Open Access
P. Suneeta , S. Vinod Kumar, K. Eraivan Arutkani Aiyanathan and S. Nakkeeran
Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore – 641 003, Tamil Nadu, India.
J Pure Appl Microbiol. 2017;11(1):241-247
https://doi.org/10.22207/JPAM.11.1.31 | © The Author(s). 2017
Received: 14/09/2016 | Accepted: 03/11/2016 | Published: 31/03/2017
Abstract

Novel Trichoderma harzianum isolate NVTH2, effective against Fusarium oxysporum f. sp. gerberae (FOG) causing wilt in Gerbera showed growth tolerance against azoxystrobin 23 % SC, kresoxim methyl 44.3% SC and carbendazim 50% WP at all the tested concentrations (50ppm, 100 ppm, 250 ppm, 500 ppm, 1000 ppm, 1500 ppm and 2000 ppm) followed by fosetyl Al 50% WP, difenoconazole 25% EC and tebuconazole 250 EC (effective against FOG) moderately tolerant at lower concentrations. The fungicides like propioconazole 25% EC, propineb 70 WP and tebuconazole 50%+ trifloxystrobin 25% WG (effective against FOG) completely resisted the growth of NVTH2. Finally, a field experiment was designed by utilizing 3 most efficient Trichoderma spp. and 4 fungicides in combination treatments. As a result, root dipping+ soil drenching with NVTH2 @5ml/lit followed by soil drenching of tebuconazole @1ml/lit at fortnight interval alternately resulted in highest yield, growth promotion and percent inhibition of wilt incidence.

Keywords

Fungicides, Gerbera, growth promotion, Trichoderma.

Introduction

Gerbera jamesonii Bolus ex Hook has very huge demand globally. The production of cut flowers has gone from 2,071 million stems in 2007 to 6,667 million stems in 2011, which ultimately increased the growth of domestic and export markets in India1. Deterioration of soil health makes Gerbera highly susceptible to soil borne diseases under protected cultivation. The major soil borne diseases of Gerbera are foot rot, wilt, root rot complex and blight2.

Trichoderma harzianum is among the various species of the Trichoderma that is considered to be the utmost effective biocontrol agent3. The additive effects of Trichoderma and compatible fungicides were studied from many years. Arunasri et al.4 reported that Trichoderma sp. which was effective against collar rot of crossandra were highly compatible with thiram (32% growth inhibition) and followed by captan (47.5%). Integration of Captan + Metalaxyl with Trichoderma harzianum and T. virens were found to be the superior to control the wilt complex of bell pepper caused by four wilt pathogens viz. Fusarium oxysporum, Phytopthora capsici, Rhizoctonia solani and Sclerotium rolfsii and promoted seedling growth5

Materials and Methods

Isolation, pathogenicity and identification of FOG
Pathogen was isolated from the infected roots of Gerbera (var. Donavan yellow) on potato dextrose agar (PDA) medium amended with 1000 ppm of streptomycin sulphate. Surface sterilisation of infected root bits was done by 0.1% mercuric chloride (HgCl2) solution for 30 seconds and subsequently washed in sterile distilled water and were incubated at room temperature (27±2°C) for 5 days. The phenotypic characterization was done according to Burgess et al.6 using a light microscope (Labomed – IVU 5100) and photographed using a Labomed camera model LX400 with an image analyser – pixelpro programme.

The pathogen F. oxysporum multiplied in potato dextrose broth, consisting of 107conidia/ml was inoculated @ 1% to sterilized potting mixture (laterite soil: sand: compost) in 3:1:1 ratio filled @ 5kg/pot.

Collection of fungal antagonists
Three most effective isolates of Trichoderma spp. against FOG were selected from the Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, India and were taken for further experimentation (Table 1).
Table (1):
Various isolates of Trichoderma collected from Department of Plant Pathology.

