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Temperature tolerance and humidity requirements of select entomopathogenic

fungal isolates for future use in citrus IPM programmes

M.A. Acheampong, C.A. Coombes, S.D. Moore, M.P. Hill

PII: S0022-2011(20)30142-7
DOI: https://doi.org/10.1016/j.jip.2020.107436
Reference: YJIPA 107436

To appear in: Journal of Invertebrate Pathology

Received Date: 27 February 2020

Revised Date: 21 May 2020
Accepted Date: 20 June 2020

Please cite this article as: Acheampong, M.A., Coombes, C.A., Moore, S.D., Hill, M.P., Temperature tolerance
and humidity requirements of select entomopathogenic fungal isolates for future use in citrus IPM programmes,
Journal of Invertebrate Pathology (2020), doi: https://doi.org/10.1016/j.jip.2020.107436

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Temperature tolerance and humidity requirements of select entomopathogenic fungal isolates
for future use in citrus IPM programmes

M.A. Acheampong1*, C.A. Coombes1, S.D. Moore1,2, M.P. Hill1

1Centre for Biological Control, Department of Zoology and Entomology, Rhodes University,
P.O. Box 94, Grahamstown 6140, South Africa

2Citrus Research International, Walmer, P.O. Box 5095, Port Elizabeth 6065, South Africa

*Corresponding author.

Email addresses: mavisagyeiwaaacheampong@gmail.com (M. A. Acheampong),

c.coombes@ru.ac.za (C.A. Coombes), seanmoore@cri.co.za (S.D. Moore), m.hill@ru.ac.za
(M.P. Hill).
Abstract
Several isolates of Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales:
Cordycipitacae) and Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales:
Clavicipitacae) have been investigated as possible microbial control agents of key citrus pests
in South Africa. Although laboratory results have been promising, field trials against foliar
pests have shown limited success. These findings highlighted the need to investigate other
biological attributes of these fungal isolates besides virulence in order to select candidates that
may be better suited for the foliar environment. Thus, this study investigated the influence of
temperature on the in vitro growth of seven indigenous local isolates and the humidity
requirements necessary to promote successful infection, in comparison with two commercial
isolates (B. bassiana PPRI 5339 and M. anisopliae ICIPE 69). All the fungal isolates grew
across a range of temperatures (8 to 34°C) and optimally between 26 to 28°C. Similarly, fungal
infection of Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) fifth instars occurred
across a range of humidity levels (12%, 43%, 75%, 98%) regardless of fungal concentration,
although external sporulation was restricted to treatments exposed to 98% relative humidity. It
was concluded that neither temperature nor humidity, when considered alone, is likely to
significantly influence the efficacy of any of the isolates in the field, given that they are active
within temperature and humidity ranges experienced in South African citrus orchards.

Keywords: Beauveria bassiana; Metarhizium anisopliae; environmental tolerance; microbial

control
1. Introduction
Indigenous entomopathogenic fungi (EPF) have shown great promise as biocontrol agents
against key insect pests of citrus in South Africa (Coombes et al., 2016; Goble et al., 2011).
However, like other microbial pest control agents, their efficacy in the field is influenced by
abiotic factors such as temperature, humidity and solar radiation (Jaronski, 2010; Ment et al.,
2017).

Temperature plays an important role in the success of entomopathogenic fungi as microbial

control agents in the field due to its potential deleterious effect on fungal growth, sporulation
and arthropod infection (Borisade and Magan, 2014; Bugeme et al., 2008; Dimbi et al., 2004;
Ekesi et al., 1999; Tumuhaise et al., 2018). High ambient humidity (usually > 90%) is also well
documented as playing not only a critical role in germination and arthropod infection by fungal
entomopathogens ( Fargues and Luz, 2000; Luz and Fargues, 1997, 1999; Santos et al., 2009;
Shipp et al., 2003; Sivasankaran et al., 1998), but also sporulation on cadavers during the
saprophytic phase (Arthurs and Thomas, 2001; Arthurs et al., 2001; Fargues and Luz, 1998;
Ment et al., 2010; Ramoska, 1984). This is pivotal for fungal survival and secondary infection
of target insect pests (Fargues and Luz, 1998; Inglis et al., 2001; Ramoska, 1984; Roy et al.,
2006).

Previous research on these fungi identified three isolates (Beauveria bassiana s.str. G Ar 17
B3 (Hypocreales: Cordycipitacae), Metarhizium anisopliae s.l. G 11 3 L6 and M. anisopliae
s.str. FCM Ar 23 B3 (Hypocreales: Clavicipitacae)) as the most virulent against fifth instar
Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), a key pest of citrus in South
Africa (Coombes et al., 2015; Goble et al., 2011). As a result further research focused
exclusively on these isolates, with promising laboratory results reported against other key citrus
insect pests: citrus thrips, Scirtothrips aurantii Faure (Thysanoptera: Thripidae), citrus
mealybug, Planococcus citri Risso (Hemiptera: Pseudococcidae) and California red scale,
Aonidiella aurantii Maskell (Hemiptera: Diaspididae) (Chartier FitzGerald, 2014;Chartier
FitzGerald et al., 2016; Upfold et al., unpublished data). Consequently, five field trials were
carried out using the top performing isolates against T. leucotreta soil-dwelling life stages
(Coombes et al., 2016). A further nine field trials were directed at foliage and fruit, against P.
citri and S. aurantii, in warmer (Limpopo Province) and cooler (Eastern Cape Province) citrus
regions, using the same isolates (Grout et al., unpublished data). Good control was recorded
against the soil-dwelling life stages of T. leucotreta, as M. anisopliae FCM Ar 23 B3 and B.
bassiana G Ar 17 B3, were capable of reducing infestation of this pest and its associated fruit
drop by up to 82% and 86%, respectively, relative to untreated trees (Coombes et al., 2016). In
contrast, these isolates, even when formulated in oil, had no significant impact against
mealybug and thrips in the two climatic regions, over two consecutive citrus seasons (Grout et
al., unpublished data). In the cooler production region, however, some success was achieved
in two corrective trials against very high P. citri infestation, where unformulated and oil-
formulated conidia of M. anisopliae FCM Ar 23 B3, reduced infestation by 30% (Grout et al.,
unpublished data).

