Open Access

S.S. Wagh, A.P. Suryawanshi and D.V. Pawar

Department of Plant Pathology, College of Agriculture, VNMKV, Parbhani-431402 (M.S.), India.
J Pure Appl Microbiol. 2017;11(3):1589-1598
https://doi.org/10.22207/JPAM.11.3.46 | © The Author(s). 2017
Received: 01/07/2017 | Accepted: 10/08/2017 | Published: 30/09/2017
Abstract

Safflower leaf spot / blight caused by Alternaria carthami (Chowdhury) is serious diseases in India. Considering the economic importance and seriousness of the disease, the present cost effective management practices against the test pathogen present in vitro studies were conducted during 2012-13. All the experiments were planned with CRD and all the treatments replicated thrice. A total six systemic fungicides @ 500, 1000 and 1500 ppm concentration and seven non-systemic / combi fungicides @ 1000, 2000 and 2500 ppm concentrations was evaluated in vitro against A. carthami, applying Poisoned food technique (Nene and Thapliyal, 1993). Results of the present studies revealed that systemic fungicides most effective than non-systemic against the test pathogen. Among systemic fungicides, average cent per cent inhibition of mycelial growth was observed in Hexaconazole (100%), followed by Propiconazole (94.07 %) and Penconazole (94.75%); Among non-systemic and combi- fungicides, significantly highest average mycelial growth inhibition was observed in Carbendazim 12 WP + Mancozeb 63 WP (85.80%), followed by Mancozeb (82.59%) and Copper-oxychloride (76.65%). Among bioagents tested T. viride recorded significantly highest mycelial growth inhibition (87.04%) of the test pathogen, followed by T. harzianum (82.59%) and T. koningii (78.89%). Among botanicals A. sativum was found most fungistatic and recorded significantly highest average mycelial growth inhibition 88.33%, followed by A. indica (78.58%), O. sanctum (73.83%) and E. globulus (66.05%).

Keywords

Alternaria carthami, safflower, fungicides, bioagents, botanicals, mycelial inhibition.

Introduction

Safflower (Carthamus tinctorious) is one of the important oilseed crops of the world valued for its highly nutritious edible oil. Safflower seed contains 25-32 % oil and rich in polyunsaturated fatty acids (Linoleic acid, 78%). Its belongs to family Compositae and believes to be native Afganistan. Safflower is known to suffer from many fungal diseases at different stages of crop growth (Bhale et al., 1998). Leaf spot/blight (Alternaria carthami), Wilt (Fusarium oxysporum F. Sp. carthami), Root rot (Rhizoctinia bataticola), Powdery mildew (Erysiphe cicnoralearum DC) and Anthracnose (Colletotrichum capsici). Among these diseases, under present situation leaf spot/blight caused by Alternaria carthami (Chowdhury) has become one of the major constraints in the production and productivity of safflower in the country in general as well as in the state of Maharashtra particularly. The disease (A. carthami) has been reported to cause 25 to 60 per cent yield losses in safflower (Singh and Prasad, 2005). The disease has been also reported to reduce drastically the seed size, seed volume test as well as per cent oil content.

Typical symptoms of the disease
(A. carthami) are appeared as irregular necrotic lesions on leaves and stem. Dark necrotic lesions 2-5 mm in diameter are firstly found on hypocotyls and cotyledons. In mature plant, small brown to dark brown concentric spot of 1-2 mm appear on leaves and brown discoloration appear on the stem, dark brown spot with concentric ring up to 1 cm in diameter appear on leaves which later developed in to large lesion (Mortensen, 1993).

Safflower cultivars / varieties presently under cultivation do not posses proven field resistance or tolerance and majority of them are more or less prone to the leaf spot / blight disease (A. carthami), under such circumstances fungicides provide the most reliable means of controlling foliage diseases. Present day public perceptions and environmental hazards are compelling to search for alternative eco-friendly disease management strategies, for which integration of various cultural, biological and chemical methods might be the solution (Barnwal et al., 2011; Jagan et al., 2013). Recently, biological control using bioagents and phytoextracts has received much attention in both conventional and organic farming to suppress plant diseases and to overcome some extent the public concerns regarding chemical fungicides (Gardener and Fravel, 2002; Samnels, 2006). Several plant extracts have been demonstrated to possess excellent antifungal properties and their exploitation as bio-fungicides has been emphasized within the broad strategy of environmental protection (Singh et al., 2013; Devi et al., 2014). An understanding of the role of environmental factors and their consequences on infection, development and spread of the pathogen / diseases is needed to develop sustainable disease management practices (Bal and Kumar, 2014; Singh et al. 2014).

Materials and Methods

In vitro evaluation of fungicides
Efficacy of six systemic fungicides and seven non-systemic / combi fungicides was evaluated in vitro at 500, 1000, 1500ppm (Systemic fungicides) and 1000, 2000 and 2500 ppm  (Non Systemic) concentration against
A. carthami, applying Poisoned food technique (Nene and Thapliyal, 1993) and using Potato dextrose agar (PDA) as basal culture medium. Based on active ingredient, requisite quantity of the test fungicides was calculated, mixed separately thoroughly with autoclaved and cooled (400C) PDA medium in conical flasks to obtain desired concentrations. This PDA medium amended separately with the test fungicides was then poured (20 ml / plate) aseptically in Petri plates (90mm dia.) and allowed to solidify at room temperature. For each of the test fungicide and its desired concentrations, three plates / treatment / replication were maintained. After solidification of the PDA medium, all the plates were inoculated aseptically by placing in the centre a 5mm culture disc obtained from actively growing 7 days old pure culture of A. carthami and incubated in an inverted position at 28±20C. Petri plates filled with plain PDA (without any fungicide) and inoculated with the pure culture disc of A. carthami were maintained as untreated control.

Observations on radial mycelial growth / colony diameter were recorded at an interval of 24 hours and continued till untreated control plates were fully covered with mycelial growth of the test pathogen. Per cent inhibition of the test pathogen with the test fungicides over untreated control was calculated by applying following formula (Vincent, 1927).

Per cent inhibition = C-T/C × 100

Where,
C = growth of the test fungus in untreated control plate
T = growth of the test fungus in treated plate

In vitro evaluation of bioagents
Seven fungal and two bacterial bioagents were evaluated in vitro against A. carthami, applying Dual Culture Technique (Dennis and Webster, 1971). Seven days old cultures of the test bioagents and test pathogen (A. carthami) grown on PDA were used for the study. Two 5mm culture discs, one each of the test pathogen and test bioagent were cut out with sterilized cork borer and placed at equidistance, exactly opposite to each other on autoclaved and solidified PDA medium in Petri plates and three plates were incubated at 28±20C. PDA plates inoculated alone with pure culture disc (5mm) of the test pathogen were maintained as untreated control.

Observations on linear mycelial growth of the test pathogen and test bioagent were recorded at an interval of 24 hours and continued till untreated control plates were fully covered with mycelial growth of the test pathogen. Per cent inhibition of the test pathogen with the test bioagent, over untreated control was calculated by applying following formula (Arora and Upaddhyay, 1978).
In vitro evaluation of plant extracts / botanicals
Aqueous extracts of 14 botanicals (as detailed under treatments) were evaluated in vitro against A. carthami. Leaf / bulb / rhizome extract of the test botanicals were prepared by grinding with mixture-cum grinder. Washed 100g each leaves / Turmeric rhizome / Onion bulb / Garlic cloves were macerated separately in 100ml distilled water (w/v) and the macerates obtained were filtered separately through double layered muslin cloth. Each of the filtrate obtained was further filtered through Whatman No. I filter paper using funnel and volumetric flasks (100ml cap.). The final clear extracts obtained formed the standard plant extracts of 100 per cent concentration. These were evaluated (each @ 10%, 15% and 20%) in vitro against A. carthami, applying Poisoned Food Technique (Nene and Thapliyal, 1993) and using Potato dextrose agar (PDA) as basal culture medium.

