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Pathogenic variability of Alternaria spp. isolates causing leaf blight of cotton.

Cotton is an important cash crop playing a significant role in the economy of the major developing countries including India. It is known as the 'King of fiber' and 'White Gold'. India has been recognized as the cradle of cotton industry and is the original home of domestication, diversification and development of particularly Asiatic cultivated cottons.. In India, leaf spot of cotton (Alternaria macrospora Zimm,) was reported for the first time by Uppal et al. (1935) which is a major contributing factor from the recent past for low productivity of cotton in Karnataka. The fungus derives food and energy from the substrate upon which they grow in nature. In order to culture the fungus in the laboratory, there is no universal substrate or artificial medium upon which all the fungi can grow and reproduce. Hence the present study was carried out to identify surface medium for the growth and sporulation by using different solid media.

MATERIALS AND METHODS

Leaves were collected from infected fields and used for isolation of the fungus in vitro. The isolation of the fungus was made by following standard tissue isolation technique. Identification of the fungus was carried out based on the morphological characters of the isolated fungus. Selection of basal medium for growth and sporulation of the fungus was done by using potato dextrose agar and studied morphological characters like length and width of conidia, number of horizontal and vertical septa and beak length were measured under 40x using Differential Image Contrast microscope. Later the measurements were compared with the standard descriptions given by Ellis (1971) regarding Alternaria macrospora and Alternaria alternata for identification of Alternaria spp. Later cultural study was carried out by inoculating the pathogen on Potato Dextrose Broth and dry mycelial weight was recorded at regular intervals inorder to know the number of days required for maximum growth of the fungus. The isolates were grown on 8 different solid media to select best media for growth viz., Potato Dextrose Agar (PDA), Potato Carrot Agar (PCA), Czapek's Dox Agar (CDA), Host Extract Agar (HEA), Oat Meal Agar (OMA), Corn Meal Agar (CMA), Sabouraud's -Dextrose Agar (SDA) and V8 Juice Agar (V8JA) and to find the difference in colony characters such as radial growth, type of colony margin, colour of margin, mycelial growth, sectoring and sporulation. The best media was found and used as a basal media for further studies. Radial growth (mm) = (length + breadth of grown mycelium)/2.

RESULTS AND DISCUSSION

Isolates collected during survey viz., Karlakatti ([A.sub.1]), Amminbhavi ([A.sub.2]), Yamkanmardi ([A.sub.3]), Marewada ([A.sub.4]), Unkal ([A.sub.5]), Chandanamatti ([A.sub.6]), Haveri ([A.sub.7]), Saundatti ([A.sub.8]), Gadag ([A.sub.9]) and Jagalur ([A.sub.10]) were isolated and pure culture was maintained and were stored in the refrigerator at 5[degrees]C for further studies. The isolates subjected to various morphological variability tests showed that, conidia were septated by 1-2 vertical and 4-6 horizontal septa (Figure 1). The isolates, [A.sub.3], [A.sub.6] and [A.sub.10] showed maximum horizontal septa of 6 followed by 5 horizontal septa in isolates, A1 and [A.sub.5]. Whereas minimum horizontal septa (4) was observed in the isolates, A4 and A8. The isolates, [A.sub.5] and [A.sub.8] showed maximum of 2 vertical septa and isolates, [A.sub.1], [A.sub.3] [A.sub.4] [A.sub.6] and [A.sub.10] showed minimum of 1 vertical septa. The isolates, [A.sub.1] [A.sub.6] and [A.sub.4] showed maximum size of 49.38 x 12.82 mm, 48.24 x 15.79 mm and 47.95 x 15.26 mm, respectively. The least size of the conidia (21.97x 13.02 mm) was observed in isolate, [A.sub.8] (Table 1a). By comparing withAlternaria macrospora structural figure described by Ellis M.B. revealed that out of 7 isolates, only two isolates viz., [A.sub.5] and A8 showed complete resemblance with Alternaria macrospora and other isolates viz., [A.sub.3] [A.sub.3], [A.sub.4] [A.sub.6] and [A.sub.10] showed no resemblance with Alternaria macrospora morphologically.

