2023.08.04.41
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Isolation and Identification of Fungal Species from the Insect Pest Callosobruchus maculatus (F.)
oor
Akmoosh 1*, Ekhlas
Al-Shareefi2, Kawther Mohammed Ali3
1 University of Babylon / Babylon/ Iraq; [email protected]; +9647818198773.
2 University of Babylon / Babylon/ Iraq; [email protected]; +9647822114389
3 University of Babylon / Babylon/ Iraq; [email protected]; +9647800351337
* Correspondence: [email protected]; Tel.: (+9647822114389)
ABSTRACT
Cowpea seed beetle Callosobruchus
maculatus (F.) (Coleoptera: Bruchidae) is one of the most common and
economically important pests of stored cereal products worldwide. Furthermore,
these beetles can act as vectors for several fungal post-harvest diseases. The
current research aimed to isolate the fungi associated with adults of C.
maculatus (F.) (Coleoptera: Bruchidae). The present study aimed to isolate
and identify fungi associated with adult insects and evaluate their potential
as biocontrol agents against the cowpea beetle, C. maculatus. In this study, we collected C.
maculatus adults from the Babylon local market and five regions (Hilah,
Mahaweel, Al-Mashrou', Al-Haswa and Al-Musayyab) with no history of insecticide
exposure. A potato dextrose agar medium was used to isolate the fungi attached
to the surfaces of adult beetles. By Morphological and microscopic
examination, Molecular identification
and Sequencing analysis, Twenty-seven genera of fungi were isolated and
identified from adult C. maculatus, including Aspergillus, Penicillium,
Candida spp, white mycelium, Curvularia, Cladosporium,
Chaetomium, Stachybotrys, Rhizopus, Drechslera, Scopulariopsis,
Paecilomyces, Mucor, Geotrichum candidum, Streptomyces,
Sarocladium, Beauveria bassiana, Talaromyces varians,
Sporothrix flocculasa, Pseudozyma flocculasa, and Isaria
fumosorosea. The molecular weights of the PCR
products for the fungi isolates ranged from 650-700 bp, and the amplified
ITS1-5.8S-ITS4 rDNA region of indigenous entomopathogenic fungal isolates
showed a high sequence similarity (99-100%) with B. bassiana and M.
anisopliae deposited in the NCBIGenebank. The phylogenetic tree analysis of
the ITS region sequences showed a high degree of similarity between the
isolates under study, ranging from 96.5-100. However, there were differences in
the lines among clades, indicating genetic variation possibly due to mating or
mutations in different environments. Our study suggests that the storage pest, C.
maculatus, would be essential in spreading fungal contaminants and
consequently increasing mycotoxin contamination in stored Cowpea.
Keywords: Callosobruchus maculatus,
Isolation, Fungi
INTRODUCTION
Controlling pests in stored grains is as economically important as
increasing the crop yield because, unlike crop damage during the growing
season, post-harvest damage of stored grains is not financially compensated.
Fungi and animal pests are the primary culprits for damage to stored grains,
globally estimated to be responsible for 20% of food losses and up to 40-50% in
some developing countries 23. Cowpea seed beetle C. maculatus (F.) (Coleoptera: Bruchidae) is one
of the most destructive pests on Cowpea and other legumes growing in tropical
and sub-tropical countries, both in fresh green crusts in fields and in stored
seeds 21. The adults are not harmful. But, the larvae of this pest
feed on Cowpea V. unguiculata (L.), chickpea, Cicer arietinum L., lentil,
Lens culinaris (Medik.), soybean, Glycine max (Mer.), and haricot beans,
Phaseolus vulgaris (L.) 17. The female adults of C. maculatus lay
their eggs in the fresh cowpeas before reaping in the field. The larvae,
hatched from these eggs, bore into the cowpea seeds, developed by feeding the
embryo of the sources and matured just about a month in storage conditions 8.
Therefore, the larvae can lead to both quantitative, due to grain weight loss
caused by larvae feeding, and qualitative, due to product alterations such as
loss of nutritious and aesthetic values, which increases the loss level in the
cowpea grain mass 1, 19. Therefore, this pest requires
great care due to the potential for severe damage.
