Fungal Biology xxx (xxxx) xxx
ts available at ScienceDirectContents lisFungal Biology
journal homepage: www.elsevier .com/locate/ funbioConidial mass production of entomopathogenic fungi and tolerance of
their mass-produced conidia to UV-B radiation and heat
Drauzio E.N. Rangel a, c, *, Mavis A. Acheampong b, Helen G. Bignayan c, d,
Hernani G. Golez c, d, 1, Donald W. Roberts c, 1
a Universidade Tecnolo
gica Federal do Parana
, Dois Vizinhos, Parana
, 85660-000, Brazil
b Department of Crop Science, University of Ghana, Legon, P.O. Box LG 44, Accra, Ghana
c Department of Biology, Utah State University, Logan, UT, 84322-5305, USA
d Bureau of Plant Industry, National Mango Research, and Development Center, Jordan, Guimaras, 5045, Philippinesa r t i c l e i n f o
Article history:
Received 17 November 2022
Received in revised form
30 April 2023
Accepted 7 July 2023
Available online xxx
Handling Editor: Dr Simon Avery
Keywords:
Entomopathogenic fungi
Mass production
Heat tolerance
UV-B radiation tolerance
Conidial viability* Corresponding author. Universidade Tecnolo
gic
Vizinhos, Parana
, 85660-000, Brazil.
E-mail address: drauzio@live.com (D.E.N. Rangel).
1 Deceased.
https://doi.org/10.1016/j.funbio.2023.07.001
1878-6146/© 2023 British Mycological Society. Publis
Please cite this article as: D.E.N. Rangel, M
tolerance of their mass-produced conidia toa b s t r a c t
We investigated conidial mass production of eight isolates of six entomopathogenic fungi (EPF), Apha-
nocladium album (ARSEF 1329), Beauveria bassiana (ARSEF 252 and 3462), Lecanicillium aphanocladii
(ARSEF 6433), Metarhizium anisopliae sensu lato (ARSEF 2341), Metarhizium pingshaense (ARSEF 1545),
and Simplicillium lanosoniveum (ARSEF 6430 and 6651) on white or brown rice at four moisture condi-
tions (75e100%). The tolerance of mass-produced conidia of the eight fungal isolates to UV-B radiation
and heat (45 
C) were also evaluated. For each moisture content compared, a 20-g sample of rice in a
polypropylene bag was inoculated with each fungal isolate in three replicates and incubated at 28 ± 1 
C
for 14 days. Conidia were then harvested by washing the substrate, and conidial concentrations deter-
mined by haemocytometer counts. Conidial suspensions were inoculated on PDAY with 0.002% benomyl
in Petri plates and exposed to 978 mW m2 of Quaite-weighted UV-B for 2 h. Additionally, conidial
suspensions were exposed to 45 
C for 3 h, and aliquots inoculated on PDAY with benomyl. The plates
were incubated at 28 ± 1 
C, and germination was assessed at 400  magnification after 48 h. Conidial
production was generally higher on white rice than on brown rice for all fungal species, except for
L. aphanocladii ARSEF 6433, regardless of moisture combinations. The 100% moisture condition provided
higher conidial production for B. bassiana (ARSEF 252 and ARSEF 3462) and M. anisopliae (ARSEF 2341)
isolates, while the addition of 10% peanut oil enhanced conidial yield for S. lanosoniveum isolate ARSEF
6430. B. bassiana ARSEF 3462 on white rice with 100% water yielded the highest conidial production
(approximately 1.3  1010 conidia g1 of substrate). Conidia produced on white rice with the different
moisture conditions did not differ in tolerance to UV-B radiation or heat. However, high tolerance to UV-
B radiation and heat was observed for B. bassiana, M. anisopliae, and A. album isolates. Heat-treated
conidia of S. lanosoniveum and L. aphanocladii did not germinate.
© 2023 British Mycological Society. Published by Elsevier Ltd. All rights reserved.1. Introduction
Concerns about the negative effects of synthetic insecticides on
human health and the environment have encouraged use of alter-
native strategies for pest control, including developing entomo-
pathogenic fungi (EPF) as biological control agents (BCA)
(Acheampong et al., 2020a; Faria and Wraight, 2007; Feng et al.,a Federal do Paran
a, Dois
hed by Elsevier Ltd. All rights rese
.A. Acheampong, H.G. Bignay
UV-B radiation and heat, Fu1994; Hatting et al., 2019; Lacey et al., 2015; Li et al., 2010; Rangel
et al., 2022; van Lenteren et al., 2018). Roberts (1973) mentioned
several potential benefits of EPF for regulation of insect pop-
ulations. Moreover, fungi are unique among insect pathogens, in
that they infect through the insect cuticle and do not need to be
ingested; therefore, they are the only microorganism group that
can infect sucking insects such as aphids and leafhoppers (Roberts
and Hajek, 1992; Sayed et al., 2019).
