1
Journal of PhytoPathology and Disease Management
Print ISSN: 3009-6111 Online ISSN: 3009-6170
Volume 13, Issue 1, 2026, Article ID 18698633
Article
Evaluation of smut resistance in selected sugarcane genotypes and their
molecular characterization using SCoT, ISSR, and RAPD analysis
Sayed H. Agag1 | Abeer H. Abbas1
1Maize and Sugar Crops Diseases Research Department, Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt
DOI:
10.5281/zenodo.18698633
ARK:
ark:/24629/PPDJ.v13i1.269
Received:
12 December 2025
Accepted:
15 January 2026
Published online:
2 February 2026
Correspondence:
Abeer H. Abbas
Maize and Sugar Crops Diseases
Research Department, Plant Pathology
Research Institute, Agricultural
Research Center, Giza, Egypt.
Email: abeer_hamdy@ymail.com
This is an open-access article distributed
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Abstract:
Sporisorium scitamineum causes sugarcane smut, which is a global problem that
seriously reduces yield and quality. The disease is most effectively managed through the
use of resistant cultivars. Molecular markers can support breeding programs by helping
to identify resistant genotypes at early stages. In this study, three sugarcane varieties
G.2003-47 (G3), G.2004-27 (G4), and the commercial cultivar G.T.54-9 (C9) were
evaluated for whip smut response under artificial inoculation and characterized using
Start codon Targeted (SCoT), inter simple sequence repeats (ISSR), and random
amplified polymorphic DNA (RAPD) markers. Disease incidence was lowest in G.2004-
27 (4%), while G.2003-47 and G.T.54-9 showed higher infection levels (13.34% and
12.88%, respectively). SCoT primers generated 25 bands with 55.33% polymorphism,
whereas ISSR and RAPD produced 37 and 32 bands with polymorphism levels of 46.3%
and 49%, respectively. Several primers across the three marker systems distinguished the
relatively resistant variety G.2004-27 from the more susceptible genotypes. These
findings indicate that SCoT, ISSR, and RAPD markers can complement phenotypic
screening and assist in the preliminary identification of whip smut–resistant sugarcane
clones for further field evaluation. Consequently, the variety G.2004-27 is recommended
as a promising source of resistance for Egyptian sugarcane breeding programs.
Additionally, SCoT markers proved to be more informative than RAPD and ISSR in
discriminating among the tested genotypes.
Keywords:
Sugarcane, whip smut, Sporisorium scitamineum, molecular markers, SCoT, ISSR, RAPD.
Article | Agag and Abbas | Molecular characterization and smut resistance evaluation of sugarcane genotypes
2 | Journal of Plant Pathology and Disease Management | Vol. 13, No. 1 | Article ID 18698633
1. Introduction
Sugarcane (Saccharum spp.) is a major economic crop in
many countries, and its productivity is constrained by
numerous diseases, among which smut is one of the most
destructive (Bhuiyan et al., 2021; Rott et al., 2000). The
disease was first reported in Natal, South Africa, in 1877
(Braithwaite et al., 2004; Rott et al., 2000), and spread to
other sugarcane-growing areas in Central, East, and West
Africa, Indonesia, Central and South America, Brazil, and
Australia. Sugarcane smut is caused by the biotrophic
basidiomycete fungus Sporisorium scitamineum (synonym
Ustilago scitaminea) and can cause severe losses in
productivity and quality of cane (Comstock, 2000). In
Egypt, the area cultivated with sugarcane during the
2023/24 season was approximately 132,762 hectares
(equivalent to 316,099 feddans), producing about 642,166
tons of sugar, which accounted for nearly 29% of the
country’s total sugar production (Annual Report for Sugar
Crops in Egypt, 2024). The characteristic feature of this
disease is the appearance of a typical "whip"-like structure,
known as a sorus, from the aerial part of a plant, consisting
of a central vascular strand surrounded by massive colonies
of dark teliospores enveloped in a thin membranous
covering (Hoy et al., 1986). Smut propagules are primarily
dispersed by wind, with additional spread via smut-
infected planting material and agricultural equipment
(Croft and Braithwaite, 2006; Rott et al., 2000). Globally,
smut control depends mainly on growing resistant cultivars,
supported by other practices within an integrated disease
management (IDM) approach. Key IDM measures include
using resistant varieties, removing diseased plants,
planting disease-free seed-cane, and treating planting
material with fungicides in infested fields. Together, these
approaches have been proven effective in controlling smut
outbreaks and associated yield losses (Bhuiyan et al.,
2021). Recently, greenhouse experiments conducted in
Egypt have demonstrated the efficacy of chemical
fungicides in preventing sugarcane smut infection and
whip formation caused by S. scitamineum, highlighting
their potential role in managing this major sugarcane
pathogen (Osman et al., 2025). Resistant varieties are
generally considered the most efficient and cost-effective
methods in controlling sugarcane smut. Resistance to S.
