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361
ISSN: 0974 - 0376
KEYWORDS
Proceedings of National Conference on
Harmony with Nature in Context of
Environmental Issues and Challenges of the 21st Century
(HORMONY - 2014)
November 28 - 30, 2014, Udaipur,
organized by
Department of Environmental Sciences,
Faculty of Earth Sciences
M. L. Shukhadia University, Udaipur - 313 001 (Rajasthan)
in association with
National Environmentalists Association, Indiawww.neaindia.org
NSave Nature to Survive
www.theecoscan.in
: Special issue, Vol. VI: 361-368: 2014 AN INTERNATIONAL QUARTERLY JOURNAL OF ENVIRONMENTAL SCIENCES
Bharat Singh Ambesh et al.,
Alternaria spp
Gel Electrophoresis
Morphological variability
MORPHOLOGICAL AND BIOCHEMICAL VARIATION OF SOME
ALTERNARIA SPECIES INFECTED ON DIFFERENT
FLORICULTURAL PLANTS
362
BHARAT SINGH AMBESH, SENPON NGOMLE, TANYA MARAK AND SRIKANTA DAS
Department of Plant Pathology, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia - 741 252, West Bengal
e-mail: [email protected]
*Corresponding author
INTRODUCTION
The genus Alternaria includes nearly 100 species of dematiaceous hyphomycetes,
it causes a range of economically important leaf spot diseases on a variety of fruits,
vegetable, field crops, ornamentals and flowers. Many reports on the existence
of variability among different Alternaria species from different hosts have been
reported by earlier workers (Pryor and Gilbertson, 2002; Pryor and Michailides,
2002; Quayyum et al., 2005; Kumar et al., 2008). The genus is characterized by
the production of dark colored conidia with longitudinal and transverse septa
which is the key taxonomic feature. Within the genus Alternaria species are defined
primarily on conidium characteristics including size, septation, presence and or
size of a beak and pattern of catenation. Because of the diversity of Alternaria
species, several sub-generic groups have been proposed. Alternaria spp. exhibit
considerable morphological plasticity that is dependent on cultural condition of
substrate, temperature, light and humidity. In addition, within any culture, there is
considerable range of variation in conidium morphology in regard to size, shape,
septation, color and ornamentation. In-spite of the diversity in the species and the
capacity of the fungus to infect a wide range of economically important plant, no
comprehensive information about the occurrence of the pathogen in flower plants
are available like tuberose, rose, marigold and gladiolus. The comprehensive
understanding of this causal organism with reference to morphological, cultural
and molecular variability within the four different host cultivars in this region which
can contribute a preliminary idea to develop an effective programme for further
detailed study for future breeding programme.
MATERIALS AND METHODS
Collection and isolation of the fungus: Leaves of different floricultural plants
showing typical symptoms of dark blight lesions were collected and isolated by
following technique below.
The infected leaf along with healthy portions cut into small bits and surface sterilized
using 2% HgCl2
for 30 sec. The bits were washed thoroughly in sterile distilled
water for three times to remove all traces of HgCl2. The surface sterilized leaf were
placed on PDA medium and incubated at 27 ±1ºC for 7 days. Apparently pure
culture developed from PDA slants.
Koch’s Postulates has proved the four floricultural plants tuberose, marigold, rose
and gladiolus were raised in pots (Elwakil et al., 2009; Jain et al., 2005). One month
old plants inoculated with spore 5×10u spores/mL suspension using an atomizer.
Disease symptom was appeared after 10-12 days on identified on external
symptoms.
Morphological studies of the fungus
Media used for maintenance and cultural studies of pathogen 1. Czapek’s Dox
Agar 2. PDA- Potato Dextrose Agar 3. Carrot Agar 4. Oatmeal Agar 5. Potato Carrot
Agar were prepared as suggested by “Ainsworth and Bisby’s Dictionary of the
fungi” by Ainsworth (1961). 20mm of each medium listed above was poured into
90mm diameter Petri plates. After solidification 5mm discs from 5-7 days old cultures
NSave Nature to Survive QUARTERLY
Alternaria spp. is an economically important
pathogen widely distributed throughout the
world and cause devastated disease on
floricultural crops. Variability among isolates
of Alternaria spp. was determined based on
conidial morphology and isozyme variability.