The genera Metarhizium and Beauveria are well-known EPF
because of their wide geographical distribution and host ranges
(Hall and Papierok, 1982; Roberts and St. Leger, 2004). They have
been intensively studied to develop commercial mycopesticidesrved.
an et al., Conidial mass production of entomopathogenic fungi and
ngal Biology, https://doi.org/10.1016/j.funbio.2023.07.001
D.E.N. Rangel, M.A. Acheampong, H.G. Bignayan et al. Fungal Biology xxx (xxxx) xxx(Faria and Wraight, 2007; Feng et al., 1994; Hatting et al., 2019;
Kassa et al., 2004; van Lenteren et al., 2018). The fungal species
Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, and
Aphanocladium album are BCA used in Integrated Pest Management
(IPM) in the Philippines to control mango-crop pests. The first three
species have been produced and commercialized worldwide for
arthropod pest control (Butt et al., 2001; Faria and Wraight, 2007;
Hatting et al., 2019; van Lenteren et al., 2018). Lecanicillium apha-
nocladii (formerly A. album) and Simplicillium lanosoniveum
(formerly V. lecanii) has also been produced in Brazil to control the
rubber tree pest, Leptopharsa heveae (Hemiptera: Tingidae) (Rangel
and Correia, 2003).
Diverse techniques for mass production of fungal mycelium or
conidia have been reported, including in vivo, submerged and sur-
face culture (Feng et al., 1994; Mascarin and Jaronski, 2016). The
method used depends on the growth requirements of the fungus
and the desired end product (Goettel and Roberts, 1992; Lomer
et al., 2001). The most common systems worldwide for conidial
production utilize sterile rice grains as the growth substrate
(Cebrail and Mehmet, 2021; Jaronski, 2014; Jenkins et al., 1998;
Kruger et al., 2014; Loera-Corral et al., 2016; Mathulwe et al., 2022;
Mun~iz-Paredes et al., 2017; Pham et al., 2010; Roswanjaya et al.,
2022; Taylor et al., 2013). In Brazil, commercial production of
M. anisopliae to control spittle bugs on sugarcane and B. bassiana to
control grasshoppers utilizes rice as the substrate because it is
simple, nutritive, and provides a large surface area for aeration and
conidia production, resulting in high yield (Alves and Pereira, 1989;
Aquino et al., 1975; Mascarin et al., 2010; Mendonça, 1992).
Although promising rice byproducts, such as husk (Mishra et al.,
2016; Sala et al., 2020) and bran or branehusk combinations,
which are cheaper substrates than rice grains, have resulted in
greater yield than the grains (Dorta et al., 1990), white or brown rice
grains are usually favored as substrates due to their higher nutrient
contents (Bich et al., 2018).
Besides the type of substrate used, themoisture content and C/N
ratio of substrates are reported to influence mass conidial pro-
duction of EPF (Aregger, 1992; Camara et al., 2022; Jenkins et al.,
1998; Mun~iz-Paredes et al., 2017; Sala et al., 2020; Teja and
Rahman, 2017). High moisture (>90%) enhances B. bassiana and
M. anisopliae conidial production on rice substrates (Aregger, 1992,
1992a; Dorta et al., 1990; Taylor et al., 2013). However, optimal
conidial production has also been obtained for some EPF (including
B. bassiana andM. anisopliae) cultivated on rice grains or husk with
40e70% moisture (Camara et al., 2022; Pham et al., 2010; Sala et al.,
2020). Furthermore, the addition of oils and supplementary carbon
sources (such as glucose, yeast extract, and coconut water) to
substrates improves conidial production of EPF (Aregger, 1992;
Camara et al., 2022; Kim et al., 2019; Safavi et al., 2007; Shah et al.,
2005; Teja and Rahman, 2017).
Ultimately, the choice of an entomopathogenic fungal isolate for
IPM should rely on (1) higher virulence; (2) greater efficiency in
mass production; and (3) performance under challenging envi-
ronmental conditions (heat, UV radiation, dry conditions, etc.)
