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Research article
Effects of Beauveria bassiana (Hypocreales) on plant growth and secondary
metabolites of extracts of hydroponically cultivated chive (Allium
schoenoprasum L. [Amaryllidaceae])
Friederike Espinoza
a
, Stefan Vidal
a
, Fanie Rautenbach
b
, Francis Lewu
c
, Felix Nchu
d
,
*
a
Department of Crop Sciences, Section of Agricultural Entomology, Georg-August University, Grisebachstrasse 6, 37077, Goettingen, Germany
b
Department of Biomedical Sciences, Cape Peninsula University of Technology, Symphony Way, Bellville, P.O. Box 1906, Bellville 7535, South Africa
c
Department of Horticultural Sciences, Cape Peninsula University of Technology, Symphony Way, Bellville, P.O. Box 1906, Bellville 7535, South Africa
d
Department of Agricultural Sciences, Cape Peninsula University of Technology, Wellington, South Africa
ARTICLE INFO
Keywords:
Biotechnology
Microbiology
Plant biology
Endophytic fungus
Chives
Total alkaloids
Plant growth
Plant cultivation
ABSTRACT
The endophytic plant-fungi symbiotic relationship can be explored to improve cultivation of targeted medicinal
plant species. The objective of this study was to assess the effects of the cultivation of chive (Allium schoeno-
prasum) in plant growth medium inoculated with the entomopathogenic fungus, Beauveria bassiana (Hypocreales).
Twelve replicates of chive seedlings were exposed to B. bassiana inoculum formulated at concentrations of 0, 1
10
5
,110
4
and 1 10
3
conidia mL
1
in a completely randomized design. We assessed plant growth parameters,
such as leaf number and plant height weekly and root length, leaf and root fresh and dry weights and secondary
metabolites three weeks post-fungal inoculation. The fungus was re-isolated from some of the leaves and roots of
the treated plants suggesting that the fungus successfully colonized the plant tissue. Generally, the results indi-
cated that the fungal inoculation had minimal effect on most of the growth parameters assessed in relation to the
control. Remarkably, plants exposed to the fungus recorded greater (p <0.05) total alkaloid, ranging from 2.98 –
3.76 mg atropine equivalent (AE)/g dry weight (DW) compared to the control plants (1.96 mg AE/g DW) for the
leaves. This study demonstrated that endophytic fungi could be used to improve the yield of active chemical
constituents in cultivated medicinal plants.
1. Introduction
High demand has bolstered trade in medicinal plants and subse-
quently, creating the need for commercial cultivation of these plants.
However, successful commercial cultivation of medicinal plants relies on
achieving consistently high quality and quantity of medicinal materials at
a reasonable cost of production. Consequently, efficient technologies and
techniques are being developed. For example, greenhouse technologies
circumvent the setbacks which are normally associated with open air
conditions, such as variations in biotic and abiotic factors, and limited
arable land (Canter et al., 2005).
A plethora of studies have examined the effects of varying levels of
specific abiotic factors on the production of secondary metabolites
(Pavarini et al., 2012). On the contrary, much fewer studies have
investigated the effects of biotic factors on nutraceutical and medicinal
plants (Gouvea et al., 2012). Some entomopathogenic fungi are
endophytic; some are easily mass-produced in vitro; some are rhizo-
spheric; they are quite ubiquitous (Vega et al., 2008,2009). An endo-
phytic fungus forms a mutually beneficial symbiotic relationship with a
plant; it lives inside a plant's tissues without causing disease to the plant,
meanwhile, boosting plant defenses and in return the plant acts as the
host (Behie and Bidochka, 2014). Metabolites produced by some endo-
phytic fungi have been reported to influence the reduction of insect in-
festations on their host plants (Jaber and Ownley, 2017). The increase in
quantity and diversity of secondary metabolites in endophyte-containing
plants are probably responsible for the reduction of insect herbivory on
plants. The endophytic fungi-plant relationship can be explored for
cultivation of targeted high value medicinal plant species with the view
to optimizing medicinal properties by increasing quantity and quality of
secondary metabolites in these plants. Beauveria bassiana, an entomo-
pathogenic fungus which occurs naturally and ubiquitously in the soil
(Keswani et al., 2013), is endophytic and an interesting candidate for the
* Corresponding author.
