology, P.B.A.; investigation, M.A.
and E.B.D.; resources, L.R. and P.B.A.; statistical analysis, M.A.; writing—original draft preparation,
M.A.; writing—review and editing, L.R., M.A. and P.B.A.; supervision, L.R. and P.B.A.; funding acqui-
sition, L.R. and P.B.A. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by U.S. Department of Agriculture (USDA), National Institute
of Food and Agriculture, Hatch project #FLA-IRC-005743. Florida Department of Agriculture and
Consumer Services contract #27971; NIFA/USDA Multi-State Hatch Research Project #FLA-IRC-
005729; S1070: The Working Group on Improving Microbial Control of Arthropod Pests.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Appl. Microbiol. 2022, 2 570

Data Availability Statement: The data presented in this study are available within this article. Raw
data are available upon request to the corresponding author ([email protected]).
Acknowledgments: We thank Southern Citrus Nurseries (Dundee, FL, USA) for providing us with
plant materials. We are also grateful to Laura Muschweck and John-Paul Fox for assistance with
treatment application and data collection.
Conflicts of Interest: The authors declare no conflict of interest.

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