Journal of Applied Research on Medicinal and Aromatic Plants 14 (2019) 100211

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Journal of Applied Research on Medicinal and Aromatic

Plants
journal homepage: www.elsevier.com/locate/jarmap

Evaluation of ashwagandha (Withania somnifera L.) Dunal accessions and T

breeding lines against leaf spot disease caused by Alternaria alternata under
subtropical condition of India
⁎
Ram P. Meena , Kuldeepsingh A. Kalariya, Parmeshwar L. Saran, Ponnuchamy Manivel
ICAR- Directorate of Medicinal and Aromatic Plants Research, Boriavi, Anand, Gujarat 387310, India

A R T I C LE I N FO A B S T R A C T

Keywords: Withania somnifera L. Dunal, is one of the important medicinal crops and extensively used in the traditional
Withania somnifera medicine systems of India. Leaf spot disease caused by Alternaria alternata is a major concern and adversely affect
Alternaria alternata the commercial cultivation as well as production of crop. The disease symptoms appear around 50 days after
Internal transcribed spacer sowing (DAS) on naturally infected plants while on artificially inoculated plants symptoms developed within 2
Accession
weeks. The associated pathogen was characterized for precise identification based on the sequencing of partial
Disease severity
Temperature
internal transcribed spacer (ITS) and actin gene considering the microscopic observations. The disease is pre-
Humidity vailing more severe at flowering stage, 90 DAS under high temperature and low relative humidity. In the present
study 328 breeding lines and 140 accessions of W. somnifera were procured under breeding program and
screened against leaf spot disease under natural field conditions for three consecutive years. The results of our
study revealed that only 12.85% of accession lines viz., RAS – 36, MWS-207, MWS-211, MWS-303, MWS-304,
RAS -10, RAS – 49, RAS – 50, MWS-336, RAS – 130, IC – 283942, RAS – 60, RAS – 61, RAS – 143, RAS – 133, RAS
– 137, MWS-139 and RAS – 41 and 7.32% of breeding lines viz., DWS-108, DSW-130, DWS-74, DWS-118, DWS-
152, DWS-279, DWS-96, DWS-37, DWS-107, DWS-133, DWS-234, DWS-311,DWS-34, DWS-41, DWS-42, DWS-
69, DWS-86, DWS-127, DWS-170, DWS-223, DWS-293, DWS-270, DWS-296 and DWS-309 were falling under the
highly resistant class. The pathogenesis as well as disease progress mechanisms influenced by environmental
factors and growth stages of crop. Maximum temperature 35 + 5 °C when coincide with low relative humidity
(66 + 5% RH1) supported the aggressiveness of the pathogen.

1. Introduction
Withania somnifera L. Dunal, commonly known as ashwagandha or
Indian ginseng is a well known herbal plant for its therapeutic prop-
erties and immensely used in the Ayurveda, Siddha and Unani systems
of medicines (Chopra et al., 1958; Murthy et al., 2008). This plant
belongs to family Solanaceae and grown widely in tropical and sub-
tropical climate of Africa, Middle East and Mediterranean region of the
world. The roots of the plants contain several bio active principal
compounds which include withaferin-A, withanolides, sterols and
phenols etc., (Bhattacharya et al., 2002). The entire plant as such pos-
sesses important medicinal value, although therapeutic properties of
the roots have been explored immensely against large number of
human as well as animal ailments like tumour (Uma devi, 1996), car-
diac disorders (Mohanty et al., 2008), cancer (Widodo et al., 2007) and
also as anti- aging agent (Singh et al., 2008). In India, the knowledge to
use the medicinal plants in traditional system of health care has been
transmitted orally by different cultural communities and also through
thousands of written scripts. The awareness about the value of natural
products for health care has led to increased demand of ashwagandha
based products. The crop is suitable for growing under warm, tropical
and subtropical climate around the word. In India, ashwagandha is
successfully grown in Madhya Pradesh, Gujarat, Utter Pradesh, Har-
yana, Rajasthan and Punjab (Bhatia et al., 1987).
The crop encounters many pest and diseases in the field condition
especially coleopteran and lepidopteran pest infestation and fungal
disease infection were observed frequently (Verma et al., 2007; Kumar
et al., 2009; Sharma et al., 2011). Among them, leaf spot disease caused
by Alternaria alternata is the most abundantly prevailing (Shivanna
et al., 2014) and significantly affect the secondary metabolites pro-
duction (Pati et al., 2008). The species of genus Alternaria included
mainly saprophytic fungi and collectively causes devastating foliar

