Performance variation in growth and sporulation of isolates of Alternaria alternata Fr. Keissler causing blight disease in Mulberry

Abstract

Mulberry Alternaria blight (Alternaria alternata) is one of the foliar devastating diseases and reduces leaf quality and yield. Studying the cultural characters of the pathogen is the first and most important basic step in identification process. The results of cultural studies revealed that PDA was the best medium for supporting the good radial growth of the isolate Aa-1, Aa-2, Aa-4 and Aa-7 with the maximum radial growth of 90.00 mm after ten days of inoculation. Whereas, isolate Aa-2 and Aa-6 yielded maximum growth of 90 mm in Sabouraud's dextrose agar medium and isolate Aa-8 and Aa-9 in oat meal agar medium. Maximum growth (90 mm) in corn meal agar (Aa-7) and in Richards synthetic agar medium isolate Aa-4, Aa-6 and Aa-10 recorded good growth. Dry weight of mycelium of the different isolates A. alternata studies revealed that isolate Aa-3 showed maximum dry mycelial weight of the fungus noticed in Host leaf extract broth (998.71 mg) which was followed by Isolate Aa-2 (997.93 mg) and Isolate Aa-5 (997.4mg) in Sabouraud's dextrose broth. Very good sporulation of the Isolate Aa-1 on Potato dextrose broth, Host leaf extracts broth, Oat meal agar broth and Corn meal broth was recorded. The Potato broth also supported good sporulation of three isolates viz: Aa-1, Aa-5 and Aa-9 whereas, by Host leaf extract broth and corn meal broth supported good sporulation of Isolate Aa-4 and Aa-5 respectively.

Full text

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International Journal of Chemical Studies 2020; 8(6): 1096-1099
P-ISSN: 2349–8528
E-ISSN: 2321–4902
www.chemijournal.com
IJCS 2020; 8(6): 1096-1099
© 2020 IJCS
Received: 22-08-2020
Accepted: 10-10-2020
SE Manjunatha
Ph. D. Scholar, College of
Agriculture, (UAS Dharwad),
Dharwad, Karnataka, India
Rajegowda
Senior Scientist and Head, KVK,
Kandali, (UAS-Bangalore),
Hassan, Karnataka, India
M Raju
Professor of Sericulture, College
of Agriculture, (UAS-Bangalore),
V.C. Farm, Mandya, Karnataka,
India
N Kiran Kumar
Associate Professor, Department
of Plant Pathology, College of
Agriculture, (UAS-Bangalore),
V.C. Farm, Mandya, Karnataka,
India
VB Sanath Kumar
Professor, Department of Plant
Pathology, College of
Agriculture, (UAS-Bangalore),
V.C. Farm, Mandya, Karnataka,
India
KR Ashoka
Assistant Professor, Department
of Soil Science and Agricultural
Chemistry, College of Agriculture
(UAS-Bangalore), V.C. Farm
Mandya, Karnataka, India
Corresponding Author:
VB Sanath Kumar
Professor, Department of Plant
Pathology, College of
Agriculture, (UAS-Bangalore),
V.C. Farm, Mandya, Karnataka,
India
Performance variation in growth and sporulation
of isolates of Alternaria alternata Fr. Keissler
causing blight disease in Mulberry
SE Manjunatha, Rajegowda, M Raju, N Kiran Kumar, VB Sanath Kumar
and KR Ashoka
DOI: https://doi.org/10.22271/chemi.2020.v8.i6p.10907
Abstract
Mulberry Alternaria blight (Alternaria alternata) is one of the foliar devastating diseases and reduces leaf
quality and yield. Studying the cultural characters of the pathogen is the first and most important basic
step in identification process. The results of cultural studies revealed that PDA was the best medium for
supporting the good radial growth of the isolate Aa-1, Aa-2, Aa-4 and Aa-7 with the maximum radial
growth of 90.00 mm after ten days of inoculation. Whereas, isolate Aa-2 and Aa-6 yielded maximum
growth of 90 mm in Sabouraud’s dextrose agar medium and isolate Aa-8 and Aa-9 in oat meal agar
medium. Maximum growth (90 mm) in corn meal agar (Aa-7) and in Richards synthetic agar medium
isolate Aa-4, Aa-6 and Aa-10 recorded good growth. Dry weight of mycelium of the different isolates A.