Various researchers have isolated fungal species from insects. Burnside 6
isolated A. flavus, A. niger and P. corylophilum from
bees. Gillian and Prest 10 and Gillian et al. 11 also
isolated various fungi from bees, identifying the species A. niger, A.
flavus, P. corylophilum, C. cladosporoides and Alternaria
sp.
Recently, Rehner & Buckley 20 used two DNA regions, rDNA
ITS-5.8s and elongation factor EF1-α, and compared 86 isolates of Beauveria
from around the world and from several hosts. In addition to the original
isolate from Scottish soil, they found a Swiss isolate labeled B. bassiana
(ARSEF 1567) from a scolytid beetle grouped in B. caledonica. Similarly,
an isolate classified as B. amorpha (ARSEF 2251), also grouped in
B. caledonica was originally from a coleopteran insect in Brazil.
This suggests that B. caledonica has the potential to be an insect
pathogen 12.
Samples Locations
Cowpea samples infested with cowpea seed beetle C. maculatus (F.)
were collected from the local market in Babylon and for five regions (Hilah,
Mahaweel, Al-Mashrou', Al-Haswa and Al-Musayyab), with no history of exposure
to insecticides.
Ready-made
dextrose potato medium
According
to the manufacturer's instructions, this medium is prepared by suspending 39 gm
of the medium in 1000 ml of distilled water with 250 mg of chloramphenicol that
prevents bacteria growth and sterilizes by autoclave at 121°C and 15 lbs
pressure. Soak for 20 minutes; after the end of the sterilization period, leave
the beaker until it cools down to 50 °C, then pour the nutrient media into
Petri dishes with a diameter of 9 cm and put them in the refrigerator until
they are used for isolation, cultivation and reproduction of the fungi used in
study 24.
Isolation of fungi from adult insects
To isolate pathogenic fungi from
the insects, the insects are sterilized with ethyl alcohol (70%) to remove the
fungus on the external surface for one minute. Then they are washed with
distilled water and then sterilized with a solution of sodium hybochlorate
NaOCl (1%) for 30 second, then washed with distilled water and placed on filter
papers and then transferred by sterile forceps to the PDA medium at the rate of
three replications, where five insects were placed In each repetition. The
dishes were incubated at a temperature of 27
± 2 for a period of 5-7 days;
after that, the fungal isolates were purified on a new nutrient medium by
taking a 0.3 cm diameter disc from the edge of the fungal colonies and
transferred this disc by the sterilized needle to the center of a plastic petri
dish containing 20 mm of PDA medium, and the plates were incubated at a
temperature of 27 ± 2 for 5-7 days. After incubation and
identification, the percentages of frequency and appearance of isolated fungi
were calculated according to the following equation: -
Percentages of frequency = Number
of isolates per species/ Total Number of isolates of all species * 100
Percentages of appearance = Number
of formations in each species of all samples/ Total Number of samples * 100
Morphological and microscopic examination
After appearance growth and
examining colonies of fungi concerning color, shape and texture (Powdery,
Granular, Cottony) as recorded, pigments are discussed on the colony surface
appearance on the foundation. Fungi isolates are examined microscopically, and the
fingerprint of the fungi in the colony is taken by adhesive tape; transparent
adhesive tape is used, touches the surface of the fungal colonies and then the
tape on a glass slide containing a drop of lactophenol cotton blue. Slides
examined under magnification 10X, 40X and 100X as described 30.
Molecular
identification
Fungal genomic DNA was extracted
from mycelia by using a Favorgen kit. The internal transcribed spacer with 5.8
s rDNA was amplified using ITS5/ITS4 universal primer for fungal isolates was
used. The PCR mixture was prepared according to table (1) and augmented on the
current System of cycler PCR (Labnet, USA) by conditions in table (2).0; (Pitt
and Hocking, 2013; Rai, 2016). The products of PCR were run on 1.5% agarose gel,
and electrophoresis was made at 70 V for 30 min. The gel was pre-stained with
0.05% ethidium bromide. The PCR bands were noticed by using an ultraviolet
transilluminator.