scitamineum is complex and modulated by multiple host-
pathogen interactions. Early in resistance work, it was
found that resistance may be linked to morphological
characteristics of sugarcane buds, making infection
difficult, thereby confirming the importance of the host's
genetic makeup in resistance (Fawcett, 1946). However,
conventional resistance evaluation methods are time-
consuming, require long crop cycles, and are highly
dependent on environmental conditions (Bhuiyan et al.,
2021). After five years of selective breeding for resistant
parents, the percentage of susceptible seedlings in Hawaii
decreased significantly from 64% at the time of the smut
intrusion to 11% (Comstock et al., 1983). In Australia,
awareness of the importance of smut as a serious
biosecurity risk led to preventive screenings in Indonesia
and Western Australia prior to the 2006 outbreak along the
southeastern coast (Croft et al., 2008a). Initial screenings
showed that over 70% of commercial and breeding
seedlings were susceptible to the pathogen (Croft et al.,
2008b), and, as a result, the strategy changed to use
resistant parents. As a result, the percentage of biparental
crosses resistant to smut increased from 0.4% to 52% from
2000 to 2007, almost doubling the number of resistant
seedlings by 2011. Through this continuous selective
breeding program, there was an impressive decrease in the
percentage of seedlings that were susceptible to smut from
over 70% in 2004 to below 10% by 2019. Molecular
approaches also provide useful support to traditional
breeding by enabling discrimination among different
sugarcane varieties based on genotype. Marker-assisted
selection (MAS) has become useful in crop breeding
programs to accelerate the selection of favorable genotypes
without relying solely on phenotypic selection (Collard
and Mackill, 2009). In previous studies on sugarcane smut,
molecular marker techniques have been used effectively to
analyze genetic diversity and assist breeding efforts to
improve resistance to the disease (Que et al., 2012; Wei et
al., 2006). Therefore, the objective of the present study was
to evaluate the response of the common sugarcane
varieties in Egypt to whip smut disease and to discriminate
between resistant and susceptible genotypes using five
start codon-targeted (SCoT) primers, eleven inter-simple
sequence repeat (ISSR) primers, and six random amplified
polymorphic DNA (RAPD) primers, thereby supporting
sugarcane breeding programs through the efficient
identification of resistant clones.
Article | Agag and Abbas | Molecular characterization and smut resistance evaluation of sugarcane genotypes
3 | Journal of Plant Pathology and Disease Management | Vol. 13, No. 1 | Article ID 18698633
2. Materials and Methods
2.1 Collection of smut samples
Samples of sugarcane smut whips were collected from the
commercial cultivar G.T.54-9 (C9) grown in the sugarcane-
growing regions of El-Minia governorate, Egypt.
2.2 Inoculum preparation
Collected smut whips were air-dried and stored at room
temperature on a laboratory bench for ve days. Teliospores
were released by manually crushing the dried whips in a large
container. Major plant debris was removed, and the
remaining material was passed through a ne-mesh screener.
Finally, the obtained spores were maintained in paper bags at
room temperature until use for inoculum preparation
(Gillaspie et al.,1983).