alternata studies revealed that isolate Aa-3 showed maximum dry mycelial weight of the fungus noticed
in Host leaf extract broth (998.71 mg) which was followed by Isolate Aa-2 (997.93 mg) and Isolate Aa-5
(997.4mg) in Sabouraud’s dextrose broth. Very good sporulation of the Isolate Aa-1 on Potato dextrose
broth, Host leaf extracts broth, Oat meal agar broth and Corn meal broth was recorded. The Potato broth
also supported good sporulation of three isolates viz: Aa-1, Aa-5 and Aa-9 whereas, by Host leaf extract
broth and corn meal broth supported good sporulation of Isolate Aa-4 and Aa-5 respectively.
Keywords: Alternaria blight, Alternaria alternate, solid media, liquid media, cultural variability
Introduction
Mulberry (Morus alba) is one of the commercial perennial plant grown in topical India. The
healthy leaves of mulberry are the only food source for silkworm (Bombyx mori L.) and it
belongs to the family Moraceae. It is cultivated in both tropical and temperate countries of the
world. Though mulberry cultivation is practiced in various climates in India, it is extensively
grown in the tropical zone covering Karnataka, Andhra Pradesh and Tamil Nadu states with
about 90 percent of area where, most of the sericulture industry is concentrated.
Healthy mulberry leaves are the one of the chief source of morine protein for Silk worm and
leaf blight incited by Alternaria alternata adversely affects the protein content and yield of
quality leaves. It manifests by the appearance of burning at leaf tip initially and spread towards
the middle which later become brownish-black. Infected leaves dries up within a week and
ultimately mulberry growth affected, which lead to poor leaf quality and decrease the leaf
yield. The disease also affects the seedling by causing seedling blight. The fungus belongs to
the Class Deuteromycetes. The spores of A. alternata are five to six horizontal septa with two
to three vertical septa and pigmented and they are produced in chains or branching chains. The
spores have a distinctive appearance that makes them easy to recognize. They are broadest
near the base and taper gradually to a short beak. The disease is common in southern part of
India during Kharif season. The disease cause severe defoliation and mortality during winter
months (Gunasekhar and Govindaiah, 1990-91) [2].
Since the information on the growth and sporulation of the fungus on different nutritional
sources is scanty, present study was undertaken to know the variation among different isolates
of Alternaria alternata in mulberry growing areas of Mandya district with respect to growth
parameters on various culture media.

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Materials and methods
The study was conducted to assess the growth variability of F.
verticillioides isolates on different media during 2017 at
Department of Plant Pathology, College of Agriculture, V.C.
Farm, Mandya.
Isolation of the different isolates of Alternaria alternata
Different isolates of Alternaria alternata leaf blight of
mulberry samples were collected from ten different locations
viz: Malavally (Aa-1), Channapatna (Aa-2), Ramanagara (Aa-
3), Magadi (Aa-4), Madduru (Aa-5), Mandya (Aa-6),
Nagamangala (Aa-7), Krishna raja Pete (Aa-8), Sri ranga
Patna (Aa-9) and Mysore (Aa-10) and the isolate name was
given accordingly. The isolation was done by standard tissue
isolation method. The Pathogen was identified by comparing
their morphological characters of vegetative and reproductive
structures on potato dextrose agar Petri plates with the
standard descriptions described by Leslie and Summerell
(2006) [3].
Growth studies of A. alternata isolates on different solid
and liquid media
The eight solid media viz; Potato dextrose agar, Host Leaf
Extract Agar, Sabouraud's dextrose agar, Takahashii's
medium, Oat meal agar, Corn meal agar, Richard's synthetic
agar and Yeast extract agar were prepared. 5 mm disc from
seven day old culture of each A. alternata isolates were placed
in the centre of Petri plates containing test media and were
incubated at 28 ± 1º C for eight days and radial growth was
measured. Similarly the same eight liquid media were used to
test the growth variability of the isolates. A 100 ml of each
respective prepared broth contained in 250 ml conical flasks
was inoculated with 5 mm disc of each isolate and incubated
at 28 ± 1º C for eight days. Later, the broth was filtered using
12.5 cm diameter Whatman No 1 filter paper discs. The
mycelial mat along with the filter paper was dried at 60ºC in a
hot air oven. Finally, the weight of the harvested mycelia mat
and sporulation were recorded. Average of dry mycelial
weight of the three treatments was calculated to compare the
effect of different liquid broth on mycelia dry weight of
fungus.