⁎
Corresponding author.
E-mail address: rp.meena@icar.gov.in (R.P. Meena).

https://doi.org/10.1016/j.jarmap.2019.100211
Received 3 October 2018; Received in revised form 25 June 2019; Accepted 27 June 2019
Available online 04 July 2019
2214-7861/ © 2019 Elsevier GmbH. All rights reserved.
R.P. Meena, et al. Journal of Applied Research on Medicinal and Aromatic Plants 14 (2019) 100211

Table 1
Description of disease infection type, scoring scale and host reaction.
Host reaction Scoring scale Description of reaction

Highly resistant (HR)
Resistant (R)
Moderately resistant (MR)
Moderately susceptible (MS)
Susceptible (S)
0 Leaves free from infection or 0 to 5% leaf area with lesions
1 Small irregular brown spots with concentric rings covering 6–10% leaf area
2 Lesions enlarging, irregular brown with concentric rings covering 11–25% leaf area
3 Lesions coalesce to form irregular and appears as a typical leaf spot covering 26–50% leaf area
4 Lesions coalesce to form irregular and appears as a typical blight symptom covering > 50% leaf area

diseases on wider host range. Fungicide spraying is one of the common
management practices followed for controlling the disease, but re-
peated use of same group of fungicides leads to development of fun-
gicides resistance as observed in many other pathogens (Golembiewski
et al., 1995). Nevertheless, roots of the plants are food storage reserved
and hence fungicide application may lead to residual toxicity.
Therefore, in the present investigation attempts were made to
identify the resistance source against leaf spot disease from the avail-
able genetic resources of W. somnifera and also to characterize the as-
sociated pathogen. Besides that the epidemiological investigations were
also attempted to assess the growth and development of the disease.
The information generated from this study will be helpful for the
breeders in crop improvement programmes and can mitigate the re-
sidual toxicity by curtailing the use of fungicides.
2. Materials and methods
2.1. Experimental site and cultivation measures
The field study and laboratory experiments were conducted at the
ICAR- Directorate of Medicinal and Aromatic Plants Research (ICAR-
DMAPR), Boriavi, Anand, Gujarat (India) for three consecutive crop
seasons during 2015-16, 2016-17 and 2017-18. The experimental site is
located at 22° 35′ N and 72° 55′ E at an altitude of about 45.1 m above
sea level. The soil was less fertile with sandy loam texture having pH
7.62 and EC 0.27 dS m−1. The crop was sown during first fortnight of
October and the area belongs to subtropical conditions.
2.2. Sample collection and development of pure culture
The infected leaf samples exhibiting disease symptoms were col-
lected from the experimental field of ICAR-DMAPR and kept separately
in aluminum foil pack. Small pieces of the freshly infected leaf were cut
off, surface sterilized with sodium hypochlorite solution (NaOCl) of
4.0% concentration by dipping the leaf bits for 3 min and followed by
three washing with the sterile distilled water. About 2 mm size of the
leaf tissues were transferred for inoculation on the sterile potato dex-
trose agar (PDA) plates using a sterile needle and kept for incubation at
25 + 2 °C in BOD incubator. The mycelia growth of fungus was ob-
served after 48 h of incubation and pure culture of the fungus was re-
trieved following the standard protocol of single spore isolation (Choi
et al., 1999).
2.3. Morphological and molecular characterization
Microscopic slides were prepared by scraping the fungal colonies
from natural substrata as well as 7 days old culture, mounted in cotton
blue and examined under microscope for morphological measurements.
For more precise and molecular characterization, total DNA was iso-
lated from the fresh mycelia grown in PD broth using the commercial
Nucleo-pore gDNA fungal/bacterial mini kit following the manu-
facturer’s instructions. Partial ITS region and Actin gene of A. alternata
were amplified using primers, ITS1 and ITS4 (White et al., 1990) and
ACT 512 F and ACT 783R (Carbone and kohn, 1999), respectively, in
PCR assay. The PCR reaction was performed with a total volume of
20 μl containing 1.5 μl of template DNA, 10.0 μl PCR master mix
(Thermo scientific) 0.5 μl of each 10 mM of forward and reverse primers
and sterile nuclease free water. The following PCR program was per-
formed following one cycle at 94 °C for 4 min, 35 cycles at 94 °C for
35 s, 56 °C for 50 s, and 72 °C for 1 min, followed by final extension at
72⁰C for 10 min. Amplicons were purified using commercial kits and
purified PCR product was sequenced in both the directions. The re-
trieved sequences were assembled and compared via BLAST with the
DNA sequences available in GenBank (http://www.ncbi.nlm.nih.gov/
genbank/).
2.4. Evaluation of accessions and breeding lines
Total 328 breeding lines and 140 accessions lines of W. somnifera
were procured from the breeding program of W. somnifera grown in
augmented randomized block design keeping three replication of each
at the experimental field, ICAR-Directorate of Medicinal and Aromatic
Plants Research, Boriavi, Anand (Gujarat). The experimental material
collected from Madhya Pradesh, Rajasthan, Utter Pradesh and Southern
part of the country. All these accessions and breeding lines were
screened during three consecutive crop seasons of 2015–16, 2016–17
and 2017–18 under the natural epiphytotic conditions. Based on the
pooled data of three years, accessions and breeding lines were cate-
gorised in five host reaction classes as described in Table 1.
2.5. Disease incidence and scoring
Disease incidence and severity on the W. somnifera accessions and
breeding lines were recorded with the standard methods considering
the whole plant as a unit. The variable measures of disease were ob-
served on 10 plants of each accession /breeding lines. The observations
on the infection type and disease severity were recorded at the weekly
intervals. The categorization of accessions and breeding lines for dis-
ease reaction was determined based on the disease severity (Fig. 1)
according to the assigned relative value of infected area under the
disease scoring scale of 0–4 as described in Table 1.
The disease incidence (DI) was calculated using the following for-
mula:
IP
Disease Incidence(DI) = × 100
TP
Where IP: number of infected plants (Plant with the visible leaf spot
disease symptoms), TP: Total number of plants observed for incidence.
Where the disease severity (DS) was calculated using the formula of
Wheeler (1969).
Sum of individual ratings x 100
Disease Severity(DS) =
No. of leaves observed x Maximum disease grade
2.6. Area under disease progressive curve (AUDPC) and statistical analysis
Observations on the disease incidence and disease severity of leaf
spot disease were recorded at weekly intervals. The area under disease
progressive curve (AUDPC) was calculated based on the disease severity
on 10 tagged plants chosen randomly in each plot, under the natural
conditions using the following formula given by Shaner and Finney
(1977).