(Acheampong et al., 2020a; Acheampong et al., 2020b; Dias et al.,
2018; Lacey et al., 2001; Licona-Ju
arez et al., 2023; Rangel et al.,
2005, 2015). Therefore, the current study investigated conidial
mass production of eight EPF isolates, A. album (ARSEF 1329),
B. bassiana (ARSEF 252 and 3462), L. aphanocladii (ARSEF 6433),
M. anisopliae (ARSEF 2341),Metarhizium pingshaense (ARSEF 1545),
and S. lanosoniveum (ARSEF 6430 and 6651) onwhite or brown rice
as substrate at four different moisture conditions. In addition, this
study evaluated the tolerance of mass-produced conidia of the
eight fungal isolates to UV-B radiation or heat (45 
C).2
2. Materials and methods
2.1. Fungal isolates
Eight fungal isolates were obtained from the USDA-ARS
Collection for Entomopathogenic Fungal Cultures (ARSEF), US
Plant, Soil, and Nutrition Laboratory, Ithaca, New York, USA. The
geographic origin and the insect host from which they were iso-
lated are listed in Table 1.
2.2. Mass production of conidia
2.2.1. Conidial production and inoculum preparation
The fungal isolates were cultured on 23 mL potato dextrose agar
(PDA, Difco Laboratories, Detroit, MI, USA) supplemented with 1%
yeast extract (1 g L1) (Technical, Difco) (PDAY) in Petri dishes
(polystyrene, 95  15 mm, Fisherbrand® Pittsburg, PA, USA). The
isolates were incubated in the dark at 28 ± 1 
C for 14 days. Conidia
were harvested from the medium surface and suspended in 0.1%
Tween 80 solution. The suspensionwas filtered through four layers
of sterile cheesecloth and immediately used for substrate
inoculation.
2.2.2. Substrate for conidial mass production
White Basmati rice (Shangri-la Health Foods, Logan, UT, USA) or
premium short grain brown rice (Premium short grain, Shangri-la
Health Foods) was used as a substrate. Four moisture conditions
were also compared using 20-g samples of rice in three replicates:
1) 100% distilled water (20 mL); 2) 100% distilled water, plus 5%
pure peanut oil (Planters®, Nabisco, East Hanover, NJ, USA); 3) 100%
distilled water plus 10% peanut oil; and 4) 75% distilled water and
25% coconut milk (Coco Premium Coconut Milk, Shangri-la Health
Foods). The peanut oil was used because we hypothesized that the
oil would prevent clumping of the rice. In addition, Aregger (1992)
studied the growth and sporulation in 25 different combinations of
water and oil, and found for Beauveria brongniartii that when oil
was added, the conidial production was generally higher, probably
due to a better granular structure of the medium. Furthermore,
growth of Isaria fumosorosea on ground corn mixed with corn oil as
a substrate produced conidia more tolerant to heat (Kim et al.,
2010). Agricultural byproducts including coconut water has also
been used in EPF mass production as nutritive additive and to in-
crease moisture content (Sahayaraj and Namasivayam, 2008);
therefore, in this study, we supplemented the rice grains with co-
conut milk.
This method was adapted from Daoust and Roberts (1983a;
1983b). Each mixture was autoclaved in a polypropylene bag
(20 30 cm, Fisherbrand®), and the top of the bag was closedwith a
cotton plug held in place with a tie wire, and autoclaved at 121 
C for
20 (white rice) or 25 (brown rice) min. The brown rice cooked
completely only after 25min in the autoclave. Once cooled, the grains
were squeezed to reduce clumps. Each bag was inoculatedwith 4mL
of the conidial suspension of each isolate (ca.107 conidiamL1) using
a sterile syringe and needle, and the point of inoculation was sealed
with tape. The inoculated rice bagswere incubated at 28± 1 
C for 14
days an optimum temperature for several insect-pathogenic fungal
species (Dimbi et al., 2004; Fargues et al., 1997; Rangel, 2000; Rangel
et al., 2010; Roberts and Campbell, 1977; Yeo et al., 2003).
2.2.3. Conidial yield and viability
Conidia were harvested after 14 days by washing the substrate
in 100 mL Tween 80 (0.1%). Twowashings were done with 50 mL of
Tween 80 (0.1%). The rice grains were squeezed in the plastic bag to
dislodge conidia from the substrate. The suspension was passed
through two layers of sterile cheesecloth to separate mycelia and
D.E.N. Rangel, M.A. Acheampong, H.G. Bignayan et al. Fungal Biology xxx (xxxx) xxx
Table 1
List of Isolates, their hosts, and geographic origin.