Results revealed that isolate [A.sub.2] showed 6 horizontal and 1 vertical septa, whereas isolates, [A.sub.7] and [A.sub.9] showed 5 horizontal and 2 vertical septa (Table 1b). Maximum conidial size (33.29 x 12.54 mm) was observed in isolate, [A.sub.2], whereas isolate, [A.sub.7] showed minimum conidial size of 21.5 x 6.87 mm. Isolate, [A.sub.9] showed conidial size of 30.22 x 7.4 mm. When the isolates were compared with Alternaria alternata structural figure described by Ellis M.B., all the three isolates viz., [A.sub.2] [A.sub.7] and [A.sub.9] showed complete resemblance morphologically.

Conidiophores of Alternaria macrospora arise singly or in groups, straight or flexuous, tapering towards the apex and septate. They are pale brown in colour, 4-9 [micro]m thick and upto 180 [micro]m in length. Conidia are solitary or in chains of two, straight or curved with the body of the conidium ellipsoidal tapering to a narrow beak and equal in length or up to twice as long as body. They are reddish brown in colour with four to nine transverse septa and several longitudinal septa (Ellis, 1971). Several attempts are made to classify Alternaria genera, several re-descriptions and revised criteria of these genera (Joly, 1964) resulted in a growing number of new species.

There was significant difference among the incubation periods. The dry mycelial weight of Alternaria macrospora gradually increased (36.53 mg) from third day of inoculation and reached maximum (287.31 mg) on sixteenth day. The data showed a declining trend from eighteenth day (275.90 mg) to thirtieth day (192.74 mg) (Table 2). The isolates exhibited variability in cultural characters when grown on 8 different solid media (Figure 2). Among ten isolates, [A.sub.4] and [A.sub.9] showed maximum radial growth (90 mm) on many of the media viz., PDA, PCA, HEA, OMA and SDA tested. Whereas, Isolate, [A.sub.2] showed least mean radial growth (61.98 mm).

Among the eight solid media, HEA (90 mm) and OMA (89.25 mm) showed maximum radial growth in all isolates. Majority of the isolates showed moderate to excellent sporulation (Table 3). SDA (80.43 mm) and PCA (79.17 mm) were on par with each other. Whereas, PDA recorded mean radial growth of 77.49 mm and V8JA (43.81 mm) showed least radial growth.

The isolates grown on different media showed varied colony characters (Table 4). [A.sub.1] [A.sub.2] [A.sub.5] [A.sub.7] and [A.sub.9] isolates showed grey colony on most of the media, whereas isolates, [A.sub.3] [A.sub.6] [A.sub.8] and [A.sub.10] showed grey and black colour colonies. The colony margin varied from grey to black in all isolates. Irregular margin was seen predominantly in the isolates viz., [A.sub.1] [A.sub.3] [A.sub.5] [A.sub.6] [A.sub.7] and [A.sub.8], whereas isolates, [A.sub.9] and [A.sub.10] showed smooth margin among the media tested. Several workers observed diversity in cultural characteristics such as growth rate, type of growth, colony colour and sporulation among different isolates of Alternaria spp. infecting sesame, sunflower and cotton (Ramegowda, 2007).

CONCLUSIONS

Morphological variability study of ten isolates revealed that, two resembled with Alternaria macrospora, five showed no resemblance with Alternaria macrospora and three isolates resembled with Alternaria alternata morphologically. Pathogen required sixteen days to attain maximum dry mycelial weight. Among the ten isolates, [A.sub.6] recorded maximum mean radial growth (90 mm) on the media. Among the eight media tested, HEA and OMA showed maximum radial growth in all the ten isolates. Majority of the isolates showed moderate to excellent sporulation with flat to raised, irregular to smooth grey colonies with presence or absence of sectoring. Though several cotton varieties and hybrids are being released from time to time, none of them has shown complete resistance to the disease. This indicates the existence of variability among the pathogens which may be attributed to weather conditions of particular location, varieties and hybrids and ability of pathogen to adopt themselves to various situation. Once genus is narrowed by morphology, symptomatology and host-specificity, then it can be used to differentiate species (Chakrabarty et al., 2007). Therefore, study of variability among the isolates will be helpful for designing Integrated Disease Management strategies.

