A study on the effectiveness of (+)-usnic acid as oral toxic sugar bait against adult male and female Anopheles gambiae

Abstract

Background
Despite the application of various tools for the control of vectors of Plasmodium falciparum, malaria remains the major killer disease in sub-Saharan Africa accounting for up to 90% of deaths due to the disease. Due to limitations of the useage of chemical insecticides such as resistance, negative impact on the environment and to nontarget organisms, the World Health Organization (WHO) requires that affected countries find alternative vector control tools. This study evaluated the effectiveness of ( +)-usnic acid (UA) as an insecticide through oral administration to male and female Anopheles gambiae as an alternative or additional active ingredient to be used in toxic sugar bait.

Methods
( +)-usnic acid was diluted using acetone at 5, 10, and 15 mg/ml concentrations in three replicates. A 5 ml mixture of 2% food dye and 10% sugar using chlorine-free water mixed with the dilutions of the ( +)-usnic acid and negative control was made containing 2% food dye and 10% sugar solution. The preparations were soaked on a ball of cotton wool and placed over the net of a cup. 5 male and 5 non-blood-fed female newly hatched starved An. gambiae Kisumu strain were introduced together into a cup and monitored for knockdown and mortalities after 4, 24 48, and 72 h. The data were analysed using a multiple linear regression model using the lm function, a base R function and a posthoc test were conducted on the significant main effects and interaction terms using the emmeans function from the emmeans R package. All analyses were performed in RStudio using base R (version 4.3.3).

Results
There was high mortality of both male and female An. gambiae after ingestion of the toxic sugar bait. 15 mg/ml usnic acid caused the highest mortality (50%) within the first 4 h compared to 5 and 10 mg/ml ( +)-UA. There was a decline in the mortality rate with increased exposure time from 24 to 72 h, however, there was a significant difference in mortality at 5, 10 and 15 mg/ml. Acute toxicity was associated with ingestion of 15 mg/ml after 24 h. 72 h post-mortality was lower in all concentrations than in the control. High mortality was observed among females over the first 4 h (60%) compared to males (40%) due to higher feeding rate of the toxic agent. The proportion of dead males and females was equal after 24 h while after 48 h, the proportion of dead males was high.There was a significantly lower mortality rate after 72 h for both males and females (0 to 13.3%). Compared to all the treatments, high mortality of males was observed.

Conclusions
The results of this study indicate that ( +)-UA when administered as oral sugar bait to An. gambiae has insecticidal properties and is a suitable ingredient to be used as a toxic agent in the novel attractive toxic sugar bait for the control of malaria vectors. ( +)-UA may be an alternative active ingredient as toxic bait in the effort to reduce and eliminate the transmission of Plasmodium falciparum in Africa.

Full text

Muhoroetal. Malaria Journal (2024) 23:311
https://doi.org/10.1186/s12936-024-05141-4
RESEARCH
A study ontheeectiveness of(+)-usnic
acid asoral toxic sugar bait againstadult male
andfemale Anopheles gambiae
Arthur Macharia Muhoro1,2*, Eric Odhiambo Ochomo2, Isaac Njangiru Kinyua3, Jackline Jeruto Kosgei2,
Laide Abbas Rasaki4 and Edit Farkas5
Abstract
Background Despite the application of various tools for the control of vectors of Plasmodium falciparum, malaria
remains the major killer disease in sub-Saharan Africa accounting for up to 90% of deaths due to the disease.
Due to limitations of the useage of chemical insecticides such as resistance, negative impact on the environment
and to nontarget organisms, the World Health Organization (WHO) requires that affected countries find alternative
vector control tools. This study evaluated the effectiveness of ( +)-usnic acid (UA) as an insecticide through oral admin-
istration to male and female Anopheles gambiae as an alternative or additional active ingredient to be used in toxic
sugar bait.
Methods ( +)-usnic acid was diluted using acetone at 5, 10, and 15 mg/ml concentrations in three replicates. A 5 ml
mixture of 2% food dye and 10% sugar using chlorine-free water mixed with the dilutions of the ( +)-usnic acid
and negative control was made containing 2% food dye and 10% sugar solution. The preparations were soaked
on a ball of cotton wool and placed over the net of a cup. 5 male and 5 non-blood-fed female newly hatched starved
An. gambiae Kisumu strain were introduced together into a cup and monitored for knockdown and mortalities after 4,
24 48, and 72 h. The data were analysed using a multiple linear regression model using the lm function, a base R func-
tion and a posthoc test were conducted on the significant main effects and interaction terms using the emmeans
function from the emmeans R package. All analyses were performed in RStudio using base R (version 4.3.3).