Species/Isolates Host/Substrate Geographic origin
Beauveria bassiana (Balsamo-Crivelli) Vuillemin
ARSEF 252 Leptinotarsa decemlineata[Coleoptera: Chrysomilidae] Orono, Maine, USA
ARSEF 3462 Soil Canada
Metarhizium anisopliae sensu lato (Metschniko) Sorokin
ARSEF 2341 Scotinophara coarctata [Hemiptera: Pentatomidae] Philippines
Metarhizium pingshaense QT Chen & HL Guo
ARSEF 1545 Scotinophara coarctata [Hemiptera: Pentatomidae] Philippines
Simplicillium lanosoniveum (F. H. Beyma) Zare & W. Gams
ARSEF 6430 Leptoharsa hevea [Hemiptera: Tingidae] French Guiana
ARSEF 6651 Leptoharsa hevea [Hemiptera: Tingidae] Brazil
Lecanicillium aphanocladii Zare & W Gams
ARSEF 6433 Leptoharsa hevea [Hemiptera: Tingidae] Brazil
Aphanocladium album (Preuss) Gams
ARSEF 1329 egg of Hypera postica [Coleoptera: Curculionidae] Franceaggregates of the substrate. To determine conidial production, an
aliquot of 10 mL was suspended in 990 mL of 0.1% Tween 80, and the
number of conidia was determined by hemacytometer counts. The
viability was assessed by inoculating 20 mL conidial suspension on
10 mL PDAY supplemented with 0.003% gentamycin (150 mg L1)
(Sigma Chemical Company, Irvine, UK) in three replicate Petri
dishes (Polystyrene, 60  15 mm), and incubation at 28 ± 1 
C for
12 h. Conidia were stained with a drop of methyl blue solution
[13 g l1 in a 85% (w/w) lactic acid solution]. Germination was
assessed at 400  magnification; conidia were considered germi-
nated when the germ tube was longer than the diameter of the
conidium (Rangel et al., 2005). A total of 300 conidia per plate were
evaluated and viability was calculated. The conidial viability of
some isolates grown on white rice with 100% moisture and stored
for 2 years at 20 
C were also determined as above.2.3. Effect of UV-B and heat on conidial germination
The effects of UV-B and heat on conidial germination were
evaluated for all eight isolates of the EPF produced on white rice
with the four moisture combinations previously mentioned. The
inoculum was prepared by suspending conidia (ca. 1  105 conidia
mL1) in 10 mL 0.1% Tween 80. The suspensions were vigorously
shaken, and 2 mL was filtered through a polycarbonate membrane
(25mmdiam., 8 mmpore size,Whatman®Nucleopore®, Clifton, NJ,
USA). A drop of 20 mL conidial suspension was inoculated on 4 mL
PDAY þ 0.002% Benomyl [25% active ingredient (Hi-Yield Chemical
Company, Bonham, TX, USA)] in three replicate Petri plates (poly-
styrene, 35 10mm), and immediately exposed to UV-B irradiation
for 2 h. PDAY plates with conidia were exposed to 978 mW m2 of
Quaite-weighted UV-B radiance (Quaite et al., 1992a;b) produced
by two TL 20 W/12 RS fluorescent lamps (Philips, Eindhoven,
Holland) [with primarily UV-B (peak at 313 nm)withminimal UV-A
radiation output], providing a total dose of 7.04 kJ m2, in a Percival
growth chamber (Boone, IA, USA) at 28 ± 1 
C. Plates were covered
with cellulose diacetate filters (JCS Industries, Le Miranda, CA, USA)
to exclude UV-C and short wavelength UV-B radiation provided by
two TL 20 W/12 RS fluorescent lamps as described by Rangel et al.
(2004). Control plates were covered with aluminum foil to block all
UV radiation. Spectral irradiance was measured as done in Rangel
et al. (2004). The DNA-damage (cyclobutane pyrimidine dimer
formation) action spectrum developed by Quaite (Quaite et al.,
1992a;b) and normalized to unity at 300 nm was used to calcu-
late the weighted UV irradiances in mWm2. The reasons for using
this action spectrum and selecting this biological spectral weight-
ing function (BSWF) are discussed in Rangel et al. (2006b).
For heat treatment, the conidial suspension (2 mL) in 20 mL test
tubes (Pirex®, NY, USA) was exposed in awater bath at 45 ± 1 
C for3
3 h, an established heat-stress condition according to (Rangel et al.,
2005; Souza et al., 2014). Then, 20 mL of the conidial suspensionwas
inoculated on 4 mL PDAY þ Benomyl (0.002%) in three replicate
Petri plates (polystyrene, 35  10 mm). Immediately after treat-
ments, the plates were inoculated with a drop of 20 mL and incu-
bated at 28 ± 1 
C. The conidial germination in the plates exposed
to UV-B or heat was assessed after 24 h of incubation for control
(non-exposed plates) and 48 h of incubation for the treatments.
Relative germination was calculated according to Rangel et al.
(2005). The experiments were repeated three times.
2.4. Conidial survival after two years under freezing temperatures
Mass-produced conidia (on white rice with 100% moisture) of
the isolates ARSEF 1545 (M. pingshaense), 2341 (M. anisopliae), 6651
(S. lanosoniveum), 6430 (S. lanosoniveum), and 6651
(L. aphanocladii) were stored at 20 
C. Then two years after pro-
duction, the conidial viability was evaluated following the method
above on PDAY medium. The conidial germination was counted
after 16 h at 28 
C. Neither of the two B. bassiana isolates (ARSEF
252 and ARSEF 3462) nor the A. album isolate (ARSEF 1329)
germinated following the 2-y storage at 20 
C.