The conidia of Alternaria spp. isolated from
tuberose, rose, marigold and gladiolus varied
in length (7.93-64.30 ìm), breadth (3.23-
17.62ìm), beak size (0.0-10.26ìm), septation
(2-8) within the host species. Their cluster
analysis showed that tuberose and gladiolus
are same cluster, marigold isolate are nearer
to tuberose and gladiolus whereas rose are
different from tuberose, marigold and
gladiolus. Cultural characteristics like colony
size (6-90cm), colour of substrates (whitish-
black), zonation (concentric ring), and growth
characteristics (cottony-fluffy growth) were also
different on different media. The isozyme
tested in native polyacrylamide gel
electrophoresis (PAGE), â- esterase produced
well resolved electrophoretic phenotypes that
could be used as markers for the isolates. In
the similarity matrix derived from hierarchical
cluster analysis indicated that tuberose and
rose isolates and gladiolus and marigold isolates
were closely related to each other through
their isozyme profile bands. So, it was
concluded from the above experiment that
Alternaria spp. isolates infected on tuberose
and gladiolus isolates were to some extent
similar, whereas marigold and rose isolates
were different among the isozyme,
morphological and cultural characters.
ABSTRACT
363
MORPHOLOGICAL AND BIOCHEMICAL VARIATION
of Alternaria spp. were cut by using a cork borer and were
placed at the centre of the plate. Each set of the experiment
was replicated thrice and the plates were incubated at
27±1ºC for 9 days. The colony diameter, color of the colony,
nature of colony margin and the zonation of the colony were
recorded.
Morphological characters of the fungal pathogen infecting
floricultural plants were studied from the culture growth on
Carrot Agar media. The slides of the selected fungal cultures
or colony were prepared in order to study the fungal
morphology such as conidial length, breadth, number of
septations and length of the beak. The prepared slides were
observed under Phase-contrast microscope. The photographs
of the observed conidia are taken and the micrometric
measurements of the conidia are done. Ocular micrometer
was calibrated and by use of micrometry (Meena et al., 2005)
Studies on Iso-enzyme and gel Electrophoresis of Esterase
Iso-enzyme
To study this, different isolates of Alternaria spp. were grown
in 250mL conical flask containing 50mL potato dextrose broth.
Table 1: Morphological characters of different Alternaria spp
Sl. Alternaria No. of No. of No. of Length of Length of Beak length Width of
No. spp on host horizontal vertical oblique conidia with conidia without (μm) conidia (μm)
septation septation septation beak (μm) beak (beak (μm)
1 Tuberose 2-7 0-2 0-1 17.28-64.30 14.44-46.98 00-17.32 4.68-16.49
2 Marigold 2-5 0-3 0 21.45-44.42 16.41-43.76 0.0-10.26 9.40-17.62
3 Rose 1-5 0-1 0-1 7.93-32.30 6.91-26.14 0.0-6.16 3.71-9.48
4 Gladiolus 2-8 0-4 0.0 15.96- 41.16 13.85-36.02 0.0-9.19 3.23-9.43
Table 2: DMRT test of morphological characters of Alternaria spp
Spp. No. of No. of No. of Length of Length of Beak Width of conidia
horizontal vertical oblique conidia with conidia without length (μm) (μm)
septation septation septation beak (μm) beak (μm)
1 4.067 a 0.400 b 0.400 a 28.847 b 24.853 b 4.125 a 7.734 b
2 3.333 b 0.933 a 0.033 b 32.992 a 28.019 a 4.974 a 13.705 a
3 2.333 c 0.200 b 0.167 b 16.741 c 13.328 c 1.724 b 6.609 c
4 4.600 a 0.967 a 0.000 b 27.368 b 23.611 b 3.678 a 6.200 c
Table 3: Cultural characteristics of the isolates of Alternaria spp. from rose on different media
Day Media Colony size Colony colour Mycelial Margin of Zonation
(Diameter) growth colony
(mm)
1day-9day Czapek’s dox 12 -58.66 Whitish- Whitish Cottony- Cottony Roundish No zonation-
colony Concentric black
Zonation
Potato Dextrose Agar 12.33 -90 Whitish-Black mycelia Cottony- Compact Roundish No zonation
Surrounded by whitish growth
Carrot Agar 11.66 -90 Whitish-Black with hyaline Thin Roundish No zonation
margin Mycelium- Cottony
Oat Meal Agar Sep-90 Whitish-White to brownish Thin Roundish No zonation
mycelium - Thin
mycelium
Potato Carrot Agar 11.33 -86.33 Whitish-Blackish with white Cottony - Blackish Roundish No zonation
margin Cottony
Table 4: Cultural characteristics of the isolates of Alternaria tagetus from marigold on different media
Day Media Colony size Colony colour Mycelial Margin of Zonation
(Diameter) growth colony
(mm)
1d-9d Czapek’s dox 10.66 -70.33 Whitish - Whitish Cottony-Fluffy Roundish No zonation- Concentric
colony zonation
Potato Dextrose Agar 8.66 -90 Whitish - Whitish Cottony - Cottony Roundish No zonation - No zonation
Carrot Agar 7.33 -81.33 Whitish -Blackish with Thin Roundish No zonation - No zonation
white margin mycelium - Cottony
Oat Meal Agar 7.66 -80.33 Whitish -Blackish with Thin Roundish No zonation - No zonation
white margin mycelium - Cottony
Potato Carrot Agar Jun-55 Whitish -Whitish with Thin Roundish No zonation- Concentric
hyaline margin mycelium - Cottony zonation
364
BHARAT SINGH AMBESH et al.,
Three flasks were used for each isolate. Each flask was
inoculated with 2 discs each of 5mm diameter cut from the
periphery of actively growing 5 day old culture grown on
PDA. The inoculated flasks were incubated at 28±ºC for 10
days. Electrophoresis of esterase isoenzyme was done in 7.5%
gel according to the method proposed by Kahler and Allard
(1970).