S. No
Name of isolate
Name of antagonist
Accession number of the isolate in NCBI
3.
NVTH1
T. harzianum
KJ803856
4.
NVTH2
T. harzianum
KJ803857
3.
TV1
T. viride
Commercial strain (not submitted)

Compatibility of Trichoderma harzianum NVTH2 with fungicides
Compatibility of Trichoderma harzianum NVTH2 with fungicides viz. difenoconazole 25% EC (Score), tebuconazole 50% + trifloxystrobin 25% WG (Nativo), azoxystrobin 23 % SC (Amistar), propineb 70 WP (Antracol), fosetyl aluminium 50% WP (Alliete), propioconazole  25% EC (Tilt), tebuconazole 250 EC (Folicur), kresoxim-methyl 44.3% SC (Ergon) and carbendazim 50% WP (Benfil) was tested by Poisoned food technique7. The minimum inhibitory concentrations (50ppm, 100 ppm, 250 ppm, 500 ppm, 1000 ppm, 1500 ppm and 2000 ppm) of systemic and contact fungicides were amended in 100 ml of Potato Dextrose Agar medium @ 15 ml/plate.  The medium without fungicide served as control. The plates were incubated at room temperature (28±2oC). Three replications were maintained for each treatment @ 10 plates per replication.

Development of liquid formulation of Trichoderma spp
The fungal antagonists viz., T. harzianum strain NVTH1, T. harzianum NVTH2 and T. viride TV1 were cultured on 1000ml of Potato Dextrose Broth and incubated in an orbital shaker at 150 rpm at room temperature (28±2°C) for 48hr. Later the liquid biomass was mixed with 1% glycerol (10ml), tween 20 (10ml) and poly vinyl pyrrolidone – 40000 ml. wt (10g) each separately8. The resultant mixture was kept in orbital shaker at 200 rpm for 5 minutes to ensure uniform blending and homogenization of the bacterial cells. Then the formulation was standardized to obtain one ml of formulation consists of 106 cfu/ml. The liquid formulation was stored at 5°C for further study.

Root dip and soil drenching with liquid formulation of Trichoderma spp. against wilt of Gerbera under protected cultivation
Field experiment was conducted during 2013-2014 in Gerbera (var. Donavan yellow) fields located at Spic Agro Biotech centre, Ooty, to assess the efficacy of liquid formulation of Trichoderma spp. (106 cfu/ ml) @ 5ml/litre and fungicides @ 1ml/lit against wilt under protected condition (polyhouse). Thirty days old plants of Gerbera were used and the experiment was laid out with 7 treatments and 3 replications in RBD. The bed size of each replication was 5m2 with 30 × 30 cm spacing (Table 2).
Table (2):
Treatment schedule for Fusarium wilt management.

Treatment Treatment details
T1 RD of seedlings with T. harzianum NVTH2 (106cfu/ml) @ 5ml/litre during planting +
*SD with NVTH2 @ 5 ml/litre at 15 DAP+
**SD with Tebuconazole 250 EC @ 1ml/lit at 30 DAP
T2 RD of seedlings with T. harzianum NVTH2 (106cfu/ml) @ 5ml/litre during planting +
*SD with NVTH2 @ 5 ml/litre at 15 DAP+
**SD with Difenoconazole 25% EC @ 1ml/lit at 30 DAP
T3 RD of seedlings with T. harzianum NVTH2 (106cfu/ml) @ 5ml/litre during planting +
*SD with NVTH2 @ 5 ml/litre at 15 DAP+
**SD with Fosetyl Al 50% WP @ 1ml/lit at 30 DAP
T4 RD of seedlings with T. harzianum NVTH2 (106cfu/ml) @ 5ml/litre during planting +
*SD with NVTH2 @ 5 ml/litre at 15 DAP+
**SD with Carbendazim 50% WP @ 1ml/lit at 30 DAP
T5 RD of seedlings with T. harzianum NVTH2 (106cfu/ml) @ 5ml/litre during planting +
*SD with NVTH2 @ 5 ml/litre at 15 days interval.
T6 RD of seedlings with T. harzianum NVTH2 (106cfu/ml) @ 5ml/litre during planting +
*SD with T. harzianum NVTH1 @ 5 ml/litre at 15 DAP+
T7 **SD with T. viride TV1 @ 5 ml/litre at 30 DAP
Untreated control

SD-Soil Drenching; RD-Root Dip; DAP-days after planting
*Soil Drenching given at 15 days interval alternately with **Soil Drenching.

