Research Articles

2018  |  Vol: 4(2)  |  Issue: 4(March-April)  |  https://doi.org/10.31024/ajpp.2018.4.2.18
Bio-efficacy of fungal and bacterial antagonists against Xanthomonas axonopodis pv. vesicatoria (Doidge) Dye in chilli (Capsicum spp.) grown in Rajasthan

Dilip Kumar Sharma

Vardhaman Mahaveer Open Universiy, Kota, Rajasthan, India

Address for corresponding Author

Dr. Dilip Kumar Sharma

Vardhaman Mahaveer Open Universiy, Kota, Rajasthan, India


Abstract

Background: The fungal and bacterial antagonist were used to study their antimicrobial efficacy against Xanthomonas axonopodis pv. vesicatoria (Doidge) Dye in chilli causing bacterial leaf spot. Objective: The present study indicates the effective control of the pathogen and reduced the disease incidence significantly in non-hazardous manner by using fungal and bacterial antagonists. Materials and methods: For the control of pathogen 5 fungal and 2 bacterial antagonists namely Gliocladium virens, Trichoderma viride, T. harzinum, Stachybhotrys atra, Penicillium chrysogenum and Bacillus subtilis, Pseudomonas fluorescence respectively used by filter paper disc assay, seeded agar method and standard blotter methods. Results:  Out of used fungal antagonists Trichoderma viride found most effective which improve seed germination and control of pathogen followed by Penicillium chrysogenum as compared to check in all the method. In filter paper method, bacterial antagonist Bacillus subtilis found effective against the pathogen followed by Pseudomonas fluorescence in control of various isolates of XAV.

Keywords: Antibacterial, chilli, fungal antagonists, bacterial antagonists, Xanthomonas axonopodis pv. vesicatoria


Introduction

Use of bio-agent for control of several plant diseases is an alternative approach in comparison of chemical fungicides because these are safe, effective and eco-friendly in comparison of chemical (Ramamoorthy et al., 2002). The most commonly used bioagents are Gliocladium virens, Trichoderma harzianum, T. veridae, Agrobacterium radiobacter, Pseudomonas fluorescens, Baccillus subtilis, soil-borne fungi, bacteria and Actinomycetes as antagonist against several pathogenic microbes (Ramamoorthy et al., 2002; Agrios, 2005; Gravel et al., 2005; Intana et al., 2008; Montealegre et al., 2010; Khare et al., 2010; Christy Jeyaseelan et al., 2012).

The antagonistic reactions include antibiosis, competition and hyper parasitism of (Cook and Baker, 1983; Leelavathi et al., 2014). It is reported that various species of Trichoderma secret approx 40 different types of antibiotics, chemical compounds and many secondary metabolites against microorganisms specially gram positive and negative bacteria. These chemicals or metabolites promote plant growth and yield of crop (Tapwal et al., 2011; Mukherjee et al., 2013; Ruano-Rosa et al., 2014).

Bacterial leaf spot (BLS) disease of sweet pepper (Capsicum annuum) caused by Xanthomonas axonopodis pv. vesicatora is recorded from several countries of eastern and southern Africa, USA, Ethiopia, Kenya, Malawi, Mozambique and South Africa (Black et al., 2001). XAV suspected from symptoms on fruit was confirmed by isolation on semi-selective media including Tween B (McGuire et al., 1986; Jones et al., 2000). In India, bacterial leaf spot disease was first reported from Pune, Maharastra in 1948 by Patel et al. in chilli. In Rajasthan, the disease caused 7.5 to 16.6 per cent loss in the yield of fruits 22-34°C and high humidity for maximum infection (Shekhawat and Chakravarti, 1976; Shekhawat and Chakravarti, 1977). It losses in marketable fruits may be more than 50% (Pohronezny et al., 1992; Anonymous, 2012). The pathogen was found seed-borne (10-15%) and occurs on infected plant debris and weeds (Jones et al., 1986). The incidence was less than 5% persisted from one season to next in crop debris or on weed hosts (Ravinkar et al., 2001).

Xanthomonas axonopodis pv. vesicatora (Doidge) Dye (syn: Xanthomonas campestris pv vesicatoria) (XAV) a gram-negative, rod-shaped bacterium attack on plant (Anonymous, 1996; Thieme et al., 2005; Anonymous, 2016). The bacterial colonies were circular, raised, yellow, mucoid colonies on Tween-80 agar medium. The isolates were gram's negative, KOH solubility test positive, levan negative, lipase activity positive, oxidase negative, starch hydrolyzing, gelatin hydrolyzing, arginine variable, did not reduce nitrate and no rotting of potato tissue occurred. The pathogen induced positive hypersensitivity reaction on tobacco leaves after infiltration and turgidity of leaves was lost within 6-10hrs followed by local necrosis and desiccation of affected leaf tissues after 36 hrs (Bradbury, 1986; Neergaard, 1986; Schaad, 1988; Agarwal, et al., 1989; Saettler et al., 1989; Mortensen, 1994a, b; Anonymous, 1996; Thieme et al. 2005; Anonymous, 2016). The pathogen XAV has been reported to be seed-borne in chilli (Neergaard, 1977; Bradbury, 1986; Richardson, 1990).