It is well known that, when selecting suitable entomopathogenic microbial agents for
mycopesticides, two important traits must be evident or enhanced for product development to
proceed: (1) high virulence against target pests and (2) tolerance to environmental stress
(Ibarra-Cortés et al., 2013; Ravensberg, 2011; Senthil Kumar et al., 2016; Shapiro-Ilan et al.,
2006; Yeo et al., 2003). Thus far, virulence has been the sole focus of research surrounding
these fungi, and not environmental tolerance, which may differ amongst species and isolates
(Fernandes et al., 2015; Inglis et al., 2001; Jaronski, 2010).

Therefore, the aim of this study was to investigate the tolerance of seven local indigenous
isolates, along with two commercial isolates for comparison, to two important abiotic factors
that may influence fungal efficacy against a particular pest in the field, temperature and
humidity. The fungal isolates selected for use had all previously reported in laboratory
bioassays above 80% mortality against T. leucotreta, as was the case with the isolates applied
in the field. Considering variability in tolerance to abiotic factors amongst species and isolates,
it may be possible that previously overlooked isolates are more suited to field application in
South African citrus orchards, in relation to temperature and humidity, especially in the foliar
environment where limited success in the field has been experienced.

2. Materials and Methods

2.1 Insect culture

The humidity requirements of the fungal isolates was not tested on the intended arboreal pests
of our research, P. citri and S. aurantii due to the following reasons: (1) it has to date proven
impossible to rear S. aurantii in the laboratory and (2) cultures of P. citri were not available
when this study was conducted. However, fifth instars of T. leucotreta were readily available
and were an equally effective host insect to test the aforementioned biological parameter,
resulting in their use in the humidity study. Fifth instars of T. leucotreta, ready to pupate within
24 h, were obtained when needed from laboratory cultures held at the Centre for Biological
Control (CBC), Rhodes University (RU), South Africa. Detailed description of the larval
rearing protocol for T. leucotreta, including its specially formulated larval diet is presented in
Moore et al. (2014).

2.2 Fungal isolates

The seven indigenous entomopathogenic fungal isolates investigated in this study, which
comprised four B. bassiana and three M. anisopliae isolates, are presented in Table 1. All
isolates were obtained from cultures held at CBC, Rhodes University, South Africa. These
isolates were originally obtained from soil samples collected from citrus orchards in the Eastern
Cape Province of South Africa, following baiting with either T. leucotreta or Galleria
mellonela (Lepidoptera: Pyralidae) (Goble et al., 2010, 2011) (Table 1). The commercial
isolates, B. bassiana PPRI 5339 and M. anisopliae ICIPE 69 were obtained from the
mycoinsecticide products, Broadband (BASF, South Africa) and Real Metarhizium 69 (Real
IPM, Kenya), respectively. All isolates including the commercial strains were cultured on
Sabouraud dextrose agar (SDA) (Merck, South Africa) for 14 d at 27°C, 60% relative humidity
(RH) and a 12 h photoperiod. Conidial suspensions (1×108 conidia/ml) of each isolate were
then passaged once through T. leucotreta fifth instars and incubated for 7 d at 27°C. Conidia
from sporulating cadavers were plated on chloramphenicol (50 mg/l) supplemented SDA
medium (SDAC) and incubated for 14 d under the same culture conditions as described above.
These cadaver-cultures were then maintained at 4°C, and served as stock cultures for this
research.

2.3 Effect of temperature on fungal growth

The stock culture of each isolate was subcultured on SDAC and incubated for 14 d at 27°C,
60% RH and a 12 h photoperiod. Conidial suspensions were then prepared from these cultures
using sterile distilled water supplemented with 0.01% Tween 20 (sdt H2O), and adjusted to
1×107 conidia/ml using a haemocytometer. For each isolate, 200 μl were then spread-plated on
SDAC plates at five replicates. Plates were incubated at 27°C, 60% RH and a 12 h photoperiod
for 3 d (72 h) to obtain mycelial mats. A total of 50 circular mycelial plugs were cut from the
five cultures for each isolate using a sterile glass Pasteur pipette (5 mm diameter). Each plug
was placed upside down in the centre of a new dish of the same medium (15 ml SDAC in a 90-
mm Petri plate). The inoculated plates were covered and sealed with Parafilm M®. All plates,
in groups of 10, with each group containing all isolates at five replications were placed in a
clear plastic storage box (32 × 24 × 16 cm). A 500 ml plastic container filled with saturated
salt solution of potassium sulphate was placed in each box to provide approximately 98% RH.
Each box was incubated for 15 d at a 12 h photoperiod for each of the 10 temperatures studied
(6, 8, 16, 20, 25, 27, 29, 32 35, 40 ± 1°C). Surface radial growth was measured at two-day
intervals starting from the third day. This was done with a ruler, following four segments
formed by two orthogonal lines, previously drawn on the bottom of each plate (Miyashira et
al., 2010). The entire experiment was replicated twice, with each replicate originating from
different cadaver-culture plates.

2.4 Effect of humidity on fungal-induced T. leucotreta mortality

2.4.1 Humidity chambers

Bioassays were conducted at four RH levels: 12, 43, 75 and 98%. Thus, saturated solutions of
LiCl, K2CO3, NaCl and K2SO4, well known to produce the aforementioned humidities,
respectively, within an enclosed system at 25 to 30°C (Greenspan, 1977; Winston and Bates,
1960) were used. Saturated solutions of these salts were prepared according to their solubility
in water, as specified by the manufacturer (Merck, South Africa). Each humidity test chamber
representing each RH level, consisted of two airtight clear plastic storage boxes (32 × 24 × 16
cm) in a KBW 240 Binder™ growth chamber (Monitoring and Control laboratories, South
Africa), with each box containing a 500 ml plastic container filled with 500 ml of the respective
salt solution at 27°C and a 12 h photoperiod. The RH inside each box within each chamber was
found to be within ± 1% of the indicated values in literature (Greenspan, 1977; Winston and
Bates, 1960), throughout a 21-day test period, monitored with a Hygrochron™ iButton (Maxim
Integrated, United States of America). Following confirmation of the humidity levels within
this system, each humidity chamber was set up 24 h before the start of the experiment to allow
stabilisation of humidity prior to their use.

