Journal of the Saudi Society of Agricultural Sciences xxx (xxxx) xxx

Contents lists available at ScienceDirect

Journal of the Saudi Society of Agricultural Sciences

journal homepage: www.sciencedirect.com

Symptomatology of Sigatoka leaf spot disease in banana landraces and

identification of its pathogen as Mycosphaerella eumusae
Milsha George a,1, K. Anita Cherian a, Deepu Mathew b,⇑
a
Department of Plant Pathology, College of Agriculture, Kerala Agricultural University, Thrissur, Kerala 680 656, India
b
Centre for Plant Biotechnology and Molecular Biology, Kerala Agricultural University, Thrissur, Kerala 680 656, India

a r t i c l e i n f o a b s t r a c t

Article history: Mycosphaerella musicola, M. fijiensis and M. eumusae are known to cause banana yellow Sigatoka, black
Received 11 March 2021 Sigatoka and Eumusae leaf spot diseases respectively. However, differentiating the causative species
Revised 4 September 2021 has been challenging and the responses of banana landraces to this pathogen were little known.
Accepted 26 September 2021
Purposive sampling surveys were conducted in different agro-climatic zones of Kerala state, India, to col-
Available online xxxx
lect the leaf samples with Sigatoka leaf spot-like symptoms, to document the symptom expression on dif-
ferent landraces and to confirm the identity of the pathogen at species level. Fields surveyed during the
Keywords:
post-monsoon period had 100% disease incidence with highest per cent disease severity of 43.90% in
DNA barcoding
Foliar disease
nine-month old cv. Palayankodan, while lowest PDS of 3.33% was observed in three-month old cv.
Fungal disease Njalipoovan, during a pre-monsoon survey. Initial symptoms were small brown streaks, later developing
ITS-rDNA into oval or elliptical brown spots with greyish centre and subsequently coalescing for leaf necrosis.
Musa Symptoms in different landraces varied across the zones. Sequencing and analysis of the ITS region from
Mycosphaerella eumusae 50 isolates collected during the survey showed 99–100% homology with M. eumusae. Phylogenetic anal-
Per cent disease severity yses further proved the species identity of the local isolates. Comparing the ITS barcodes of the three spe-
cies of Mycosphaerella along with the local isolates confirmed that the pathogen inciting Sigatoka leaf spot
in Kerala state, India, is Mycosphaerella eumusae.
Ó 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction cercospora fijiensis (M. Morelet) Deighton) and Mycosphaerella

Sigatoka leaf spot caused by the fungal complex Mycosphaerella
spp. leads to 11–80% yield losses in banana by reducing the photo-
synthetic tissues through necrotic leaf lesions (Shanthiyaa et al.,
2013). Among the landraces, French plantain cv. Nendran (AAB),
grown extensively in southern most states of India and especially
in Kerala, is highly prone to this disease (Selvarajan et al., 2001).
Three species of ascomycetous fungi, Mycosphaerella musicola R.
Leach. Ex. J.L. Mulder (anamorph Pseudocercospora musae (Zimm.)
Deighton), Mycosphaerella fijiensis M. Morelet (anamorph Pseudo-
⇑ Corresponding author.
E-mail address: deepu.mathew@kau.in (D. Mathew).
1
Current address: Department of Agriculture, Karunya Institute of Technology and
Sciences, Kaurunya Nagar, Coimbatore
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
eumusae Crous & Mourichon (anamorph Pseudocercospora eumusae
Crous & Mourichon) have been reported as the causal agents of dif-
ferent Sigatoka leaf spot diseases viz., yellow Sigatoka, black Siga-
toka and Eumusae leaf spot, respectively (Arzanlou et al., 2007).
Among these, yellow and black Sigatoka are considered as the
major leaf spot diseases of banana, making banana farming less
profitable and driving the popular cultivars out from cultivation.
However, (Carlier et al., 2000) have reported the presence of Eumu-
sae leaf spot in South India; causing 20–40% yield losses.
The symptoms produced by all the three species of pathogens
were similar and confusing. Initial symptom of the disease is the
appearance of pale yellow or dark brown indistinct linear streaks
lying parallel to the veins on the lower leaves. In advanced stages,
these streaks enlarge to form necrotic lesions with yellow halo and
light grey centres (Stover, 1972). These necrotic lesions later coa-
lesce, causing complete drying of the leaves and defoliation, lead-
ing to delayed flowering, reduction in the number of hands and
fingers, premature ripening and peel splitting of the fruits
(Surridge et al., 2003). Similarity of symptoms of the three Sigatoka
diseases along with the difficulty in artificial culturing of the
pathogens limit their identification. Based on visual symptoms,

https://doi.org/10.1016/j.jssas.2021.09.004
1658-077X/Ó 2021 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: M. George, K. Anita Cherian and D. Mathew, Symptomatology of Sigatoka leaf spot disease in banana landraces and identification
of its pathogen as Mycosphaerella eumusae, Journal of the Saudi Society of Agricultural Sciences, https://doi.org/10.1016/j.jssas.2021.09.004
M. George, K. Anita Cherian and D. Mathew
Eumusae leaf spot has mostly been mistaken for yellow Sigatoka
and also for black Sigatoka. Considering the rapid replacement of
yellow Sigatoka leaf spot by Eumusae leaf spot due to its aggressive
nature and dominance in South India (Carlier et al., 2000), a culture
independent species-level molecular detection technique was nec-
essary. Further, severe outbreak of the disease during the monsoon
season in Kerala state necessitated the pathogen identification and
design of management strategies. The characteristic and conserved
nucleotide variations help in the species-level identification in
Mycosphaerella (Surridge et al., 2003). Hence, this study was taken
up with the objectives to collect the leaf samples with Sigatoka leaf
spot-like symptoms, to document in detail the variation in symp-
toms in different landraces and to confirm the identity of the
pathogen associated with each symptom at species level based
on the unique barcodes present in the ITS-rDNA region.
2. Materials and methods
2.1. Survey and collection of samples
Purposive sampling surveys were conducted in different agro-
climatic zones of Kerala state, India, viz., Malappuram, Palakkad
(Northern zone), Ernakulam, Thrissur (Central zone), Wayanad
(High range zone) and Thiruvananthapuram (Southern zone) to
document the symptoms occurring in the field under natural con-
ditions, to assess disease severity, to record the youngest leaf spot-
ted (YLS) and to identify the pathogen associated with Sigatoka leaf
spot disease complex. In Thrissur district, intensive surveys were
conducted twice in 18 selected panchayats, during April-May
(pre-monsoon) and July-August 2018 (post-monsoon).
The data collection was carried out based on the procedure fol-
lowed by (Selvarajan et al., 2001). Twenty five plants were selected
randomly from each plot to assess the per cent disease severity and
the youngest leaf spotted. Variations in the symptoms observed in
different landraces in each location were also recorded. Tissues
with advanced leaf spot symptoms were cut into 30
40 cm size
and brought to the laboratory in a cool box.
The youngest leaf spotted was identified by counting the posi-
tion of the youngest leaf showing at least ten leaf spots. Disease
severity was assessed using a standard score chart (Meredith and
Lawrence, 1969) using the 0–6 scale (Supplementary Table 1).
Per cent disease severity ðPDSÞ was calculated
P
nb
100
using the formula;
ðN  1ÞT
where n = number of leaves in each grade, b = grade, N = Number of
grade used in the scale, T = total number of leaves.
Temperature and rainfall data for each location for the period of
survey, were also recorded and correlation of disease severity with
the weather parameters and age of the plants was analyzed using
SPSS v.16.0.
2.2. Symptomatology
Infected leaves of the landraces were closely observed for the
development of symptoms from the initial spots to mature lesions.
Variations in symptoms in each landrace in each zone were also
recorded.
2.3. Isolation of DNA and PCR amplification
Fungi associated with the symptoms were characterized by PCR
amplification of the ITS-rDNA region using specific primer combi-
nation and sequencing (Surridge et al., 2003). DNA was isolated
from approximately 1.0 g of the leaf lesions, replicated thrice, from
2
Journal of the Saudi Society of Agricultural Sciences xxx (xxxx) xxx
50 samples belonging to different landraces, using DNeasy Plant
Mini Kit (Qiagen, India) following manufacturer’s protocol. Ther-
mal cycling of the isolated DNA was performed using ITS1 and
ITS4 primer combination reported for the ITS-rDNA region of Myco-
sphaerella (White et al., 1990; Carlier et al., 2000; Crous et al., 2002;
Udugama, 2002). Reaction was carried out in 25 mL volume com-
prising 12.5 mL of PCR master mix (Takara Emerald AmpÒ GT PCR
Master Mix), 0.5 mL each of the primers, 2 mL of DNA (50 ng/ mL)
and 9.5 mL of distilled water. Thermal cycling included initial
denaturation at 94 °C for three min. followed by 35 cycles compris-
ing denaturation at 94 °C for 45 sec., annealing at 60.4 °C for 45
sec., elongation at 72 °C for one min. and final elongation at
72 °C for 10 min. Product was electrophoresed on one per cent
agarose gel and the amplicons were eluted using DNA elution kit
(GeNeiTM Gel Extraction Kit) and sequenced (AgriGenome Pvt.
Ltd., Kochi, Kerala, India).
2.4. Sequence and barcode analyses
Contigs were generated from the paired-end reads and ITS-rDNA
sequences of 50 isolates were deposited in NCBI GenBank. The
sequences were aligned using Clustal Omega program to identify
the unique barcodes to differentiate among the species. Phyloge-
netic tree was generated from the distance matrix by the UPGMA
method, with 1000 bootstrap replicates using MEGA 6.0
(Surridge et al., 2003).
3. Results
3.1. Incidence and severity of the disease
Sigatoka leaf spot disease was severe in all the fields surveyed.
Of the 50 samples collected, Thrissur, Thiruvananthapuram,
Ernakulum, Wayanad, Malappuram and Palakkad districts had
19, 12 (six each from Northern and Southern regions), 3, 5, 7 and
4 samples, respectively. Disease severity varied with landrace,
age of the plant, zone and the season. In Thrissur district, highest
per cent disease severity (PDS) of 43.90% was recorded on nine-
month old Mysore Poovan in Pudukkad panchayat with YLS of
3.78. The lowest PDS of 3.33% was recorded on three-month old
Njalipoovan variety in Thalikulam panchayat with YLS of 10.00
(Table 1).
In Malappuram district (Northern zone), the highest PDS of
16.78% was recorded on nine-month old Nendran with YLS of
6.12 at Vengara panchayat while the lowest PDS of 4.57% was
recorded on five-month old Nendran with YLS of 8.78 at Perinthal-
manna (Table 2). In Palakkad district (Northern zone), the highest
PDS was recorded on eight-month old Nendran with YLS of 6.89 at
Pattambi and the lowest PDS of 4.53% was noticed on five-month
old Nendran with YLS of 7.00 in Vattapara panchayat. Among the
three panchayaths surveyed in Ernakulam district (Central zone),
highest PDS of 18.76% was recorded on seven-month old Nendran
with YLS of 6.00 in Pothanicad panchayat and the lowest PDS was
in eight-month old Nendran with YLS of 5.70 at Kalady panchayat.
In Wayanad district (High range zone), the highest PDS of 23.41%
was seen in Ambalavayal panchayat on eight-month old Nendran
with YLS of 4.98 while lowest PDS of 14.01% was recorded on
seven-month old Nendran at Lakkidi panchayat with YLS of 6.01.
In Thiruvananthapuram district (Southern zone), the highest PDS
of 29.40% was noticed on nine-month old Nendran at Peringam-
mala panchayat with YLS of 5.34 and lowest PDS (21.34%) was
on seven-month old Nendran at Nedumangad panchayat with
YLS of 6.00. Rainfall and the age of the plants had significant pos-
itive correlation with PDS whereas temperature had negative cor-
relation (Figs. 1–3).
M. George, K. Anita Cherian and D. Mathew Journal of the Saudi Society of Agricultural Sciences xxx (xxxx) xxx