2.3 Varietal response
Three sugarcane cultivars were used: G.2003-47 (G3; CP 55-
30 ♀ × EI 85-1696 ♂) and G.2004-27 (G4; CP 55-30 ♀ × ROC 22
♂), both newly registered varieties developed by the Egyptian
Sugarcane Breeding Program, and G.T.54-9 (C9; NCo 310 ♀ ×
F 73-925 ♂), which is widely used as a commercial cultivar in
Egypt. Cuttings of the three sugarcane cultivars were
obtained from the Sugar Crops Research Institute (SCRI),
Agricultural Research Center, Giza, Egypt. The cuts of each
variety, containing one bud, were soaked in a fungal spore
suspension (4 g per 1 L of water) for 2 hours. Three cuts were
sown in 35 cm diameter pots containing clay soil. The
greenhouse experiment was arranged in a Randomized
Complete Design (RCD) with ve replicates for each variety.
The experiment was repeated twice (2024 and 2025). The
greenhouse of the Maize disease and Sugar crops Research
section at the Plant Pathology Research Institute, ARC, Giza
was used for the experiment.
2.4 Disease assessment
Disease symptoms, such as whip-like structures, assessed six
months after sowing and used to estimate disease incidence
(Firehun et al., 2009).
Disease incidence (DI %) = Number of infected tillers
Number of total tillers ×100
2.5 Molecular identication of the causal organism
After using liquid nitrogen to grind one gram of S.
scitamineum fungus spores into a ne powder in a mortar,
DNA was extracted using the Bio Basic DNA Extraction Kit.
2.5.1 PCR reactions and conditions
Specic primers bE4 (5-CGCTCTGGTTCATCAACG-3) and
bE8(5-TGCTGTCGATGGAAGGTGT-3) (Albert and Schenck,
1996), the amplication reaction was carried out in 15 µl
includes 2.4 µl master mix Solis Biodyne, 1 µl of each primer
(10 µm concentration), 10 µl sterilized distilled water and 0.5
µl DNA. Applied Biosystems 2720 Thermal cycler was used in
a PCR reaction at 96 °C for 6 minutes. As initial denaturation,
then 35 cycles of 94 °C for 1 minute, 52 °C for 1 minute, and
72 °C for 1 minute. And nal extension 72 °C for 7 minutes.
2.6 Molecular markers
Three types of molecular markers were used in this study: ve
SCoT primers, eleven ISSR primers, and six RAPD primers
(Table 1).
2.6.1 DNA extraction
One gram of sugarcane leaves of the three varieties were
collected after two months post inoculation, then washed
with sterilized distilled water and ground with liquid
nitrogen to obtain a ne powder. DNA extraction was carried
out using the Biobasic DNA extraction kit. The quality and
quantity of the extracted DNA were measured by running the
DNA on a 1% agarose gel. Biobasic Inc., Canada, added 5 5 µL
Ethidium bromide (10mg/mL) alongside a 100 bp DNA ladder
(Solis Biodyne) for 1.5 hours.
2.6.2 PCR reactions and conditions
PCR reactions were performed in a total volume of 15 µL using
an Applied Biosystems 2720 thermal cycler. Cycling
conditions were optimized separately for each molecular
marker system as follows: Start Codon Targeted (SCoT)
markers. PCR amplication was carried out with an initial
denaturation at 94 °C for 4 min, followed by 45 cycles of
denaturation at 94 °C for 40 s, annealing at 45 °C for 1 min,
and extension at 72 °C for 2 min, with a nal extension at 72
°C for 7 min.
Article | Agag and Abbas | Molecular characterization and smut resistance evaluation of sugarcane genotypes
4 | Journal of Plant Pathology and Disease Management | Vol. 13, No. 1 | Article ID 18698633
Table 1: List of SCoT, ISSR and RAPD primers and their sequence.
Molecular marker
Sequence
SCoT
1
CAACAATGGCTACCACGC
2
CAACAATGGCTACCACGT
3
CAACAATGGCTACCAGCA
4
ACGACATGGCGACCAACG
5
CACCATGGCTACCACCAG
ISSR
1
AG9C
2
AC9T
3
GA9A
4
GA9T
5
GA9C
6
CA9G
7
AC9C
8
AC9G
9
TA10T
10
CA9A
11
CA9T
RAPD
1
CACGGCGAGT
2
GTCGATGTCG
3
AAGCCTCCCC
4
CGTCGCCCAT
5
GGGTTTGGCA
6
AGCGAGCAAG
2.6.2.1 Inter-Simple sequence repeat markers
PCR amplication consisted of an initial denaturation at 94
°C for 4 min, followed by 45 cycles of 94 °C for 40 s, 47 °C for
1 min, and 72 °C for 2 min, and a nal extension at 72 °C for 7
min.
2.6.2.2 Random Amplied Polymorphic DNA markers
PCR amplication was performed with an initial
denaturation at 94 °C for 4 min, followed by 45 cycles of
denaturation at 94 °C for 40 s, annealing at 37 °C for 1 min,
and extension at 72 °C for 2 min, with a nal extension at 72°C
for 7 min.
2.6.3 Gel electrophoresis
A 15µl of PCR product was loaded onto a 1.7% agarose gel
(Biobasic Inc., Canada) containing 5µl of Ethidium bromide
(10 mg /ml) in an electrophoresis tank (13cm x 16 cm)
containing 1X TAE buer. The PCR product was run for 1.5
hours at 60 °C. The rst and end lanes of the comb were
loaded with Thermo Scientic O Gene Ruler Ready-to-Use
100 bp plus DNA ladder, containing 14 discrete DNA
fragments ranging from 100 bp to 3000 bp. After the DNA
fragment ran on the gel, it was exposed to UV light using a
Hero Lab UV-40 S/L transilluminator, and then the image
was captured manually with a Sony Cyber-shot camera.
2.7 Statistical analysis
The percentage of plants exhibiting whip-like shape was used
to calculate the disease incidence, and an analysis of variance
(ANOVA) was calculated using Minitab 17 statistical
software. In SCoT, ISSR, and RAPD markers, each scorable
band was treated as a single locus; a data matrix was
generated by scored as present (1) or absent (0). The
percentage of polymorphic bands and the total number of
bands were calculated based on Ng and Tan (2015). For every
molecular marker, a cluster analysis dendrogram and a
similarity matrix were calculated using the Dice coecient,
using the SPSS program version 16. Principal component
analysis (PCA) was performed with the Past statistical
package version 4.03.
3. Results
3.1 Morphological identication of the causal organism
Infected sugarcane plants exhibited typical smut symptoms
characterized by the emergence of a whip-like sorus from the
shoot apex. The sorus consisted of a central vascular core
surrounded by abundant dark, powdery teliospores enclosed
within a thin membranous sheath (Figure 1A). These
symptoms were consistent with sugarcane smut caused by S.
scitamineum. Microscopic examination of teliospores
Article | Agag and Abbas | Molecular characterization and smut resistance evaluation of sugarcane genotypes
5 | Journal of Plant Pathology and Disease Management | Vol. 13, No. 1 | Article ID 18698633
collected from mature smut whips revealed dark brown to
black, globose to subglobose spores with thick walls (Figure
1B). The observed teliospore morphology was consistent with
published descriptions of S. scitamineum, supporting the
morphological identication of the pathogen prior to
molecular conrmation (Hoy et al., 1986; Rott et al., 2000).
Figure 1: Field symptoms on sugarcane plants showing characteristic whip-like, blackened structures emerging from
the shoot apex (A). (B) Microscopic view of the pathogen showing numerous rounds to oval, thick-walled teliospores.
The spores appear brownish and are densely aggregated, consistent with the teliospores of the sugarcane smut fungus.
3.2 Molecular identication of the causal organism
PCR amplication using the species-specic primers bE4 and
bE8 produced a single amplicon of approximately 459 bp,
conrming the identity of the causal organism as S.
scitamineum. (Figure 2).
3.3 Varietal response to whip smut
Three sugarcane varieties were evaluated for their
response to whip smut under artificial inoculation.
Disease incidence differed among the tested varieties. The
lowest disease incidence was recorded in G.2004-27 (G4),
with 4% infected plants, whereas higher disease incidence
was observed in G.T.54-9 (C9) and G.2003-47 (G3), with
values of 12.88% and 13.34%, respectively. Disease
incidence values represent the mean of two independent
experiments.
3.4 Molecular diversity of sugarcane varieties
3.4.1 SCoT marker analysis
Five SCoT primers yielded 25 scorable bands, averaging 5.0
per primer. Of these, 55.33% were polymorphic, with an
average of 2.8 polymorphic bands per primer (Table 2).
Figure 2: Agarose gel electrophoresis of PCR amplification using bE4/bE8 primers for detection of Sporisorium
scitamineum. Lane L, 100-bp DNA ladder; lane S, fungal DNA sample showing the expected 459-bp fragment.
Article | Agag and Abbas | Molecular characterization and smut resistance evaluation of sugarcane genotypes
6 | Journal of Plant Pathology and Disease Management | Vol. 13, No. 1 | Article ID 18698633
Polymorphic information content (PIC) values ranged
from 0.15 to 0.37, with a mean value of 0.25. Banding
patterns obtained with SCoT primers 2 and 5
differentiated G.2004-27 (G4) from the other two varieties
(Figure 3). Primer 1 grouped G.2003-47 (G3) and G.2004-
27 (G4) together, with 86% similarity (Figure 4). Cluster
analysis based on combined SCoT data grouped the three
varieties into two main clusters. G.2004-27 (G4) clustered
with G.T.54-9 (C9), whereas G.2003-47 (G3) formed a
separate cluster (Figure 5).
Table 2: Total number of bands, polymorphic bands, percentage of polymorphic
bands, and PIC values for SCoT primers.
SCoT Primer
TNB
PB
PPB
PIC
1
4
2
50
0.22
2
4
2
50
0.22
3
6
2
33.33
0.15
4
6
5
88.33
0.37
5
5
3
60
0.33
Total
25
14
-
-
Average
5.0
2.8
55.33
0.25
A5
B1
Figure 3: Agarose gel electrophoresis profiles generated by Start
Codon Targeted (SCoT) markers in three sugarcane varieties.
Lanes are labeled as follows: L, 100-bp DNA ladder; G3, sugarcane
variety G.2003-47; G4, sugarcane variety G.2004-27; C9,
commercial variety G.T.54-9. The upper panel shows amplification
patterns obtained with SCoT primers 1 and 2, and the lower panel
shows amplification patterns obtained with SCoT primers 4 and 5.
Differences in banding patterns among the varieties indicate
polymorphism detected by the SCoT markers.
Figure 4: Dendrograms derived from SCoT marker analysis. (A)
Cluster analysis based on SCoT primer 5 grouped sugarcane
varieties G.2003-47 (G3) and G.T.54-9 (C9) into one cluster,
whereas G.2004-27 (G4) formed a separate cluster. (B) Cluster
analysis based on SCoT primer 1 grouped G.2003-47 (G3) and
G.2004-27 (G4) together in the same cluster.
Article | Agag and Abbas | Molecular characterization and smut resistance evaluation of sugarcane genotypes
7 | Journal of Plant Pathology and Disease Management | Vol. 13, No. 1 | Article ID 18698633
Figure 5: Dendrogram showing cluster analysis of sugarcane varieties G.2003-47 (G3),
G.2004-27 (G4), and G.T.54-9 (C9) based on combined data from all SCoT primers.
3.4.2 ISSR marker analysis
Eleven ISSR primers produced 37 scorable bands, averaging
3.4 per primer. Sixteen bands (46.3%) were polymorphic,
with an average of 1.8 polymorphic bands per primer (Table
3). PIC values ranged from 0.09 to 0.44, with a mean value
of 0.21. Several ISSR primers differentiated G.2004-27 (G4)
from the other two varieties (Figure 6). Primers 2 and 3
grouped G.2003-47 (G3) and G.2004-27 (G4) together with
complete similarity (Figure 7). Cluster analysis based on
combined ISSR data separated the three varieties into two
clusters, with G.2003-47 (G3) and G.T.54-9 (C9) grouped
together and G.2004-27 (G4) forming a separate cluster
(Figure 8).
Table 3: Total number of bands, polymorphic bands, percentage of polymorphic bands, and PIC values for ISSR primers.
ISSR Primer
TNB
PB
PPB
PIC
1
5
1
20
0.09
2
2
1
50
0.22
3
3
1
33.33
0.15
4
6
4
66.66
0.33
5
5
3
60
0.27
6
3
1
33.33
0.15
7
1
0
0
0
8
1
0
0
0
9
3
3
100
0.44
10
5
1
20
0.09
11
3
1
33.33
0.15
Total
37
16
-
-
Average
3.4
1.8
46.3
0.21
Figure 6: Agarose gel electrophoresis profiles generated by ISSR markers in three sugarcane varieties. Lanes
are labeled as follows: G.2003-47 (G3), G.2004-27 (G4), and G.T.54-9 (C9). Banding patterns obtained with
ISSR primers 3, 4, and 5 show polymorphic profiles among the tested varieties.
Article | Agag and Abbas | Molecular characterization and smut resistance evaluation of sugarcane genotypes
8 | Journal of Plant Pathology and Disease Management | Vol. 13, No. 1 | Article ID 18698633
A4
B3
Figure 7: Dendrograms derived from ISSR marker analysis. (A) Cluster analysis based on ISSR primer 4 grouped
sugarcane varieties G.2003-47 (G3) and G.T.54-9 (C9) into one cluster, whereas G.2004-27 (G4) formed a separate
cluster. (B) Cluster analysis based on ISSR primer 3 grouped G.2003-47 (G3) and G.2004-27 (G4) together in the same
cluster.
Figure 8: Dendrogram showing cluster analysis of sugarcane varieties G.2003-47 (G3), G.2004-27 (G4), and G.T.54-9
(C9) based on combined data from all ISSR primers.
3.4.3 RAPD marker analysis
Six RAPD primers yielded 32 scorable bands, averaging 5.33
per primer. Sixteen bands (49%) were polymorphic, with an
average of 2.66 polymorphic bands per primer (Table 4). PIC
values ranged from 0.07 to 0.30, with a mean value of 0.20.
Banding patterns obtained with RAPD primers 4 and 5
differentiated G.2004-27 (G4) from the other varieties, whereas
primer 2 grouped G.2003-47 (G3) and G.2004-27 (G4) together
(Figures 9–10). Cluster analysis based on combined RAPD
data grouped G.2003-47 (G3) and G.T.54-9 (C9) together,
while G.2004-27 (G4) formed a separate cluster (Figure 11).
Table 4: Total number of bands, number of polymorphic bands, percentage of polymorphic bands, and
polymorphic information content (PIC) for RAPD primers.
RAPD Primer
TNB
PB
PPB
PIC
1
8
5
62.5
0.30
2
6
3
50
0.07
3
4
1
25
0.11
4
5
2
40
0.20
5
3
2
66.6
0.30
6
6
3
50
0.22
Total
32
16
----
----
Average
5.33
2.66
49
0.20
Article | Agag and Abbas | Molecular characterization and smut resistance evaluation of sugarcane genotypes
9 | Journal of Plant Pathology and Disease Management | Vol. 13, No. 1 | Article ID 18698633
Figure 9: Agarose gel electrophoresis profiles generated by RAPD markers in three sugarcane varieties. Lanes are
labeled as G.2003-47 (G3), G.2004-27 (G4), and G.T.54-9 (C9). Banding patterns obtained with RAPD primers 2, 4,
and 5 show polymorphisms among the tested varieties.
A5
B2
Figure 10: Dendrograms derived from RAPD marker analysis. (A) Cluster analysis based on RAPD primer 5 grouped
sugarcane varieties G.2003-47 (G3) and G.T.54-9 (C9) into one cluster, whereas G.2004-27 (G4) formed a separate
cluster. (B) Cluster analysis based on RAPD primer 2 grouped G.2003-47 (G3) and G.2004-27 (G4) together in the same
cluster.