2.5. Statistical analyses
Differences among isolates in conidial production and germi-
nation on white and brown rice under different moisture content,
as well as the viability of two-year-old stored conidia among iso-
lates, and conidial tolerances to heat and UVwere assessed using an
analysis of variance of a one-way factorial in a randomized block
design in which experimental trials defined blocks. Assessment of
the effects of substrate and moisture combination on conidial
viability for each isolate were assessed using an analysis of variance
of a two-way factorial in a randomized block design in which
experimental trials defined blocks.
Pairwise comparisons of isolate means were calculated using
TukeyeKramer adjustment to control experimentewise Type I er-
ror at the 0.10 level. Data were square root transformed prior to
analysis to better meet assumptions of normality and homogeneity
of variance. Calculations were done using Proc MIXED in the SAS
System for Windows Version 9.0.
3. Results and discussion
Conidia produced by the eight fungal isolates were generally
higher onwhite rice than on brown rice substrate, regardless of the
moisture levels, except for L. aphanocladii ARSEF 6433, which had
similar production on both substrates (Fig. 1). Even though this
D.E.N. Rangel, M.A. Acheampong, H.G. Bignayan et al. Fungal Biology xxx (xxxx) xxxresult was unexpected, as brown rice (with its nutritious bran
component) (Dorta et al., 1990) is more nutritious thanwhite rice, a
similar finding was reported by Pham et al. (2010). Similar results
were observed with M. anisopliae, in which white rice was more
productive than brown rice (Daoust and Roberts, 1983a). However,
Sujatha et al. (2016) reported higher conidial production for an
isolate of L. lecanii on brown rice compared to white rice. However,
white rice is relatively cheaper than brown rice, hence more
economical for use as a substrate.
Conidial production on white rice with 100% water varied
among isolates (F7,22 ¼ 43.28; P < 0.001). This moisture condition
provided higher conidial production for B. bassiana (ARSEF 252 and
ARSEF 3462) and M. anisopliae (ARSEF 2341) isolates. In contrast,
the addition of 10% peanut oil enhanced conidial yield for one
isolate of S. lanosoniveum (ARSEF 6430) (Fig. 1). This result was not
entirely surprising given that previous mass production studies forFig. 1. Production of entomopathogenic fungi grown on white rice (open bars) and brown ri
production on white rice and 100% water varied among isolates (F7,22 ¼ 43.28; P < 0.001).
4
several EPF species (including B. bassiana and M. anisopliae) using
rice and other cereals as substrates reported higher conidial pro-
duction at higher moisture conditions (substrate to water ratio
generally  1:1) (Aregger, 1992; Damir, 2006, 2006a; Daoust and
Roberts, 1983b; Dorta et al., 1990; Magalha~es and Fraza~o, 1996).
Conidial production has also been optimized for several EPF under
similar mass production systems, at lower moisture level of sub-
strates (40e70%) than the present study (Camara et al., 2022; Pham
et al., 2010; Sala et al., 2020). Similarly, the insignificant effect of oil
on conidial production, with the exception of S. lanosoniveum
ARSEF 6430, contradicts findings from previous mass production
systems, where the addition of oil, even at concentrations lower
than those used in this study, enhanced conidial production
(Camara et al., 2022; Dorta et al., 1990). These results, althoughwith
few isolates, support the isolate-dependent moisture and nutrientce (closed bars) with different moisture combinations at 28 ± 1 
C for 14 days. Conidial
D.E.N. Rangel, M.A. Acheampong, H.G. Bignayan et al. Fungal Biology xxx (xxxx) xxxrequirements of EPF (Mun~iz-Paredes et al., 2017; Shah et al., 2005;
Taylor et al., 2013; Teja and Rahman, 2017).
Among the isolates, conidia produced per gram of rice substrate
were higher for B. bassiana (ARSEF 252 and ARSEF 3462),
S. lanosoniveum (ARSEF 6430 and ARSEF 6651), and A. album (ARSEF
1329) but lower for M. anisopliae (ARSEF 2341), M. pingshaense
(ARSEF 1545), and L. aphanocladii (ARSEF 6433). The isolate with
the highest conidial production was B. bassiana ARSEF 3462 on
white rice with 100% moisture condition, obtaining approximately
1.3  1010 conidia g1 of the substrate. This result concurs with the
generally higher conidial production of Beauveria thanMetarhizium
species, under the same growing conditions (Liu et al., 2003;
Petlamul and Prasertsan, 2012). Conversely, higher conidial pro-
duction has been noted for M. anisopliae compared to those of
B. bassiana using the same substrates and water volumes (Damir,
2006) and on cadavers of insect hosts (Marques et al., 2000).