Table 1. PCR mixture
Table 2. PCR conditions
Sequencing analysis
Ten isolates of fungal species from
this study PCR products are subjected to sequencing analysis. Direct sequencing
analysis was performed on the 20 μl PCR product of AFU5S primer, sent to the Macrogen
Laboratory in Korea. Afterward, the DNA sequencing data for different fungi
isolates are compared with the gene bank using the NCBI Blast nucleotide
database.
Phylogenetic tree
The phylogenetic tree is analyzed
by using Mega version 6 software program with an unweighted pair group method
with arithmetic mean (UPGMA) tree type based on sequences data of fungal
species amplified by ITS5/ITS4 primer for 10 isolates.
RESULTS
Isolation of fungi from adult insects
The results
indicate (table 3) that the highest occurrence of isolated fungi from C.
maculatus was observed in the fungus A. niger, with a rate of
46.05%, followed by Penicillium sp. and A. flavus, which had
occurrence rates of 12.91% and 12.33%, respectively. On the other hand, Aspergillus
nidulans and Pseudozyma flocculasa had the lowest occurrence rates, at
0.15% and 0.16%, respectively, among the other isolated fungi. The remaining
fungi showed varying occurrence rates ranging from 0.22% to 5.16%.
The results also
showed that the highest percentage of occurrence was found in the fungi
isolated from C. maculatus males, specifically those isolated from the
Al-Musayyib region (3.72%), followed by the Al-Hilla and Al-Mahawil regions,
where the fungal presence was recorded at 3.70% in each. At the same time, the
fungi isolated from C. maculatus female showed an occurrence rate of
3.69% among all insect samples collected from the study locations (Al-Musayyib,
Al-Hassoah, Al-Mishrak, Al-Mahawil, and Al-Hilla). Among the fungi isolated
from C. maculatus in this study, two fungi, namely B. bassiana
and I. fumosorosea, were selected as biological agents for conducting
further experiments.
NO.
Table 3. Percentages for the emergence of
fungal species in males & females of C. maculatus adults.
Molecular
diagnosis of Fungi
DNA extraction and
PCR assay
Ten isolates of
fungi species in this study were subjected to DNA extraction. The ITS5 and ITS4
are universal primer pairs that targeted the sequences place of the
ITS1-5.8S-ITS2 gene of the fungi isolates, which was used to discriminate fungi
to the species level. The molecular weights of the PCR products for the fungi
isolates under study ranged from 650-700 bp., as there are apparent differences
in the molecular weight of fungi species when ITS5 and ITS4 primers were used.
Fig shows agarose gel electrophoresis of PCR products for fungi species.
Figure 1. Agarose gel electrophoresis of PCR products for
ITS1-5.8S-ITS2 gene of fungi species. Lane M= molecular
marker 1500 bp.
Sequencing
analysis
PCR was done for
ten fungi isolates for the ITS region; direct sequencing analysis was performed
on the 20 µl PCR product of the ITS region, which was sent to the Macrogen
Laboratory in Korea. After obtaining the sequence of the nitrogenous bases of
the sent isolates, they are matched with the series of reference samples in the
gene bank using the NCBI Blast Nucleotide Database to confirm the highest
proportion of the genus and species name for each isolate.
Table (4) shows the
Results of molecular diagnosis for fungi isolates under study comparison with
reference strains in NCBT by Telomorphe name, and the Anamorphe name offsets it.
The table showed that the molecular diagnosis using the nitrogenous bases
sequence of some samples was identical to the phenotypic diagnosis using
traditional laboratory methods except for several isolates; some have not been
diagnosed with conventional species-level approaches, which are analyzed by
molecular methods.
Table 4. Results of molecular diagnosis for fungi
isolates under study comparison with reference strains in NCBT.