3.4.4 Combined marker analysis and principal component
analysis
Combined analysis of SCoT, ISSR, and RAPD marker data
revealed 82% similarity between G.2003-47 (G3) and
G.T.54-9 (C9), whereas similarity between G.2004-27 (G4)
and G.T.54-9 (C9) was 76%. Cluster analysis based on
merged marker data grouped G.2003-47 (G3) and G.T.54-9
(C9) into one cluster, while G.2004-27 (G4) formed a
separate cluster (Figure 12). Principal component analysis
(PCA) combining varietal response and molecular
marker data showed that the first principal component
explained 91.34% of the total variation (eigenvalue =
148.60), while the second component explained 8.65%
(eigenvalue = 14.08), as illustrated in the scree plot
(Figure 13). The PCA ordination separated G.2004-27 (G4)
from G.2003-47 (G3) and G.T.54-9 (C9), which clustered
together (Figure 14).
Article | Agag and Abbas | Molecular characterization and smut resistance evaluation of sugarcane genotypes
10 | Journal of Plant Pathology and Disease Management | Vol. 13, No. 1 | Article ID 18698633
Figure 11: Dendrogram showing cluster analysis of
sugarcane varieties G.2003-47 (G3), G.2004-27 (G4), and
G.T.54-9 (C9) based on combined data from all RAPD
primers.
Figure 12: Dendrogram showing cluster analysis based on
combined SCoT, ISSR, and RAPD marker data.
Figure 13: Scree plot derived from principal component
analysis (PCA) of three sugarcane varieties, G.2003-47
(G3), G.2004-27 (G4), and G.T.54-9 (C9), based on
combined varietal response and molecular marker data.
Figure 14: Principal component analysis (PCA) ordination of
three sugarcane varieties, G.2003-47 (G3), G.2004-27 (G4),
and G.T.54-9 (C9), based on combined varietal response and
molecular marker data.
4. Discussion
Previous studies in Egypt have primarily focused on chemical
and integrated management strategies for sugarcane smut
under local conditions (Osman et al., 2025). In this context,
the present study complements earlier work by emphasizing
host varietal response and molecular discrimination among
sugarcane genotypes. Morphological characterization of
infected plants revealed the distinctive whip-like sorus
emerging from the shoot apex, accompanied by abundant
dark, powdery teliospores, which is diagnostic of smut
caused by S. scitamineum. Microscopic results revealed
abundant thick-walled teliospores that were globose to
subglobose, in agreement with previously published
morphological descriptions of the pathogen (Hoy et al., 1986;
Rott et al., 2000). Based on these features, the pathogen was
tentatively identied, and subsequently conrmed by
molecular analyses. Molecular identication using species-
specic primers targeting the bE gene complex yielded a
single amplicon of the expected size (≈459 bp), conrming
the presence of S. scitamineum. PCR assays targeting primers
derived from the bE gene have been shown to provide a
reliable and specic means of identifying S. scitamineum,
particularly when morphological characteristics alone do not
allow clear discrimination (Albert and Schenck, 1996). The
agreement between morphological and molecular
identication in this study supports the reliability of the
pathogen conrmation approach used before resistance
evaluation. Evaluation of varietal response under articial
inoculation revealed apparent dierences in disease
incidence among the tested sugarcane varieties. The variety
G.2004-27 (G4) consistently recorded the lowest disease
incidence, while G.T.54-9 (C9) and G.2003-47 (G3) exhibited
higher, yet comparable, infection rates. Such variation in
smut incidence among sugarcane genotypes has been widely
reported and is largely attributed to dierences in host
Article | Agag and Abbas | Molecular characterization and smut resistance evaluation of sugarcane genotypes
11 | Journal of Plant Pathology and Disease Management | Vol. 13, No. 1 | Article ID 18698633
genetic background, which aect pathogen infection,
colonization, and subsequent disease development (Bhuiyan
et al., 2021; Fawcett, 1946). The relatively lower disease
incidence observed in G4 suggests a higher level of tolerance
or resistance under the conditions of this study. However,
resistance expression in sugarcane is known to be inuenced
by environmental factors and the long crop cycle
highlighting the need for further eld validation. Molecular
marker analysis provided additional resolution in
dierentiating the tested varieties. All three-marker systems