Materials and methods

Seed treatment with fungal antagonists in SBM

For the study of effect of antagonists on germination and control of pathogen, the fungal antagonist as Gliocladium virens, Trichoderma viride, T. harzinum, Stachybhotrys atra and Penicillium chrysogenum were used against  seed-borne infection of Xanthomonas campestris pv. vesicatoria in chilli. The pure cultures of G. virens, T. viride, T. harzinum, were obtained from Agriculture Research Station (ARS), Navgaon (Alwar) and S. atra and P. chrysogenum were isolated by serial dilution method from the chilli and tomato field soils. All the fungal cultures were raised on PDA (Potato dextrose agar) for the each treatment. In 12 days old culture plates of all the fungal bio-agent, 20 ml of double sterilized distilled water was added to and suspension were obtained in conical flask. The naturally infected seed for each pathogen (100 seeds/treatment in triplicate) were taken soaked in spore suspension of the antagonist for 4 hrs. The seeds were incubated on moistened blotters by standard blotter method (Anonymous, 1985) and per cent seed germination, seedling symptoms incidence of the pathogen and inhibition of the pathogen were recorded on 8th day. The percent control of pathogens was calculated by the following formula:

In vitro evaluation in filter paper disc assay

The pure culture of bacterial antagonists was obtained from Agriculture Research Station (ARS), Durgapura, Jaipur. A lawn on nutrient agar medium with test pathogen was prepared using L-rods and incubates at 30±2°C for 20 min for the better growth of the test pathogen. A disc of (6 mm) Whatman filter paper, sterilized in oven at 140°C for 1 hr was impregnate with culture of antagonist namely Bacillus subtilis and Pseudomonas fluorescence placed on the already prepared lawn of the pathogen. In seeded agar method, wells of 6 mm diameter were yielded on (already seeded) nutrient agar medium using sterilized cork borer and crude suspension of fungal antagonists were place in it. The diameter or zone of inhibition was recorded up to 6 days in intervals of 24 hrs at 25 ± 2°C for each test agent and activity index was calculated. The clearing or zone of inhibition and activity index was calculated by given formula (Bahadauria and Kumar, 2004).

Results and discussion

Seed treatments using fungal antagonists in SBM

The significant improvement in seed germination in infected seeds was observed after treating the seeds with Trichoderma viride (87.5 and 90%) followed by Penicillium chrysogenum (85 and 87.5%) as compared to check (65.5 and 70%) in chilli seed samples ac. nos. Ca-1227 and Ca-1234 respectively infected with XAV. There was reduction in the incidence of the pathogen after treating T. viride (25 and 27.5%) followed by P. chrysogenum (42.5 and 37.5%) in both the samples showed the reduction of pathogen as compared to check (67.5 and 62.5%) respectively. The maximum per cent control of the pathogen shown by T. viride (62.96 and 56%) followed by P. chrysogenum (37.33 and 40%) in both the samples respectively (Table 1). The per cent control of the pathogen was statistically significant (P< 0.05) and relative per cent control of pathogen was as follows:

Trichoderma viride> Penicillium chrysogenum> Gliocladium virens> T. harzinum> Stachybhotrys atra

Table 1.  In vitro effect of seed treatment of fungal antagonists on seed germination and control of Xanthomonas campestris pv. axanopodis in chilli

S. No.

Fungal antagonists

Seed Samples

Ca-1227

Ca-1234

Seed germination (%)

Incidence of pathogen (%)

Control of pathogen (%)

Seed germination (%)

Incidence of pathogen (%)

Control of pathogen (%)

1.

Check

65.5 (55.24)

67.5 (55.24)

0 (0.00)

70.0 (56.79)

62.5 (52.24)

0 (0.00)

2.

Trichoderma viride

87.5** (69.30)

25.0** (30.0)

62.96**  (51.59)

90.0 (71.56)

27.5 (31.63)

56.00 (48.64)

3.

Gleocladium virens

82.5 (65.27)

37.5**  (37.76)

44.44** (41.55)

80.0 (63.44)

40.0 (39.23)

36.00 (35.15)

4.

T. harzianum

77.5 (61.68)

45.0 (42.13)

33.3 (35.24)

77.5 (61.68)

47.5 (43.57)

24.00 (28.59)

5.

