Bio-efficacy of fungicides, bioagents and plant extracts/ botanicals against Alternaria carthami, the causal agent of Alternaria blight of Safflower (Carthamus tinctorius L.)

A total of eleven fungicides namely: mancozeb 75 WP, chlorothalonil 75 WP, copper oxychloride 50 WP, thiram 75 WP, captan 50 WP, difenconazole 25 EC, carbendazim 50 WP, hexaconazole 5 EC, propiconazole 25 EC, penconazole 10 EC and metalyxil + mancozeb (Ridomil) 68 WP were evaluated (at 500, 1000 and 1500 ppm each) in vitro against Alternaria carthami. All the fungicides tested caused significant inhibition at all three concentrations tested over untreated control. The average inhibition recorded with the test fungicides was ranged from 28.39% (chlorothalonil) to 94.44% (carbendazim). However, carbendazim recorded significantly highest average mycelial growth inhibition (94.44%). The second and third best fungicides found were mancozeb (85.43%) and thiram (83.33%). These were followed by hexaconazole (82.95%), ridomil (82.83%) and difenconazole (77.16%). All the six fungal and one bacterial bioagents/antagonists evaluated in vitro were found antifungal/antagonistic against A. carthami. However, T. viride was found most effective and recorded significantly highest mycelial growth inhibition (94.07%) of the test pathogen over untreated control. The second and third best bioagents/antagonists found were T. hamatum and T. koningii which recorded mycelial growth inhibition 85.18 and 81.11%, respectively. All the eleven plant extract/botanicals were evaluated in vitro (each at 10, 15 and 20%) against A. carthami. The mean percentage mycelial growth inhibition recorded with all the test botanicals was ranged from 19.26 (A. racemosus) to 62.47 (A. sativum) per cent. However, A. sativum was found most fungistatic which recorded significantly highest mean mycelial growth inhibition (62.47%). The second and third best botanicals found were D. metal (49.87%) and C. longa (46.91%). Thus, all the botanicals tested were found fungistatic antifungal and caused significant inhibition of A. carthami over untreated control.


INTRODUCTION
Safflower (Carthamus tinctorius L.) is one of the important oilseed crops of the world valued for its highly nutritious edible oil.Safflower seed contains about 25-32% oil.It belongs to family compositae and believes to be native of Afganistan.Safflower is known to suffer from many fungal diseases at different stage of crop growth (Bhale et al., 1998).Leaf spot/blight (Alternaria carthami), wilt (Fusarium oxysporum f. sp.carthami), root rot (Rhizoctonia batatiocla), powdery mildew (Erysiphe cichoracearum DC), anthracnose (Colletotrichum capsici).
Out of several diseases reported on safflower, Alternaria blight caused by A. carthami is one of the most important diseases.This disease was first reported by Chowdhury (1944) at Pune in India.In India, disease plays an important role in safflower cultivation and responsible to cause 25 to 60% yield losses every year.In general, the season was suitable for the development of foliar diseases particularly for Alternaria leaf spot/ blight development as there were rains after sowing in Maharashtra and a disease severity from 10 to 95% was observed.In Marathwada region of Maharashtra, Alternaria leaf spot intensity of 30 to 40%, while in Karnataka and Andhra Pradesh maximum intensity of Alternaria leaf spot upto 25 and 20%, respectively The disease has been reported to cause seed yield losses to the tune of 10-25% (Indi et al., 1988).Under severe conditions, it has been reported to cause 50% loss in yield (Indi et al., 1986).An extensive survey work carried out by Deokar et al. (1991) revealed the predominance of Alternaria leaf spot disease on safflower in the traditional safflower growing areas in the scarcity zone of Maharashtra.
The Alternaria leaf spot symptoms appears as small brown to dark spots with concentric rings appears first on lower leaves and later spread on upper ones.These spots increases in number and size coalesce and form large irregular lesions.The center of mature spot usually becomes lighter in colour.Infected seed may show dark sunken lesions on the testa at the floret end.Seed may rot or seedling damping off and brown spots up to 5 mm in diameter appear on cotyledons.It has therefore become highly essential to have a good knowledge of such association, method of detection as well as way to control them.Considering economic importance of the disease, the present investigation was undertaken.