2
R.P. Meena, et al. Journal of Applied Research on Medicinal and Aromatic Plants 14 (2019) 100211

Fig. 1. Leaf spot disease scoring scale on ashwagandha (W. somnifera) based on the disease severity and host reaction, where: 0- Highly resistant, 1- Resistant, 2-
Moderately resistant, 3- Moderately susceptible and 4- Susceptible.

Fig. 2. Leaf spot disease of W. somnifera (A.) Typical symptoms of Alternaria leaf spot disease (B.) Morphology of the fungal pure culture (C.) Conidia of the Alternaria
alternata observed under the compound microscope and (D.) Gel profile of amplified Actin and ITS region of the pathogen where Lane M; 100bp DNA ladder, 1; Actin
and 2; sowing amplified ITS region.

up to 80% under natural conditions on W. somnifera genetic resources.

AUDPC= ∑ (Yi+ Yi+1) / 2 × (ti+1- ti)

Where, 3.2. Morphological and molecular characterization

Yi = Disease severity at time ti,
Yi+1 = Disease severity at time ti+1
The weekly weather data of temperature and relative humidity were
taken as an average of seven days and simple correlation of the disease
incidence with the environmental factors was performed in SAS soft-
ware (SAS.1.).
3. Results
3.1. Symptoms and pathogenicity
The leaf spot disease symptoms initially appear as minute brown to
dark brown necrotic spots and noticed around 45–50 DAS. The disease
symptoms were characterized by concentric brown spots surrounded
with water soaked yellow halo prominent on the upper surface of the
older leaves. Koch’s postulates for the isolated pathotype were estab-
lished on the W. somnifera plants and initial symptoms of the disease
appeared after 8–9 days on the inoculated plants under control condi-
tions. The percent of disease incidence was 60–90% and severity varied
The fungal mycelia was of dark brown to greyish colour and the
conidia were muriform type, pale to black olivaceous colour with
20–38 × 11–16 μm size. The fungus produced conidia in long branched
chains. The associated causal agent of leaf spot disease on W. somnifera
based on the microscopic observations, cultural and morphological
descriptions was identified as A. alternata (Fig. 2).
The retrieved sequence contigs of the ITS rDNA and partial Actin
gene assembled to generate the consensus sequences. The BLASTN
search revealed that consensus sequences of ITS and Actin gene of the
isolate (AWS-1) showed > 99% similarity with the Alternaria alternata
isolates. The sequences were submitted to NCBI GenBank with the ac-
cession number as MK695680 and MK695681, respectively of the
partial ITS and Actin genes.
3.3. Evaluation of accessions and breeding lines
The host pathogen interaction reactions (Resistant or Susceptible)
were categorised in five classes based on the 0–4 disease scoring scale