Statistical analysis
All the experiments were statistically analyzed independently. The treatment means were compared by Duncan’s Multiple Range-Test (DMRT) 9. The package used for analysis was IRRISTAT version 92-1 developed by the International Rice Research Institute, Biometrics unit, The Philippines.

RESULTS AND DISCUSSION

Symptomatology of Fusarium wilt
The symptoms associated with wilt were yellowing of lower most leaves, and subsequently spread to entire plant. Affected leaves droop down and finally wilted. In later stages of the crop the presence of black discolouration in collar areas and brownish discolouration in petioles was observed. Wilting of the entire plant occurred within 3 to 4 weeks after infection (Figure 1). Similar report was made by Garibaldi et al.10

Identification of the Pathogen
The mycelium of the fungal culture on PDA medium was initially white and later turned light pink to dark pink in different isolates. Macroconidia was sparse, and fusoid, 2-3 septate and measured 16.0-29.0 x 2.5-4.2 µm.  Microconidia were abundant, hyaline, continuous, ovoid and measured 3.8-8.5 x 2.0-3.5 µm. Chlamydospores were hyaline and spherical, measured 4.0 – 7.5 µm in diameter. Based on these phenotypic characters, the pathogen was confirmed as Fusarium oxysporum f. sp. gerberae (KJ570974). The morphological characters were similar with the descriptions made by Booth11.

Pathogenicity
Inoculation of F. o. f. sp. gerberae (FOG) in to the healthy Gerbera seedlings of var. Bellwater white (30 days old) expressed the typical symptoms of wilt of Gerbera after 15 days of inoculation. Infected plants showed typical stunting of the plants and yellowing of leaves with brown to black streaks noticed in the crown portion and petioles of the plant. No symptoms were observed in un-inoculated control plants. Similar pathogenicity results were recorded by Garibaldi and Minuto12.

Compatibility of Trichoderma harzianum isolate NVTH2 with fungicides
In the present study, tebuconazole 250 EC which was the most effective fungicide against FOG13 was compatible with biocontrol agent T. harzianum isolate NVTH2 only at lower concentrations and was highly compatible with azoxystrobin 23 % SC, kresoxim methyl 44.3% SC and carbendazim 50% WP at all the tested concentrations. It was followed by fosetyl Al 50% WP, difenoconazole 25% EC and tebuconazole 250 EC moderately tolerant at lower concentrations. The fungicides like propioconazole 25% EC, propineb 70 WP and tebuconazole 50%+ trifloxystrobin 25% WG (effective against FOG)13 resisted the growth of NVTH2 to 100% (Table-3).

Table (3):
Compatibility of Trichoderma harzianum (NVTH2) with fungicides.