An appropriate quantity of each test aqueous extract (100 %) was separately mixed thoroughly with autoclaved and cooled (400C) PDA medium in conical flasks (250ml cap.) to obtain desired concentrations of 10, 15 and 20 per cent. The PDA medium amended separately with the test aqueous extract was then poured (20ml / plate) into sterile glass Petri plates (90mm dia.) and allowed to solidify at room temperature. For each test botanical extract and their respective concentrations, three plates / treatment / replication were maintained and all the treatments were replicated thrice. Upon solidification of the amended PDA medium, all the treatment plates were aseptically inoculated by placing in the centre a 5mm mycelial disc obtained from a week old actively growing pure culture of A. carthami. Plates containing plain PDA without any botanical extract and inoculated with mycelial disc of the test pathogen served as untreated control. All these plates were then incubated at 28± 20C temperature for a week or till the untreated control plates were fully covered with mycelial growth of the test pathogen.

Observations on radial mycelial growth / colony diameter of the test pathogen were recorded treatment-wise at 24 hours interval and continued till mycelial growth of the test pathogen was fully covered in the untreated control plates. Per cent inhibition of mycelial growth over untreated control was calculated by applying the formula given by Vincent (1927).

RESULTS AND DISCUSSION

Disease management strategies
In vitro evaluation of systemic fungicides
Mycelial inhibition
Results (Table 1 and Plate 1) revealed that all the systemic fungicides tested (each @ 500, 1000 and 1500ppm) significantly inhibited mycelial growth of A. carthami, over untreated control. Further, per cent mycelial inhibition was increased with increase in concentrations of the fungicides tested represented in Fig. 1.
Table (1):
In vitro bioefficacy of systemic fungicides against A. carthami.

Tr. No. Treatments % Inhibition* at ppm Av. (%)
500 1000 1500
T1 Carbendazim
50 WP
38.52
(22.66)
49.26
(29.51)
53.70
(32.48)
47.16
(28.22)
T2 Propiconazole 25 EC 94.22
(70.44)
100
(89.99)
100
(89.99)
98.07
(83.47)
T3 Hexaconazole 5 EC 100
(89.99)
100
(89.99)
100
(89.99)
100
(89.99)
T4 Difenconazole 25 EC 83.70
(56.87)
89.08
(63.00)
93.33
(68.95)
88.70
(62.94)
T5 Penconazole 10 EC 90.19
(64.40)
94.07
(70.17)
100
(89.99)
94.75
(74.85)
T6 Thiophanate methyl 70WP 27.41
(15.91)
41.48
(24.50)
44.63
(26.50)
37.84
(22.30)
T15 Control
(Untreated)
00.00
(00.00)
00.00
(00.00)
00.00
(00.00)
00.00
(00.00)
S.E. + 0.69 0.45 0.32
C.D.

(P = 0.01)

2.08 1.37 0.97

*: Mean of three replications, Dia: Diameter, Av.: Average
Figures in parentheses are arcsine transformed values

Plate 1. In vitro effect of systemic fungicides at various concentrations on growth and inhibition of A. carthami
Fig. 1. In vitro bioefficacy of systemic fungicides against A. carthami

Average mycelial growth inhibition recorded with the test systemic fungicides was ranged from 37.84 (Thiophanate methyl) to 100 (Hexaconazole) per cent. However, it was cent per cent with Hexaconazole (100%), followed by Propiconazole (94.07%) and Penconazole (94.75%) and Difenconazole (88.70%). Whereas, it was comparatively minimum with Thiophanate methyl (37.84%) and Carbendazim (47.16%).

These results are in conformity with the earlier findings of those workers who reported systemic fungicides viz., Hexaconazole, Propiconazole, Penconazole, Difenconazole, Thiophanate methyl and Carbendazim at various concentrations had significantly inhibited mycelial growth of A. carthami infecting safflower (Murumkar et al., 2009; Taware et al., 2014), A. helianthi infecting sunflower (Amaresh and Nargund, 2004), A. alternata infecting sesame (Bavaji et al., 2012).

In vitro evaluation of non-systemic and combi- fungicides
Mycelial inhibition
Results (Table 2, Plate 2 and Fig. 2) revealed that all non-systemic and combi- fungicides tested (each @ 1000, 2000 and 2500 ppm) significantly inhibited mycelial growth of A. carthami, over untreated control. Further, per cent mycelial inhibition was increased with increase in concentrations of the fungicides tested.