3
 R.P. Meena, et al.

Table 2
Categorization of ashwagandha (W. somnifera) accessions/breeding lines against leaf spot disease.
S. No. Reaction Infection type Genetic materials

Accessions (140) Breeding lines (328)

1 0 Highly resistant (HR) RAS – 36, MWS-207, MWS-211, MWS-303, MWS-304, RAS -10, RAS – 49, RAS – 50, MWS-336,
RAS – 130, IC – 283942, RAS – 60, RAS – 61, RAS – 143, RAS – 133, RAS – 137, MWS-139, RAS
– 41 (18)
2 1 Resistant (R) MWS-213, RAS-134, MWS-334, RAS-62, MWS-315, MWS-219, RAS-66, MWS-117, RAS-20,
MWS-333, MWS-335, MWS-124, MWS-130, MWS-132, MWS-135, MWS-218, MWS-323, MWS-
324, MWS-327, MWS-332, RAS-7, RAS-51, RAS-140, RAS – 22, RAS – 59, RAS – 39, MWS-310,
MWS-316, RAS – 34, MWS-210, RAS – 33, RAS – 21, MWS-330, MWS-321, MWS-205, MWS-
131, MWS-204, MWS-221, MWS-222, MWS-227, MWS-302, MWS-309, MWS-314, MWS-322,
JA-20, RAS – 11, RAS – 142, MWS-226, RAS – 13, RAS – 52, JA-134, RAS – 42, RAS – 65, MWS-
206 (54)
3 2 Moderately resistant IC-310595, MWS-201, MWS-114, RAS-37, MWS-108, MWS-141, MWS-306, MWS-329, Red
(MR) Barries, RAS-30, RAS-40, RAS-135, IC-310620-B, MWS-223, RAS-58, RAS-141, MWS-209,
MWS-203, RAS-53, IC-283662, BNM-42, IC-283966, RAS-16, RAS-35, RAS-136, MWS-208,
MWS-319, MWS-214, MWS-216, MWS-217, MWS-312, MWS-318, MWS-328 (33)
DWS-108, DSW-130, DWS-74, DWS-118, DWS-152, DWS-279, DWS-96, DWS-37, DWS-107,
DWS-133, DWS-234, DWS-311,DWS-34, DWS-41, DWS-42, DWS-69, DWS-86, DWS-127,
DWS-170, DWS-223, DWS-293, DWS-270, DWS-296, DWS-309. (24)
DWS-11, DWS-23, DWS-30, DWS-31, DWS-35, DWS-36, DWS-39, DWS-40, DWS-43, DWS-47,
DWS-48, DWS-56, DWS-65, DWS-67, DWS-68, DWS-80, DWS-82, DWS-83, DWS-85, DWS-87,
DWS-88, DWS-89, DWS-90, DWS-95, DWS-97, DWS-98, DWS-104, DWS-106, DWS-109, DWS-
114, DWS-116, DWS-122, DWS-132, DWS-140, DWS-146, DWS-128, DWS-129, DWS-182,
DWS-131, DWS-134, DWS-141, DWS-148, DWS-150, DWS-151, DWS-153, DWS-154, DWS-
155, DWS-158, DWS-163, DWS-84, DWS-169, DWS-183, DWS-184, DWS-185, DWS-186,
DWS-217, DWS-221, DWS-222, DWS-232, DWS-233, DWS-234, DWS-235, DWS-238, DWS-
243, DWS-244, DWS-250, DWS-264, DWS-266, DWS-251, DWS-274, DWS-280, DWS-294,
DWS-318, DWS-325, DWS-326, DWS-327, DWS-328. (77)
DWS-12, DWS-15, DWS-24, DWS-32, DWS-172, DWS-19, DWS-25, DWS-45, DWS-81, DWS-
91, DWS-93, DWS-94, DWS-103, DWS-105, DWS-115, DWS-38, DWS-92, DWS-99, DWS-123,
DWS-126, DWS-190, DWS-13, DWS-9, DWS-17, DWS-21, DWS-276, DWS-70, DWS-100, DWS-
157, DWS-117, DWS-137, DWS-143, DWS-161, DWS-168, DWS-174, DWS-176, DWS-181,

4
DWS-187, DWS-196, DWS-203, DWS-175, DWS-312, DWS-191, DWS-314, DWS-211, DWS-
162, DWS-313, DWS-218, DWS-167, DWS-216, DWS-230, DWS-245, DWS-206, DWS-236,
DWS-219, DWS-220, DWS-224, DWS-231, DWS-237, DWS-246, DWS-247, DWS-248, DWS-
249, DWS-253, DWS-212, DWS-262, DWS-263, DWS-265, DWS-283, DWS-213, DWS-290,
DWS-291, DWS-292, DWS-295, DWS-297, DWS-317, DWS-320, DWS-317, DWS-322, DWS-
324, DWS-10, DWS-29, DWS-53, DWS-54, DWS-66, DWS-77, DWS-110, DWS-135, DWS-156,
DWS-164, DWS-165, DWS-177, DWS-195, DWS-258, DWS-278, DWS-282, DWS-284, DWS-
287, DWS-289, DWS-307, DWS-323, DWS-78, DWS-208, DWS-269, DWS-28, DWS-121, DWS-
147, DWS-189, DWS-194, DWS-18, DWS-111, DWS-180, DWS-241, DWS-27, DWS-73, DWS-
75, DWS-145, DWS-201, DWS-215, DWS-261, DWS-281, DWS-286, DWS-300, DWS-22, DWS-
136, DWS-144, DWS-159, DWS-199, DWS-209, DWS-228, DWS-304, DWS-138, DWS-298,
DWS-321, DWS-49, DWS-59, DWS-305, DWS-26, DWS-101, DWS-210, DWS-226, DWS-242,
DWS-315 (142).
4 3 Moderately susceptible RAS-14, RAS-15, RAS-138, RAS- 67, RAS-29, RAS-23, RAS-44, RAS-45, MWS-313, IC-286632, DWS-225, DWS-310, DWS-188, DWS-239, DWS-8, DWS-44, DWS-20, DWS-260, DWS-142,
(MS) K-86, RAS-55, MWS-101, RAS-139, MWS-202, RAS-56, MWS-307, MWS-100, RAS-47, RAS-64, DWS-197, DWS-302, DWS-214, DWS-252, DWS-160, DWS-149, DWS-6, DWS-113, DWS-120,
RAS-32, MWS-301, MWS-311, RAS-57, RAS-28, IC-310620-A (26) DWS-124, DWS-198, DWS-271, DWS-200, DWS-227, DWS-299, DWS-166, DWS-125, DWS-
179, DWS-204, DWS-229, DWS-254, DWS-277, DWS-301, DWS-193, DWS-55, DWS-102,
DWS-308, DWS-4, DWS-50, DWS-119,DWS-139, DWS-178, DWS-202, DWS-207, DWS-240,
DWS-255, DWS-267, DWS-306, DWS-1, DWS-58, DWS-64, DWS-112, DWS-173, DWS-273,
DWS-275, DWS-51, DWS-46, DWS-268, DWS-288, DWS-62, DWS-63, DWS-79, DWS-272,
DWS-3, DWS-5, DWS-256, DWS-16, DWS-72, DWS-171, DWS-259, DWS-285 (70).
5 4 Susceptible (S) RAS-27, MWS-325, RAS-18, RAS-31, RAS-46, RAS-54, RAS-48, RAS-63, RAS-38 (9) DWS-71, DWS-316, DWS-60, DWS-303, DWS-52, DWS-257, DWS-57, DWS-76, DWS-205,
DWS-7, DWS-61, DWS-33, DWS-2, DWS-192, DWS-14 (15).
Journal of Applied Research on Medicinal and Aromatic Plants 14 (2019) 100211
R.P. Meena, et al. Journal of Applied Research on Medicinal and Aromatic Plants 14 (2019) 100211

Table 3
Frequency distribution of the response to leaf spot disease infection among the accessions/ breeding lines of ashwagandha (W. somnifera).
Response Disease scoring Accessions accession Accessions (%) Breeding lines accession Breeding lines (%)

Highly resistant (HR) 0 18 12.85 24 7.32

Resistant (R) 1 54 38.57 77 23.47
Moderately resistant (MR) 2 33 23.57 142 43.30
Moderately susceptible (MS) 3 26 18.57 70 21.34
Susceptible (S) 4 9 6.42 15 4.57
Total 140 100 328 100
S.Em. ± 0.966 3.03
CV (%) 5.98 8.01

S.Em. ± = standard error; CV = coefficient of variation.