Sporulation studies of A. alternate isolates on different
media
Sporulation was estimated by dissolving the eight days old 5
mm disc of pathogen culture grown on different media, in 10
ml of distilled water and shaken well to obtain the spores in
solution. The spores were counted with the help of compound
microscope under 10x magnification. The results were
expressed as very good, good, fair, poor and no sporulation on
the basis of the following scale as mentioned in Table 1.
Table 1: Details of expression of sporulation of mulberry blight
pathogen
Sporulation
Representation
No of conidia/10x microscopic field
Very good
++++
>40
Good
+++
26-40
fair
++
11-25
Poor
+
1-10
No
-
0
Results and discussion
Effect of different solid media on the mycelial radial
growth of Mulberry blight pathogen (A. alternata)
The results of cultural studies revealed that PDA was the best
medium for supporting the good radial growth of the isolate
Aa-1, Aa-2, Aa-4 and Aa-7 with the maximum radial growth
of 90.00 mm after ten days of inoculation. Whereas, isolate
Aa-2 and Aa-6 yielded maximum growth of 90 mm in
Sabouraud’s dextrose agar medium. Isolate Aa-8 and Aa-9 in
oat meal agar medium. Maximum growth (90 mm) in corn
meal agar ( Aa-7) and in Richards synthetic agar medium Aa-
4, Aa-6 and Aa-10 was recorded, which was on par with
isolate Aa-1(89.67 mm), Aa-9 (88.93) and Aa-8 (88.58 mm)
in Richards synthetic medium followed by Aa-10 in Oat meal
agar ( 89. mm), Aa-3 in (88.44) Potato dextrose agar medium.
Whereas least growth of isolate Aa-1was shown on Corn meal
agar (64.46 mm) which was followed by Isolate Aa-2 (76.11),
Aa-3 (71.67) in Takahashii,s medium (Table 2). The results of
the cultural studies on A. alternata causing leaf blight of
turmeric showed that among 10 solid media evaluated, radial
growth of A. alternata was maximum on potato dextrose and
Richards's agar, while maximum sporulation was observed in
host leaf extract agar (Gorawar et al. 2006) [1].
Nagrale et al. (2013) [5] observed that the fungus A. alternata
causing fungal blight in gerbera, produced profuse mycelium
on potato dextrose agar (PDA) with an average width of 4.42
µm in diameter, conidiophores, conidia and intercalary
chlamydospores measured as 42.26 x 4.29 µm, 47.16 x 13.49
µm and 7.22 µm in diameter, respectively. The synthetic
media viz., Leonions’s agar, Glucose-peptone agar and
Sabourand’s agar and non-synthetic media, Oat meal agar and
PDA were excellent for the mycelial growth and conidial
production of A. alternata. These results were in agreement
with findings of Poornima and Hegde (2014) [6] wherein they
opined that PDA and Oat meal agar media were the best
media for the growth and sporulation of Cercospora beticola
Sacc. These results were in validation with the findings of
Poornima (2010) [7] wherein she reported that PDA and Oat
meal agar supported the growth of C. betiocola.
.
Effect of different liquid media on the dry mycelial weight
of Mulberry blight pathogen ( A. alternata )
Dry weight of mycelium of the different isolates of mulberry
blight pathogen ( A. alternata) was studied in eight different
broth media to choose the best broth medium that would
support maximum growth. The average dry mycelial weight
of the fungus was recorded, ten days after inoculation (Table
3). The results of cultural studies revealed that isolate Aa3
showed maximum dry mycelial weight of the fungus as
evaluated in Host leaf extract broth (998.71 mg) which was
followed by Isolate Aa2 (997.93 mg) and Isolate Aa-5
(997.4mg) in Sabouraud’s dextrose broth.
Isolate Aa-4 (997.33mg) and Isolate Aa-5 (997.49mg) in oat
meal broth and isolate Aa-7 (996.44mg) in Yeast Extract
broth. Remaining all other isolates documented dry mycelial
weight was on par with each other. Whereas, lowest dry
weight of mycelia was in isolate Aa-8 (757.17mg) on Host
Leaf extract broth which was followed by isolate Aa-9
(758.30 mg) on Richard’s synthetic broth medium
The findings of Reddy et al., 2019 are in line with the various
culture media tested that revealed preference of A. alternata
to PDB exhibit black to brownish colour colony, maximum
growth with excellent sporulation, with large sized conidia
(42.15 - 47.16 μm). These results were contradictory with the
findings of Shranamma (2012), [11] wherein she reported that
highest mean dry mycelial weight was obtained in Richards
synthetic broth and none of the media favoured sporulation of
Cercoapora musae.
Sporulation of the different isolates of Mulberry blight
pathogen A. alternata
To understand good supporting media for sporulation of A.
alternata a study was carried out on different broth media and

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International Journal of Chemical Studies http://www.chemijournal.com
the results are presented in the table 4. A very good
sporulation of the Isolate Aa-1 on Potato dextrose broth, Host
leaf extracts broth, Oat meal agar broth and Corn meal broth
was recorded. The Potato dextrose broth also supported good
sporulation of three isolates viz; Aa-1, Aa-5 and Aa-9.
Host leaf extract broth and corn meal broth supported good
sporulation of Isolate Aa-4 and Aa-5 respectively. Whereas,
Takahashis broth poorly supported and just sporulation was
observed in Isolate Aa-4, Aa-8 and Aa-10 and remaining all
other isolates no sporulation was recorded. Similar trend was
observed on Richard’s synthetic broth, a good sporulation of
isolate Aa-10, Aa-1 and Aa-5 and remaining other isolates
with no sporulation on the above said media. Finally on Yeast
extract broth all isolates produced no sporulation except
isolate Ac-9 and Ac-10 which were poor sporulated. Findings
of Ramjegathesh and Ebenezar (2012) [9], revealed that the
isolates produced light brown conidia with muriform shape,
but the isolates varied in the size (length, width and beak
length), abundance of the conidia, the number of cells per
conidium and sporulation times.
It was found that mycotoxin production and spore formation
of A. alternata are regulated by light in opposite ways.
Whereas spore formation was largely decreased under light
conditions, the production of AOH was stimulated 2- to 3-fold
(Pruss et al., 2014) [8]. Similar results were found with the
findings of Reddikumar et al. (2015) [10] wherein they
observed varied coloured colonies of A. caricae on PDA and
Cooks No.2 media with varied pigmentation and excellent
sporulation on PDA medium. These results were not in tune
with the results of Sharanamma (2012) [11] wherein there was
no sporulation of Cercospora musae in PDA medium.
Sporulation of A. alternata f. sp. sphenocleae using the
sporulation medium (S-medium) technique was rapid.
Conidia were produced within 24 h and continuous
sporulation was still observed until 120 h. The best primary
agar media for conidia production were PDA, 1/2 PDA and
VJA, while water agar was the poorest of A. alternata f. sp.
sphenocleae conidia (Masangkay et al., 2000) [4].
Table 2: Radial growth of isolates of A. alternata on different solid media
Sl. No.
Solid media
Radial growth (mm) (8-DAI)
ISOLATES
Aa-1
Aa-2
Aa-3
Aa-4
Aa-5
Aa-6
Aa-7
Aa-8
Aa-9
Aa-10
1.
Potato Dextrose Agar
90.00
90.00
88.44
81.51
90.00
81.00
90.00
81.11
85.83
78.65
2.
Host Leaf Extract Agar
82.83
81.67
84.87
87.33
84.74
76.67
87.50
85.64
80.33
86.54
3.
Sabouraud’s Dextrose Agar
87.62
90.00
81.33
82.77
88.31
90.00
82.11
84.48
76.19
82.54
4.
Takahashii's Medium
77.96
76.11
71.67
82.19
84.64
75.38
87.55
81.62
88.29
80.00
5.
Oat Meal Agar
84.22
87.79
82.91
88.11
85.84
85.87
85.20
90.00
90.00
89.00
6.