The Rm (Relative mobility) value of band(s) in gel was estimated.
Distance of the band from origin
Rm value =
Distance of buffer front
Estimation of Phenol and total carbohydrate of the fungal mat
was estimated by the method of Mahadevan and Sridhar (1982)
and total sugars were determined by using anthrone reagent
(Hedge and Hofreiter). Prepare a standard curve using different
concentrations of catechol for phenol. Standard curve was
prepared with different concentration of dextrose for sugar.
The color intensity was measured at 630 nm in a
spectrophotometer.
RESULTS AND DISCUSSION
The morphological characters were different among the 30
observations taken within the same host (Table 1). In case of
tuberose, length of conidia with beak varied between 17.28-
64.30ìm, length of conidia without beak varied between 14.44-
46.98ìm, beak length varied between 0.0-17.32ìm, width of
conidia varied between 4.68-11.71ìm. Horizontal septation
varied between 2-7, vertical septation varied between 0-2 and
oblique septation varied between 0-1. The marigold isolate
showed a varied length of conidia with beak varied between
21.45-44.42ìm, length of conidia without beak varied between
16.41-43.76ìm, beak length varied between 0.0-10.26ìm,
width of conidia 9.40-17.71ìm. Similarly, horizontal septation
varied between 2-5, vertical septation varied between 0-3 and
oblique septation 0-1. The rose isolate showed a varied length
of conidia without beak 6.91-26.14ìm, varied beak length
0.0-6.16ìm, varied length of conidia with beak 7.93-32.30ìm,
width of conidia varied between 3.71-9.48μm varied number
of horizontal septation 1-5, vertical septation varied between
0-1 and oblique septation varied between 0-1. Similarly, in
gladiolus, length of conidia with beak varied between 15.96-
41.16ìm, length of conidia without beak varied between 13.85-
36.02ìm, beak length varied between 0.0-9.19ìm, width of
conidia varied between 3.23-9.43ìm. Horizontal septation
varied between 2-8, vertical septation 0-4 and no oblique
septation was observed. So, the result noted that the Alternaria
spp. effect on different host produced different type of
morphological variability. Muthulakhsmi (1990) and Cuervo-
Parra et al. (2011) reported A. alternata produced both beaked
and unbeaked conidia This conidial morphology of Alternaria
spp. isolates in different hosts was in accordance with those
described by Simmons (1952) in rose and Keisuke et al. (2000)
on marigold. However, Kaul and Saxena (1989) also reported
that spore dimensions are not useful in distinguishing the
Alternaria spp. strains. The cluster analysis of morphological
characters showed that tuberose and gladiolus were in similar
cluster and nearer to marigold but rose is in different cluster.