Stachybotrys atra

72.5 (58.37)

65.0 (53.73)

03.70 (10.94)

72.5  (58.37)

55.0 (47.87)

12.00 (18.64)

6.

Penicillium chrysogenum

85.0** (67.21)

42.5 (40.69)

37.33 (36.05)

87.5 (69.30)

37.5 (37.76)

40.00 (38.59)

 

CD at 5%

CD at 1%

5.62

7.70

18.39

25.20

10.62

14.55

9.16

12.55

16.55

18.97

15.58

21.35

Values are the mean of 3 replicates; values in parentheses are angular transformed values.

In vitro evaluation of bacterial antagonists in filter paper disc assay

Against all the 18 morphologically and biochemically different isolates of XAV two bacterial antagonists namely Bacillus subtilis and P. fluorescence were used. The maximum zone of inhibition around the disc, in filter paper disc assay method was shown by the antagonist B. subtilis (15.60 to 16.10 mm) followed by P. fluorescence (11.50 to13.00 mm). The activity index was 2.60 to 2.68 and 1.9 to 2.16 in case of B. subtilis and P. fluorescence respectively (Figure 1A, Table 2). In the present study T. viride gave best control against XAV in chilli followed by P. chrysogenum. In in vitro evaluation of bacterial antagonist against pathogen B. subtilis showed potential control followed by P. fluorescence in filter paper disc method.

Table 2. In vitro evaluation of antimicrobial activity of bacterial antagonists against isolates of Xanthomonas axonopodis pv. vesicatoria in chilli

S. No.

Test Isolates

Bacterial Antagonists

Pseudomonas fluorescence (Pf)

Bacillus subtilis (Bs)

Inhibition zone (mm)

Activity Index

Inhibition zone (mm)

Activity Index

1

Ca-XCV-1

12.80

2.13

15.60

2.60

2

Ca-XCV-2

12.10

2.01

15.90

2.65

3

Ca-XCV-3

13.00

2.16

15.50

2.58

4

Ca-XCV-4

11.90

1.98

15.60

2.60

5

Ca-XCV-5

11.50

1.91

16.00

2.66

6

Ca-XCV-6

12.80

2.13

16.10

2.68

7

Ca-XCV-7

11.50

1.91

15.90

2.65

8

Ca-XCV-8

11.60

1.93

15.80

2.63

9

Ca-XCV-9

12.10

2.01

15.60

2.60

10

Ca-XCV-10

12.20

2.03

16.00

2.66

11

Ca-XCV-11

12.80

2.13

15.80

2.63

12

Ca-XCV-12

12.70

2.11

15.90

2.65

13

Ca-XCV-13

12.90

2.15

15.70

2.61

14

Ca-XCV-14

11.90

1.98

16.00

2.66

15

Ca-XCV-15

12.80

2.13

15.60

2.60

16

Ca-XCV-16

12.90

2.15

16.00

2.66

17

Ca-XCV-17

13.00

2.16

16.10

2.68

18

Ca-XCV-18

12.80

2.13

15.90

2.65

*Diameter of filter paper disc (6 mm) included inhibition zone in check (IZ = Inhibition Zone; AI = Activity Index).

Figure 1. Control of Xanthomonas campestris pv. vesicatoria isolated from seeds of chilli by Bacillus subtilis (Bs) and Pseudomonas fluorescence (Pf) on seeded nutrient agar medium. Note the zone of inhibition around the paper discs

 

In earlier studies, P. fluorescence found effective against several bacterial pathogens such as X. c. pv. citri, the inducer of citrus canker (Unnamalai and Gnanamanickam, 1984), X. oryzae pv. oryzae, causing bacterial blight of rice (Sivamani et al., 1987), Erwinia carotovora sub sp. carotovora (Kloepper et al., 1980), X. c. pv. malvacearum causing bacterial leaf sheath blight of cotton (Mondal et al., 1999) and X. axonopodis pv. vignaerditae causing bacterial leaf spot in mungbean (Dutta et al., 2005). It was observed that seed treatment with phylloplane bacteria Bacillus spp. reduced the X. axonopodis pv. vignaeraditae (Borah et al., 2000). A biformulation prepared (Plb. 3 isolate of Bacilluis spp. + inert carrier (talc) at different ratio + carboxy methyl cellulose) and 10% of total weight (spore power and talc as sticker) was found to be effective in producing inhibition zone of varying sized in spot test against different pathovars of X. a. pv. campestris (13 mm), X. a. pv. citri (20 mm),  X. a. pv. malvacearum (10 mm), X. a. pv. mangiferaeindicae (16 mm) and X. oryzae pv. oryzae (18 mm) (Patro et al., 2004).