E-mail address: felixnchu@gmail.com (F. Nchu).
Contents lists available at ScienceDirect
Heliyon
journal homepage: www.cell.com/heliyon
https://doi.org/10.1016/j.heliyon.2019.e03038
Received 12 June 2019; Received in revised form 18 September 2019; Accepted 10 December 2019
2405-8440/©2019 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Heliyon 5 (2019) e03038
evaluation of endophytic fungal colonization on the medicinal properties
of plants.
Plants belonging to the genus Allium including garlic, onion, and
Chinese chive are known for their medicinal properties; they have
proven pharmacological and nutraceutical activities. They contain
organosulfur compounds such as allicin and phenolic compounds like
gallic acid, quercetin, coumaric acid, and ferulic acid (Zeng et al.,
2017;Kucekova et al., 2011). Phenolic compounds are bioactive sub-
stances that show antioxidant, anticancer, anti-inflammatory, and
antimicrobial activity, and prevent chronic disease (Vlase et al., 2013).
Allium schoenoprasum, which is commonly known as chive, is a
perennial plant that is used as a popular seasoning and is cultivated
globally (Zeng et al., 2017). Phytochemical analyses of
A. schoenoprasum extracts revealed that its water extract contains
flavonoid compounds, glycosides, and saponins while the methanol
and ethyl acetate extracts contain alkaloids, flavonoids, glycosides,
and saponins (Sihombing et al., 2018). Chive leaves have great me-
dicinal values, such as positive effects on the circulatory system by
reducing blood pressure and antimicrobial effects (Vlase et al., 2013).
Chive leaf infusions, often rich in potassium, are used for treatment of
kidney stone disease by dissolving calcium oxalate in kidneys (Tripathi
et al., 2013). Kucekova et al. (2011) demonstrated that chive flower
extract obtained by solid-liquid extraction have a great effect on
human keratinocyte cell line (HaCaT) by decreasing cell proliferation,
perhaps, due to their phenolic compounds. It is worth-mentioning that
chives may contain many of the bioactive phenolic compounds such as
gallic acid, coumaric acid, ferulic acid, and rutin that are commonly
found in other Allium spp.
Hence, the objective of this study was to assess plant growth of
chive (A. schoenoprasum) and secondary metabolites of chive leaves
and roots following cultivation in a plant growth medium inoculated
with conidia of an endophytic entomopathogenic fungus (B. bassiana)
under greenhouse conditions. This is the first study on the experi-
mental inoculation of chive with an endophytic fungus, and its sub-
sequent effects on plant growth and production of secondary
metabolites.
2. Material and methods
2.1. Fungus
An indigenous B. bassiana strain (SM3) that was previously isolated
from a soil sample collected from the Cape Winelands in South Africa
was used in this study. This strain was identified using molecular and
morphological techniques as described in Moloinyane and Nchu (2019).
The strain is being maintained at Cape Peninsula University of Tech-
nology in Bellville, South Africa. The fungus was cultivated on
half-strength potato dextrose agar (PDA); 0.02 g/L of ampicillin (Sig-
ma-Aldrich), and 0.04 g/L streptomycin (Sigma-Aldrich) in 9 cm and 14
cm diameters petri dishes. Fungal cultures were incubated at 25 Cin
the dark for four weeks. Using a spatula, mature four-week-old
B. bassiana conidia obtained from PDA plates were transferred to 2 L
glass bottles containing sterile 0.01% Tween 80 (Polysorbate,
Sigma-Aldrich, South Africa) and sterile water. Bottles were capped,
mixed by shaking for 5 min and by using a magnetic stirrer (at 20 C
and 300 rpm for 30 min) to homogenous conidial suspensions. The
conidia inoculum concentration was enumerated using a haemocy-
tometer (Bright-Line, Sigma-Aldrich, South Africa) and observed with a
light microscope at 400X magnification. In order to obtain the desired
concentration (1 10
5
conidia mL
1
), the volume of sterile 0.01%
Tween was increased or conidia were added to the glass bottle. This was
followed by 10-fold serial dilutions to obtain lower concentrations of
conidial inoculum suspensions; 1 10
4
,110
3
conidia mL
1
.A
conidial germination test to determine conidial viability was carried out
according to the method described by Inglis et al. (2012) and high spore
germination of over 90% was obtained.