Nonetheless, few isolates were used in this study; thus, theFig. 2. Conidial viability of entomopathogenic fungi grown on white rice (open bars) and b
5
observed differences in conidial yield between isolates may be
ascribed to the inherent characteristics of each isolate.
For B. bassiana ARSEF 252, conidial production was affected by
the interaction of substrate and moisture combination (F3,19 ¼ 3.17;
P ¼ 0.048) (Fig. 1A). Production was higher on white rice than
brown for water and coconut milk. White rice was not shown to be
either better or worse than brown for peanut oil 10% or peanut oil
5%, although the white rice mean was greater than the brown rice
mean. On brown rice, 100% water was significantly higher than
peanut oil 10%; neither peanut oil 5% nor coconut milk could be
distinguished from any other moisture combination. On white rice,
water, coconut milk, and peanut oil 5% all are significantly higher
than peanut oil 10%; water is significantly higher than peanut oil
5%; and coconut milk cannot be distinguished from either water or
peanut oil 5%.
For B. bassiana ARSEF 3462, the conidial production was greater
on white rice (F1,19 ¼ 73.74; P < 0.001) (Fig. 1B). Production variedrown rice (closed bars) with different moisture combinations at 28 ± 1 
C for 14 days.
D.E.N. Rangel, M.A. Acheampong, H.G. Bignayan et al. Fungal Biology xxx (xxxx) xxx
Fig. 3. Conidial viability of entomopathogenic fungi grown on white rice with 100%
moisture at 28 ± 1 
C and stored for 2 years at 20 
C.due to moisture combination and the production was greatest on
water (F3,19 ¼ 35.53; P < 0.001). There was no evidence of inter-
action between substrate and moisture combination effects
(interaction; F3,19 ¼ 1.17; P ¼ 0.346).
For M. pingshaense ARSEF 1545, evidence of an interaction be-
tween substrate and moisture combination effects on production
was found (F3,14 ¼ 3.10; P ¼ 0.061) (Fig. 1C). Conidial production
was higher on white rice than brown for peanut oil 5% and coconut
milk. White rice was not shown to be either better or worse than
brown for water or peanut oil 10%; however, the white rice mean
was greater than the brown rice mean. The figure also illustrates
little difference in production among coconut milk, peanut oil 5%,
and peanut oil 10% on brown rice, while productionwith water was
significantly higher (based on pairwise mean comparisons). Pro-
duction on white rice, water, and coconut milk was significantly
higher than peanut oil 10%; peanut oil 5% cannot be distinguish
from any other moisture combination. Main effects of both sub-
strate and moisture combination were significant (P < 0.001 for
both). Production was greater on white rice. Production was also
greater for water compared to the other three moisture combina-
tions (based on pairwise mean comparisons); differences among
peanut oil 5%, peanut oil 10%, and coconut milk could not be
distinguished.
ForM. anisopliae ARSEF 2341, the conidial production varied due
to moisture combination (F3,7.08 ¼ 16.95; P ¼ 0.001) (Fig. 1D). There
was weak evidence that production was greater on white rice than
on brown (F1,11.1 ¼ 4.31; P ¼ 0.062). No evidence was found of an
interaction between substrate and moisture combination effects
(F3,7.08 ¼ 0.42; P ¼ 0.746).
For S. lanosoniveum ARSEF 6430, the conidial production was
greater on white rice with peanut oil 10% (F1,16 ¼ 25.61; P < 0.001)
(Fig. 1E). There was no evidence of production differences among
the studied moisture combinations (F3,16.1 ¼ 0.42; P ¼ 0.742) or of
interaction between substrate and moisture combination effects
(F3,16.1 ¼ 0.85; P ¼ 0.489).
For S. lanosoniveum ARSEF 6651, the conidial production was
greater on white rice (F1,16 ¼ 11.54; P ¼ 0.004) (Fig. 1F). The sta-
tistical evidence of differences in production among moisture
combinations are weak. Coconut milk was greater than either
peanut oil 5% or water; no other distinctions among moisture
combination means were apparent (F3,15.1 ¼ 3.02; P ¼ 0.063). There
was no evidence of interaction between substrate and moisture
combination effects (F3,15.1 ¼ 0.71; P ¼ 0.561).
For A. album isolate ARSEF 1329, the production was greater on
white rice (F1,11.8 ¼ 18.97; P ¼ 0.001) (Fig. 1G). Production varied
due to moisture combination (F3,11.8 ¼ 3.39; P ¼ 0.055). There was
no evidence of interaction between substrate and moisture com-
bination effects (F3,11.8 ¼ 0.12; P ¼ 0.946).