Plate 2. In vitro effect of non-systemic and combi- fungicides at various concentrations on growth and inhibition of A. carthami
Fig. 2. In vitro bioefficacy of non-systemic and combi- fungicides against A. carthami

Table (2):
In vitro bioefficacy of non-systemic and combi- fungicides against A. carthami.

Tr. No. Treatments % Inhibition* at ppm Av. (%)
1000 2000 2500
T7 Chlorotholonil 46.30
(27.82)
50.74
(30.49)
52.96
(31.98)
50.00
(30.10)
T8 Ridomil MZ
72 WP
51.67
(31.23)
53.89
(32.60)
56.11
(34.13)
53.89
(32.65)
T9 Carbendazim 12 WP + Mancozeb 63 WP 83.15
(56.62)
84.81
(58.03)
89.44
(63.48)
85.80
(59.38)
T10 Copper-oxychloride 50WP 71.67
(46.23)
77.74
(50.72)
80.54
(53.64)
76.65
(50.20)
T11 Copper-hydroxide
77 WP
66.30
(41.24)
70.74
(45.03)
71.67
(45.77)
69.57
(44.01)
T12 Mancozeb
75 WP
80.00
(53.84)
81.85
(54.95)
85.93
(59.24)
82.59
(56.01)
T13 Propineb
70 WP
57.22
(34.90)
61.48
(37.93)
63.52
(39.43)
60.74
(37.42)
T14 Curzate M8
72 WP
64.62
(40.54)
66.48
(41.67)
68.52
(43.25)
66.54
(41.82)
T15 Control
(Untreated)
00.00
(00.00)
00.00
(00.00)
00.00
(00.00)
00.00
(00.00)
S.E. + 0.46 0.65 0.63
C.D. (P = 0.01) 1.37 1.94 1.87

*: Mean of three replications, Dia: Diameter, Av.: Average
Figures in parentheses are arcsine transformed values


Plate 2.
In vitro effect of non-systemic and combi- fungicides at various concentrations on growth and inhibition of A. carthami

Fig. 2.
In vitro bioefficacy of non-systemic and combi- fungicides against A. carthami

Average mycelial growth inhibition recorded with the test non-systemic and combi- fungicides was ranged from 50.00 (Chlorotholonil) to 85.80 (Carbendazim 12 WP + Mancozeb 63 WP) per cent. However, it was highest average mycelial growth inhibition with Carbendazim 12 WP + Mancozeb 63 WP (85.80%), followed by Mancozeb (82.59%), Copper-oxychloride (76.65%), Copper-hydroxide (69.57%), Curzate (66.54%), Propineb (60.74%), Ridomil MZ (53.89%) and Chlorotholonil (50.00%).

Thus, six systemic, six non-systemic and two combi- fungicides tested were found fungistatic against A. carthami. However, on the basis of order of merit systemic fungicides viz., Hexaconazole, Propiconazole, Penconazole and Difenconazole and non-systemic / combi fungicides Carbendazim 12 WP + Mancozeb 63 WP (SAAF 75 WP), Mancozeb, Copper-oxychloride, Copper-hydroxide and Curzate etc were found most promising against A. carthami, causing leaf spot / blight in safflower.

Non-systemic fungicides viz., Mancozeb, Copper-oxychloride, Copper hydroxides, Propineb and Chlorothalonil and Combi- fungicides (systemic+contact) viz., Carbendazim+Mancozeb, Metalaxyl+Mancozeb were also reported to cause cent-per cent or significantly maximum mycelial growth inhibition of Alternaria leaf spot / blight causing fungal pathogens such as A. carthami infecting safflower (Murumkar et al., 2009; Taware et al., 2014), A. helianthi infecting sunflower (Mathivanan and Prabhavathy, 2007; Mesta et al., 2009).

In vitro evaluation of bioagents
The results obtained on mycelial growth and inhibition of A. carthami with seven fungal and two bacterial antagonists are presented in Table 3, Plate 3 and Fig. 3.

Table (3):
In vitro bioefficacy of bioagents against A. carthami.