2.2. Plants
Chive (A. schoenoprasum) seedlings, were purchased from Stodels
Nurseries (Pty) Ltd in Bellville, Western Cape Province, South Africa.
Plants were maintained in the greenhouse at Cape Peninsula University
of Technology in Bellville, South Africa at 23–25 C, 60%–80% RH and
13/11 natural light/dark regime. Individual chive plants were separated
from a clump and one plant was transplanted to each 10 cm diameter pot
containing a substrate mixture of one-third of river sand, one-third of
vermiculite, and one-third of perlite by volume. The substrate materials
were sterilized using 1% sodium hypochlorite for 1h before rinsing with
sterile distilled water. The plants were fed with water soluble, formulated
hydroponic fertilizer, Nutrifeed (Starke Ayres Pty. Ltd., South Africa).
The fertilizer was dissolved in sterile distilled water at a concentration of
10 g/5 L and 100 mL of the mixture was added to each plant once a week.
Each plant was watered with 100 mL reverse osmosis water once a week.
2.3. Inoculation of plants
Three conidial inoculum suspensions (1 10
5
,110
4
,110
3
conidia mL
1
) were prepared as described above. All chive plants had a
uniform age and twelve chive plants with a similar leaf length were
randomly allocated to each of the treatments (T1 ¼110
5
,T2¼1
10
4
,T3¼110
3
conidia mL
1
). Ninety milliliters of each of the conidial
concentrations was added manually and separately to the root system of
each plant of the same treatment using a hand-held plastic dispenser at
one week following the commencement of the experiment. Twelve plants
were used as the control and treated with 90 mL of sterile 0.01% Tween
80 water.
2.4. Plant growth measurements
Leaf length (cm) from the soil surface to the top of the highest leaf and
leaf number of each plant were measured weekly for three weeks. Leaf
length increment (cm) was calculated as the difference between leaf
length at weeks one and three and percentage growth was calculated as
follows: 100 x the leaf length increment divided by week one leaf length.
Similarly, leaf number was counted and leaf number increment and %
increase were calculated. At the end of the experiment, three weeks post
fungal inoculation, root length and fresh weights of plants were recorded.
Root length (cm) was measured from the hypocotyl-root junction to the
root tip with a ruler. Dry weights of the plants were determined by
placing plants in paper bags in a drying oven at 35 C for 7 days.
2.5. Fungal colonization
To determine fungal colonization of leaf tissue, three sections of leaf
(5 5 mm) as well as root (5 mm length) sections were collected from
each plant soon after harvesting. The excised material was surface ster-
ilized by first dipping in 70% ethanol for 3 s and then rinsing in sterile
distilled water for 1 min. Thereafter, the sterile leaf and root sections
were placed on solid half-strength PDA, incubated in the dark at 25 C,
and were checked for outgrowth of B. bassiana in root and leaf tissues
under stereo microscope after one and two weeks. Efficiency of surface
sterilization was evaluated by placing drops of the previously used 70%
ethanol and distilled water for surface sterilization on plates containing
solid half-strength PDA, and then incubated and checked for fungal
outgrowth.
2.6. Analysis of secondary metabolites in leaf and root extracts
Chive plants were harvested at three weeks post treatment and the
leaves and roots were separated, placed in separate paper bags and dried
in a drying oven at 35 C for 7 days and the dry leaves and roots of each
plant were ground separately using a ceramic mortar and pestle for 2 min
and 1 min, respectively.