Results There was high mortality of both male and female An. gambiae after ingestion of the toxic sugar bait. 15 mg/
ml usnic acid caused the highest mortality (50%) within the first 4 h compared to 5 and 10 mg/ml ( +)-UA. There
was a decline in the mortality rate with increased exposure time from 24 to 72 h, however, there was a significant
difference in mortality at 5, 10 and 15 mg/ml. Acute toxicity was associated with ingestion of 15 mg/ml after 24 h.
72 h post-mortality was lower in all concentrations than in the control. High mortality was observed among females
over the first 4 h (60%) compared to males (40%) due to higher feeding rate of the toxic agent. The proportion of dead
males and females was equal after 24 h while after 48 h, the proportion of dead males was high.There was a signifi-
cantly lower mortality rate after 72 h for both males and females (0 to 13.3%). Compared to all the treatments, high
mortality of males was observed.
Conclusions The results of this study indicate that ( +)-UA when administered as oral sugar bait to An. gambiae
has insecticidal properties and is a suitable ingredient to be used as a toxic agent in the novel attractive toxic sugar
Open Access
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Malaria Journal
*Correspondence:
Arthur Macharia Muhoro
arthmacharia@yahoo.com
Full list of author information is available at the end of the article
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Muhoroetal. Malaria Journal (2024) 23:311
Background
Malaria is still a burden in African regions and accounts
for 95% of malaria-related deaths (580,000 annually)
where approximately 80% are children. Approximately
249 million cases of malaria were reported in the world in
2022 in 85 countries [1]. Anopheles gambiae is the main
vector of Plasmodium falciparum that causes malaria in
sub-Saharan Africa due to its high abundance and lon-
gevity, strong preference to feed on human blood and
high vectorial capacity [2–5].
e most effective methods used to control the spread
of malaria are to prevent mosquito bites or reduce their
population [6]. To control mosquitoes, methods such as
reducing or eliminating their breeding sites, using natural
predators, genetically modifying mosquitoes and apply-
ing pathogenic microorganisms such as entomopath-
ogenic fungi (e.g., Lagenidium, Coelomomyces and
Culicinomyces) or bacteria (e.g., Bacillus thuringiensis)
have been used [7]. Despite the continuous application
of these methods, the challenge to eliminate malaria
through vector control still exists due to resistance of the
target mosquitoes to the chemical agents, human and
mosquito behaviour factors and climate change [8–14].
e possibility of transmission of infectious agents by
the vector between humans and non-human hosts also
underlines the importance of eliminating malaria. e
spread of other species of mosquitoes to other new geo-
graphical areas, such as Anopheles stephensi in Africa,
can be a major challenge to control malaria [15, 16].
However, some lessons can be learned from countries
that have eliminated malaria in sub-Saharan Africa to
attain a malaria-free continent status [17].
erefore, there is a need for new mosquito control
strategies to overcome the limitations of the existing
vector control methods and other indirect factors that
hinder the expected goal of eliminating malaria. Novel
mosquito control methods are needed to address the
limitations of current intervention strategies in elimi-
nating malaria [18, 19]. One such potential method
is the Attractive Toxic Sugar Bait (ATSB), which uses
sugar bait to lure and kill insects, a method with origins
that dates back to 77 CE [20]. Toxic sugar baits (TSB)
with arsenic and boric acid as a toxic agent was used to
kill termites and mosquitoes, respectively [21, 22]. e
concept has been used successfully to control mosqui-
toes and other insects of medical importance based on
the sugar-seeking natural behaviour observed among
mosquitoes sandflies and blackflies [23–27]. However,
arsenic is an exceptionally toxic element that causes
serious health problems to humans in case of long-term
application and accumulation similarly to other heavy
metals [28].