In talc based formulation of P. fluorescens in glass house, revealed that bacterization of rice seed with the formulation followed by its two foliar sprays gave 61% disease control (Thind and Singh, 2004). Local isolation of P. fluorescens showed potential bio-control agent against R. solanacearum (Nath et al., 2004). A strain of P. putida isolation from pepper fruits was able to inhibit a wide spectrum of pathogens including 5 pathovars of P. syringae in in vitro study. P. fluorescence and Bacillus polymyxa have been found to delay the development and reduce the incidence of bacterial wilt (Shekhawat et al., 1983; Shekhawat et al., 1992; Mishghi et al., 1992). Several fluorescent pseudomonads were isolated from cotton rhizosphere (Laha, 1994). Out of 48 isolates only 9 were found antagonistic to Xanthomonas campestris pv. malvacearum (Xcm). In cotton B. subtilis has been reported as an excellent antagonistic against Xcm (Safiazov et al., 1995).

P. fluorescence produces various compounds that suppress the growth of R. solanacearum and induces systemic resistance in the plant (Li et al., 2011; Park et al., 2009; Alsohim et al., 2014). B. subtilis induce systemic resistance; improve plant growth in plants by secreting by several types of lipopeptides or secondary metabolites (Ongena et al., 2007; Kloepper et al., 2004; Bernal et al., 2002). Pre-application of biocontrol agents prevent the disease attack successfully (Ippolito and Nigro, 2000; Yendyo et al., 2017).

In in vitro study T. harzianum showed antimicrobial properties against both bacteria and fungi. It showed maximum antagonistic activity on A. terreus, A. fumigatus, A. clavatus and on clinical isolates namely Staphylococcus aureus, E. coli and Klebsiella. The minimum inhibitory concentration of T. harzianum on fungal isolates ranges from 100 150 μl/ml and for bacterial isolates ranges from 50 100 μl/ml of media. At 100μl/ml concentrations A. fumigatus, A. flavus, A. candidus, Cladosporium, Rhizopus were found to be inhibited whereas A. niger, Fusarium graminarium, F. semitectum, A. terreus were found to inhibit by the T. harzianum extract at 150 μl/ml (Leelavathi et al., 2014). Strain of Trichoderma showed a various degree of inhibition to various plant pathogens. T. harzianum tested against S. aureus, Proteus, E. coli, Klebsiella and found to be affective at 100 μl/ml concentrations (Parshikov et al. 2002; Fethi Bel Haj 2008; Nashwa et al., 2008; Jegathambigai et al., 2009). The MIC ranges from 100 150 μl/ml for fungal isolates and 50-100μl/ml for bacterial isolates (Leelavathi et al., 2014).

In another studies R. solanacearum controlled by B. subtilis and T. harzianum in potato or tobacco or tomato diseases (Lemessa and zeller, 2007; Sharma 2007; Aliye et al., 2008; Maketon et al., 2008; Ji et al., 2008; Chen et al., 2012). B. subtilis was used against infection of Arabidopsis roots by Pseudomonas syringae (Bais et al., 2004). Black rot caused X. c. pv. campestris in brassica controlled by strain of Bacillus subtilis in Zimbabwe (Monteiro et al., 2005; Wulff et al., 2002). In evaluation of spray programs containing famoxadone plus cymoxanil, acibenzolar-Smethyl and B. subtilis compared to copper sprays for management of bacterial spot on tomato was found effective against Xcv (Robert, 2008). Endophytic Streptomyces spp. and T. harzianum used as biocontrol agents of rice against bacterial leaf blight (Harman et al., 2004; Hastuti et al., 2012).

Conclusion

The fungal and bacterial antagonist were used to study their antimicrobial efficacy against Xanthomonas axonopodis pv. vesicatoria (Doidge) Dye and indicates the effective control of the pathogen and reduced the disease incidence significantly in non-hazardous manner by using fungal and bacterial antagonists. Out of used fungal antagonists Trichoderma viride found most effective which improve seed germination and control of pathogen followed by Penicillium chrysogenum as compared to check in all the method. In filter paper method, bacterial antagonist Bacillus subtilis found effective against the pathogen followed by Pseudomonas fluorescence in control of various isolates of XAV. The treatment also improved seed germination and control of pathogen as compared to check.

Acknowledgement

Author is also grateful to Prof. Ashok Sharma, vice-chancellor, VMOU, Kota; Prof. kailash Agrawal, University of Rajasthan, Jaipur, Principal, Agrawal P.G. College, Jaipur, for their valuable guidance and faculty members of P.G. Department of Botany, Agrawal PG College, Jaipur for valuable support. The author is also thankful to all the scientists whom work is cited and could not acknowledge unknowingly and persons that directly or indirectly engaged in writing in this paper and during practical work.

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