Table 1
Severity of Sigatoka leaf spot disease at different locations surveyed in Thrissur district, Kerala state, India.

Sl. No. Location of survey April-May 2018 (pre-monsoon) July-August 2018 (post-monsoon)
Cultivar Age of plants (MAP)* PDS** YLS*** Cultivar Age of plants (MAP)* PDS** YLS***
1. Wadakkanchery Nendran 5.00 12.72 6.70 Mysore Poovan 7.00 30.08 6.90
2. Chelakkara Nendran 5.00 12.33 9.00 Nedunendran 7.00 43.23 3.80
3. Erumapetty Nendran 3.00 7.33 9.20 Nendran 8.00 36.92 4.40
Kadali 3.00 5.42 7.52 Pooja Kadali 7.00 23.02 6.40
Poovan 5.00 13.49 8.00 Nendran 7.00 34.85 6.30
4. Vellangallur Nendran 4.00 6.12 7.40 Nendran 8.00 43.88 4.00
5. Meloor Nendran 3.00 7.48 10.30 Nendran 5.00 38.23 6.10
6. Pudukkad Nendran 8.00 19.41 7.70 Mysore Poovan 8.00 42.07 5.30
7. Kodakkara Nendran 6.00 15.87 7.00 Njalipoovan 4.00 26.98 7.10
Nendran 5.00 32.79 6.00
8. Mattathur Kadali 4.00 10.85 7.00 Kadali 5.00 35.61 5.40
9. Nenmanikkara Nendran 5.00 13.85 10.10 Nendran 8.00 39.92 6.60
10. Vellanikkara Nendran 4.00 11.09 10.80 Nendran 6.00 26.08 6.80
11. Mala Nendran 3.00 6.40 10.40 Nendran 7.00 34.53 5.50
12. Chowannur Nendran 4.00 10.13 11.50 Nendran 9.00 40.12 5.80
13. Kodariyur Nendran 2.00 5.57 10.12 Robusta 7.00 38.28 6.90
Nendran 7.00 41.87 5.40
14. Nattika Nendran 3.00 7.98 7.80 Njalipoovan 5.00 31.44 6.30
15. Thalikulam Nendran 5.00 7.86 11.7 Njalipoovan 6.00 23.58 7.20
Njalipoovan 3.00 3.33 10.00
16. Puzhakkal Nendran 7.00 10.34 11.60 Nendran 8.00 40.49 4.90
17. Thekkumkkara Mysore Poovan 5.00 8.35 8.12 Mysore Poovan 9.00 42.57 4.10
18. Nadathara Nendran 7.00 8.36 11.62 Mysore Poovan 9.00 43.90 3.78