S. No Fungicides Mycelial growth (mm2)*
Lower Moderate Recommended Higher
dosages dosages dosages dosages
50 ppm 100 ppm 250 ppm 500 ppm 1000 ppm 1500 ppm 2000 ppm
1 Tebuconazole 250 EC 30.00 21.00 0.00 0.00 0.00 0.00 0.00
(66.67) (76.67) (100.0) (100.0) (100.0) (100.0) (100.0)
2 Propioconazole25% EC 0.00 0.00 0.00 0.00 0.00 0.00 0.00
(100.0) (100.0) (100.0) (100.0) (100.0) (100.0) (100.0)
3 Difenoconazole 25% EC 14.00 8.00 0.00 0.00 0.00 0.00 0.00
(84.44) (91.11) (100.0) (100.0) (100.0) (100.0) (100.0)
4 Azoxystrobin 23 % SC 67.00 62.00 53.00 47.00 33.00 26.00 19.00
(25.56) (31.11) (41.11) (47.78) (63.33) (71.11) (78.89)
5 Propineb70 % WP 0.00 0.00 0.00 0.00 0.00 0.00 0.00
(100.0) (100.0) (100.0) (100.0) (100.0) (100.0) (100.0)
6 Fosetyl aluminium 80% WP 63.00 55.00 41.00 36.00 0.00 0.00 0.00
(30.00) (38.89) (54.44) (60.00) (100.0) (100.0) (100.0)
7 Kresoxim-Methyl 44.3%SC 90.00 78.00 61.00 58.00 51.00 49.00 42.00
(0.00) (13.33) (32.22) (35.56) (43.33) (45.56) (53.33)
8 Tebuconazole 50% + 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Trifloxystrobin 25% WG (100.0) (100.0) (100.0) (100.0) (100.0) (100.0) (100.0)
9 Carbendazim 50%WP 90.00 90.00 82.00 62.00 56.00 41.00 39.00
(0.00) (0.00) (8.99) (31.11) (37.78) (54.44) (56.67)
10 Control 90.00 90.00 90.00 90.00 90.00 90.00 90.00

*Values are mean of three replications.

In a column, means followed by a common letter are not significantly different at the 5% level by DMRT

Bhai and Thomas14 reported that mancozeb and copper oxychloride were highly compatible with Trichoderma harzianum which was effective against rhizome rot of cardamom. Tapwal et al.15 reported that T. viride effective against Armillaria mellea is compatible with the fungicide captaf and blue copper only to some extent.

Effect of Trichoderma spp. and fungicides on the management of Fusarium wilt and Plant growth promotion in polyhouse
Delivery of T. harzianum isolate NVTH2 through root dipping & soil drenching @ 5ml/litre+ soil drenching of Tebuconazole @ 1ml/lit recorded the lowest wilt incidence of 3.28% with highest mean flower yield of 52.33 numbers/m2 and also increased the root length and plant height. It was found significant with the application of treatment T6 in growth promotion and yield parameters. Likewise, treatment T1 also favored the early flowering and increased flower parameters, followed by treatment T6 over the untreated control (Table-4 and 5).

Table (4):
Effect of Trichoderma spp. + fungicides on wilt incidence, growth characters and flower yield of Gerbera under protected cultivation.

S. No. Treatment module Wilt incidence* Root length (cm)* Plant height (cm)* No. of flowers/m2*
1. RD-NVTH2 @ 5ml/lit+ 3.28a(73.33) 21.23a 43.20a 52.33a
**SD-NVTH2@5ml/lit+
***SD-Tebuconazole@1ml/lit
2. RD-NVTH2 @ 5ml/lit+ 6.16d(49.91) 19.30c 39.93c 47.00d
**SD-NVTH2@5ml/lit+
***SD-difenoconazole@1ml/lit
3. RD-NVTH2 @ 5ml/lit+ 9.25e(24.79) 18.63d 38.43d 45.38e
**SD-NVTH2@5ml/lit+
***SD-Fosetyl Al @1ml/lit
4. RD-NVTH2 @ 5ml/lit+ 10.22f(16.91) 18.66d 37.56e 44.20e
**SD-NVTH2@5ml/lit+
***SD-Carbendazim@1ml/lit
5. RD-NVTH2 @ 5ml/lit+ 5.29c(56.99) 19.13c 39.20c 49.00c
**SD-NVTH2@5ml/lit
6. RD-NVTH2 @ 5ml/lit+ 4.23b(65.60) 20.33b 42.20b 51.40b
**SD-NVTH1 @5ml/lit+
***SD-TV1 @5ml/lit
7. Untreated Control 12.30g 18.23d 36.20f 38.33f

*Values are mean of three replications
Means followed by a common letter are not significantly different at 5% level by DMRT
Data in the parenthesis are per cent reduction over control
RD-Root dipping; SD-soil drenching; **SD applied alternate to ***SD at fortnight interval

Table (5):
Effect of Trichoderma spp. + fungicides on flowering in Gerbera under protected cultivation (wilt management trial).