2.4.2 Inoculation of T. leucotreta fifth instars with fungal isolates

Only two fungal isolates, and their control, were assayed at a time, due to the large number of
insects that needed to be assessed during evaluation. Conidial suspensions of each isolate were
obtained following the same procedure described in section 2.3, and adjusted to 1×105 and
1×107 conidia/ml. Viability of each isolate was determined by plating triplicate 100 μl aliquots
of suspensions on SDAC plates for 24 h at 27°C, after which, the number germinated (conidia
with germ tubes) out of 300 conidia per plate were assessed. Viability in all concentrations
applied exceeded 90% for all isolates used in their respective bioassays.

For each concentration of an isolate, 40 T. leucotreta fifth instars were evenly placed in four
90 mm, sterile plastic Petri plates. Insects in each plate were topically inoculated with
approximately 1 ml (two sprays) of the inoculum using a 500 ml spray bottle. Control insects
were treated with sdt H2O only. Insects in their respective Petri plates were dried under a
laminar flow hood. Each larva was then placed in a glass vial using sterile blunt-tipped forceps,
and corked with sterile cotton wool. A total of 120 larvae per isolate (including the control)
were incubated in each box of each humidity chamber. Humidities inside these chambers were
again, monitored with a Hygrochron™. The saturated solution of NaCl was replaced with new
solution weekly, as the stoppered cotton wool on top of vials tended to absorb precipitate of
this salt after about 10 d incubation, which could influence fungal efficacy in this treatment.
The number of live and dead larvae, pupae and adults were recorded daily, ceasing 10 d after
first adult eclosion was recorded, approximately 20-21 d post inoculation. Cadavers were
removed daily and death due to mycosis was verified as follows: insects were surfaced
sterilised in 0.5% bleach (3.5% NaOCl) with 0.01% Tween 20 for 1 min, followed by two
rinses (1 min each) in sterile distilled water. Fresh solutions were used for each treatment to
avoid cross contamination. Insects from each treatment were then transferred to sterile plastic
Petri plates lined with Whatman Filter papers, moistened with 1 ml sterile distilled water and
incubated for 4 d at 27°C, 60% RH and a 12 h photoperiod. Insects on moistened filter papers
were kept apart and examined after 3 d for fungal outgrowth. The experiment was replicated
three times for each concentration and humidity level, with each replicate originating from a
different passaged culture of the same isolate.

2.5 Statistical analyses

All statistical analyses were conducted in R version 3.4.0 (R Core Team, 2016). Regression
analysis was conducted for each isolate/temperature combination because the radial
measurements from the third to the fifteenth day fitted the linear model, y = vt +b, where the
slope of the model (v), indicates the growth rate (velocity in mm/day) at incubation
temperature, t (Ouedraogo et al., 1997). The growth rates under the different temperatures were
subsequently fitted to a modified β function (Bassanezi et al., 1998), to determine the
minimum, maximum and optimal temperatures for growth of each isolate. The modified β
function is given by the following equation:

Tb3 × ( Topt ― Tmin

) Tb3

Y(T) = TYopt × ( T ― Tmin

Topt ― Tmin ) Tmax ― Topt
× ( Tmax ― T
Tmax ― Topt )

where Y (T) is the fungal growth in mm/day (dependent variable) and T is the incubation
temperature (independent variable). Tmin, Tmax and Topt are the minimum, maximum and the
optimal temperatures for fungal growth, respectively. TYopt is the fungal growth at the optimal
temperature (Topt) and the shape parameter, Tb3, determines the temperature range around Topt
at which the growth rates stay near TYopt. “For low Tb3 values, such as 0.1, a broad temperature
range exists, whilst for high values, such as 3.0, the curve sharply declines when temperature
differs only slightly from Topt” (Bassanezi et al., 1998). Tmin was fixed at 6°C, as no radial
growth occurred for any isolate at this temperature (6°C). This also improved the model fit and
standard error of the estimated parameters. All parameter estimates, and their standard errors,
were attained using the 10 values of Y (mm/day) obtained from 10 replicates (5 from each
repeat) at each temperature. TYopt, Topt, Tmax and Tb3 were estimated using the Levenberg-
Marquardt non-linear least square method (Bates and Watts, 1988; García-Fernández et al.,
2008), and accomplished using the “nlsLM” function from the minipack.lm package. Pairwise
comparisons between isolates were made by conducting a Pairwise t-test with Bonferroni
adjustment (to control type 1 errors) for each estimated parameter.

Only larval and pupal mortality were analysed because adult mortality was extremely low (<
2%) and never resulted from mycosis. Data were not adjusted for natural mortality due to low
control mortalities (5-8%) and high confirmed mycosis (> 93%) on fungal treated pupae/larvae
across all the humidity levels tested. For each concentration tested, mortality and survival data
for all isolates and controls at each humidity level tested over the 21-day period were pooled
for the three replicates, resulting in a sample size of 120 insects per treatment for analysis. The
number of dead larvae in all treatments at each humidity were grouped with the number of dead
pupae as one developmental stage (dead pupae). The number of larvae and pupae alive in all
treatments at each humidity were also grouped as one outcome (live pupae). These two
outcomes were firstly, combined for both concentrations tested and fitted to a logistic
regression in a generalised linear model with binomial error distributions to determine an
interaction effect. Secondly, for each concentration tested, data for all treatments at each
humidity level tested were fitted to a logistic regression. Where statistical differences amongst
treatments in the model were found, pairwise comparisons with Tukey’s HSD contrast (P <
0.05), was performed. This was accomplished using the “ghlt” function from the multcomp
package (Hothorn et al., 2017). The efficacy of each fungal isolate across the humidity levels
tested, was also analysed. The number of dead larvae and pupae, live larvae and pupae in each
humidity level were grouped as described above. Data were fitted to logistic regressions and
contrasted using Tukey’s HSD test. For graphical representation, all outputs in proportions
were converted into percentages. The LT50 (time required to kill 50% larvae/pupae) value for
each isolate at each humidity level in the highest test concentration was computed; pupal
mortality data (dead and live pupae/day) pooled for the three replicates, were fitted to a logistic
regression model. The LT50 values and their confidence interval (P < 0.05), were then obtained
from this model using the “dose.p” function from the MASS package (Venables and Ripley,
2002).