Tr. No. Treatments Colony Dia. of test pathogen * (mm) % Inhibition
Fungal antagonists
T1 Trichoderma viride 11.67 87.04
(60.54)
T2 T. harzianum 15.67 82.59
(55.70)
T3 T. hamatum 23.33 74.07
(47.80)
T4 T. koningii 19.00 78.89
(52.08)
T5 T. longibrachiatum 27.33 69.63
(44.13)
T6 T. (Gliocladium) virens 31.67 64.81
(40.41)
T7 Aspergillus niger 39.83 55.74
(36.60)
Bacterial antagonists
T8 Bacillus subtilis 36.33 59.63
(29.63)
T9 Pseudomonas fluorescens 45.05 49.33
(33.87)
T10 Control (Untreated) 90.00 00.00
(00.00)
S.E. + 0.78 0.75
C.D. (P = 0.01) 2.30 2.22

* : Mean of three replications, Dia.: Diameter,
Figures in parentheses are arcsine transformed values

Plate 3. In vitro effect of bioagents on growth and inhibition of A. carthami
Fig. 3. In vitro bioefficacy of bioagents against A. carthami

Results (Table 3) revealed that all the bioagents evaluated exhibited fungistatic / antifungal activity against A. carthami and significantly inhibited its growth, over untreated control. Of the antagonists tested, T. viride was found most effective with highest mycelial growth inhibition (87.04%) of the test pathogen. The second and third most inhibitory antagonists found were T. harzianum and T. koningii with and inhibition of 82.59 and 78.89 per cent, respectively. There were followed by T. hamatum (74.07%), T. longibrachiatum (69.63%), T. virens (64.81%), Bacillus subtilis (59.63%), A. niger (55.74%) and P. fluorescens (49.33%). Thus, the bioagents viz., T. viride, T. harzianum, T. koningii, T. hamatum were found most potential antagonists against A. carthami (Fig. 3).

These results are in conformity with the earlier findings of those workers who reported bioagents viz., T. viride, T. harzianum, T. koningii and T. hamatum had significantly inhibited mycelial growth of A. carthami infecting safflower (Taware et al., 2014), A. brassicae infecting rapeseed-mustard (Reshu and Khan, 2012), A. alternata / A. sesame infecting sesame (Akbari and Parakhia, 2007; Savitha et al., 2011).

The fungistatic / antifungal action exerted by the species of Trichoderma and A. niger against A. carthami and other species of Alternaria may be attributed to their production of volatile and non-volatile substances, cell wall degrading enzymes (glucanases, B1, 3 glucanase), the phenomenon of competition, lysis and antibiosis.

In vitro evaluation of plant extracts / botanicals
Results (Table 4) revealed that all the 14 botanicals tested (each @ 10, 15 and 20%) exhibited a wide range of radial mycelial growth of A. carthami (Plate 4 and Fig. 4) and it was decreased drastically with increase in concentrations of the test botanicals from 10 to 20 per cent.

Plate 4. In vitro effect of phytoextract at various concentrations on growth and inhibition of A. carthami
Fig. 4. In vitro bioefficacy of botanicals against A. carthami

Table (4):
In vitro bioefficacy of botanicals against A. carthami.

Tr. No. Treatments Av. (mm) % Inhibition Av. (%)
10 15 20
T1 Onion
(Allium cepa)
44.22 50.37
(23.12)
55.56
(33.75)
57.78
(35.30)
50.87
(30.72)
T2 Ghaneri
(Lantana camera)
44.28 46.30
(27.58)
50.37
(30.24)
55.74
(33.87)
50.80
(30.56)
T3 Tulsi
(Oscimum sanctum)
23.56 65.74
(41.10)
76.49
(49.89)
79.26
(52.43)
73.83
(47.81)
T4 Gliricidia
(Gliricidia sepium)
77.78 07.04
(40.37)
14.07
(80.87)
19.63
(11.32)
13.58
(44.19)
T5 Neem
(Azardirachta indica)
19.28 72.96
(46.86)
79.26
(52.43)
83.52
(56.64)
78.58
(51.98)
T6 Garlic
(Allium sativum)
10.50 83.70
(56.87)
88.52
(62.27)
92.78
(68.15)
88.33
(62.43)
T7 Bouganveillia
(B. spectabilis)
72.06 14.44
(83.04)
19.44
(11.21)
25.93
(15.03)
19.94
(36.43)
T8 Datura
(Datura metal)
41.33 51.48
(30.98)
56.67
(34.52)
61.30
(37.80)
56.48
(34.43)
T9 Eucalyptus
(Eucalyptus globulus)
30.56 59.63
(36.60)
65.93
(41.24)
72.59
(46.55)
66.05
(41.46)
T10 Periwinkle
(Vinca rosea)
51.17 35.93
(21.05)
47.96
(28.66)
50.37
(30.24)
44.75
(26.65)
T11 Turmeric
(Curcuma longa)
32.33 53.89
(32.61)
60.74
(37.40)
67.41
(42.39)
60.68
(37.47)
T12 Custard apple
(Annona squimosa)
64.78 21.48
(12.41)
27.04
(15.69)
35.56
(20.83)
28.03
(16.31)
T13 Parthenium
(P. hysterophorus)
59.11 25.74
(14.91)
35.37
(20.71)
41.85
(24.74)
34.32
(20.12)
T14 Karanj
(Pongamia pinnata)
53.44 32.96
(19.24)
41.85
(24.74)
47.04
(28.06)
40.62
(24.01)
T15 Control (Untreated 90.00 00.00
(00.00)
00.00
(00.00)
00.00
(00.00)
00.00
(00.00)
S.E. + 0.64 0.41 0.67
C.D. (P = 0.01) 1.86 1.18 1.94

* : Mean of three replications, Dia.: Diameter, Av.: Average, Conc.: Concentration,
Figures in parentheses are arcsine transformed values

Mycelial inhibition
Average mycelial growth inhibition recorded with the test botanicals was ranged from 13.58 (G. maculata) to 88.33 (A. sativum) per cent. However, it was highest with A. sativum (88.33%), followed by A. indica (78.58%), O. sanctum (73.83%), E. globulus (66.05%), C. longa (60.68%), D. metal (56.48%), Allium cepa (50.87%) and L. camera (50.80%). Rest of the test botanicals recorded average mycelial growth inhibition in the range of 28.03 to 44.75 per cent; whereas, it was significantly least with G. sepium (13.58%) and B. spectabilis (19.94%).

Thus, on the basis of antifungal activity, the botanicals found most effective in the order of merit were A. sativum, A. indica, O. sanctum, E. globules, C. longa, D. metal, Allium cepa, L. camera, V. rosea and P. pinnata, which after their further confirmation could be exploited for control of leaf spot / blight of safflower (A. carthami).          

These results are in conformity with the earlier findings of those workers who reported plant extracts / botanicals viz., A. sativum, A. indica, O. sanctum, E. globules, C. longa, at various concentrations had significantly inhibited mycelial growth of A. carthami infecting safflower (Ranaware et al., 2010; Usha et al., 2012; Taware et al., 2014).

In the present study, of the 14 phytoextracts A. sativum extract was found most effective which caused substantial inhibition (> 88%) of A. carthami. The antifungal activity of A. sativum has been attributed to the presence of diallyl sulphide and other compounds like alllisatin I, II and garli phytocide (Cavallito et al., 1974; Sharma and Prasad, 1980).

A number of phytoextracts / botanicals have been studied and reported with potential inhibitory action against many phytopathogenic fungi, bacteria and viruses. The presence of various secondary metabolites viz., alkaloids, quaternary alkaloids, cumarins, flavanoids, steroids / terpenoids, phenolics etc with potential antifungal activity were reported in various plant extracts (Alice,1984; Aswal, et al.,1984; Abraham et al.,1986; Chopra et al., 1992).

Declarations

ACKNOWLEDGMENTS
I greatly acknowledge to my Research Guide, Dr. A. P. Suryawanshi Professor, Department of Plant Pathology, College of Agriculture, Latur and Head, Dr. K. T. Apet,, Department of Plant Pathology, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani for their support during research work for the completion of my Ph.D. degree programme.

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