For S. lanosoniveum ARSEF 6433, the conidial production was
greater onwhite rice (F1,21¼8.87; P¼ 0.007) (Fig.1H). Therewas no
evidence of differences in production among moisture combina-
tions (main effect of moisture combination); (F3,21 ¼ 0.97;
P ¼ 0.426) or of interaction between substrate and moisture com-
bination effects (F3,21 ¼ 0.91; P ¼ 0.452).
The conidial germination after 12 h of incubation did not vary
greatly between isolates grown on either type of rice or moisture
combination, with relative germination exceeding 80%. The
exception was A. album ARSEF 1329, whose germination on brown
rice and 10% peanut oil was lower than conidia produced on white
rice (Fig. 2). Conidial germination was tested to determine the
culture conditions affected viability. We demonstrated in our pre-
vious studies that conidial viability can change when conidia are
produced on certain culture medium or physical conditions
(Oliveira et al., 2018; Oliveira and Rangel, 2018; Rangel et al., 2004),
for example, conidia of Metarhizium robertsii produced on cadavers6
of Zophobas morio (Coleoptrea: Tenebrionidae) germinated less
than conidia produced on PDAY medium. In addition, conidia of
M. robertsii (ARSEF 23 and ARSEF 2575) germinated faster when
produced on Emerson or Czapek media (Rangel et al., 2004). ARSEF
2575 also germinated faster on minimal medium (Czapek medium
without sucrose) supplemented with lactose (Oliveira et al., 2018)
or when conidia were produced on PDA medium under the white
or blue light (Oliveira et al., 2018).
The viability of conidia stored at 20 
C for 2 years decreased
significantly for M. pingshaense and M. anisopliae (ARSEF 1545 and
ARSEF 2341) but not S. lanosoniveum (ARSEF 6651 and ARSEF 6430)
and L. alphanocladii (ARSEF 6433) (Fig. 3). The result of the 2-y
conidial viability test broadly matches those of other authors:
Conidial viability of EPF species are lost over time depending on
storage temperature, but effects are slower for some isolates
(Roswanjaya et al., 2022; Sy et al., 2016; Taylor et al., 2013). The
conidial viability of EPF was better maintained at lower tempera-
tures (typically -20e4 
C), if long-term storage (1 y) is required
(Daoust and Roberts, 1983b; Kim et al., 2019; Marques et al., 2000;
Oliveira et al., 2011; Sy et al., 2016). Although the B. bassiana isolates
in the present were not viable after the 2-y storage, the viability of
millet and rice-mycotized grains of an isolate of this species
exceeded 85%, when stored for 2 years at 4 
C (Kim et al., 2019).
Similarly, pure conidia of B. bassiana isolates remained viable
(100%) for 24 and 80 months when glycerol-frozen at 20 
C and
stored at 7 
C, respectively (Marques et al., 2000; Oliveira et al.,
2011).
Mycelial growth on different culturemedia (Rangel et al., 2006a;
Rangel et al., 2004, 2008, 2012, 2015) or mycelial growth exposed to
certain biotic or abiotic stress conditions (Dias et al., 2020, 2021,
2022; Medina et al., 2020) can greatly influence the stress tolerance
of the produced conidia. However, all isolates exhibited no evident
differences between themoisture combinations studied with white
rice for conidial relative germination when exposed to heat
(F4,8 ¼ 1.48; P ¼ 0.295) (Fig. 4) or UV-B radiation (Fig. 5). The
exception was A. album (ARSEF 1329) whose conidia were more
tolerant to UV-B radiation when white rice was moistened with
water or peanut oil 0.5% than conidia produced on white rice
moistened with peanut oil 0.5% or coconut milk (Fig. 5)
(F3,6 ¼ 14.17; P ¼ 0.004).
D.E.N. Rangel, M.A. Acheampong, H.G. Bignayan et al. Fungal Biology xxx (xxxx) xxxHigh tolerance to heat was observed for the B. bassiana (ARSEF
252 and 3462), M. anisopliae (ARSEF 2341), M. pingshaense (ARSEF
1545), and A. album (ARSEF 1329) (Fig. 4), with no differences in
relative germination (F4,8 ¼ 1.48; P ¼ 0.295). However,
S. lanosoniveum (6430 and 6651) and L. aphanocladii (6433) were
very vulnerable to heat at 45 
C and did not germinate (Fig. 4).