F. Espinoza et al. Heliyon 5 (2019) e03038
2
The spectroscopic method described by Fadhil and Reza (2007) was
used to determine total alkaloids in the plant extracts. Briefly, 100 mg of
the chive leaf and root materials were extracted separately with 10 mL of
aqueous ethanol (mixture of 60% ethanol and 40% water) for 2 h,
centrifuged (4000 x g for 10 min) and the supernatant was used in the
assay. Two milliliters of the extract supernatant and atropine standard
solutions were mixed with 5 mL sodium phosphate buffer and 12 mL
bromocresol green solution. Thereafter, 12 mL of chloroform was added
to the solution and the solution was mixed vigorously using a vortex
mixer. The absorbance at 417 nm was determined and the concentration
of mg atropine equivalent per g dry weight (mg AE/g DW) in the sample
using a standard curve of atropine was calculated.
The total polyphenol content of the aqueous ethanol extracts of dried
leaf and root dried materials of the chives were determined by the Folin-
Ciocalteu method (Singleton et al., 1999;Swain and Hills, 1959).The
method of Swain and Hills (1959) was adapted for the plate reader. Using
a 96-well microplate, 25
μ
L of the sample was mixed with 125
μ
L
Folin-Ciocalteu reagent (Merck, South Africa) and diluted 1:10 with
distilled water. After 5 min, 100
μ
L (7.5%) aqueous Na
2
CO
3
(Sigma-Al-
drich, South Africa) was added to the well. The plates were incubated for
2 h at room temperature before the absorbance was read at 765 nm using
a Multiskan plate reader (Thermo Electron Corporation, USA). The
standard curve was prepared using 0, 20, 50, 100, 250 and 500 mg/L
gallic acid in 10% ethanol and the results were expressed as mg gallic
acid equivalents per g dry weight (mg GAE/g DW).
The flavonol content of the aqueous ethanol extracts of dried leaf and
root materials of the chives were determined using quercetin 0, 5, 10, 20,
40, and 80 mg/L in 95% ethanol (Sigma-Aldrich, South Africa) as stan-
dard. In the sample wells, 12.5
μ
L of the crude aqueous extracts were
mixed with 12.5
μ
L 0.1% HCl (Merck, South Africa) in 95% ethanol, and
225
μ
L 2% HCl and incubated for 30 min at room temperature. The
absorbance was read at 360 nm, at a temperature of 25 C(Mazza et al.,
1999).The results were expressed as mg quercetin equivalent per g dry
weight (mg QE/g DW).
2.7. Statistical analysis
All data were analyzed with Paleontological Statistics (PAST)
(Hammer et al., 2001). Plant growth and secondary metabolite data were
compared using one-way ANOVA at p ¼0.05 level of significance. The
significant difference between the means was determined with the
Tukey-pairwise test (P ¼0.05). Chive plants were arranged in a
completely randomized design. Each treatment contained 12 replicate
chive plants. The results are presented as mean SE.
3. Results
3.1. Re-isolation of fungus
B. bassiana was re-isolated from chive leaf and root samples from all
fungal treatments, but not the controls. Fungal outgrowth on chive leaves
was found in the fungus inoculated treatments after two weeks, but not
from control treated plants (T1 ¼33%, T2 ¼22%, T3 ¼33%, Control ¼
0% of leaf samples). The root samples of chive plants showed less fungal
outgrowth in all treatments than the leaf samples (T1 ¼11%, T2 ¼11%,
T3 ¼11%, Control ¼0% of root samples). Again, B. bassiana was also not
detected in the root samples from control treated plants. Sterility of the
re-isolation process was confirmed due to no outgrowth on petri dishes
containing 70% ethanol and distilled water.
3.2. Chive growth
Generally, plant growth increased over time in both test and control.
However, there was no significant difference (df ¼3, 44; p >0.05) in the
leaf length between the B. bassiana treated and the control plants for all
weeks (Table 1). Interestingly, the only exception was the root length;
root lengths were statistically different among treatments (df ¼3, 44; p <
0.05) with the highest concentration of conidial suspension yielding the
shortest root length (Figure 1). While there were no significant differ-
ences (df ¼3, 44; p >0.05) among treatments for the number of leaves or
leaf number increment, chives exposed to the highest concentration of
conidial suspensions showed a slightly greater percentage increase in leaf
number compared to the control and the other two fungus treatments
from first to third week post-treatment (Table 2). Although root and leaf
fresh and dry weights were comparatively lower in the fungus treated
plants compared to their control counterparts, the differences were not
significant (df ¼3, 44; p >0.05) (Table 3).