3 Results

3.1 Effect of temperature on fungal growth

None of the tested fungal isolates grew at 6, 35 or 40°C. All the M. anisopliae isolates had a
similar growth-temperature range to the B. bassiana isolates, growing between 16 and 32°C,
with the exception that no growth occurred at 8°C for the former species, unlike the latter.

The optimum temperatures for growth (Topt), ranged from 26.19 to 28.31°C, and did not differ
amongst six out of the seven local isolates and ICIPE 69 (Table 2). At the optimal temperatures,
the three local M. anisopliae isolates, G 11 3 L6, FCM Ar 23 B3 and G OL R8, recorded
significantly higher radial growth rates (TYopt) (3.15 to 3.42 mm/day) than their counterpart B.
bassiana isolates (0.69 to 2.44 mm/day) and the two commercial isolates (1.72 to 2.55
mm/day). Fungal growth rate-temperature curves were generally well described by the
modified β function used in this study (Figure 1). The shape parameter (Tb3) values were
generally low for all isolates, and ranged from 0.38 to 1.32 (Table 2). The maximum
temperature for fungal growth, Tmax, varied amongst fungal isolates and species, ranging
between 32.05 to 32.91°C and 32.45 to 33.93°C for B. bassiana and M. anisopliae isolates,
respectively (Table 2).

3.2 Effect of humidity on fungal-induced T. leucotreta pupal mortality

3.2.1 Interaction effect on T. leucotreta pupal mortality

The interaction between humidity, concentration and treatment did not significantly influence
T. leucotreta pupal mortality (LRT, χ2 = 10.84, df = 27, P = 0.99). However, significant effects
were found for the main (individual) effects: treatment (LRT, χ2 = 2176.67, df = 9, P < 0.001),
concentration (LRT, χ2 = 193.99, df = 1, P < 0.001) and humidity (LRT, χ2 = 20.88, df = 3, P
< 0.001).

3.2.2 Fungal-induced mortality of T. leucotreta at 1×107 conidia/ml

Control pupal mortalities were significantly lower (5.0-5.8%) than pupae treated with fungal
isolates in all bioassays (Table 3). Significant differences were found amongst treatments at
each humidity level: 12% RH (LRT, χ2 = 291.77, df = 9, P < 0.001); 43% RH (LRT, χ2 =
332.52, df = 9, P < 0.001); 75% RH (LRT, χ2 = 354.86, df = 9, P < 0.001) and 98% RH (LRT,
χ2 = 352.11, df = 9, P < 0.001). High pupal mortalities were recorded in the three local M.
anisopliae isolates, G 11 3 L6, FCM Ar 23 B3 and G OL R8, regardless of humidity. Mortalities
in these isolates, ranged from 77.50 to 83.33% at 12% RH; 82.50 to 90.00% at 43% RH; 80.83
to 91.67% at 75% RH; and 82.50 to 90.83% at 98% RH (Table 3). For each of these three
isolates, pupal mortalities did not differ across the humidity levels tested, except for G 11 3 L6,
whose mortality at 75% RH, (91.67%) was significantly higher than at 12% RH, (77.50%) (P
< 0.001). In addition, pupal mortalities for the three local M. anisopliae isolates did not differ
significantly from those realised in the two commercial isolates at each humidity level tested
(Table 3). All the local B. bassiana isolates, showed reduced virulence against T. leucotreta,
with pupal mortalities ranging from 40.00 to 63.33% across the humidity levels tested. Their
efficacy did not differ significantly, within and across all humidity levels tested (Table 3).
Mycosis of pupal cadavers exceeded 96% for all isolates tested.
The LT50 values for the three M. anisopliae isolates, did not vary much across the humidity
levels tested and ranged from 5.50 to 6.66 d at 12%, 4.46 to 5.16 d at 43%, 4.08 to 5.32 d at
75% and 4.15 to 5.16 d at 98% RH (Table 4). LT50 values of the B. bassiana isolates were
typically 2-3 fold longer than the M. anisopliae isolates at each humidity level. However, their
values, in a similar pattern to the M. anisopliae isolates, did not vary much across the humidity
levels tested (Table 4).

3.2.3 Fungal-induced mortality of T. leucotreta at 1×105 conidia/ml

Control pupal mortalities were significantly lower than pupae treated with fungal isolates, and
ranged between 5.00 to 8.33%. Significant differences were found amongst treatments at each
humidity level tested: 12% RH (LRT, χ2 = 217.70, df = 9, P < 0.001); 43% RH (LRT, χ2 =
247.74, df = 9, P < 0.001); 75% RH (LRT, χ2 = 246.79, df = 9, P < 0.001) and 98% RH (LRT,
χ2 = 240.50, df = 9, P < 0.001) (Table 5). Similar to the results obtained in the highest
concentration, T. leucotreta pupal mortality for most isolates did not vary across the tested
humidity levels, and were greatest for G 11 3 L6, FCM Ar 23 B3, G OL R8, ICIPE 69 and
PPRI 5339. However, mortalities were, slightly lower (11-30%) at this concentration (Table
5). Pupal mortalities for the local B. bassiana isolates never exceeded 48% (Table 5). Mycosis
of pupal cadavers for all tested isolates (B. bassiana and M. anisopliae) was high ranging
between 94 and 96%.