*Months after planting, ** Per cent disease severity, ***Youngest leaf spotted.

Table 2
Severity of Sigatoka leaf spot disease at different locations in districts other than Thrissur, Kerala state, India.

Sl. No. District Location Variety MAP* PDS** YLS***

1. Malappuram Vattumkulam Nendran 5 6.50 7.33
2. Perinthalmanna Nendran 5 4.57 8.78
3. Vengara Nendran 10 16.78 6.12
4. A.R. Nagar Nendran 9 10.23 6.81
5. Palakkad Malampuzha Nendran 3 3.62 8.01
6. Srikrishnapuram Nendran 7 11.03 6.25
7. Vattapara Nendran 5 4.53 7.00
8. Pattambi Nendran 8 12.50 6.89
9. Thrithala Nendran 7 10.13 6.31
10. Ernakulam Kalady Nendran 7 14.34 5.70
11. Kothamagalam Nendran 8 12.96 6.69
12. Pothanicad Nendran 7 18.76 6.00
13. Wayanad Lakkidi Nendran 7 14.01 6.01
14. Ambalavayal Nendran 8 23.41 4.98
15. Sultan Bethery Nendran 9 19.08 5.67
16. Thiruvanathapuram Nedumangad Nendran 7 21.34 6.00
17. Neyyattinkara Nendran 6 21.79 6.10
18. Peringammala Nendran 9 29.40 5.34

*Months after planting, ** Per cent disease severity, *** Youngest leaf spotted.

Fig. 1. Influence of rainfall on disease severity.