Conidial UV tolerance varied among isolates (F7,14 ¼ 11.22;
P < 0.001) (Fig. 5). Accordingly, the isolates ARSEF 1329 (A. album),
ARSEF 3462, ARSEF 252 (B. bassiana), ARSEF 2341 (M. anisopliae),
and ARSEF 1545 (M. pingshaense) are considerably more UV
tolerant than ARSEF 6651 and 6430 (S. lanosoniveum), and ARSEF
6433 (L. aphanocladii), and within each of these two groups, dif-
ferences among moisture conditions could not be distinguished.
Dias et al. (2018) reported that ARSEF 252 and ARSEF 6651
exhibited similar UV tolerance when conidia are produced on PDA,Fig. 4. Relative germination of conidia produced on white rice with different moisture com
treatments within each isolate did not differ statistically (F4,8 ¼ 1.48; P ¼ 0.295).
7
and exposed to simulated solar radiation at a Quaite-weighted
irradiance of 1335 mW m2 for 2 h.
In summary, conidial productions were generally higher on
white rice than on brown rice for all fungal species, except for
L. aphanocladii (ARSEF 6433), regardless of moisture combinations.
The 100% moisture condition provided higher conidial production
for B. bassiana (ARSEF 252 and ARSEF 3462) and M. anisopliae
(ARSEF 2341) isolates, while the addition of 10% peanut oil
enhanced conidial yield for S. lanosoniveum ARSEF 6430. B. bassiana
ARSEF 3462 showed greater promise for future studies, as this
isolate yielded the highest conidial production (approximately
1.3  1010 conidia g1 of substrate) on white rice with 100% water
and exhibited greater tolerance to UV radiation and heat.
S. lanosoniveum isolates ARSEF 6430 and 6651 also yielded high
quantity of conidia, remained viable for 2 years, but were extremely
sensitive to UV radiation and heat. The virulence of these isolatesbinations at 28 ± 1 
C exposed to wet heat (45 
C) for 3 h. Tolerance to heat among
D.E.N. Rangel, M.A. Acheampong, H.G. Bignayan et al. Fungal Biology xxx (xxxx) xxx
Fig. 5. Relative germination of conidia produced on white rice with different moisture combinations at 28 ± 1 
C were exposed for 2 h to 978 mW m2 of Quaite-weighted UV-B
radiance [with primarily UV-B (peak at 313 nm) and minimal UV-A radiation output] providing total dose of 7.04 kJ m2. Variation in UV-B tolerance was observed among isolates
(F7,14 ¼ 11.22; P < 0.001). The isolates ARSEF 1329, ARSEF 3462, ARSEF 252, and ARSEF 2341 were more UV-B tolerant than the others.toward targeted insect pests ought to be prioritized in future
development of these isolates into mycopesticides, followed by the
biological traits obtained in this study.
Declaration of competing interest
The authors declare that they have no conflict of interest.
Author statement
Relevant CRediT roles: Conceptualization: DWR, HGG, and
DENR; Data curation: DENR; Formal analysis; DENR, DWR, HGG,
HGB, and MAA; Funding acquisition: DWR; Investigation: HGG,
HGB, MAA, and DENR; Methodology: HGG, HGB, MAA, and DENR;
Project administration: DWR; Resources: DWR; Supervision: DWR;8
Validation: DENR; Roles/Writing - original draft: DENR, MAA, and
HGB; Writing - review & editing: DENR, MAA, and HGB. The au-
thors HGB, MAA, and DENR read and approved the manuscript, the
authors DWR and HGG are deceased.
Acknowledgments
We are grateful to Susan Durham (Utah State University, Logan,
UT, USA) for the statistical analyses. This research was supported by
grants from FulbrightePhilippine Agriculture Scholarship Program
for fellowship for H.G.B. and H.G.G. We sincerely thank the National
Council for Scientific and Technological Development (CNPq) of
Brazil for PhD fellowships GDE 200382/02e0, PQ1D 302100/
2018e0 and PQ1D 302282/2022e0 and the S~ao Paulo Research
Foundation (FAPESP) 2010/06374e1 and 2013/50518e6 for D.E.N.R.
D.E.N. Rangel, M.A. Acheampong, H.G. Bignayan et al. Fungal Biology xxx (xxxx) xxxThis research was supported by grants from the United States
Department of Agriculture (USDA, APHIS), Utah Department of
Agriculture and Food, and the Community/University Research
Initiative of Utah State University. This article is part of the “Fungal
growth, development, and stress responses” special issue for the
XIII International Fungal Biology Conference (IFBC) & IV Interna-
tional Symposium on Fungal Stress (ISFUS), which is supported by
the Fundaç~ao de Amparo a Pesquisa do Estado de Sa~o Paulo
(FAPESP) grant 2021/13614e3 and the Coordenaça~o de Aperfei-
çoamento de Pessoal de Nível Superior (CAPES) grant
88887.680665/2022e00.References
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