4 Discussion

The need to consider tolerance to key field environmental factors, importantly, temperature,
humidity and solar radiation in mycopesticide product development has been emphasised
(Fernandes et al., 2015; Ravensberg, 2011; Tumuhaise et al., 2018). In our case, neglecting the
aforementioned, and concentrating only on the three most virulent EPF isolates (Coombes et
al., 2015) resulted in limited success against citrus arboreal pests (Grout et al., unpublished
data). Consequently, seven initially selected promising EPF isolates have been evaluated for
tolerance to abiotic constraints, to select the most suitable candidate for product development,
especially, for citrus foliar environment. The UV-tolerance of these isolates is presented and
discussed in Acheampong et al. (2020), and this study focused on the temperature and humidity
requirements.
The temperature-growth curve of all fungal isolates tested, except G Ar 17 B3, conformed to
that found for most fungi: bell-shaped, asymmetrical at the optimum temperatures and skewed
to the lower temperatures (Thomas and Jenkins, 1997). In addition, the mesophilic growth
characteristics of B. bassiana and M. anisopliae isolates in this study corroborate previous
reports of these species. Their optimal temperatures for growth were well within the 25 to 30°C
found for either of these two species from different geoclimatic origins and isolation habitats
across the globe (e.g. Chandra Teja and Rahman, 2016; Ekesi et al., 1999; Fargues et al., 1997a;
Fernández-Bravo et al., 2016, 2017; Oreste et al., 2011; Ouedraogo et al., 1997; Tumuhaise et
al., 2018).

Although the thermal optima for the majority of the isolates investigated was 27°C, the growth
response near this temperature did not vary amongst isolates. At the optimal temperatures, M.
anisopliae isolates developed the fastest and thus may be considered the better growing of the
two species. This finding is in accordance with those of Ekesi et al. (1999) who, in in vitro
growth studies of these species from different origins and isolation habitats (insect hosts and
soil), recorded higher growth rates for the M. anisopliae strains than the B. bassiana strains at
an optimum temperature of 25°C. Bugeme et al. (2008), using isolates from soil and insect
hosts, reported higher growth rates for nine M. anisopliae isolates than for the two B. bassiana
isolates tested, at all temperatures studied (20-35°C). Similarly, while investigating the thermal
tolerance of several Italian, Albanian and Algerian soil isolates of M. anisopliae and B.
bassiana, Oreste et al. (2011) reported higher growth rates for the former than the latter at their
optimal temperatures of 26-29°C. Nonetheless, the converse does exist where the growth rates
of some isolates of both species were on a par (García-Fernández et al., 2008), highlighting the
significant intra- and inter-species variation in temperature-growth pattern of these species.
Although faster and better growth rates are important for (1) disease development especially
due to fluctuating field conditions (Goettel and Inglis, 1997; Inglis et al., 2001), and (2)
optimisation during mass production of a product (Jaronski, 2013); the lower and upper thermal
limits, which influence survival outside an arthropod host are considered more critical for
persistence.

The upper thermal limit, predicted by the model never exceeded 34°C for any isolate, which is
in agreement with the 32°C reported for the commercial B. bassiana isolate PPRI 5339 (Arena
et al., 2018). The thermal limits of EPF have been speculated to be related to their geoclimatic
origins and habitats (Roberts and Campbell, 1977), with some evidence to support this
(Bidochka et al., 2001, 2002; Fernandes et al., 2008; Rangel et al., 2005; Vidal et al., 1997).
Conversely, weak to no correlation between in vitro temperature profile of entomopathogenic
fungi and their geoclimatic origins or isolation habitats abound (Fargues et al., 1997a;
Ouedraogo et al., 1997; Devi et al., 2005; Fernández-Bravo et al., 2016, 2017). Therefore,
although with few isolates, this study is in support of the latter case. For instance, the
commercial M. anisopliae isolate, ICIPE 69, originally isolated from a warmer tropical country
[(Democratic Republic of Congo, with mean monthly air temperature ≥18°C (Chen and Chen,
2013; Tumuhaise et al., 2018)] had a similar temperature profile as the candidate isolates from
a cooler South African province (Eastern Cape). Likewise, the commercial B. bassiana isolate,
PPRI 5339, originally isolated from a cassid beetle in a warmer region in South Africa
(KwaZulu Natal Province) (Arena et al., 2018), had similar optimal and cardinal temperatures
as the candidate soil isolates from the Eastern Cape.

Historical temperature data from 2008 to 2017, at four out of the five orchards within the
Eastern Cape where field trials of the candidate EPF had been conducted (Coombes et al.,
2016) were analysed. Their average annual air temperature was approximately 18°C
(https://www.wunderground.com). Temperature in spring and summer months ranged between
20 to 39°C, averaging approximately 20°C; and soil temperature during these months, at 10
cm depth, where soil-dwelling stages of T. leucotreta occur and would be controlled by EPF,
was 19°C and ranged between 13 to 24°C (Coombes et al., 2016). A similar analysis in a
warmer province (Limpopo), where the EPF field trials against S. aurantii and P. citri were
conducted (Grout et al., unpublished data), revealed slightly higher temperatures. For instance,
the annual air temperature was approximately 22°C, with spring and summer temperatures
between 23 to 41°C (https://www.wunderground.com). These data suggest that temperatures
in spring and summer months, which favour field infestations of our targeted key pests (T.
leucotreta, S. aurantii and P. citri), fall within the estimated thresholds reported in this work
i.e., 6 to 34°C. Therefore, a mycoinsecticide product from these isolates, particularly the fastest
growers, G 11 3 L6 and FCM Ar 23 B3, is anticipated to be active within the optimal
temperature ranges for survival of these pests.