3.3. Quantification of secondary metabolites
Results from the chemical analysis showed that the secondary metab-
olite concentrations were statistically greater (p <0.05) in the leaves than
Table 1. Leaf length (cm), total plant growth (cm; the difference between week one and week three), and percentage growth (%; the percentage change from week one
to week three) of chive treated with different concentrations of Beauveria bassiana at one, two and three weeks after inoculation in the greenhouse at CPUT, Bellville,
South Africa. N ¼12.
Leaf length (cm)
Treatment
a
Week one (baseline) Week two Week three Total plant growth (cm) Percentage growth (%)
T1 19.10 0.70 25.20 1.00 30.50 1.10 11.40 1.20 59.70
T2 19.60 1.10 25.50 0.80 32.50 1.40 12.801.40 65.80
T3 20.30 1.10 26.80 0.80 33.60 0.80 13.30 1.30 65.50
Control 21.20 1.10 26.90 0.90 33.50 1.60 12.30 1.50 61.10
a
T1 ¼110
5
,T2¼110
4
,T3¼110
3
B bassiana conidia mL
1
. There was no significant difference (df ¼3, 44; p >0.05) in the leaf length between the treated
and the control plants at weeks 1, 2 and 3 post treatment.
Figure 1. Root length (cm) of chive plants. treated with different concentra-
tions of Beauveria bassiana three weeks after inoculation in the greenhouse at
CPUT, Bellville, South Africa. N ¼12. Bars with the same lowercase letters in
the same column are not significantly different following Tukey test at the p <
0.05 level of significance and error bars represent standard error. T1 ¼110
5
,
T2 ¼110
4
,T3¼110
3
B.bassiana conidia mL
1
.
F. Espinoza et al. Heliyon 5 (2019) e03038
3
in the roots, irrespective of whether plants were exposed to fungus or not
(Table 4). However, while noticeably greater polyphenol and flavonol
contents in the leaves were yielded by plants exposed to fungal inoculum at
a concentration of 10
4
B. bassiana conidia mL
-1
(T2) compared to the other
plants, the difference was not significant (p >0.05). Remarkably, the
alkaloid content in the leaves of fungus exposed plants were significantly
(df ¼3, 8; F ¼3.06; p <0.05) greater than that of the control plantswith T2
being about twice that of the control plant. Otherwise, there was no dif-
ference in the polyphenol and flavonol concentrations among treatments
(df ¼3, 8; F
polyphenol
¼0.44; F
flavonols
¼1.33; p >0.05).
4. Discussion
The B. bassiana isolate used in the present study was able to endo-
phytically colonize chive plants. The re-isolation of the fungus from chive
leaf samples showed that the fungus was systemic, i.e., inoculum was
transferred from the growth medium to the leaves. This is the first record
of successful experimental inoculation and colonization of B. bassiana in
chives. Previous studies have reported the colonization of B. bassiana in
other plant species with different inoculation methods (Akutse et al.,
2013;Quesada-Moraga et al., 2009).
Inoculation with B. bassiana did not improve the growth of chives
over the control treatment (Tables 1,2, and 3). In fact, the control plants
had slightly higher biomass and root growth compared to the treated
plants. These results differ from those reported in previous studies, which
showed that B. bassiana promotes plant growth of cassava (Manihot
esculenta), faba bean (Vicia faba) and cotton (Gossypium hirsutum)(Lopez
and Sword, 2015;Greenfield et al., 2016;Jaber and Enkerli, 2016a and
b). However, Lewis et al. (2001) reported no significant difference in the
growth of maize (Zea mays) plants exposed to seed treatments with
B. bassiana and the corresponding control treatment. Jaber and Enkerli
(2016b) reported inconsistent endophyte-induced plant growth promo-
tion across sampling dates following foliar inoculation of faba bean
plants with B. bassiana (Naturalis®), B. brongniartii (BIPESCO2 and 2843)
and M. brunneum (BIPESCO5). The root lengths varied significantly
among treatments, and the shortest length was observed in plants
Table 2. Number of leaves, increase of leaf number (the difference in the number of leaves between week one and week three), and percentage increase (%; the
percentage change in the number of leaves from week one to week three) of leaves of chive treated with different concentrations of Beauveria bassiana at one, two and
three weeks after inoculation in the greenhouse at CPUT, Bellville, South Africa. N ¼12.