Fig. 3. Effect of different weather factors on development of leaf spot disease of W. somnifera observed under the field condition.

Table 4 Table 5
Correlation values between the leaf spot disease severity on W. somnifera and Relationship of environmental factors with disease severity, AUDPC of leaf spot
the environmental factors. disease development in ashwagandha (W. somnifera).
S. No. Weather factors Correlation coefficient ‘R’ values Ob. Weeks Max T Mini T RH1 (%) RH2 (%) Disease AUDPC
(2 weeks (°C) (°C) severity (%)
1. Maximum temperature 0.87013 after sowing)
2. Minimum temperature 0.78768
3. Relative humidity I −0.67419 1 30 10.4 95.86 40.71 0 0
4. Relative humidity Il −0.83969 2 26.2 9.3 89.86 38.29 0 0
3 26.8 12 81 44.71 0 25.2
4 31.1 13.5 92.57 39.29 7.2 92.57
5 31.4 11.1 90.14 37.14 19.25 156.06
for leaf spot disease in W. somnifera and following the same scale, the
6 28.7 10.2 81.29 29.57 25.34 191.62
328 breeding lines and 140 accessions were evaluated for three con- 7 32.2 16.1 75.71 38.71 29.41 216.82
secutive years (Appendix-1 & 2). In our study the results revealed that 8 33.4 13.8 80.71 26.86 32.54 235.06
only 12.85% accessions viz., RAS-36, MWS-207, MWS-211, MWS-303, 9 36.1 14.1 77.14 23.14 34.62 249.16
MWS-304, RAS-10, RAS-49, RAS-50 & MWS-336 and 7.32% breeding 10 33.6 15.8 71 26.71 36.57 261.97
11 33.9 12.6 68.86 19.57 38.28 281.82
lines viz., DWS-108, DSW-130, DWS-74, DWS-118, DWS-152, DWS-279,
12 37.3 18.8 70.71 25.57 42.24 306.81
DWS-96, DWS-37, DWS-107, DWS-133, DWS-234, DWS-311,DWS-34, 13 40.8 20.8 66 19.57 45.42 336.59
DWS-41, DWS-42, DWS-69, DWS-86, DWS-127, DWS-170, DWS-223, 14 37.3 20.6 88.29 29.29 50.75 –
DWS-293, DWS-270, DWS-296 & DWS-309 accessions fall under the S.Em. 1.11 1.00 2.62 2.21 4.73 –
CV (%) 12.75 26.45 12.19 26.41 68.51 –
highly resistant class. Out of 140 accessions, 6.42% and among the
SD 4.178 3.761 9.81 8.28 17.69 –
breeding lines 4.57% were recorded highly susceptible (Table 2). The
other accessions and breeding lines of W. somnifera fall under resistant, Ob. Weeks = observation week; Max T = maximum temperature; Mini
moderately resistant and moderately susceptible classes as presented in T = minimum temperature; RH1= relative humidity 1; RH2= relative hu-
Table 2 and details of the total accessions is presented in Table 3. midity 2; AUDPC = area under disease progress curve; S.Em. = standard error;
CV = Coefficient of Variation & SD = Standard deviation.
3.4. Etiology and AUDPC
temperature was coincided with the +75% indicating the positive
The effects of various weather parameters were evaluated and dis- correlation with the higher temperature (Tables 4 and 5). AUDPC for
ease development was measured at the weekly interval (Fig. 3). The the disease was also analysed based on the three years pooled data on
progress of leaf spot disease was found to be higher when maximum leaf spot disease severity. The disease severity observation at the con-
temperature of 28 +4 °C and an average 70% or more relative humidity stant interval could identify the progress of disease development. The
(RH) coincided with the vulnerable stage of the plants. The prompt AUDPC value was highest when the maximum temperature 35+5 °C
growth in disease development was recorded when 30+2 °C maximum coincided with the 66+5% RH1 for the leaf spot disease. The disease