Both males and females require sugar as the main
source of energy, however, males exclusively feed on
sugar and the females occasionally seek sugar dur-
ing their lifetime and only seek blood from animals
to obtain protein for egg production [29]. Anopheles
gambiae naturally feeds on preferred sugar sources of
glucose, fructose and gulose which contributes to its
fitness and survival for both males and females. Simi-
lar results on preference for types of sugar have been
observed in a baseline study in Kenya on the sugar-
feeding patterns in mosquitoes [30, 31].
e concept behind toxic sugar bait is to lure both
male and female mosquitoes that exhibits a natural
feeding behaviour of sugar. Toxic agents are added to
the sugar, and when mosquitoes consume them, they
are poisoned. Boric acid, dinotefuran and ivermectin
are among the common synthetic insecticide chemi-
cals used in sugar bait which have shown promising
results in killing mosquitoes [32–37]. Although the
chemicals used have been demonstrated to be potential
oral insecticides, they have a limitation of being toxic
to the environment and non-target organisms espe-
cially when used outdoor [38]. Non-toxic plant and
microbial-based products such as microencapsulated
garlic oil, eugenol, spinsyns, erythritol, B. thuringiensis
var. israelensis (Bti) and sodium ascorbate have been
used as toxic agents in sugar bait and shown to be safe
[39–43]. e use of plant-based toxic agents has been
emphasized by Rezende etal. [38].
Lichens are composed of one or more photosynthetic
partners, fungi and other indeterminate numbers of
microscopic organisms that live together [44]. However,
this definition is limited since it is based on limited
knowledge about the role of other microorganisms on
the lichen [45]. ey are known to produce over 1000
unique secondary metabolites. e amount of usnic
acid produced by lichens varies and factors such as the
algal partner and season have been shown to have a sig-
nificant relationship [46, 47]. ese metabolites exhibit
various biological activities that include insecticidal
bait for the control of malaria vectors. ( +)-UA may be an alternative active ingredient as toxic bait in the effort
to reduce and eliminate the transmission of Plasmodium falciparum in Africa.
Keywords Mosquitoes, Vector, Lichen secondary metabolites, Bioactive substances, Usnic acid, Enantiomer, Kenya,
East Africa
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Muhoroetal. Malaria Journal (2024) 23:311
properties [48–51]. Various lichen secondary metabo-
lites have been used against insects’ larval and adult
stages and shown to be potential insecticide [52].
Usnic acid exhibits biological properties and it is a
potential candidate to be explored as a potential insecti-
cide to be used as an oral insecticide in a toxic sugar bait
(TSB) [53–56]. Usnic acid (UA) is a chiral molecule and it
is known to occur in two forms in nature: ( +)-usnic acid
and (−)-usnic acid, the two isomers may exhibit differ-
ent biological activities, hence their efficacy as insecticide
need to be further investigated and determined [57, 58].
ere is limited knowledge and studies on the poten-
tial of ( +)-UA as an oral insecticide against An. gambiae,
therefore, this study aimed to determine the effectiveness
of different concentrations of ( +)-UA on both male and
female adult stages of An. gambiae by performing labora-
tory bioassay experiments for various lengths of time and
concentrations.
Methods
is study aimed to determine the killing ability of the
3 concentrations of ( +)-usnic acid (5, 10 and 15mg/ml)
mixed with 10% sugar solution and 2% food dye as an
oral insecticide. Insecticidal property was determined by
measuring the knockdown effect of male and female An.
gambiae at 4, 24, 48, and 72h.
Mosquitoes
e mosquito, Anopheles gambiae Kisumu strain, used
in this study was reared at Kenya Medical Research Insti-
tute Insectary (KEMRI). Its susceptibility to pyrethroids
is known and has been used for bioassay studies as a con-
trol in insecticide resistance and other bioassay studies
[59].
e conditions of the insectary were maintained at
270C and relative humidity of 78% ± 10, and 12:12 L/D.
Adult females were fed on bovine blood by use of a mem-
brane feeding machine. Males and females were fed on a
10% sugar solution adlibitum.
( +)‑usnic acid
e active compound used in the experiments in this
study was ( +)-usnic (IUPAC name: 2,6-diacetyl-7,9-dihy-
droxy-8,9b-dimethyl-1,3(2H,9bH)- dibenzo-furandione
(Fig.1). It is more soluble in acetone than in water [60].
It is a natural compound produced by various taxa of
lichens that belong e.g., to the genera Cladonia, Usnea,
Lecanora, Ramalina, Parmelia and Evernia that are
widely distributed all over the world [54, 61–63]. e
( +)-usnic acid was supplied from Phytolab where purity
was certified [64].