S. No. Treatment module Days taken for flower bud initiation* Days taken for flower bud opening* Length of flower stalk(cm)* Flower Diameter (cm)*
1. RD-NVTH2 @ 5ml/lit+ 82.20a 99.30a 36.60a 9.60a
**SD-NVTH2@5ml/lit+
***SD-Tebuconazole@1ml/lit
2. RD-NVTH2 @ 5ml/lit+ 84.00b 105.00cd 30.40d 8.30bc
**SD-NVTH2@5ml/lit+
***SD-difenoconazole@1ml/lit
3. RD-NVTH2 @ 5ml/lit+ 85.00c 109.00e 28.80e 7.90cd
**SD-NVTH2@5ml/lit+
***SD-Fosetyl Al@1ml/lit
4. RD-NVTH2 @ 5ml/lit+ 87.00d 111.33f 28.00e 7.50de
**SD-NVTH2@5ml/lit+
***SD-Carbendazim@1ml/lit
5. RD-NVTH2 @ 5ml/lit+ 83.66b 104.00c 32.10c 8.60b
**SD-NVTH2@5ml/lit
6. RD-NVTH2 @ 5ml/lit+ 83.00b 101.00b 35.70b 9.20a
**SD-NVTH1 @5ml/lit+
***SD-TV1 @5ml/lit
7. Untreated Control 91.00e 119.00g 26.10f 7.10f

*Values are mean of three replications
Means followed by a common letter are not significantly different at 5% level by DMRT
Data in the parenthesis are per cent reduction over control
RD-Root dipping; SD-soil drenching; **SD applied alternate to ***SD at fortnight interval

The efficiency of managing soil borne pathogen FOG can be maximised with the combined use of fungicides and antagonistic fungi but this can be achieved only if both are compatible with each other. Gaur and Sharma16 integrated T. viride -1 or T. harzianum (TG-1) with metalaxyl and cymoxanil 8% + mancozeb 64% to control root rot in cotton. Seed treatment with T. harzianum + soil treatment with neem cake powder + foliar spray with carbendazim had reduced the Fusarium wilt of tomato and increased the the yield and growth parameters like shoot & root length and fresh weight & dry weight of the plants over the control17.

Many triazole compounds have good fungicidal and plant growth regulating activities. In the triazole fungicide (difenoconazole), thirteen novel triazole analogs of difenoconazole containing 1, 3-dioxolane rings have been synthesized and they express plant-growth regulatory activity18.  Seedling inoculation with three isolates of Trichoderma spp. (1, 2 and 3) significantly increased the growth and controlled the wilt pathogen (F. o. f. sp. gerberae) of Gerbera plants as compared to control19.

CONCLUSION

Present experiment was undertaken by keeping in view of the hasty incidence of Fusarium oxysporum f. sp. gerberae in Gerbera plants. Initially the antagonistic fungi (NVTH2) was tested for its compatibility with the chemical fungicides at all types of dosages. Mostly it was compatible at lower dosages with fungicides. Later, the management module was developed at field level by combining both the Trichoderma spp. and fungicides against the FOG which finally resulted in the efficient module of RD + SD with NVTH2 @ 5ml/lit+ SD with Tebuconazole @ 1ml/lit at fortnight interval alternatively.