The microclimate within cuticular folds of insects has been suggested by many authors as
sufficient to ensure in vitro germination and infection by fungal entomopathogens (Doberski,
1981; Fargues et al., 1997b; Inglis et al., 2001; Wraight et al., 2000). Likewise, RH in the
boundary layer near the leaf surface, has been experimentally shown to be higher than ambient
humidity, which facilitates insect infection by fungal entomopathogens (Boulard et al., 2002;
Shipp et al., 2003; Vidal, et al., 2003; Wraight et al., 2000). Thus, the need for prolonged
periods of high ambient humidity required for arthropod infection can no longer hold true for
all entomopathogenic fungal isolates (Fargues et al., 1997b; Inglis et al., 2001; Jaronski, 2010).
Accordingly, ambient humidity, previously thought to be less than required for infection (12 to
75%), did not affect the virulence of certain EPF (Doberski, 1981; Fargues et al., 1997b; James
et al., 1998; Marcandier and Khachatourians, 1987; Ramoska, 1984; Wraight et al., 2000; Vidal
et al., 2003). This has been corroborated in field studies in desert (Kooyman and Godonou,
1997) and semi-arid regions in Africa (Arthurs et al., 2001), although isolates had been
formulated in oils (that confer anti-desiccant properties). The ambient humidity range of 12 to
98% in this study had no effect on the insecticidal efficacy of all local entomopathogenic fungal
isolates under investigation or the commercial isolates, at both concentrations studied, which
is in line with the findings in the above-mentioned studies.

External sporulation on insect cadavers following fungal induced-infection, is important for

fungal establishment in the applied environment, and therefore, also for persistent control
(Arthurs et al., 2001; Gottwald and Tedders, 1984; Inglis et al., 2001; Roy et al., 2006). Even
though internal sporulation of cadavers might have occurred in this study as reported in
Rhammatocerus schistocercoides (Rehn) (Orthoptera: Acrididae) (Magalhães et al., 2000) and
observed in Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) (B. Luke, personal
communication, May 12, 2020), external sporulation in glass vials was observed only on pupae
incubated at 98% RH. This observation, corroborates results from cadaver sporulation studies
recorded elsewhere against other pest species, both in the laboratory and the field. Ramoska
(1984) investigated the influence of humidity on B. bassiana infectivity to Blissus leucopterus
(Say) (Hemiptera: Blissidae), and observed sporulation only from cadavers incubated above
75% RH. When sporulation was quantified in the same study, these infective conidia were
found only from cadavers previously incubated at humidity levels above 75%. In a field
observation study, M. acridum infected cadavers of various species of grasshoppers sporulated
only after a few days of rainfall, with humidity near saturation (> 96%) in Sahel (a semi-arid
region) (Arthurs et al., 2001). Although the effect of humidity on cadaver sporulation was not
assessed in the present study, the lower LT50 values of the virulent M. anisopliae isolates when
corroborated in the field, particularly against arboreal pests, may circumvent the need for
horizontal transmission by sporulating cadavers. Thus, assuming ambient humidity may not be
suitable for sporulation, substantial control would have been achieved to reduce the pest
population. Furthermore, humidity in sheltered areas within tree canopies (such as boundary
layers of leaves) may be higher than the prevailing ambient humidity, and hence more likely
to ensure sporulation. In citrus IPM programmes, several complementary control options are
incorporated simultaneously to reduce pest populations below economic thresholds (Moore and
Hattingh, 2004, 2012; Moore et al., 2016). Thus, these isolates, once developed into
mycoinsecticides, will mainly be used to augment other control options for these pests.

Historical RH data (2008 to 2017) from the four selected orchards, as done for temperature,
revealed an average annual ambient humidity of approximately 75%
(https://www.wunderground.com). Ambient RH in spring to summer months was 76% and
typically ranged between 88 to 100%; daily humidity levels in these months ranged from
approximately 66% during the day to 81% during the night. Humidity in orchards in warmer
citrus producing regions tends to be lower. For instance, the annual ambient humidity in
Limpopo, where the fungal field trials against S. aurantii and P. citri were conducted (Grout et
al., unpublished data), was approximately 55%, with spring and summer humidity usually
between 73 to 100%, and averaging 58% (https://www.wunderground.com). The results from
this study, previous field trials (Coombes et al., 2016), and humidity anticipated in citrus
orchards, indicate that once developed into mycoinsecticides, ambient humidity will definitely
not limit the efficacy of the virulent isolates in this study against T. leucotreta. In previous
laboratory studies, the virulent isolate, FCM Ar 23 B3, at 1×107 conidia/ml, showed similarly
good control potential against P. citri and S. aurantii, reducing crawlers of the former species
and adults (on leaf buds) of the latter by 68% and 60%, respectively (Chartier Fitzgerald, 2014;
Chartier Fitzgerald et al., 2016). In addition, even at the lower dose tested in this study, which
is more reflective of true field inoculum, mortality was not affected across the humidity levels,
again supporting that humidity is unlikely to be a limiting factor in the field. Therefore,
provided the efficacy of this isolate can be reproducible in the field as obtained for T. leucotreta
(Coombes et al., 2016), it can be deduced from this study that similarly (to T. leucotreta), its
efficacy against P. citri and S. aurantii could not have been affected by ambient humidity, but
more likely UV radiation. This assumption may be supported by the following: (1) its high
sensitivity to UV radiation (Acheampong et al., 2020); and (2) the good control of soil-dwelling
life stages of T. leucotreta by this same isolate in the upper 10 cm depth of soil where UV
radiation is virtually non-existent (Coombes et al., 2016).

In summary, all the local tested isolates exhibited highly similar temperature profiles and were
equally capable of inducing fungal mortality in T. leucotreta fifth instars across a range of
humidity levels, which fall within the temperature and humidity ranges likely experienced in
South African citrus orchards. Thus, these abiotic factors alone are unlikely to influence the
efficacy of these isolates in the field and, coupled with their similar performance to each other
and the commercial isolates, used against similar arboreal pests (Hatting et al., 2019), virulence
may be prioritised. In general, the M. anisopliae isolates grew faster and induced higher insect
mortality than the B. bassiana isolates. Future applied research focused on these M. anisopliae
isolates is thus validated with respect to temperature and humidity.

Acknowledgements
This work was supported by Citrus Research International [Project 1143] and the South African
Research Chairs Initiative of the Department of Science and Technology and the National
Research Foundation (NRF) of South Africa [grant number: 199609]. Any opinion, finding,
conclusion or recommendation expressed in this material is that of the authors and the NRF
does not accept any liability in this regard. Dr Christian Monaco (IFREMER, France), Dr
Shelly Edwards, Clarke van Steenderen, and Eric Kamau of Rhodes University for assistance
with statistical analysis are acknowledge as well as the support of Rhodes University.

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Tables
Table 1: Isolation details of the indigenous entomopathogenic fungal isolates of interest in this
study.

(Locality, coordinates,
Fungal Species RU Accession codea
soil type and date isolated)b
M. anisopliae s.l. G 11 3 L6 Mosslands (33°23′54″S; 26°25′41″E)
Oakleaf Caledon, April 2008
M. anisopliae s.str. FCM Ar 23 B3c Arundel (33°30′57″S; 25°39′11″E)
Loam, April 2008
M. anisopliae s.l. G OL R8 Olifantskop (33°37′14″S; 25°40′49″E)
Unknown, July 2008
B. bassiana s.str. G Ar 17 B3c Arundel (33°30′57″S; 25°39′11″E)
Loam, May 2008
B. bassiana s.str. G B Ar 23 B3c Arundel (33°30′57″S; 25°39′11″E)
Loam, May 2008
B. bassiana s.l. G 14 2 B5 Mosslands(33°23′54″S; 26°25′41″E)
Oakleaf Richie, May 2008
B. bassiana s.l. FCM 10 13 L1 Mosslands (33°23′54″S; 26°25′41″E)
Oakleaf Caledon, April 2008
aCultures stored at Centre for Biological Control, Rhodes University (Grahamstown, South Africa).
bOrigin of fungal isolates (farm name, co-ordinates, soil type and year of isolation) (reproduced from Goble et al.,
2011).
cSpecies identity confirmed based on BLAST analysis of ITS regions; Genbank accession numbers for FCM Ar 23

B3, G Ar 17 B3 and G B Ar 23 B3 are KF834188, KF834186 and KF834187, respectively (Chartier Fitzgerald,
2014; Chartier Fitzgerald et al., 2016).
Table 2: Estimated parameters (± SE) from the modified β functiona fitted to vegetative growth data of B. bassiana and M. anisopliae
isolates.

Estimated parametersf
Fungal isolate
Topt (°C)b TYopt (mm/day)c Tmax (°C)d Tb3e
M. anisopliae G 11 3 L6 26.33 (0.11) bc 3.42 (0.10) a 33.93 (0.14) a 0.98 (0.05) ab
M. anisopliae FCM Ar 23 B3 26.90 (0.11) abc 3.15 (0.08) a 32.98 (0.16) bc 0.74 (0.07) ab
M. anisopliae G OL R8 26.27 (0.10) c 3.23 (0.06) a 33.45 (0.16) ab 0.93 (0.07) ab
M. anisopliae ICIPE 69 27.27 (0.21) abc 2.55 (0.03) b 32.50 (0.12) cde 0.48 (0.06) b
B. bassiana G Ar 17 B3 28.31 (0.83) a 0.69 (0.02) f 32.07 (0.05) e 0.38 (0.16) b
B. bassiana G B Ar 23 B3 28.24 (0.17) ab 2.44 (0.09) c 32.05 (0.02) e 0.35 (0.04) b
B. bassiana G 14 2 B5 27.44 (0.59) abc 1.81 (0.03) d 32.91 (0.40) bcd 1.32 (0.45) a
B. bassiana FCM 10 13 L1 26.91 (0.58) abc 1.46 (0.05) e 32.43 (0.17) cde 0.96 (0.19) ab
B. bassiana PPRI 5339 26.19 (0.20) c 1.72 (0.02) d 32.07 (0.03) de 0.61 (0.05) ab
aThe modified β function is given by Y(T) = TYopt ⁄ ((T -Tmin) / (Topt - Tmin)) ^ (Tb3 * ((Topt - Tmin) / (Tmax -Topt))) *((Tmax - T) / (Tmax -Topt)) ^ Tb3, where Y (T) is
the fungal growth in mm/day (dependent variable) and T is the incubation temperature (independent variable) (Bassanezi et al., 1998). Tmin is the minimum
temperature for fungal growth, and was fixed at 6°C.
bT
opt is the optimal temperature for fungal growth.
cTY
opt is the fungal growth at the optimal temperature.
dT
max is the maximum temperature for fungal growth.
eTb3 is the shape parameter which influences the temperature range around T
opt in which the curve stays near TYopt.
fMeans within columns with the same letter are not significantly different (Pairwise t-test, P > 0.05 with Bonferroni correction factor).
Table 3: Mean pupal mortality (± SE) of T. leucotreta fifth instars exposed to M. anisopliae and B. bassiana isolates at 1×107
conidia/ml and incubated at four RH levels (12, 43, 75 and 98%) at 27°C, 12 h photoperiod, over a 21-day period.

Mean mortality (%) at the four humidity levels testeda

Fungal isolate/treatment 12% 43% 75% 98%
M. anisopliae G 11 3 L6 77.50 (2.50) Bab 85.83 (2.21) ABa 91.67 (0.83) Aa 85.83 (0.83) ABa
M. anisopliae FCM Ar 23 B3 83.33 (3.01) Aa 90.00 (2.89) Aa 91.67 (2.21) Aa 90.83 (1.67) Aa
M. anisopliae G OL R8 80.00 (2.50) Aa 82.50 (1.44) Aa 80.83 (2.21) Aab 82.50 (2.50) Aa
M. anisopliae ICIPE 69 75.83 (2.21) Aabc 83.33 (1.67) Aa 82.50 (1.44) Aa 85.83 (2.21) Aa
B. bassiana G Ar 17 B3 40.00 (4.33) Ad 47.50 (3.82) Ab 46.67 (5.07) Ad 45.83 (2.21) Ab
B. bassiana G B Ar 23 B3 57.50 (3.82) Acd 58.33 (2.21) Ab 63.33 (2.21) Abd 60.00 (2.89) Ab
B. bassiana G 14 2 B5 60.00 (1.44) Abd 57.50 (1.44) Ab 59.17 (1.67) Acd 60.83 (2.21) Ab
B. bassiana FCM 10 13 L1 50.83 (2.21) Ad 55.83 (2.21) Ab 55.00 (2.89) Ad 58.33 (2.21) Ab
B. bassiana PPRI 5339 78.33 (3.33) Aab 80.00 (2.50) Aa 76.67 (5.47) Aabc 82.50 (2.89) Aa
Control 5.00 (1.44) Ae 5.8 (0.83) Ac 5.00 (1.44) Ae 5.00 (1.44) Ac
a Meanswithin each row with the same uppercase letter are not significantly different (Tukey’s HSD test, P > 0.05). Means within each column with the
same lower case letter are not significantly different (Tukey’s HSD test, P > 0.05).
Table 4: Mean number of days required for 50% pupal mortality (LT50) (95% CI) after exposure of T. leucotreta fifth instars to M.
anisopliae and B. bassiana isolates at 1×107 conidia/ml and incubated at four RH levels (12, 43, 75 and 98%) at 27°C, 12 h
photoperiod, over a 21-day period.

LT50 (95% CI)a at the various humidity levels

Fungal isolate/treatment 12% 43% 75% 98%

M. anisopliae G 11 3 L6 6.66 (5.97-7.35) 4.76 (4.13-5.39) 4.16 (3.65-4.67) 4.45 (3.78-5.12)
M. anisopliae FCM Ar 23 B3 5.50 (4.87-6.13) 4.46 (3.93-4.99) 4.08 (3.57-4.59) 4.15 (3.62-4.68)
M. anisopliae G OL R8 5.89 (5.20-6.58) 5.16 (4.45-5.87) 5.32 (4.59-6.05) 5.16 (4.45-5.87)
M. anisopliae ICIPE 69 6.60 (5.86-7.34) 5.08 (4.39-5.77) 5.23 (4.54-5.92) 4.82 (4.19-5.45)
B. bassiana G Ar 17 B3 21.82 (19.70-23.94)b 17.32 (16.09-18.55)b 17.74 (16.45-19.03)b 18.19 (16.79-19.58)b
B. bassiana G B Ar 23 B3 12.88 (12.06-13.70) 12.53 (11.73-13.33) 10.77 (10.03-11.51) 11.85 (11.07-12.63)
B. bassiana G 14 2 B5 11.95 (11.19-12.71) 12.82 (11.99-13.64) 12.19 (11.39-12.99) 11.48 (10.69-12.26)
B. bassiana FCM 10 13 L1 15.61 (14.53-16.69) 13.24 (12.32-14.16) 13.40 (12.42-14.38) 11.94 (11.04-12.84)
B. bassiana PPRI 5339 6.17 (5.44-6.89) 5.84 (5.13-6.55) 6.52 (5.78-7.26) 5.31 (4.62-5.99)
a Means based on 3 replicates from logistic mortality curves.
b LT values are an extrapolation of the data set due to insufficient data.
50
Table 5: Mean pupal mortality (± SE) after exposure of T. leucotreta fifth instars to M. anisopliae and B. bassiana isolates at 1×105
conidia/ml and incubated at four RH levels (12, 43, 75 and 98%) at 27°C, 12 h photoperiod, over a 21-day period.

Mean mortality (%) at the four humidity levels testeda

Fungal isolate/treatment 12% 43% 75% 98%
M. anisopliae G 11 3 L6 62.50 (6.61) Bab 69.17 (5.83) ABa 80.00 (2.89) Aa 80.83 (3.01) Aa
M. anisopliae FCM Ar 23 B3 67.50 (6.61) Aa 73.33 (3.01) Aa 71.67 (2.21) Aa 72.50 (1.44) Aa
M. anisopliae G OL R8 66.67 (3.01) Aa 64.17 (4.41) Aab 71.67 (3.33) Aa 64.17 (3.01) Aab
M. anisopliae ICIPE 69 66.67 (6.01) Aa 75.83 (2.21) Aa 71.67 (3.33) Aa 75.00 (1.44) Aa
B. bassiana G Ar 17 B3 25.00 (2.89) Bd 31.67 (3.63) ABc 38.33 (3.63) ABb 40.83 (3.01) Ac
B. bassiana G B Ar 23 B3 38.33 (4.41) Acd 42.50 (2.89) Ac 43.33 (2.21) Ab 43.33 (4.64) Ac
B. bassiana G 14 2 B5 45.00 (4.33) Abc 44.17 (3.63) Abc 45.00 (5.78) Ab 47.50 (1.44) Abc
B. bassiana FCM 10 13 L1 40.00 (5.77) Acd 40.83 (4.17) Ac 39.17 (3.01) Ab 37.50 (1.44) Ac
B. bassiana PPRI 5339 65.83 (7.95) Aa 68.33 (3.63) Aa 70.83 (3.63) Aa 70.83 (3.01) Aa
Control 5.83 (2.21) Ae 5.00 (1.44) Ad 6.67 (3.01) Ac 8.33 (2.21) Ad
a Meanswithin each row with the same uppercase letter are not significantly different (Tukey’s HSD test, P > 0.05). Means within each
column with the same lower case letter are not significantly different (Tukey’s HSD test, P > 0.05).
Figure

Figure 1: Effect of temperature on the colony growth rate of five B. bassiana and four M.
anisopliae isolates. Lines show the nonlinear model (modified β function, Bassanezi et al.,
1998) fitted to average growth rates under the various temperatures tested, to predict the
minimum, maximum and optimum temperatures for these isolates.
Highlights

 All fungal isolates grew within temperature ranges experienced in citrus orchards
 All isolates infected T. leucotreta fifth instars across a range of humidity levels
 Temperature and humidity are unlikely to affect efficacy of isolates in the field
 M. anisopliae isolates grew faster and were more virulent than B. bassiana isolates