Phylogenetic tree
The phylogenetic
tree was analyzed by using Mega 6 software program with an unweighted pair
group method with arithmetic mean (UPGMA) tree type based on sequences data of
ITS region amplified by ITS5/ITS4 primers pair for 9 isolates. The results of
phylogenetic tree analysis for entomopathogenic fungi were observed in five
groups. T. varians, B. bassiana, P. flocculosa, A.
flavus and Sarocladum sp. (Figure 2).
Figure 2. Phylogenetic tree based
on ITS region sequences for Fungi isolates understudies.
Drawing the
phylogenetic tree of the ITS region sequences for the isolates under study
shows a high degree of similarity between species, ranging from 96.5 to 100 %.
The difference in sequence among any clades offers approximately 0.0-3.5 %. The
weighted pair group method with arithmetic mean (UPGMA) tree type was a common
type of phylogenetic tree to determine the diversity of pathogenic fungi.
Therefore, many studies have used this type of tree.
DISCUSSION
Isolation of fungi from adult insects
Twenty-seven
genera of fungi were isolated and identified from adult C. maculatus,
including Aspergillus, Penicillium, Candida spp, white mycelium, Curvularia,
Cladosporium, Chaetomium, Stachybotrys, Rhizopus, Drechslera, Scopulariopsis,
Paecilomyces, Mucor, Geotrichum candidum, Streptomyces, Sarocladium, Beauveria
bassiana, Talaromyces varians, Sporothrix flocculasa, Pseudozyma flocculasa,
and Isaria fumosorosea.
Most of
them belonged to Aspergillus, from which seven species were isolated. The
reason for this is that Aspergillus possesses small reproductive units in large
numbers, allowing for long-distance dispersal and the ability to form specific
structures to resist unfavorable environmental conditions for its growth 25.
These findings are consistent with 26, who isolated several fungal
genera from the mosquito Gx. quinquefasciatus, including Aspergillus,
Fusarium, Penicillium, and Trichoderma. These results also agreed
with 23, who isolated several fungal genera from the Tribolium
castaneum, including Aspergillus spp., Cladosporium spp., Hyphopichia
burtonii, Penicillium spp., Mucor spp., Rhizopus spp., Cephaliophora spp.,
Alternaria alternate, Monascus sp., Fusarium, Nigrospora, Beauveria,
Chaetomium, Coprinellus, Irpex, Lichtheimia, Trichoderma, Byssochlamys,
Cochliobolus, Cunninghamella, Mortierella, Polyporales, Rhizomucor and Talaromyces
Among the
fungi isolated from C. maculatus insects, two fungi, Beauveria
bassiana and Isaria fumosorosea, were found. These fungi were
utilized as biological agents in this study for insect control, as they have
been used to combat various insects.
The selection of B. bassiana in the current study agreed with
Ozdemir et al.'s 27, who used B. bassiana in combating the
insect C. maculatus, additionally, I. fumosorosea was chosen to
control the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae),
which is consistent with the findings of the current study on biological
control.
Molecular
diagnosis of Fungi
The molecular weights of the PCR
products for the fungi isolates under study ranged from 650-700 bp. As there
are apparent differences in the molecular weight of fungi species by ITS5 and
ITS4 primers were used, this agrees with most references that used
ITS1-5.8S-ITS2 region of fungal rDNA by ITS5 and ITS4 primer 22.
While several references were used for
ITS1 and ITS4 primer for ITS1-5.8S-ITS2 region of fungal rDNA to identify,
entomopathogenic fungi [5,18,4] were isolated twenty-one entomopathogenic
fungi, the resulting from the electrophoresis of the PCR product using the
universal primer for the amplification of ITS rRNA gene of bands with a size of
650 bp. Also, Gebremariam et al.,9 used ITS1 and ITS4 primer
to identify Beauveria bassiana and Metarhizium anisopliae with a
band size of 545 bp. for two species. In another study 15, for the
first time, the primer binding site (PBS) marker system was used to
discriminate among the entomopathogenic fungi species successfully.