Fungi secure food and energy from substrate upon which
Table 5: Cultural characteristics of the isolates of Alternaria polyanthi from tuberose on different media
Day Media Colony size Colony colour Mycelialgrowth Margin ofcolony Zonation
(Diameter)(mm)
1d-9d Czapek’s dox 7.33 -90 Whitish ThinMycelium-Thin mycelium Roundish No zonation
colony Potato Dextrose Agar 7.66 -90 Whitish Cottony - Fluffy Roundish No zonation
Carrot Agar 8 -90 Whitish Fluffy - Fluffy Roundish No zonation
Oat Meal Agar 7 -90 Whitish Fluffy - Fluffy Roundish No zonation
Potato Carrot Agar 7.33 -90 Whitish Fluffy - Fluffy Roundish No zonation
Table 6: Cultural characteristics of the isolates of Alternaria fasciculatus from gladiolus on different media
Day Media Colony size Colony colour Mycelial Margin of Zonation
(Diameter) growth colony
(mm)
1d-9d Czapek’s dox 8 - 45.66 Whitish -Dull white Cottony Roundish - Irregular with No zonation
colony white margin
Potato Dextrose Agar 14.66 -77.33 Whitish - Whitish Cottony Roundish No zonation
Carrot Agar 12.66 -62.66 Whitish - Blackish Cottony Roundish No zonation
with white
margin
Oat Meal Agar 8.33 -90 Whitish - Whitish Thin mycelium Roundish No zonation
Potato Carrot Agar 6.66 -38 Whitish - Whitish Cottony Roundish No zonation
Table 7: Relative mobility (Rm) values of â- esterase isozyme in different isolates of Alternaria spp.
Sr. No. Isolates Rm value No. of bands
1 Tuberose 0.7185 0.575 0.4625 0.4375 0.375 0.275 6
2 Rose 0.625 0.575 0.4625 0.4375 0.375 0.25 0.1 7
3 Gladiolus 0.4375 0.375 0.275 0.1875 4
4 Marigold 0.4375 0.375 2
365
they live in nature. They furnish nutrient from culture medium
for their growth. All medium are not equally good for all fungi,
nor there can be universal artificial substrate upon which they
grow. Studies of liquid media revealed that Alternaria solani
growth was best on Potato Dextrose Broth followed by
Czapeck’s medium and sporulation was maximum on Potato
1. Tuberose 2. Marigold 3. Rose 4. Gladiolus
Figure 1: Morphological dendrogram of different Alternaria spp
Figure 2: Dendrogram of â-esterase isomers of different isolate of Alternaria spp.
Dextrose Agar (Somappa et al. 2013). So, the cultural
variability also showed different characters among the four
isolates grown on different media like Potato Dextrose agar,
Czapek’s Dox, Carrot agar, Oatmeal agar and Potato Carrot
agar. The size of colony was increased with increase of
incubation period. Rose isolate showed maximum colony
Figure 3: Dendrogram of phenol isomers of different isolate of Alternaria spp.
1. Rose 2. Tuberose 3. Gladiolus 4. Marigold
MORPHOLOGICAL AND BIOCHEMICAL VARIATION
366
Figure 4: Dendrogram of Carbohydrate isomers of different isolate of Alternaria spp.
1. Rose 2. Tuberose 3. Gladiolus 4. Marigold
Czapek’s dox PDA Carrot agar Oat meal agar Potato carrot agarIsolate
Gladiolus
Marigold
Rose
Tuberose
Plate 1: Cultures of Alternaria spp. in different media
BHARAT SINGH AMBESH et al.,
367
TR=Tuberose, R= Rose, G= Gladiolus, MG = Marigold
Plate 2: Isomers of â-esterase isomers of different isolate of Alternaria
spp.
diameter on potato dextrose agar/carrot agar/oat meal agar
(Table 3), marigold isolate on potato dextrose agar (Table 4)
and tuberose isolate produced maximum colony diameter in
all the five media (Table 5). Whereas gladiolus isolate showed
maximum growth on oat meal agar at 9 days after incubation
among the five media (Table 6). Color of the colony showed
different colors on different media particularly whitish, greyish
and brownish which was changed to black with increase in
the age of the fungal cultures in every media. Gladiolus isolate
produced cottony growth in every media with a few
exceptions whereas tuberose produced fluffy growth. Isolates
of rose and marigold produced cottony growth with few
exception of thin mycelium growth of rose and fluffy growth
of marigold on some media few days after incubation. Different
media showed same margin of the colony i.e. roundish of
different isolates. These four Alternaria spp. produces no
zonation in all the media with a few exception or substrate by
the four isolates from 24 hours after incubation (Plate 1).
Generally Alternaria species produce some toxin on the culture
media which was noted by a zonation around the growth of
mycelia of the fungus. To find out any difference in toxin
production on different media zonation of the four isolates
were observed and no zonation was noted within the four
media by the four isolates with a few exception. This result
indicate that the tuberose and gladiolus isolate does not able
to produce any toxin within the five media used upto 9 days
after incubation. Whereas other two isolates i.e. rose and
marigold able to produce toxin on czapek’s dox and potato
carrot agar media within 9 days after incubation.
Relative mobility (Rm) values â-Esterase isozyme in different
isolates of Alternaria spp.In case of â- esterase, tuberose
produces six bands, rose produces seven bands and gladiolus
produces four bands and marigold produces two bands. The
biochemical analysis of phenol shows that the highest amount
of total phenol was found in gladiolus isolate (2.347 mg/g)
followed by tuberose isolate (2.176 mg/g) and rose isolate
(2.101 mg/g) and minimum in marigold isolate (1.924 mg/g)
among the four isolates tested. The biochemical analysis of
carbohydrate showed that the highest amount of carbohydrate
was in tuberose isolate (141.20 mg/g) followed by rose isolate
(132.25 mg/g) and gladiolus isolate (124.5 mg/g) and minimum
in marigold isolate (112.54 mg/g) among the four isolates tested.
The dendrogram of these four isolate produce two clusters in
which tuberose and marigold were similar and both of them
were nearer to gladiolus isolate and rose came in under
separate cluster. So, it can be concluded from this study that
Alternaria spp. of four host cultivars exists high morphological
variability and biochemical variability among the isolates
themselves. So, the direct and reliable technique is analysis of
DNA. It has many strains, quite different to each other in
physiological, ecological and pathological characters.
REFERENCES
Ainsworth, G. C. 1961. Dictionary of Fungi. Common Wealth
Mycological Institute, Kew, Swerry. England, pages. p. 547
Cuervo-Parra, J. A., Sanchez-Lopez, V., Ramirez-Suero, M. and
Ramirez-Lepe, M. 2011. Morphological and molecular
characterization of Moniliophthora roreri causal agent of frosty pod
rot of cocoa tree in Tabasco, Mexico. Plant Pathol. J. 10: 122-127.
Elwakil, M. A., El-Refai, I. M. Awadallah, O. A. El-Metwally, M. A.
and Mohammed 2009. Seed borne pathogens of faba bean in Egypt:
Detection and pathogenicity. Plant Pathol. J. 8: 90-97.
Jain, S., Verma, S. K. and Saha, M. D. 2005. Pathological studies on
Alternaria alternate (Fr.) Keiss. Causing leaf blight of pear. Plant Pathol.
J. 4: 51-53.
Kaul, A. K. and Saxena, H. K. 1988. Physiologic specialization in
Alternaria solani causing early blight of potato I. Cultural and
physiologic variability. Indian J. Mycology and Plant Pathology. 18(2):
128-132.
Keisuke, T., Toyozo, S. and Hiroki, K. 2000. Marigold Leaf Spot
Caused by Alternaria tagetica New to Japan. J. Gen. Plant Pathol. 66:
294-298 .
Kumar, V., Haldar, S., Pandey, K. K., Singh, R. P., Singh, A. K. and
Singh, P. C. 2008. Cultural, morphological, pathogenic and molecular
variability amongst tomato isolates of Alternaria solani in India. World
J. Microbiol. Biotechnol. 24: 1003-1009.
Mahadevan, A. and Sridhar, R. 1982. Methods in physiological plant
pathology. II. Ed. Sivakami Publ. pp. 157- 159.
Meena, P. D., Chattopadhyay, C., Kumar, V. R., Meena, R. L. and
Rana, U. S. 2005. Spore behaviour in atmosphere and trends in
variability of Alternaria brassicae population in India. J. Mycol. Plant
Pathol. 35: 511.
Muthulakhsmi, P. 1990. Studies on fruit rot of chillies (Capsicum
annum L.) caused by Alternaria tenuis Nees, M.Sc. Thesis Tamil
Nadu Agricultural University, Coimbatore, India
Pryor, B. M. and Gilbertson, R. L. 2002. Molecular phylogenetic
relationship amongst Alternaria species and related fungi based upon
analysis of nuclear ITS and mt SSU rDNA sequences. Mycol. Res.
104: 151-160.
Pryor, B. M. and Michailides, T. J. 2002. Morphological, pathogenic
and molecular characterization of Alternaria isolates associated with
Alternaria late blight of Pistachio. Phytopathol. 92: 406-416.
Quayyum, H. A., Dobinson, K. F. and Traquair, J. A. 2005. Conidial
morphology, virulence, molecular characterization, and host-
parasite interactions of selected Alternaria panax isolates on
American ginseng. Can. J. Bot. 83: 1133-1143.
Simmons, S. A. 1952. Alternaria fasciculata (Gooke and Eil.) Jones
MORPHOLOGICAL AND BIOCHEMICAL VARIATION