Corn Meal Agar
64.46
81.13
77.31
86.14
88.43
84.74
90.00
88.28
75.86
85.50
7.
Richard's Synthetic Agar
89.67
87.57
88.00
90.00
85.94
90.00
86.20
88.58
88.93
90.00
8.
Yeast Extract Agar
86.09
81.89
85.51
85.80
84.64
80.34
86.80
87.69
88.50
85.56
F
**
**
**
**
**
**
**
**
SE m±
1.45
1.57
1.62
1.61
1.44
1.6
0.91
1.11
1.19
0.93
CD @ P=0.01
6.10
6.61
6.83
6.8
6.04
7.03
3.83
4.69
5.01
3.92
** Significant at 1% level DAI- Days after Incubation
Table 3: Dry mycelial weight of isolates of A. alternata in different liquid media
Sl. No.
Liquid media
Dry mycelial weight (mg) (8-DAI)
ISOLATES
Aa1
Aa2
Aa3
Aa4
Aa5
Aa6
Aa7
Aa8
Aa9
Aa10
1
Potato Dextrose broth
980.61
959.02
950.58
948.00
856.00
960.00
928.21
967.10
890.11
851.22
2
Host Leaf Extract broth
880.44
785.52
998.71
955.43
848.00
789.00
800.19
757.17
889.08
865.18
3
Sabouroud’s Dextrose broth
896.50
997.93
820.77
896.03
997.41
982.18
936.11
852.07
987.18
950.41
4
Takahashii's broth
997.28
854.50
992.33
980.07
980.00
886.00
951.08
977.15
897.81
866.11
5
Oat Meal broth
884.41
867.44
988.27
997.33
997.49
986.33
987.03
969.03
882.12
954.22
6
Corn Meal broth
964.43
931.17
994.33
895.17
841.83
867.74
951.13
987.87
855.18
984.26
7
Richard's Synthetic broth
988.33
887.04
929.67
986.25
870.10
785.18
955.10
968.27
758.30
897.07
8
Yeast Extract broth
930.00
988.87
981.33
956.28
984.11
901.07
996.44
856.00
797.74
805.21
F
**
**
**
**
**
**
**
**
**
**
SE m±
1.06
0.80
1.79
0.93
1.09
0.96
0.83
0.74
0.71
0.47
CD @ P=0.01
4.45
3.37
7.57
3.92
4.57
4.02
3.52
3.12
2.97
2.0
** Significant at 1% level; DAI- Days after Incubation
Table 4: Sporulation of isolates of A. alternata on different liquid media
Sl. No.
Liquid media
Sporulation (8 DAI)
ISOLATES
AC1
AC2
AC3
AC4
AC5
AC6
AC7
AC8
AC9
AC10
1
Potato Dextrose broth
+++
++
+
+
+++
++
+
++
+++
+
2
Host Leaf Extract broth
+++
+
+
+++
+
+
+
+
++
+
3
Sabouroud’s Dextrose broth
+
+
++
+
_
_
+
+
++
+
4
Takahashii's broth
_
_
_
+
_
_
_
+
-
+
5
Oat Meal broth
+++
+
+
+
+
+
+
+
-
-
6
Corn Meal broth
+++
+
+
+
+++
_
+
+
+
++
7
Richard's Synthetic broth
+
_
_
_
+
_
_
_
-
++
8
Yeast Extract broth
_
_
_
_
_
_
_
_
+
+
DAI- Days after Incubation
Poor sporulation (+) (1-10 conidia); Fair sporulation (++) (11-25 conidia); Good sporulation (+++) (26-40 conidia); Very good
sporulation (++++) (>40 conidia); No sporulation (-); Number of conidia per microscopic field under 10x considered for
categorization

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Conclusion
The variation was noticed among all the isolates of A.
alternata in present study with regard to mycelia growth and
amount of sporulation on different culture media tested.
Majority of the isolates showed better mycelia growth and
sporulation in Potato dextrose medium and Oat meal medium
with variations. The present findings also deciphered that
these two media are better for obtaining rich mycelia growth
of A. alternata and could aide in molecular studies. Further,
the variation among the isolates needs to be authenticated by
virulence and molecular studies.
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