REFERENCES

(1.) Uppal, B. N., Patel, M. K., Kamat, M. M. The fungi of Bombay Dept. of Agric., Bull. No. 176, 1935; 28.

(2.) Ellis, M. B. Dematiaceous Hypomycetes, Commonwealth mycological institute, Kew, Surrey, England, 1971; 495-496.

(3.) Joly, P. Le genre Alternaria. Encyclopediemycologique XXXIII, P. Lechevalier, Paris, France, 1964.

(4.) Ramegowda, G. Disease scenario in Bt cotton with special reference to Alternaria leaf spot. M. Sc. (Agri.) Thesis, Univ. Agric. Sci., Dharwad, Karnataka (India), 2007.

(5.) Chakrabarty, P. K., Chavhan, R. L., Sable, S. V., Narwade, A. V., Monga, D. and Khadi, B. M. Development of sensitive molecular diagnostic tools for detection of economically important fungal pathogens of cotton. Invited paper presented In: World Cotton Res. Conf.-4, 2007.

K.D. Sangeetha [1], S.A. Ashtaputre [1], G.H. Anil [1], G.R. Guru Prasad [2] and T.S. Ramya [1]

[1] Department of Plant Pathology, University of Agricultural Sciences, Dharwad--580 005, India.

[2] Department of Plant Pathology, University of Agricultural Sciences, Raichur 584 101, Karnataka, India.

(Received: 13 June 2015; accepted: 19 September 2015)

* To whom all correspondence should be addressed. Tel.: +91-8904607157; E-mail: sangu931@gmail.com
Table 1(a). Morphological variability of isolates of Alternaria spp

Name of the      Number of      Number         Size of         Beak
isolate          horizontal   of vertical      conidia        length
                   septa         septa        (Length x     ([micro]m)
                                              Breadth)
                                             ([micro]m)

Karlakatti           5             1        49.38 x 12.82     29.15

Yamkanmardi          6             1        32.8 x 17.17      32.84

Marewada             4             1        47.95 x 15.26     33.52

Unkal                5             2         28 x 12.31       63.02

Chandanamatti        6             1        48.24 x 15.79     27.47

Saundatti            4             2        21.97 x 13.02     74.03

Jagalur              6             1         26.99 x 7.61     30.98

                                            Descriptions
                                           of Ellis M.B.
                                             regarding
                                             Alternaria
                                             macrospora
                                               Zimm.

Name of the       Overall     Number of      Number of
isolate            length     horizontal   vertical septa
                     of         septa
                  conidia
                 ([micro]m)

Karlakatti         78.53

Yamkanmardi        65.64

Marewada           81.47

Unkal              91.02         4-9            1-5

Chandanamatti      75.71

Saundatti            96

Jagalur            57.97

                    Descriptions
                   of Ellis M.B.
                     regarding
                     Alternaria
                     macrospora
                       Zimm.

Name of the         Beak length        Overall     [TEXT INCOMPLETE
isolate                                 length       IN ORIGINAL
                                      ([micro]m)       SOURCE.]

Karlakatti                                         [TEXT INCOMPLETE
                                                     IN ORIGINAL
                                                       SOURCE.]

Yamkanmardi                                        [TEXT INCOMPLETE
                                                     IN ORIGINAL
                                                       SOURCE.]

Marewada                                           [TEXT INCOMPLETE
                                                     IN ORIGINAL
                                                       SOURCE.]

Unkal            Equal or twice the     90-180     [TEXT INCOMPLETE
                 length of conidia                   IN ORIGINAL
                                                       SOURCE.]

Chandanamatti                                      [TEXT INCOMPLETE
                                                     IN ORIGINAL
                                                       SOURCE.]

Saundatti                                          [TEXT INCOMPLETE
                                                     IN ORIGINAL
                                                       SOURCE.]