Leaf number (cm)
Treatment
a
Week one (baseline) Week two Week three Increase of leaf number Percentage increase (%)
T1 9.50 0.60 11.70 0.5 13.40 1.00 3.90 41.10
T2 8.60 0.70 10.70 0.80 11.70 1.10 3.10 36.00
T3 8.50 0.50 9.80 0.80 11.70 0.90 3.20 37.60
Control 10.80 1.00 13.00 1.30 14.70 1.40 3.90 36.10
a
T1 ¼110
5
,T2¼110
4
,T3¼110
3
B bassiana conidia mL
1
. There was no significant difference (df ¼3, 44; p >0.05) in the leaf number between the treated
and the control plants at weeks 1, 2 and 3 post treatment.
Table 3. Fresh weight (g) and dry weight (g) of roots and leaves of chive plants treated with different concentration of Beauveria bassiana entomopathogenic endophytic
fungi three weeks after inoculation in the greenhouse at CPUT, Bellville, South Africa. N ¼12.
Fresh weight (g) Dry weight (g)
Treatment
a
Roots Leaves Roots Leaves
T1 4.10 0.60 5.90 0.90 0.48 0.09 1.16 0.33
T2 3.70 0.90 5.20 1.30 0.45 0.14 0.99 0.41
T3 3.80 0.70 5.20 0.90 0.40 0.09 1.03 0.35
Control 4.90 0.80 6.40 1.00 0.62 0.15 1.18 0.47
a
T1 ¼110
5
,T2¼110
4
,T3¼110
3
B. bassiana conidia mL
1
. There was no significant difference (df ¼3, 44; p >0.05) in the dry and fresh weights between
the treated and the control plants.
Table 4. Content of polyphenols (mg GAE/g DW), flavonols (mg QE/g DW), alkaloids (mg AE/g DW) in leaf and root samples of chives inoculated with Beauveria
bassiana entomopathogenic endophytic fungi in the greenhouse at Bellville, South Africa. Data are the mean of 3 replicate chive plants. GAE ¼gallic acid equivalent, QE
¼quercitin equivalent, AE ¼atropine equivalent, DW ¼Dry weight.
Treatment
a
Polyphenols (mg GAE/g DW) Flavonols (mg QE/g DW) Alkaloids (mg AE/g DW)
Leaves
T1 7.41 0.68aA 3.25 0.40aA 3.14 0.37aA
T2 8.16 1.40aA 4.30 0.41aA 3.76 0.62aA
T3 6.78 0.84aA 2.90 0.66aA 2.98 0.04aA
Control 7.00 0.43aA 3.41 0.55aA 1.96 0.02bA
Roots
T1 3.55 0.65aB 0.70 0.23aB 1.13 0.04aB
T2 3.70 0.66aB 0.65 0.06aB 1.03 0.05aB
T3 3.81 0.78aB 1.08 0.16aB 1.06 0.03aB
Control 4.03 0.46aB 0.96 0.25aB 1.09 0.05aB
a
T1 ¼110
5
,T2¼110
4
,T3¼110
3
B. bassiana conidia mL
1
. Means with the same lowercase letters in the same column, for roots or leaves, are not
significantly different when leaf or root data for the different treatments are separately compared using Tukey test at the p <0.05 level of significance. Means with the
same uppercase letters in the same column are not significantly different when leaves and roots for corresponding treatments were compared using Tukey test at the p <
0.05 level of significance.