5
R.P. Meena, et al.
progress curve changed abruptly at the initial stage of the infection and
gradually increased over the period. When crop reached at the mature
stage, the infected leaves dried up first and following by leaf shedding.
4. Discussion
Leaf spots are the most commonly prevailing diseases in the sola-
neceous crops and imposed major constraints in the production of ve-
getable and oil crops. The genus Alternaria consisting opportunistic
pathogenic species and have recorded to cause leaf spot, blight and
other diseases on over 380 plant host species (Simmons, 1995; Chavan
and Korekar, 2011). The aerial pathogens mainly destroy the active leaf
area and may directly reduce the photosynthetic activities thus redu-
cing the productivity (Balasubrahmanyam and Kolte, 1980). It has been
reported from the previous studies that A. alternata causes leaf spot
disease on W. somnifera (Mishra et al., 2018) but for more precise
identification of the pathogen, ITS and Actin genes were amplified and
sequenced in the present study. The pathogen may affect the quanti-
tative characteristics viz., root biomass as well as secondary metabolites
production in many crops (Carson, 1985; Meena, 2012; Pati et al.,
2008) and also the photosynthesis activities (Sharma et al., 2014). The
pathogen A. alternata produced conidia in branched chain as earlier
illustrated (Simmons, 1995). The pathogenicity of the pathogen proved
in earlier investigation and similar results reported that initial disease
symptoms appeared within 2 weeks on artificially inoculated plants but
in the field conditions the symptoms observed about 45–50 DAS. The
disease incidence was higher under the higher regime of the tempera-
ture and the disease intensity gradually increased with the plant age as
reported in case of the leaf blight disease of mustard (Sinha et al.,
1992). W. somnifera is one of the most important medicinal plants and
leaf spot is one of the imposing factors for production of the crop. The
restriction on use of the chemical fungicides for management of the
diseases in medicinal plants hinders the economical production.
Therefore, resistant breeding is one of the thrust and unexplored area
which significantly reduces the need of chemicals and eliminate the
negative effects. In order to identify the resistant sources in W. somni-
fera against leaf spot disease, 140 accessions and 328 breeding lines
were screened under field conditions in subtropical area of Gujarat. The
standard disease scoring parameters were followed and 18 accessions
and 24 breeding lines were identified as highly resistant where scoring
was < 1, against the leaf spot disease.
It is evident from the study that the pathogen A. alternata showed
aggressiveness under certain circumstances and it was observed that
when the maximum temperature 35+5 °C coincides with the relatively
low relative humidity (66+5% RH1) the progress of the disease de-
velopment was reported higher (Awasthi and Kolte, 1994). Higher
temperature accelerates the sportulation of the fungus and conse-
quently disease development progressed with higher rate. It was ob-
served in the earlier studies, where 28+2 °C temperature accelerate the
sporulation of Alternaria porri (Meena, 2012) which was negatively
correlated with the increasing relative humidity and higher sugar
content (Bashi and Rotem, 1976). In another study the maximum
temperature was highly positively correlated while minimum tem-
perature and high relative humidity were negatively correlated with the
leaf spot severity in mustard (Meena et al., 2011; Sangeetha and
Siddaramaiah, 2007).
5. Conclusions
In conclusion, screened accessions and breeding lines against leaf
spot of W. somnifera provided the source of resistance for breeding
programmes to develop high yielding varieties with disease resistance
for the crop growing areas. The maximum temperature 28 +4 °C and an
average of 70% or more relative humidity (RH) coincided with the
vulnerable stage of the plants favoured the progress of disease devel-
opment. The resistant cultivars provided scope of minimising the use of
6
Journal of Applied Research on Medicinal and Aromatic Plants 14 (2019) 100211
the chemical fungicides in management of the diseases which will help
in mitigating the residual and negative effects on the health of end
users.
Ethical approval
This article does not contain any studies with human participants or
animals performed by any of the authors.
Funding
The author (s) received the financial support from its institutional
research grant of ICAR- DMAPR, Anand for the research work.
Acknowledgments
The work has been funded by ICAR- Directorate of Medicinal and
Aromatic Plants Research (DMAPR), Anand, from its institutional re-
search grant. The authors express their sincere thanks and gratitude to
the Director, ICAR-DMAPR, for extending research facilities for con-
ducting the present study.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jarmap.2019.100211.
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