Jagalur                                            [TEXT INCOMPLETE
                                                     IN ORIGINAL
                                                       SOURCE.]

Table 1(b). Morphological variability of isolates of Alternaria spp

S.           Name of       Number of    Number     Size of
No.          the isolate   horizontal   of         conidia
                           septa        vertical   (Length x
                                        septa      Breadth)
                                                   ([micro]m)

[A.sub.2]    Amminbhavi    6            1          33.29 x 12.54

[A.sub.7]    Haveri        5            2          21.5x6.87

[A.sub.9]    Gadag         5            2          30.22x7.4

                                       Descriptions of Ellis
                                          M.B. regarding
                                       Alternaria alternata
                                          (Fr.) Keissler

S.           Beak         Overall      Number of    Number of
No.          length       length       horizontal   vertical
             ([micro]m)   of           septa        septa
                          conidia
                          ([micro]m)

[A.sub.2]    26.37        56.37

[A.sub.7]    25.32        46.82        1-8          0-4

[A.sub.9]    24.04        56.26

             Descriptions of Ellis M.B.
                      regarding
             Alternaria alternata (Fr.)
                       Keissler

S.           Beak               Overall      Resemblance
No.          length             length       towards
                                ([micro]m)   Alternaria
                                             alternata (Fr.)
                                             Keissler

[A.sub.2]                                    Complete
                                             Resemblance
[A.sub.7]    Short or           20-63        Complete
             more than one                   Resemblance
             third the length
             of conidia
[A.sub.9]                                    Complete
                                             Resemblance

Table 2. Effect of incubation period on dry
mycelial weight of Alternaria macrospora

Incubation period    Mycelial dry
(days)               weight (mg)

2                       36.53
4                       77.56
6                       93.70
8                       169.88
10                      192.42
12                      229.17
14                      272.55
16                      287.31
18                      275.90
20                      267.53
22                      262.30
24                      258.20
26                      234.15
28                      207.65
30                      192.74
S. Em. [+ or -]          0.57
CD at 1%                 2.20

Table 3. Cultural variability of growth and sporulation often
isolates of Alternaria spp. on different solid media

                                       Radial growth (mm)

S.            Isolates          PDA            PCA         CDA
No.

[A.sub.1]     Karlakatti        77.50          63.77       71.23
                                +++            ++          ++
[A.sub.2]     Amminbhavi        60.33          82.50       65.00
                                +++            ++++        +++
[A.sub.3]     Yamkanmardi       75.00          61.00       73.37
                                +++            +++         +++
[A.sub.4]     Marewada          90.00          90.00       90.00
                                ++++           ++++        ++++
[A.sub.5]     Unkal             77.17          73.33       83.33
                                +++            +++         +++
[A.sub.6]     Chandanamatti     76.77          84.83       77.83
                                +++            +++         ++
[A.sub.7]     Haveri            81.27          90.00       75.43
                                +++            ++++        +++
[A.sub.8]     Saundatti         73.33          69.83       75.33
                                +++            +++         +++
[A.sub.9]     Gadag             90.00          90.00       71.42
                                ++++           ++++        +++
[A.sub.10]    Jagalur           73.50          86.38       56.46
                                +++            +++         ++
              Mean              77.49          79.16       73.94
                                Isolates (I)   Media (M)   I x M
              S. Em. [+ or -]   0.27           0.24        0.78
              CD at 1%          1.09           0.97        3.09

                                 Radial growth (mm)

S.            Isolates          HEA     OMA     CMA
No.

[A.sub.1]     Karlakatti        90.00   82.50   43.60
                                ++++    +++     ++
[A.sub.2]     Amminbhavi        90.00   90.00   25.00
                                ++++    +++     +++
[A.sub.3]     Yamkanmardi       90.00   90.00   34.27
                                +++     +++     +++
[A.sub.4]     Marewada          90.00   90.00   31.34
                                +++     +++     ++
[A.sub.5]     Unkal             90.00   90.00   36.17
                                ++++    +++     ++
[A.sub.6]     Chandanamatti     90.00   90.00   65.83
                                +++     +++     +++
[A.sub.7]     Haveri            90.00   90.00   83.83
                                ++++    ++++    +++
[A.sub.8]     Saundatti         90.00   90.00   44.33
                                +++     ++++    +++
[A.sub.9]     Gadag             90.00   90.00   90.00
                                +++     ++++    ++++
[A.sub.10]    Jagalur           90.00   90.00   67.67
                                +++     +++     +++
              Mean              90.00   89.25   52.20

              S. Em. [+ or -]
              CD at 1%

                                Radial growth
                                     (mm)       Mean

S.            Isolates          SDA     V8JA
No.

[A.sub.1]     Karlakatti        82.50   42.50   69.20
                                ++      +++
[A.sub.2]     Amminbhavi        45.47   37.50   61.98
                                ++      +++
[A.sub.3]     Yamkanmardi       86.00   35.00   68.08
                                +++     ++
[A.sub.4]     Marewada          90.00   56.27   78.45
                                ++++    ++
[A.sub.5]     Unkal             71.27   34.60   69.48
                                ++      +++
[A.sub.6]     Chandanamatti     85.00   82.30   81.57
                                ++++    +++
[A.sub.7]     Haveri            82.43   39.92   79.11
                                +++     +++
[A.sub.8]     Saundatti         81.67   40.17   70.58
                                +++     +++
[A.sub.9]     Gadag             90.00   36.08   80.94
                                ++++    ++++
[A.sub.10]    Jagalur           90.00   33.77   73.47
                                ++++    +++
              Mean              80.43   43.81   73.29

              S. Em. [+ or -]
              CD at 1%

++: Moderate sporulation
PCA--Potato carrot agar
meal agar

+++: Good sporulation
CDA--Czapeck's dox agar
SDA--Sabouraud's dextrose agar

++++: Excellent sporulation
HEA--Host extract agar
V8JA--V-8 Juice agar
PDA--Potato dextrose agar
OMA--Oat meal agar
CMA--Corn

Table 4. Cultural diversity of ten isolates of Alternaria spp

                              Colony charcters--colour, margin,
                              type of margin, mycelia growth

S.            Isolates        PDA                 PCA
No.           Media

[A.sub.1]     Karlakatti      Dark grey,          Whitish grey,
                              irregular margin,   irregular margin.
                              raised mycelium     raised mycelium

[A.sub.2]     Amminbhavi      Grey, irregular     Grey, irregular
                              margin, raised      margin. Flat
                              mycelium            mycelium

[A.sub.3]     Yamkanmardi     Grey white,         Grey, irregular
                              irregular margin,   margin, raised
                              raised mycelium     mycelium

[A.sub.4]     Marewada        Grayish black,      Grey, smooth
                              smooth margin,      margin. Flat
                              flat mycelium       mycelium

[A.sub.5]     Unkal           Black white,        Grey, smooth
                              irregular margin,   margin, raised
                              raised mycelium     mycelium

[A.sub.6]     Chandanamatti   Black grey,         Whitish grey,
                              smooth margin.      irregular margin.
                              Flat mycelium       raised mycelium

[A.sub.7]     Haveri          Black, irregular    Grey, irregular
                              margin, raised      margin. Flat
                              mycelium            mycelium

[A.sub.8]     Saundatti       Grey, irregular     Grey, irregular
                              margin, raised      margin, raised
                              mycelium            mycelium

[A.sub.9]     Gadag           White grey,         Grey, smooth
                              smooth margin.      margin. Flat
                              Flat mycelium       mycelium

[A.sub.10]    Jagalur         Grayish black,      Grayish black,
                              irregular margin.   irregular margin.
                              Raised              Flat mycelium
                              mycelium

                       Colony charcters--colour, margin,
                        type of margin, mycelia growth

S.            CDA                 HEA                OMA
No.

[A.sub.1]     Ashy white,         Grayish black,     Grey white,
              irregular margin,   smooth margin,     irregular margin,
              raised mycelium     raised mycelium    raised mycelium