F. Espinoza et al. Heliyon 5 (2019) e03038
4
exposed to the highest concentration of fungal treatment, which may
suggest that fungus might have had a negative effect on root length. In a
more recent study, B. bassiana inoculation had a positive influence on
plant growth parameters including root length of common beans (Phae-
seolus vulgaris)(Afandhi et al., 2019). Nevertheless, the colonization of
plant tissues by fungal endophytes can be influenced by many factors,
such as inoculation method, species and fungal strain (Muvea et al.,
2014;Afandhi et al., 2019). Fungal endophytes might affect the nutrient
cycle and uptake of nutrients from the soil by plants (Saikkonem et al.,
2015).
In this study, chemical analysis revealed that the leaves and roots of
both B. bassiana-exposed and unexposed chive contained polyphenols,
alkaloids and flavonols, and also that these secondary metabolites were
significantly more concentrated in the leaves than in the roots. Chives
and other Allium species, such as onion and garlic contain polyphenols,
alkaloids, flavonoids, glycosides and organosulfur (Gulfraz et al., 2014;
Soto et al., 2016;Sihombing et al., 2018). Furthermore, higher alkaloid
content was detected in the leaves of plants inoculated with B. bassiana
than in the control treated plants (Table 4). In a previous study, which
involved the same fungal strain (strain: SM3) used in this study,
drenching potted grapevine plants with its conidial suspension induced
higher production of anti-insect volatile compounds including Naph-
thalene in the fungus-exposed plants compared to the control (Moloi-
nyane and Nchu, 2019). The better yield of alkaloids in fungus-treated
plants could be due to the synthesis of secondary metabolites by fungus in
the plant tissues (Alvin et al., 2014;Lugtenberg et al., 2016). Also, en-
dophytes can potentially induce host plants to accumulate secondary
metabolites (Lugtenberg et al., 2016). Zhang et al. (2012) reviewed a
wide range of bioactive alkaloids that are produced by endophytic fungi.
Lozano-Tovar et al. (2013,2017) reported that B. bassiana produce sec-
ondary metabolites that can induce antifungal activity. B. bassiana pro-
duces several biological active metabolites of the class of alkaloids such
as tennelin, bassianin, pyridovericin, and pyridomacrolidin (Patocka,
2016). In the current study, since the specific alkaloid compounds were
not detected, it is not possible to establish with certainty whether the
higher total alkaloid content detected in the fungus-treated plants in this
study was due to the direct production of alkaloids by B. bassiana or the
fungus physiologically influenced the plant cells to produce more alka-
loids. It is worth mentioning that fungal endophytes can produce my-
cotoxins in their host that are potentially harmful to livestock and
humans (Azevedo et al., 2000).
In conclusion, this study demonstrated that endophytic entomopa-
thogenic fungi could be used to improve the yield of alkaloids in me-
dicinal plants. In order to further understand the influence of fungal
endophytes on plant production of bioactive compounds, future studies
involving detailed phytochemical elucidation of the bioactive constitu-
ents of fungus-treated plants are warranted.
Declarations
Author contribution statement
Friederike Espinoza: Conceived and designed the experiments; Per-
formed the experiments; Wrote the paper.
Stefan Vidal, Francis Lewu: Analyzed and interpreted the data; Wrote
the paper.
Fanie Rautenbach: Performed the experiments; Analyzed and inter-
preted the data.
Felix Nchu: Conceived and designed the experiments; Contributed
reagents, materials, analysis tools or data; Wrote the paper.
Funding statement
This work was supported by the exchange scholarship INSPIRE (In-
ternational Science Promoting Innovation and entrepreneurship) which
is a project of the Erasmus Mundus Action 2 program, awarded to Miss
Friederike Espinoza. Research was funded through Prof Nchu’s CPUT
University Research Fund grant (R166).
Competing interest statement
The authors declare no conflict of interest.
Additional information
No additional information is available for this paper.
Acknowledgements
This study was part of a collaborative student exchange project be-
tween Georg-August University Goettingen, Germany and Cape Penin-
sula University, South Africa.
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