SCoT, ISSR, and RAPD detected polymorphisms among the
genotypes, demonstrating their suitability for assessing
genetic variation in sugarcane. Our results indicate that SCoT
markers yielded higher polymorphism (55.33%) compared to
ISSR and RAPD, suggesting that gene-targeted markers
(SCoT) are more eective in detecting genetic variation
related to functional traits in sugarcane. SCoT markers target
conserved regions anking the start codon and have been
reported to be informative for detecting functional genetic
variation associated with agronomically important traits,
including disease response (Collard and Mackill, 2009). The
markers (ISSR and RAPD) showed clear genetic
dierentiation among the three tested varieties, although the
resulting clustering patterns diered only slightly between
marker systems. Such variations are expected, as each marker
type targets distinct genomic regions and therefore captures
dierent aspects of underlying genetic diversity. Similar
marker-dependent clustering patterns have been reported
previously in studies of sugarcane smut and other
pathosystems, highlighting the value of using multiple
marker systems to obtain a more comprehensive view of
genetic relationships (Que et al., 2012). When marker data
were combined, cluster analysis consistently separated
G.2004-27 from G.2003-47 and G.T.54-9, which clustered
together. This pattern was further supported by principal
component analysis, in which the rst principal component
explained most of the total variation and clearly
distinguished G.2004-27 from the other two varieties. The
agreement among pathogenicity assessments, cluster
analysis, and principal component analysis (PCA) reinforces
the link between molecular marker proles and disease
response, indicating that the genetic variation detected by
these markers is closely associated with smut susceptibility
and resistance. The specic bands identied in G.2004-27 can
be considered as candidate markers for dierentiating
resistant genotypes, pending further validation on larger
populations. Overall, the results demonstrate that an
integrated approach combining varietal response assay and
molecular marker analysis oers a robust framework for
assessing sugarcane responses to whip smut. While common
markers such as SCoT, ISSR, and RAPD do not directly target
resistance genes, they are eective in separating genotypes
with dierent disease reactions and are therefore suitable for
preliminary screening in breeding programs. These
approaches facilitate the more ecient identication of
sugarcane clones worthy of further assessment under eld
conditions when used alongside traditional resistance
evaluation methods (Bhuiyan et al., 2021; Collard and
Mackill, 2009).
5. Conclusion
Future studies should focus on validating these potential
markers using a larger segregating population (e.g., F2 or
RILs) derived from the cross between G.2004-27 and
susceptible parents to establish tight linkage with the smut
resistance gene.
Declarations
Funding Information: The authors received no external
funding for this article.
Conicts of Interest: The authors declare that they have no
known competing financial interests or personal relationships
that could have inuenced the work reported in this paper.
Ethics Approval and Consent to Participate: Not applicable.
This research did not involve human participants or animal
subjects.
Consent for Publication: Not applicable.
Data Availability Statement: The data that support the
ndings of this study are available from the corresponding
author upon reasonable request.
Declaration of Generative AI and AI-assisted Technologies:
The authors conrm that they have not used any articial
intelligence (AI) tools or technologies for the generation of
text, images, or data in the preparation of this manuscript.
CRediT Authorship Contribution Statement: Both
authors, Sayed H. Agag and Abeer H. Abbas, contributed
equally to this work. They jointly collaborated on the study’s
Article | Agag and Abbas | Molecular characterization and smut resistance evaluation of sugarcane genotypes
12 | Journal of Plant Pathology and Disease Management | Vol. 13, No. 1 | Article ID 18698633
conception, design, and methodology. Both authors
performed data collection and analysis, co-wrote the initial
manuscript, and participated in the critical revision and nal
approval of the published version.
Acknowledgment: Not applicable.
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