[A.sub.2]     Grey, smooth        Grayish black,     Grayish black,
              margin, raised      smooth margin,     irregular margin,
              mycelium            raised mycelium    raised mycelium

[A.sub.3]     Grey, irregular     Black, irregular   Grey, irregular
              margin, raised      margin, raised     margin, raised
              mycelium            mycelium           mycelium

[A.sub.4]     Grayish black,      Black, smooth      Black, smooth
              irregular margin,   margin, raised     margin, raised
              raised mycelium     mycelium           mycelium

[A.sub.5]     Grey, smooth        Grey, irregular    Grey, irregular
              margin, raised      margin, raised     margin, raised
              mycelium            mycelium           mycelium

[A.sub.6]     Grey, irregular     White, smooth      Black, irregular
              margin, raised      margin, flat       margin, raised
              mycelium            mycelium           mycelium

[A.sub.7]     Grayish black,      Grey, smooth       Grey, irregular
              irregular margin.   margin. Raised     margin, raised
              raised mycelium     mycelium           mycelium

[A.sub.8]     Ashy grey,          Grayish black,     Grey white,
              irregular margin.   smoothmargin.      irregular margin.
              raised mycelium     raised mycelium    raised mycelium

[A.sub.9]     Grey, smooth        White, smooth      Grey black,
              margin. Flat        margin. Flat       smooth margin.
              mycelium            mycelium           Flat mycelium

[A.sub.10]    Grey black,         Black, smooth      Grey, smooth
              smooth margin,      margin. Raised     margin. Flat
              raised mycelium     mycelium           mycelium

                  Colony charcters--colour, margin,
                   type of margin, mycelia growth

S.            CMA                  SDA               V8JA
No.

[A.sub.1]     Black, irregular     Dark grey,        Black,
              margin, distrorted   irregular         irregular
              mycelium             margin, flat      margin, raised
                                   mycelium          mycelium

[A.sub.2]     Black, irregular     Grey, irregular   Grey, smooth
              margin, distrorted   margin, raised    margin, flat
              mycelium             mycelium          mycelium

[A.sub.3]     Black, irregular     Grayish black,    Black,
              margin, distorted    irregular         irregular
              mycelium             margin, raised    margin, flat
                                   mycelium          mycelium

[A.sub.4]     Black, irregular     Black,            Black grey,
              margin, distorted    irregular         irregular
              mycelium             margin, raised    margin,
                                   mycelium          raised
                                                     mycelium

[A.sub.5]     Grey, distorted      Grayish black,    Black,
              margin, raised       irregular         irregular
              mycelium             margin, raised    margin,
                                   mycelium          raised
                                                     mycelium

[A.sub.6]     Grey, irregular      White grey,       Grey black,
              margin, distorted    irregular         irregular
              mycelium             margin. raised    margin.
                                   mycelium          raised
                                                     mycelium

[A.sub.7]     Grey, irregular      Black,            Grey black,
              margin. Distorted    irregular         irregular
              mycelium             margin, raised    margin.
                                   mycelium          raised
                                                     mycelium

[A.sub.8]     Black, irregular     Black grey,       Black,
              margin, raised       irregular         irregular
              mycelium             margin.           margin,
                                   Flat mycelium     raised
                                                     mycelium

[A.sub.9]     Grey, smooth         White grey,       White grey,
              margin, distorted    smooth margin.    irregular
              mycelium             Flat mycelium     margin. Flat
                                                     mycelium

[A.sub.10]    Black, irregular     Black, smooth     Grey, smooth
              margin, distorted    margin. Flat      margin. Flat
              mycelium             mycelium          mycelium

PDA--Potato dextrose agar, PCA--Potato carrot agar, CDA--Czapeck's
dox agar, HEA--Host extract agar, OMA--Oat meal agar, CMA--Corn meal
agar, SDA--Sabouraud's dextrose agar, V8JA--V-8 Juice agar
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Author:Sangeetha, K.D.; Ashtaputre, S.A.; Anil, G.H.; Prasad, G.R. Guru; Ramya, T.S.
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Mar 1, 2016
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