The PCR sequencing
of the ITS region of rRNA is currently regarded as the standard method for
phylogenetic analyses and identification of fungal species. It provides an
excellent tool for identifying fungi species that do not present typical
morphological characteristics 28,14.
Some studies were using sequence
analysis of ITS region by using ITS1 and ITS4 primers or ITS5 and ITS4 primers
for identification of entomopathogenic fungi such as Imoulan et al.,13
were isolated and identified several species of Beauveria. Species
identification using only the ITS region of
rDNA as a DNA barcode reached its resolution limit within Beauveria.
While Lu et al.,16 four entomopathogenic fungi strains were
isolated and identified as Lecanicillium attenuatum, Beauveria
bassiana, Lecanicillium longisporum, and Akanthmyces lecanii based
on rDNA-ITS sequence analysis. Also, Yang et al.,22 isolated
four genes of entomopathogenic fungi from soil samples collected from different
localities of southern China as Beauveria bassiana, Cordyceps
fumosorosea, Aspergillus nomius and Akanthmyces attenuatus.
Results of the phylogenetic tree
coincided with several recent studies that show a high degree of similarity and
homogeneity in the ITS region among entomopathogenic fungi. Al-Shindah et al.,29
isolated twenty-one different fungal isolates from samples of infected insects
from other areas in Salah El-Din Governorate in Iraq. They showed the similarity
percentage between the isolates of entomopathogenic fungi and their conformity
with the globally registered fungal species in NCBI. The similarity ratio
reached 97.51 - 99.80% with the globally registered strains, confirming the
diagnosis's accuracy. While Gebremariam et al.,9 used
sequences of ITS1-5.8S-ITS4 rDNA region of all indigenous entomopathogenic
fungal isolates showed 99–100% sequence similarity with B. bassiana and M.
anisopliae deposited in NCBI/Genebank.
Although there is
a high similarity to the fungi isolates in this study in matching the
nucleotide sequences, they are genetically different isolates because the match
was not 100%. This may be due to matings or mutations in fungi due to their
presence in different environments 2,3.
CONCLUSIONS
Twenty-seven genera of fungi were
isolated and identified from adult C. maculatus, including Aspergillus,
Penicillium, Candida spp, white mycelium, Curvularia,
Cladosporium, Chaetomium, Stachybotrys, Rhizopus, Drechslera,
Scopulariopsis, Paecilomyces, Mucor, Geotrichum candidum,
Streptomyces, Sarocladium, Beauveria bassiana, Talaromyces
varians, Sporothrix flocculasa, Pseudozyma flocculasa,
and Isaria fumosorosea
The molecular weights of the PCR
products for the fungi isolates ranged from 650-700 bp, and the amplified
ITS1-5.8S-ITS4 rDNA region of indigenous entomopathogenic fungal isolates
showed a high sequence similarity (99-100%) with B. bassiana and M.
anisopliae deposited in the NCBIGenebank. The phylogenetic tree analysis of
the ITS region sequences showed a high degree of similarity between the
isolates under study, ranging from 96.5-100. However, there were differences in
the lines among clades, indicating genetic variation possibly due to matings or
mutations in different environments
Two fungi, Beauveria bassiana and
Isaria fumosor osea, were selected as biological agents based on their
occurrence rates in male and female Callosobruchus maculatus adults.
Funding: This research received no external funding
Data Availability Statement: Data Availability Statements in the
"Bionatura Research Data Policies" section at https://www.revistabionatura.com/policies.html.
Acknowledgments: The authors would like to thank all the Advanced Mycology
Laboratory at the College of Science for Women / University of Babylon for
their collaboration.
Conflicts of Interest: The authors declare no conflict of interest.
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Received: 26 September 2023 / Accepted: 15 November 2023 /
Published:15 December 2023
Citation: Akmoosh N, Al-Shareefi E, Mohammed Ali K. Isolation and
Identification of Fungal Species from the Insect Pest Callosobruchus
maculatus (F.). Revis Bionatura 2023;8 (4) 41. http://dx.doi.org/10.21931/RB/2023.08.04.41
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