Preparation oftheusnic acid bait insugar solutions
A stock solution of usnic acid was prepared by dissolv-
ing 500mg of usnic acid powder in 10ml of acetone and
labelled as 50mg/ml usnic acid (UA). is was diluted to
5mg/ml, 10mg/ml, and 15mg/ml ( +)-usnic acid solu-
tion in acetone and for each of the concentrations, 10%
sugar solution and 2% food dye solution were added. e
negative control was prepared by mixing 10% sugar solu-
tion and 2% food dye.
Bioassay todetermine susceptibility
Bioassay experiments were performed according to Allan
et al. [65] and Stewart et al. [66] with a slight modifi-
cation that did not affect the outcome of the results.
Newly hatched 5 males and 5 non-blood-fed females
that were starved before the experiment (only water was
provided) were aspirated using an aspirator and gen-
tly blown together in paper coffee cups. e opening of
the cups was secured with an insecticide-free net. ey
were allowed to acclimatize for one hour due to the shock
caused by aspiration and being in the new environment.
Each coffee cup was labelled according to the concentra-
tion of the ( +)-UA and a negative control.
Each concentration of the usnic acid was introduced on
a ball of cotton wool and placed on top of the net of each
cup. e temperature and humidity of the bioassay room
were recorded at the time of the start of the experiment.
e knockdown effect was observed after 4, 24, 48, and
72h. e knocked down and dead or moribund mosqui-
toes were aspirated out and their sex was determined.
eir abdominal status was also determined by observa-
tion using a light microscope. A coloured and extended
abdomen was used as a basis to confirm that the toxic
agent was ingested.
The data wasanalysed using R program
e data was analysed using a multiple linear regres-
sion model approach that included main effects for
Fig. 1 Molecular structure of ( +)-usnic acid
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Muhoroetal. Malaria Journal (2024) 23:311
Concentration (Conc.), Sex, and Time, as well as interac-
tion terms of Conc.*Time, Conc.*Sex, and Time*Sex. A
Posthoc test was then conducted on the significant main
effects and interaction terms using the emmeans func-
tion from the emmeans R package. All analyses were per-
formed in RStudio using base R (version 4.3.3).
The specic code forthemodel used was
Model <—lm(TotalDeaths ~ Conc + Time + Sex + Conc*Ti
me + Conc*Sex + Time*Sex, group_data).
Code forthePosthoc analysis
lsmeans < -emmeans(model, ~ Conc)
lsmeans_df <—as.data.f rame(lsmeans)
lsmeansT < -emmeans(model, ~ Time)
lsmeansT_df <—as.data.frame(lsmeansT)
lsmeansCT < -emmeans(model, ~ Conc:Time)
lsmeansCT_df <—as.data .frame(lsmeansCT)
lsmeans_df; lsmeansT_df; lsmeansCT_df
Results
e total mortality of both male and female mosqui-
toes as a percentage was high after ingestion of the
( +)-UA toxic sugar bait (TSB). Higher mortality (50%)
was observed at 15mg/ml compared to 5 and 10mg/ml
within the first 4h, however, the mortality rate declined
over the next 24, 48 and 72h (Fig.2). is indicates that
the extension of the exposure time has no significant
difference in the mortality but there is a considerable
difference when mortality was compared to the concen-
tration of 5,10, and 15mg/ml. Despite the decrease in
mortality after 4h of exposure, lower concentrations (5
and 10mg/ml) caused mortality higher than 15mg/ml
after 24h, this indicates that 15mg/ml ( +)-UA caused
higher acute toxicity. At 10mg/ml, mortality was lower
(10%) for the first 4 h and sustained at the same rate
(36.7%) for the next 24 and 48h (Fig.2). In all the treat-
ments, increased higher mortalities were observed for
the first 4, 24 and 48h. After 72h, lower mortalities were
observed compared to the control and the treatments.
To determine the individual mortalities of both male
and female target mosquitoes, the mortality percentage
was considered between 4 to 72 h. Higher mortality of
females was observed at 4h (60%) while the males were
40% (Fig.3). is indicates a higher proportion of intake
of ( +)-UA toxic sugar bait for both males and females.
Also, both males and females exhibited a considerable
susceptibility to the ( +)-UA poison that was in the sugar
as a result of a higher feeding rate. ere was a high
mortality rate at 5mg/ml and 10mg/ml for the first 4h
with equal proportions of death rate for both males and
females. After 24h, there were equal mortality rates at
5mg/ml and 10mg/ml for both males and females and
a relatively higher death rate at 10mg/ml for males after
48h. After 72 h, there were significantly lower mortal-
ity rates for both males and females (0 to 13.3%). Higher
Fig. 2 Post-exposure mortalities (%) of Anopheles gambiae after 4, 24, 48, and 72 h in different concentrations of ( +)-UA
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Muhoroetal. Malaria Journal (2024) 23:311
mortality of males was observed after 48h compared to
females in all three treatments (Fig.3).
Discussion
e sugar-seeking behaviour of mosquitoes has been
demonstrated in many studies including recovery of spe-
cific types of sugar from plant sources and specific plant
types[30, 31, 67]. is natural behaviour observed among
male and female mosquitoes can be utilized as a point
of vulnerability for their control by adding poison to the
sugar bait. e success of this ATSB has been shown to
reduce the mosquito population, biting rates, vecto-
rial capacity and malaria prevalence. In Mali, there was
approximately a 57% reduction in mosquito catch, an
89% reduction in entomological inoculation and a 30%
reduction in malaria prevalence based on epidemiologi-
cal modelling prediction on the ATSBs [25, 26, 33, 34, 68,
69].
In this study susceptibility of An. gambiae after oral
administration of ( +)-UA was observed at different con-
centrations, there was high mortality among female mos-
quitoes the first 4h (60%) (Fig.3), although intake of the
sugar bait was also high among males(40%). is indi-
cates that both males and females readily took the sugar
bait, hence the toxic agent is suitable as an ingredient
of the TSBs against newly hatched male and female An.
gambiae mosquitoes investigated in this study. When the
total mortalities for male and female mosquitoes were
evaluated, 50% mortality was observed after ingestion
of 15mg/ml ( +)-UA toxic sugar bait after the 4h. Com-
pared with other studies, 4% boric acid resulted in 100%
mortality of Aedes aegypti and An. stephensi within 24h
in laboratory conditions. Studies by Allan etal. however
confirmed a variation in susceptibility among males and
females where female Culex quinquefasciatus was less
susceptible [65, 70].
e application of deltamethrin as a toxic agent in
ATSB demonstrated a significant toxicity effect on Ae.
aegypti, with mortality rates ranging from 8.33% to
97.44% within 24h of exposure [71]. A study to evalu-
ate the effect of ATSB against Ae. aegypti by use of boric
acid indicates that there was no significant difference in
the proportion of uptake of the ATSB for both males and
females and the mortality decreased after 24h. However,
females engorged more than males [72].
When Bti was used as an active ingredient in ATSB,
mortality rates were higher after 48 h, 97% for Ae.
aegypti, 98% for Aedes albopictus, and 100% for Cx.
quinquefasciatus. is indicates that Bti has an optimum
mortality effect after 24h compared to synthetic insec-
ticides when used as an oral poison [43]. However, a
study to determine the toxicity of nano-formulated oral
Fig. 3 Mortality (%) of both males and females Anopheles. gambiae after oral ingestion of ( +)-UA
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Muhoroetal. Malaria Journal (2024) 23:311
sugar bait on An. gambiae indicates that the use of nano-
ATSB cypermethrin demonstrated a high and prolonged
efficacy after 72h of delayed mortality assessment [73].
erefore, it implies that the application of nanotechnol-
ogy using the various active ingredients that have insecti-
cidal potential as an insecticide in ATSB may provide the
desired prolonged efficacy and safety to the non-target
arthropods. Also exposure to field collected Ae. albopic-
tus to boric acid as ATSB for 48h and later determina-
tion of delayed mortality of 93.3–100.0% for up to 7days
resulted in total mortality of both males and females [74].
e use of naturally occurring sugar (erythritol) as a toxic
agent with or without another toxicant has been shown
to be effective in increasing the mortality of adult Ae.
aegypti by 90% within 72h when mixed with other toxic
agents like boric acid, Bti andspinosyn [41].
is study confirmed that the 10% sugar was capable of
inducing feeding and sustaining uptake of the toxic agent
as observed in the control experiments. is has also
been confirmed in other studies to determine the impact
of sugar concentrations on engorgement to the target
mosquitoes where mosquitoes were more engorged
regardless of the sugar concentration between 10 to 70%.
However, high concentrations of sugar resulted in a high
number of engorged Ae. aegypti [75].
is study also confirms that in the event that mos-
quitoes are knocked down faster, within the first 4 to
72h, it would have the advantage of killing the mosqui-
toes before they can transmit the infectious agent. is
claim agrees with a similar study where bacterial second-
ary metabolites (spinosyns) have been used as toxicants
in TSBs. e impact has been shown to reduce vectorial
capacity when the mean knockdown time of mosquitoes
is lower [42].
Based on the promising results of the current new
mosquito control method (ATSB), this study aimed at
exploring the potential of ( +)-UA as oral toxic sugar bait
to kill both male and female An. gambiae. It provides the
knowledge that increasing concentrations of ( +)-UA
acid have the potential to kill target mosquitoes from 4
to 72h post-exposure and the degree of susceptibility of
both male and female mosquitoes under laboratory con-
ditions. e susceptibility of laboratory-reared An. gam-
biae to ( +)-UA as an oral insecticide via sugar bait has
not been evaluated. is study demonstrated its promis-
ing potential as an effective oral insecticide.
Limitations
e experiments in this study were conducted under
laboratory conditions, thus it did not use field-collected
or resistant mosquitoes for bioassay experiments. Fur-
thermore, the effects of ( +)-UA on non-target organisms
were not determined. No further mortality recordings
were performed after 72h of exposure. No other sources
of meal were provided to determine feeding preferences
among male and female mosquitoes.
Conclusions
Laboratory experiment on the susceptibility of An. gam-
biae to 5, 10 and 15mg/ml ( +)-UA when administered as
oral sugar bait has first been demonstrated by this study.
Mortality for both males and females is higher in the
first 4h of exposure and continues up to 72h. erefore,
this study has confirmed that ( +)- UA can be used as an
ingredient in the novel attractive toxic sugar bait for the
control of malaria vectors in the current effort to search
for new tools to reduce malaria transmission in African
countries.
Abbreviations
ATSBs Attractive toxic sugar baits
Bti Bacillus thuringiensis Var. israelensis
IUPAC International Union of Pure and Applied Chemistry
SERU Scientific and Ethics Review Unit
TSBs Toxic sugar Bait
UA Usnic acid
WHO World Health Organization
NACOSTI National Commission for Science, Technology and Innovation
Acknowledgements
The authors wish to thank Johnson Onguka for insectary maintenance, and
Joan Wanjiku and Diana Wagura for technical help. Abigael Onyango for
quality control, and Polo Brian for corresponding with the ethics review team.
National Commission for Science, Technology and Innovation for the permis-
sion to carry out this study.
Author contributions
The study protocol and laboratory bioassay procedures experiments were
designed by A.M. who wrote the manuscript, E.O. supervised the experiments
and guidance in drafting the study protocol, E.F. initiated the research con-
cept, supervised the research and revised the manuscript, I.N. guided in the
analytical work of the active ingredients, L.R. analysed the data. J.J. guided the
bioassay experiments. All authors read and approved the final manuscript.
Funding
Laboratory work, purchase of ( +)-usnic acid and the cost of travelling were
funded by Stipendium Hungaricum Scholarship (2020–2024) support. The
procurement of reagents was funded by the National Research Development
and Innovation Fund, grant number NKFI K 124341.
Availability of data and materials
No datasets were generated or analysed during the current study.
Declarations
Ethics approval and consent to participate
This study was approved by the scientific and ethics review unit of KEMRI
protocol number SERU04-06–423/4610.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1 Doctoral School of Biological Sciences, Hungarian University of Agriculture
and Life Sciences, Páter K. U. 1, 2100 Gödöllő, Hungary. 2 Kenya M edical
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Muhoroetal. Malaria Journal (2024) 23:311
Research Institute-Centre for Global Health Research (KEMRI-CGHR), P.O.
Box 1578, Kisumu 40100, Kenya. 3 Institute of Pharmacodynamics and Biophar-
macy, University of Szeged, Eötvös U. 6, 6720 Szeged, Hungary. 4 Department
of Crop Sciences, North Carolina State University, Raleigh, NC 27695, USA.
5 HUN-REN Centre for Ecological Research, Institute of Ecology and Botany,
Alkotmány u. 2–4, 2163 Vácrátót, Hungary.
Received: 25 June 2024 Accepted: 11 October 2024
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