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: bharatambesh@gmail.com

*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.

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