References
  1. Jafar Naqvi, G. Editorial. Floriculture Today. 2011; 16: 8.
  2. Padghan, P. R. and Gade, R. M. Biomanagement of root rot complex of Gerbera(Gerbera jamesonii Bolus). Ann. Pl. Protec. Sci. 2006; 14:134-138.
  3. Gao, K. X., Liu, X. G., Liu, Y. H., Zhu, T. B., Wang, S. L. Potential of Trichoderma harzianum and T.atroviride to control Botryosphaeriaberengeriana f. sp. piricola, the cause of apple ring rot. J. Phytopathol. 2002; 150: 271-276.
  4. Arunasri, P., Chalam, T. V., Reddy, N. P. E. and Reddy, S. T., Collar rot disease of Crossandra induced by Sclerotium rolfsii and its management: a critical review. International J. of Applied biology and Pharmaceutical technology., 2011; 2(2): 307.
  5. Rather, R. T., Razdan, K. V., Tewari, A. K., Shanaz, E., Bhat, A. Z., Hassan, G. M., Wani, A. T., Integrated Management of Wilt Complex Disease in Bell Pepper (Capsicum annuum L.). Journal of Agricultural Science. 2012; 4 (7): 141-147.
  6. Burgess, L. W., Nelson, P. E., Toussoun, T. A., Forbes, G. A. Distribution of Fusarium species in sections Roseum, Arthrosporiella, Gibbosum and Discolor recovered from grassland, pasture and pine nursery soils of eastern Australia. Mycologia. 1989; 80: 815-824.
  7. Grover, R. K., Moore, J. D. Toxicometric studies of fungicides against brown rot organisms Sclerotinia fructicola and S. laxa. Phytopathology.1962; 52: 876- 880.
  8. Somasegaran, P. and Hoben, H. J., Methods in legume Rhizobium technology, University of Hawaii, NiFTAL Project and Mircen. Department of Agronomy and Soils, 1985; pp 451.
  9. Gomez, K. A. and Gomez, A. A. Statistical Procedure for Agricultural Research. 1984; John Wiley and Sons, New York.
  10. Garibaldi, A., Minuto, A., Bertetti, D., Gullino, M.L. Fusarium Wilt of Gerbera in Soil and Soilless Crops in Italy. Pl. Dis., 2004; 88: 311.
  11. Booth, C. The genus Fusarium. Common wealth Mycological Institute, Kew Surrey,  UK., 1971;P 142-143.
  12. Garibaldi, A., and Minuto, A. Fusarium wilt of gerbera in Spain in soilless crops. Pl. Dis., 2007; 91: 638.
  13. Suneeta, P., Eraivan, A. A. K., Nakkeeran, S. In vitro management of Fusarium wilt of Gerbera by novel Trichoderma spp. and fungicidal molecules. International Journal of Agricultural Science and Research. 2016; 6(5): 327-332.
  14. Bhai, R. S. and Thomas, J. Compatibility of Trichoderma harzianum (Rifai.) with fungicides, insecticides and fertilizers. Indian Phytopath., 2010; 63(2):145-148.
  15. Tapwal, A., Kumar, R., Gautam, N. and Pandey, S. Compatibility of Trichoderma viride for selected fungicides and botanicals. International journal of Plant pathology, 2012; 3(2): 89-94.
  16. Gaur, R. B. and Sharma R. N. Biocontrol of Root Rot in Cotton and Compatibility of Potential Bioagents with fungicides. Indian Journal of Plant Protection. 2010; 38(2): 176-182.
  17. Singh, R., Biswas, K. S., Nagar, D., Singh, J., Singh, M. and Mishra, K. Y. Sustainable Integrated Approach for Management of Fusarium Wilt of Tomato Caused by Fusarium oxysporum f. sp. lycopersici (Sacc.) Synder and Hansen. Sustainable Agriculture Research. 2015; 4(1): 138-147.
  18. Xu, S. S., Friesen, T. L. and Mujeeb-Kazi, A. Seedling resistance to tan spot and Stagonospora nodorum blotch in synthetic hexaploid wheats. Crop Sci., 2004; 44: 2238-2245.
  19. Jamwal, S. and Jamwal, A. Management of root rot complex of Gerbera caused by Fusarium oxysporum f. sp. gerberae and Pythium irregualre by Trichoderma spp. Ann. Pl. Protec. Sci., 2012; 20(1):160-163.

Article Metrics

Article View: 2993

Share This Article

© The Author(s) 2017. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.