3
M. George, K. Anita Cherian and D. Mathew
Fig. 2. Influence of age of plants on disease severity.
Fig. 3. Influence of temperature on disease severity.
3.2. Symptom variability with cultivar and location
3.2.1. Symptoms on cv. Nendran (AAB)
Nendran is the widely grown commercial cultivar in Kerala
state of India. In Nendran, six types of symptoms were noticed.
Type 1 symptom initiated as small light green or yellow spots on
the lower surface of the leaves. These spots then changed into faint
brown visible streaks, later expanding in length and breadth. Sub-
sequently, they became linear in shape with pointed tips and
appeared adjacent to the veins of the leaves (Fig. 4a). The mature
spots had greyish centre with brown margin surrounded by promi-
nent yellow halo. On the lower most leaves, these mature spots
coalesced, resulting in complete necrosis of the leaves.
Type II symptoms started as small, light yellow coloured round
spots on the lower surface of the leaves. During the next stage of
infection, the spots enlarged and turned to dark brown streaks
on the lower side of the leaves. These streaks later appeared as lin-
ear dark brown spots on the inter-veinal areas of the upper surface
of the leaves. In advanced stages, the streaks changed to oval spots
with greyish centre and prominent brown margin (Fig. 4b). These
spots coalesced together, causing complete leaf drying. These
symptoms were similar to those described for Eumusae leaf spot
by (Carlier et al., 2000).
Type III symptoms started with small, yellow coloured round
spots in the lower surface of the leaves. Later, the spots changed
to small, faint brown streaks on the lower leaf surface, appearing
4
Journal of the Saudi Society of Agricultural Sciences xxx (xxxx) xxx
as dark brown streaks on the upper surface. In the next stage, these
streaks expanded both in length and breadth and became oval in
shape. Chains of these oval spots were noticed along the veins on
the leaf edges (Fig. 4c) and later spread towards the midrib. The
mature oval spots had greyish centre with definite light brown
margin surrounded by yellow halo. In the final stage of infection,
the mature spots coalesced, causing complete necrosis of the
leaves. Type III symptoms were commonly observed in cv.
Nendran.
Type IV symptoms were initially visible as faint brown streaks
on the adaxial leaf surface. In the next stage, these streaks turned
rusty brown on the adaxial surface, which then developed into oval
or elliptical brown spots surrounded by yellow halo. The mature
spots had greyish centre surrounded by irregular dark brown bor-
der having black pin head like fruiting bodies embedded in it
(Fig. 4d). The spots appeared more along the leaf veins than on
the inter-veinal spaces. Upon heavy infection, the spots coalesced
leading to complete necrosis of the leaves, destroying the func-
tional green tissues.
Type V symptoms appeared initially as light faint brown rusty
streaks on the lower surface of the leaves. Later, these streaks
started to appear on the upper surface also. These streaks then
increased in length rather than breadth and appeared as brownish
black elongated streaks instead of oval elliptical spots, without
prominent yellow halo. In advanced stages, streaks coalesced caus-
ing necrosis of the leaves (Fig. 4e).
M. George, K. Anita Cherian and D. Mathew
Fig. 4. Variation in symptoms on banana cv. Nendran (AAB).
Type VI symptoms were noticed during the post-monsoon sur-
veys. The initial visible symptoms appeared as faint rusty streaks
on the lower surface of the leaves. In the next stage, these streaks
started appearing as black, oval shaped spots along the veins on the
upper surface of the leaves. The chain of spots when matured had
greyish centre with well-defined black margin surrounded by yel-
low halo. These spots coalesced from the edge to the midrib, caus-
ing necrosis and drying up of the leaves (Fig. 4f). During the post-
monsoon surveys in different zones, the symptoms noticed in cv.
Nendran were consistent.
3.2.2. Symptoms on other cultivars
Among other cultivars, two types of symptoms each were
observed in cvs. Mysore Poovan (AAB)(Palayamkodan) and Njali-
poovan (AB), while one type of symptom was seen in cvs. Robusta
(AAA) and Kadali (AA). In Mysore Poovan, Type I symptom were
initiated as light brown streaks on the lower surface of the leaves
and corresponding mild yellow colour appeared on the upper sur-
face. In the next stage, brown streaks appeared on the upper sur-
5
Journal of the Saudi Society of Agricultural Sciences xxx (xxxx) xxx
face of the leaves which subsequently turned irregularly oval
with greyish centre and no definite margins. They later coalesced
causing complete necrosis of the leaves (Fig. 5a). Lesions were
more prominent along the inter-veinal spaces rather than on the
veins. These symptoms were noticed during pre-monsoon survey.
Type II symptoms were initiated as small rusty brown streaks
on the lower surface of the leaves which later appeared as black
thin streaks on the upper surface. These black streaks increased
in length and got coalesced along the line of the veins even before
maturation, giving an unusual black appearance to the foliage.
Water soaked lesions were found on the under surface of the leaves
corresponding to the black streaks on the upper surface. These
symptoms were more common during post-monsoon season
(Fig. 5b).
In Njalipoovan, Type I symptoms were noticed during the pre-
monsoon survey. Yellowish orange streaks were observed on the
lower side of the leaves. Then the streaks assumed a rusty brown
appearance and dark brown lesions were observed along the veins.
In the next stage, brown coloured lesions changed to elongated lin-
M. George, K. Anita Cherian and D. Mathew Journal of the Saudi Society of Agricultural Sciences xxx (xxxx) xxx

Fig. 5. Variation in symptoms on other cultivars of banana a. Type I symptom in Mysore Poovan, b. Type II symptom in Mysore Poovan, c. Type I symptom in Njalipoovan, d.
Type II symptom in Njalipoovan, e. Type I symptom in Kadali, f. Type I symptom in Robusta.

ear spots with blended tips and prominent yellow halo. Mature
lesions on the leaves were elongated, having greyish centre and
broad brown margins. They coalesced along the veins and caused
drying up of the whole leaf (Fig. 5c).
Type II symptoms appeared as yellowish orange elongated
streaks on the lower side of the leaves. Then they changed to
brown and started appearing as slightly depressed streaks on the
upper surface of the leaves. The streaks then expanded in size,
became elliptical or oval with corresponding water soaked lesions
on the lower surface of the leaves. Lesions coalesced before matu-
ration and caused complete leaf drying (Fig. 5d). On the dried
leaves, greyish areas were noticed with embedded black pin
headed fruiting bodies. This type of symptoms were more preva-
lent during the post-monsoon period.
6
In cv. Kadali, Type I symptoms appeared in both pre-monsoon
and post-monsoon periods. Light brownish black streaks were seen
on the lower surface of the leaves with corresponding black streaks
along the veins on the upper surface. These streaks increased in
length rather than breadth and became slightly depressed. Greyish
centre with black margin surrounded by yellow halo was observed
in mature spots which later coalesced and caused leaf necrosis
(Fig. 5e).
Also in cv. Robusta, Type I symptoms were noticed during both
pre-monsoon and post-monsoon seasons. Light brown streaks
appeared initially on the leaf tips which later spread uniformly
on the lamina. These initial streaks then changed to linear black
streaks on the upper leaf surface. The slightly depressed streaks
were present along the margins as well as in the inter-veinal areas,
M. George, K. Anita Cherian and D. Mathew
Fig. 6. PCR amplification of ITS-rDNA region from the isolates collected from a. Thrissur (S1-S19), b. North Thiruvananthapuram (S20-S25), Southern Thiruvananthapuram
(S26-S31), Ernakulam (S32-S34), c. Wayanad (S35-39), Malappuram (S40-S46), Palakkad (S47-S50).
Fig. 7. Phylogenetic tree showing the relatedness of different Mycosphaerella species, generated using the sequences of ITS-rDNA region.
giving the leaf an unusual black appearance. The mature lesions
had greyish centre with thick black margin surrounded by irregular
yellow halo and coalesced before maturation (Fig. 5f).
3.3. Analysis of ITS-rDNA region
From the 50 samples collected during the survey, ITS-rDNA
region was PCR amplified (Fig. 6). A product of 580 bp was ampli-
fied in each sample using ITS1 and ITS4 primer combination and
were Sanger sequenced. BLASTn analysis revealed 99–100%
sequence homology with Mycosphaerella eumusae. Homology of
the ITS-rDNA sequences of the isolates along with their GenBank
accession numbers are presented in Supplementary Table 2. The
sequences included partial sequence of 18S rDNA, full ITS 1, 5.8S
rDNA and ITS 2 and partial 28S rDNA.
3.4. Phylogenetic analysis
Phylogenetic analysis of the ITS-rDNA sequences of the isolates
along with the sequences of M. musicola, M. fijiensis and M. eumusae
retrieved from GenBank, had shown that all the isolates form a
monophyletic group. The phylogram formed two clusters, first
cluster accommodating local isolates, M. eumusae accession, M.
fijiensis accessions and one Pseudocercospora securinegae accession
and the second cluster accommodating M. musicola accessions.
First cluster housed two sub-clusters, first sub-cluster accommo-
dating local isolates and M. eumusae accessions, and second sub-
cluster accommodating M. fijiensis and Pseudocercospora securine-
gae accessions. The phylogram shows that the isolates are more
7
Journal of the Saudi Society of Agricultural Sciences xxx (xxxx) xxx
related to M. eumusae (Fig. 7). Bootstrap values for all the clusters
were above 85%.
The phylogenetic tree generated for the 50 isolates had shown
that they belong to different sub-clusters, indicating variability
among M. eumusae isolates collected from different zones (Fig. 8).
However, the bootstrap values were below 80% showing that the
clustering is non-reliable since it is generated on minimal varia-
tions in the sequences.
3.5. Barcode analysis
The characteristic nucleotide variations among the three spe-
cies compared in the barcode analysis had clearly shown that the
local isolates belong to M. eumusae. The clade containing isolates
of M. eumusae and local isolates got differentiated by 10 base pairs
from M. fijiensis clade and by four base pairs from M. musicola
clade. Based on the first identical base in all alignments which
had a motif of GGC at nucleotide 50 from the 50 , there observed a
gap of one base pair at position 75 and nine base pairs at 81–90
in M. fijiensis (Fig. 9). A gap of four base pairs was observed in M.
musicola at position 460–463 (Fig. 10).
4. Discussion
This study has revealed that the Sigatoka-like leaf spots from all
the agro-climatic zones of Kerala are caused by Mycosphaerella
eumusae, confirming the presence of Eumusae leaf spot disease in
the state. Disease incidence was observed in all the fields through-
out the year, which might be due to the presence of conidia and
M. George, K. Anita Cherian and D. Mathew Journal of the Saudi Society of Agricultural Sciences xxx (xxxx) xxx

Fig. 8. Phylogenetic tree of the 50 local isolates of Mycosphaerella sp., generated using the sequences of ITS-rDNA region.

ascopsores in the leaf debris, serving as the source of inoculums.
Similar observations were also made by (Jacome et al., 1991). It
was also observed that the spore germination and germ tube
development were strongly influenced by the rainfall and temper-
8
ature. Disease severity was higher and the youngest leaf spotted
was lower during the post-monsoon survey compared to the pre-
monsoon survey. Presence of moisture on leaf surface during the
rainy season favours the germination and release of spores thereby
M. George, K. Anita Cherian and D. Mathew Journal of the Saudi Society of Agricultural Sciences xxx (xxxx) xxx

Fig. 9. Variation in sequences of Mycosphaerella eumusae compared to M. fijiensis.

Fig. 10. Variation in sequences of Mycosphaerella eumusae compared to M. musicola.

increasing the severity of the disease (Jacome et al., 1991). The pos-
itive correlation of disease severity with rainfall and its negative
correlation with the temperature point to the favourable condi-
tions for spore germination and fast spread, causing higher losses
during post-monsoon season (Mourichon et al., 1990; Torres
et al., 2000).
Type I and II symptoms observed in Nendran were similar to the
Eumusae leaf spot symptoms reported by (Carlier et al., 2000).
Type III symptoms were similar to that reported by (Selvarajan
et al., 2001); who reported the Type IV symptoms in cv. Rasthali
also. The mature lesions appeared streak like rather than spots in
type V symptoms. Type VI symptoms were noticed during the
post-monsoon season; similar to Type III except that they were
darker due to the excessive sporulation.
In cv. Mysore Poovan, Type I symptoms with mature and irreg-
ularly oval spots having no definite margins were seen in the pre-
monsoon season, whereas post-monsoon season had Type II symp-
toms. In cv. Njalipoovan, pre-monsoon season had Type I symp-
toms with elongated linear spots having blend tips and
prominent yellow halo, whereas post-monsoon Type II symptom
had yellowish orange streaks subsequently changing to water-
soaked elliptical spots which coalesced before maturity, causing
complete necrosis. In cv. Robusta, the mature spots were slightly
depressed and black with greyish centre. These symptoms were
similar to those described for black Sigatoka disease by