In vitro efficacy of fungicides
Efficacy of eleven fungicides namely: mancozeb 75 WP, chlorothalonil 75 WP, copper oxychloride 50 WP, thiram 75 WP, captan 50 WP, difenconazole 25 EC, carbendazim 50 WP, hexaconazole 5 EC, propiconazole 25 EC, penconazole 10 EC, metalaxyl + mancozeb (Ridomil) 68 WP were evaluated (500, 1000, 1500 ppm each) in vitro against A. carthami by Poisoned food technique (Nene and Thapliyal, 1993).Based on active ingredient, the requisite quantity of each fungicide was calculated and mixed thoroughly with autoclaved and cooled (40°C) Potato Dextrose Agar medium (PDA) in conical flasks to obtain desired concentrations of 500, 1000 and 1500 ppm.Plain PDA medium without fungicides served as untreated control.Fungicide amended PDA medium was then poured aseptically in Petri plates (90 mm dia) and allowed to solidify at room temperature.After solidification of the medium, all the plates were inoculated aseptically with 5 mm culture disc of the test fungus obtained from a week old actively growing pure culture of A. carthami.The disc was placed on PDA in inverted position in the centre of the Petri plate and plates were incubated at 27 + 1°C.Each treatment was replicated thrice.Observations on radial mycelial growth/colony diameter were recorded at 24 h interval and continued till the untreated control plate was fully covered with mycelial growth of the test fungus.Per cent mycelial growth inhibition of the test pathogen with the test fungicides over untreated control was calculated by applying the formula given by Vincent (1927).
Where, C = Growth (mm) of test fungus in untreated control plates T = Growth (mm) of test fungus in treated plates

In vitro efficacy of bioagents
Six fungal antagonists namely: Trichoderma viride, T. harzianum, T. hamatum, T. koningii, T. lignorum, Gliocladium virens and one bacterial antagonist namely: Pseudomonas fluorescens 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 fungus (A.carthami) grown on (PDA, NA) were used for the study.Discs (5 mm dia) of PDA along with culture growth of the test fungus and bioagents were cut out with sterilized cork borer.Then two culture discs, one each of the test fungus and bioagent were placed at equidistance and exactly opposite with each other on solidified PDA medium in Petri plates under aseptic conditions and plates were incubated at 27 + 1°C PDA.The plates inoculated in centre only with culture disc of test fungus were maintained as untreated control and all the treatments were replicated thrice.
Observations on linear mycelial growth of the test fungus and bioagents were recorded at an interval of 24 h and continued till untreated control plates were fully covered with mycelial growth of the test fungus.Per cent inhibition of the test fungus over untreated control was calculated by applying the formula given by Arora and Upaddhay (1978).
An appropriate quantity of each plant extract (100%) was separately mixed thoroughly with autoclaved and cooled (40°C) PDA medium in conical flasks (250 ml cap) to obtain desired concentrations (10,15 and 20%).Sterilized and cooled PDA medium amended separately with plant extract was then poured (15 to 20 ml/plate) into sterile glass Petri plates (90 mm dia.) and allowed to solidify at room temperature.Each plant extract and its respective concentrations were replicated thrice.The plates containing PDA without any plant extract were maintained as untreated control.Upon solidification of PDA, all the treatment and control plates were aseptically inoculated by placing in the centre a 5 mm mycelial disc obtained from a week old actively growing pure culture of A. carthami.Plates containing plain PDA and inoculated with mycelial disc of test fungus served as untreated control.All these plates were then incubated at 27 + 1°C temperature for a week or till the untreated control plates were fully covered with mycelial growth of the test fungus.
Observations on radial mycelial growth/colony diameter of the test fungus were recorded treatment wise at 24 h interval and continued till mycelial growth of the test fungus 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).

In vitro evaluation of fungicides
A total of eleven fungicides namely: mancozeb 75 WP, chlorothalonil 75 WP, copper oxychloride 50 WP, thiram 75 WP, captan 50 WP, difenconazole 25 EC, carbendazim 50 WP, hexaconazole 5 EC, propiconazole 25 EC, penconazole 10 EC and metalaxyl + mancozeb (Ridomil) 68 WP were evaluated (at 500, 1000 and 1500 ppm each) in vitro against A. carthami, applying poisoned food technique and using potato dextrose agar (PDA) as basal medium.Effect of these fungicides on radial mycelial growth and inhibition of the test pathogen over untreated control were recorded and the results obtained are presented in Table 1, Plate-I A B C, and Figures 1, 2 and 3.

Mycelial growth/colony diameter
Result (Table 1) revealed that all the fungicides tested covered a wide range of radial mycelial growth/colony diameter of the test pathogen, depending upon their concentrations used (Plate I).
At 500 ppm, radial mycelial growth of pathogen was ranged from 5.00 (carbendazim) to 68.33 (chlorothalonil) mm.However, it was the maximum with chlorothalonil (68.33 mm) and this was followed by copper oxychloride (49.66 mm), Captan (46.00 mm), both of which were at par.Significantly least mycelial growth was recorded with carbendazim (5.00 mm) followed by mancozeb (17.33 mm), ridomil (19.33 mm), hexaconazole (21.00 mm), all three of which were at par.difenconazole (23.16 mm), propiconazole (24.83 mm) and penconazole (25.16 mm) all three of which were at par (Plate I (A) and Figure 1).