9
M. George, K. Anita Cherian and D. Mathew
(Meredith and Lawrence, 1969). In cv. Kadali; the lesions appeared
as black streaks rather than spots having a greyish centre. Present
study had clearly shown that the symptoms of Sigatoka leaf spot
disease differs with cultivar, location and season.
(Carlier et al., 2000) followed by (Devi and Thangavelu, 2014)
have reported that the ITS region is efficient to distinguish among
M. musicola; M. fijiensis and M. eumusae. The phylogenetic analysis
of the local isolates with three species of Mycosphaerella indicated
that the local isolates collected during the survey formed a sub-
cluster with M. eumusae indicating that they are closely related
to M. eumusae. The local isolates formed a monophyletic group;
pointing their lower genetic divergence and suggesting that the
Sigatoka leaf spot pathogens are originated from a common ances-
tor (Devi and Thangavelu, 2014). The alignment has revealed
unique nucleotide gaps in different species. In M. fijiensis, a unique
gap was seen at the nucleotide 75 from the 50 of the locus followed
by the second gap of 9 nucleotides at 81–90 bp whereas M. musi-
cola had a gap of 4 nucleotides at 460–463 bp. These results were
similar to the reports of (Surridge et al., 2003).
5. Conclusion
The weather conditions prevailing in Kerala state, India, are
very much conducive for the development and spread of the Siga-
toka disease of banana. Species identification of the pathogen
based on the symptoms have been challenging for this disease.
Purposive sampling surveys conducted in different districts repre-
senting the agro-climatic zones of the state has revealed that the
diseases incidence is complete with PDS range of 3.33–43.90%.
Variability in symptoms of Eumusae leaf spot in different landraces
during different seasons is detailed. ITS-rDNA region of the isolates
had 99–100% sequence homology to M. eumusae. Aligning the
sequences of M. eumusae and local isolates along with the
sequences of M. fijiensis has revealed 10 barcodes to differentiate
both species. Similarly, four barcodes were identified to differenti-
ate M. eumusae from M. musicola. Based on the symptomatology
and the analysis of ITS-rDNA region, the fungus causing Sigatoka
leaf spot in the state is identified as Mycosphaerella eumusae Crous
& Mourichon (anamorph Pseudocercospora eumusae Crous & Mouri-
chon). The unique barcodes described in this work can be used to
identify Mycosphaerella spp. inciting Sigatoka leaf spot disease in
banana.
6. Sequence submission
ITS-rDNA sequences from the 50 isolates are deposited in Gen-
Bank and the accession numbers are presented in Supplementary
Table 2.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
10
Journal of the Saudi Society of Agricultural Sciences xxx (xxxx) xxx
Acknowledgements
Authors thank Prof. Vimi Louis and Mrs. Namitha P.M. (Banana
Research Station, KAU), and Prof. Beena S. (Department of Plant
Pathology, College of Agriculture, KAU) for the assistance in this
research work.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jssas.2021.09.004.
References
Arzanlou, M., Abeln, E.C.A., Kema, G.H.J., Waalwijk, C., Carlier, J., de Vries, I., Guzman,
M., Crous, P.W., 2007. Molecular diagnostics for the Sigatoka disease complex of
banana. Phytopathology. 97 (9), 1112–1118. https://doi.org/10.1094/PHYTO-
97-9-1112.
Carlier, J., Zapater, M.F., Lapeyre, F., Jones, D.R., Mourichon, X., 2000. Septoria leaf
spot of banana: a newly discovered disease caused by Mycosphaerella eumusae
(anamorph Septoria eumusae). Phytopathology. 90 (8), 884–890. https://doi.org/
10.1094/PHYTO.2000.90.8.884.
Crous, P.W., Groenewald, J.Z., Aptroot, A., Braun, U., Mourichon, X., Carlier, J., 2002.
Integrating morphological and molecular data sets on Mycosphaerella, with
specific reference to species occurring on Musa. In: Mycosphaerella Leaf Spot
Diseases of Bananas: Present Status and Outlook. Proceedings of the Workshop
on Mycosphaerella Leaf Spot Diseases, San José, Costa Rica, 20–23 May 2003, pp.
43–57.
Devi, P.G., Thangavelu, R., 2014. Genetic diversity analyses of Mycosphaerella
eumusae causing Septoria leaf spot disease of banana in India. Indian
Phytopathol. 67 (4), 388–398.
Jacome, L.H., Schuh, W., Stevenson, R.E., 1991. Effect of temperature and relative
humidity on germination and germ tube development of Mycosphaerella fijiensis
var. difformis. Phytopathology. 81 (12), 1480–1485.
Meredith, D.S., Lawrence, J.S., 1969. Black leaf streak disease of bananas
(Mycosphaerella fijiensis): Symptoms of disease in Hawaii, and notes on the
conidial state of the causal fungus. Trans. Br. Mycol. Soc. 52 (3), 459–IN16.
https://doi.org/10.1016/S0007-1536(69)80130-0.
Mourichon, X., Fullerton, R.A., Prahl, K.S., 1990. Geographical distribution of the two
species Mycosphaerella musicola Leach (Cercospora musae) and M. fijiensis
Morelet (C. fijiensis), respectively agents of Sigatoka disease and black leaf
streak disease in bananas and plantains. Fruits. 45 (3), 213–218.
Selvarajan, R., Uma, S., Sathiamoorthy, S., 2001. Etiology and survey of banana leaf
spot diseases in India. In: Advancing Banana and Plantain R&D in Asia and the
Pacific Volume 10, Proceedings of the 10th INIBAP-ASPNET Regional Advisory
Committee Meeting held at Bangkok. In: Molina, A.B., Roa, V.N., Maghuyop, M.A.
G. (Eds.), International Network for the Improvement of Banana and Plantain
(INIBAP). – Asia and the Pacific Network, Los Baños, Laguna, Philippines, pp. 94–
102.
Shanthiyaa, V., Karthikeyan, G., Raguchander, T., Prabakar, K., 2013. Prevalence of
banana yellow Sigatoka disease caused by Mycosphaerella musicola in Tamil
Nadu. J. Mycol. Plant Pathol. 43 (4), 414–418.
Stover, R.H., 1972. Banana leaf spot disease caused by Mycosphaerella musicola:
contrasting features of Sigatoka and black leaf streak control. Plant Disease Rep.
55, 437–439.
Surridge, A.K.J., Viljoen, A., Crous, R.W., Wehner, F.C., 2003. Identification of the
pathogen associated with Sigatoka disease of banana in South Africa.
Australasian Plant Pathol. 32 (1), 27–31. https://doi.org/10.1071/AP02058.
Torres, C.M.C., Ganzales, G.A.Y., Zapata, J.C., 2000. Intensity of black Sigatoka
(Mycosphaerella fijiensis) and yellow Sigatoka (Mycosphaerella musicola) in Musa
AAB. Infomusa 9, 16–19.
Udugama, S., 2002. Septoria leaf-spot disease of banana Mycosphaerella eumusae: a
new record for Sri Lanka. Ann. Sri Lanka Dep. Agric. 4, 337–343.
White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. PCR protocols. In: Innis, M.A., Gelfand,
D.H., Sninsky, J.J., White, T.J. (Eds.), Amplification and direct sequencing of
fungal ribosomal RNA genes for phylogenetics. Academic Press, London, pp.
315–322.