Radial mycelial growth
Results (Table 3, Plate III) revealed that all the botanicals /plant extracts tested exhibited a wide range of radial mycelial growth of test pathogen, depending up on their concentration used and it was decreased with increase in concentration of the botanicals tested.At 10%, radial mycelial growth of the test pathogen was ranged from 36.66 (A.sativum) to 75.33 (A.racemosus) mm.However, it was maximum with A. racemosus (75.33 mm).This was followed by O. sanctum (74.66 mm), P. hysterophorus (71.66 mm), L. innermis (69.33 mm), A. indica (68.33 mm), three of which were at par and Z. officinale (64.33 mm), L. camera (60.66 mm), A. vasica (55.66 mm), C. longa (51.33 mm) of which were at par.comparatively least mycelial growth was recorded with the botanicals viz., A. sativum (36.66 mm) and D. metal (47.66 mm) Plate III A, Figure 5.At 15 per cent, radial mycelial growth of the test pathogen was ranged from 34.00 (A.sativum) to 73.33 (A.racemosus) mm.However, it was maximum with A. racemosus (73.33 mm).This was followed by O. sanctum (72.33 mm) both of which were at par and P. hysterophorus (67.33 mm), A. indica (66.33 mm), L. innermis (64.66 mm).All of which were at par to each other and Z. officinale (58.33 mm), L. camera (57.66 mm), A. vasica (53.66 mm) Plate III.In vitro efficacy of plant extract/botanicals at 10% (A), 15% (B) and 20% (C) on growth and inhibition A. carthami.
of which were at par.Significantly least mycelial growth was recorded with the botanicals A. sativum (34.00 mm), D. metal (44.66 mm) and C. longa (47.33 mm) (Plate III B, Figure 6).
At 20%, radial mycelial growth of the test pathogen was ranged from 30.66 mm (A.sativum) to 70.00 mm (O.sanctum).However, significantly highest mycelial growth was maximum with O. sanctum (70.00 mm).This was followed by A. racemosus (69.00 mm) both of which were at par and P. hysterophorus (63.00 mm), A. indica (61.00 mm), L. innermis (58.33 mm), Z. officinale (56.33 mm).All of which were at par to each other and L. camera (53.33 mm), A. vasica (52.00 mm), both of which were at par.Less than 50 mm growth was recorded with the botanicals C. longa (44.66 mm) and D. metal (43.00 mm) both of which were at par.Significantly least mycelial growth was recorded with the botanical A. sativum (30.66 mm), as compared to untreated control was 90.00 mm (Plate III C, Figure 7).
The mean percentage radial mycelial growth recorded with the plant extracts tested (at 10, 15 and 20% each) was ranged from 33.77 mm (A.sativum) to 72.66 mm (A.racemosus).However, significantly highest mean mycelial growth was recorded with A. racemosus (72.66 mm).This was followed by O. sanctum (72.33 mm) both of which were at par and P. hysterophorus   (45.10 mm).Significantly least mean mycelial growth was recorded with A. sativum (33.77 mm).This was followed by D. metal (45.10 mm).The mean mycelial growth was recorded in untreated control was 90.00 mm.
Mean percentage mycelial growth inhibition recorded with all the test botanicals was ranged from 19.26 (A.racemosus) to 62.47 (A.sativum) per cent.However, A. sativum was found most fungistatic and recorded significantly highest mean mycelial growth inhibition (62.47%).The second and third best plant extracts found were D.
Fig ca

Figure 4 .
Figure 4.In vitro efficacy of different bioagents on mycelial growth and inhibition of A. carthami

Figure 5 .
Figure 5.In vitro efficacy of 10% plant extracts on mycelial growth and inhibition of A. carthami.

Figure 6 .
Figure 6.In vitro efficacy of 15% plant extracts on mycelial growth and inhibition of A.carthami .

Figure 7 .
Figure 7.In vitro efficacy of 20% plant extracts on mycelial growth and inhibition of A.carthami .

Table 1 .
agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 In vitro efficacy of fungicides at different concentrations on mycelial growth and inhibition of A. carthami.

Table 2 .
Microbiol.R In vitro efficacy of different bioagents on mycelial growth and inhibition of A. carthami.Mean of three replications.Figures in parenthesis are arc sine transformed value. *

Table 3 .
In vitro efficacy of different plant extracts/botanicals on mycelial growth and inhibition of A. carthami.Means of three replications.Figures in parenthesis are arc sine transformed value. *: