Bio-efficacy of Antagonists and its Culture Filtrates against Black Mould Rot [Aspergillus niger Van Tieghem] of Garlic (Allium sativum L.)

Garlic (Allium sativum L.) is the most important bulbous crop after onion, belonging to Amaryllidaceae family (Mishra et al., 2014). Garlic is cultivated in India as the second major bulbous crops next to onion, covering an area of 227.39 thousand hectares with a total production of 1181.37 thousand MT having productivity of 5195 kg per hectare (Anon., 2015a). Garlic crop suffers from many diseases post-harvest diseases viz., white rot (Sclerotium cepivorum Berk), blue mold rot (Penicillium spp.), black mold (A. niger), pink rot (Pyrenochaeta terrestis Hansen), internal bulb rot (Macrophomina phaseolina (Tassi) Goid, basal rot (Fusarium oxysporum f. sp. cepae) and neck rot (Botrytis allii Munn) (Anonymous., 2015b), which cause considerable losses, apart from reduction in crop yields. The diseases also pose harmful effects during harvesting, post harvesting, processing and marketing stages, which lower the quality and export potentiality of the crop that significantly cause the economic loss (Mishra et al., 2014). 

Black mould rot is caused by Aspergillus niger, is the most common fungi causing food spoilage and bio deterioration of other materials (Samson et al., 2004). Black mold is responsible for incurring losses to the extent of 12-25 percent due to various bulb rots and total losses during storage of 5-6 months as high as 30-40 per cent (Maini and Chakravarti, 2000). Nandeesha et al., (2013) reported that Trichoderma spp. inhibited 81.36 per cent growth of A. niger by dual culture method. Trichoderma spp. is well documented as effective biological control agent of plant diseases (Harman et al., 1980; Sivan et al., 1984 and Coley-Smith et al., 1991). Therefore, the present investigation was carried out to evaluate bio-efficacy of antagonists and culture filtrates of Trichoderma spp., against black mold rot of garlic.   

The present study was carried out in the Department of Plant Pathology, B.A. Collage of Agriculture, Anand Agricultural University, Anand, Gujarat during  2015-16. The Completely Randomized Design (CRD) was used for the experimentation.
 
Collection and isolation of pathogen
 
Garlic bulb showing typical symptoms of black mold rot were brought to the laboratory and pathogen isolated by tissue isolation method under aseptic conditions. The pure culture of pathogen was obtained by single spore isolation and maintained on Potato Dextrose Agar (PDA) medium. The pathogen was identified by morphological characteristics and molecular studies. The molecular identification was carried out by DNA barcoding using ITS region sequencing. Various antagonists viz., Trichoderma viride, T. harzianum, T. virens, T. atroviride, T. asperellum, T. fasciculatum, Pseudomonas fluorescens and Bacillus subtilis were available in the Department of Plant Pathology, B.A. College of Agriculture, Anand Agricultural University, Anand, Gujarat screened to test their efficacy against A. niger.
 
Dual culture method
 
The dual culture method (Dennis and Webster, 1971) was used to test the antagonists against A. niger. The antagonists and pathogen were grown on PDA medium. Sterilized PDA medium (20 mL) was poured aseptically in 90 mm diameter sterilized Petri plate. Mycelial disc of 5 mm diameter cut from the periphery of both antagonist and test pathogen and were placed at 50 mm apart from each other in Petri plates. The test pathogen was grown alone in petri plate for comparison. Each treatment was repeated three times. The Petri plates were incubated at 25±1°C in BOD incubator. The observations on mycelial growth (mm) and per cent growth inhibition of test pathogen were recorded after 7 days of incubation. The per cent growth inhibition (PGI) of pathogen in each treatment was calculated by formula given by Asalmol et al., (1990).
 
Bulb dip method
 
Pre- inoculation and post-inoculation treatment was employed by using bulb dip method (Chastagner and DeBauw, 2011). In pre-inoculation, healthy, mature and uniform sized garlic bulbs were first randomly pin-pricked and then dipped in the spores suspension (106 spores/mL) and LCF of Trichoderma spp. for 5 min and allowed to air dried overnight and then inoculated with the test pathogen (106 spores/mL). The interval between spore suspension treatment and inoculation was kept 12 hours and were incubated at room temperature. Control was maintained separately with pathogen. Each treatment was replicated three times. The per cent rot severity was recorded on 7^th and 14^th day after inoculation with the help of formula given by Siyom and Yusuf (1990). In post-inoculation, the procedure detailed in pre-inoculation was followed except that the bulbs were first inoculated with pathogen and then with antagonists.
 
Effect of liquid culture filtrate (LCF) of Trichoderma spp. against A. niger
 
Bioefficacy of liquid culture filtrate of different Trichoderma spp. were evaluated by “Poisoned food technique” suggested by Nene and Thapliyal (1979) against A. niger in-vitro. Conical flask containing 100 mL of sterilized Potato Dextrose Broth (PDB) medium was inoculated with 5 mm diameter mycelial disk cut from margin of seven days old culture of six Trichoderma spp. The flasks containing individual bio-agents were incubated in an orbital shaker cum BOD incubator at 27±1°C for 21 days. After 21 days of incubation the culture filtrates were filtered separately by what man filter paper No. 1 to remove mycelial mat and the spores were removed by vacuum filter. The LCF of Trichoderma spp. (300 µL/ mL of PDA) were added to PDA medium separately and after solidification; 5 mm discs of A. niger were inoculated in the centre of each Petri plates separately. Petri plates inoculated with A. niger, without amendment of filtrate served as a control. The inoculated Petri plates were incubated in BOD incubator at 27±2ºC. Each treatment was repeated three times and the per cent mycelial growth inhibition of the pathogen was recorded after 7 days of incubation. Further, pre and post inoculation treatment was given to bulbs through bulb dip method as described earlier in antagonist study.

Dual culture method
 
The lowest mycelial growth i.e. 24.33 mm with highest percent mycelial growth inhibition i.e. 72.96 was recorded in T. asperellum, which was at par with T. harzianum. While mediocre effect on per cent mycelial growth inhibition of A. niger was noticed in T. atroviride and T. virens i.e. 64.07 and 53.96. Meanwhile, Per cent lowest mycelial growth inhibition was observed in T. fasciculatum which were at par with P. fluorescens. The results are presented in Table 1 and Plate 1.





Result similar to the present investigation was reported by Prajapati (2016). He observed highest mycelial growth in T. asperellum i.e. 78.89 per cent followed by T. harzianum and T. viride i.e. 4.44 and 71.86, after seven days of incubation against A. niger. Nandeesha et al., (2013) reported that all the fourteen isolates of Trichoderma sp. inhibited (81.36%) growth of A. niger in groundnut by dual culture method. Lone et al. (2012) and Bhushan et al., (2013) reported maximum inhibition zone created by T. harzianum against A. niger (75%) followed by Cladosporium spherospermum (Link) (72.2%) and F. oxysporum (25%) in common walnut whereas Bacillus subtilis against A. flavus and A. terreus was 1.0 cm and against A. niger was 0.9 cm and the minimum zone of 0.8 cm was recorded against A. fumigates and F. oxysporum by the bacterium in bajra, respectively by using dual culture method. Bagwan (2011) reported that the Trichoderma spp. effectively inhibited the growth of A. niger up to 98 per cent in groundnut isolate by dual culture method. Wang et al. (2008) found four isolates viz., Bacillus amyloliquefaciens (EXWB3), Bacillus subtillis strains EXWB1, EXWB2 and EXWB4 effective against eight post-harvest pathogens including B. cinerea, A. alternata, F. oxysporum, A. niger, T. roseum, Penicillium sp. and Cladosporium sp. on melon fruits. Among the Bacillus subtillis strains EXWB1 was found to be the most effective in controlling the fungal rots. Prajapati et al., (2015) reported that Trichodema asperellum showed maximum mycelial growth inhibition against Sclerotium rolfsii.
  
Bulb dip method
 
All the bioagents were further tested on the bulbs following pre- and post-inoculation treatments and results are presented in Table 2 and Plate 2. In pre-inoculation treatment, Trichoderma asperellum found significantly superior in reducing the black mold rot severity i.e. 0.49 per cent both on 7^th and 14^th day after inoculation followed by T. harzianum, T. viride and T. atroviride. Whereas T. virens and T. fasciculatum gave mediocre effect while B. subtilis and P. fluorescens found least effective in reducing black mold rot severity on 7^th and 14^th day after inoculation over control. In Post- inoculation treatment, the trend similar to that observed in pre-inoculation was noted in post-inoculation treatment. Trichoderma asperellum found significantly superior in reducing the per cent black mold rot severity i.e. 10.13 and 15.81 followed by T. harzianum (13.81 and 24.82%), T. viride (17.85 and 24.71%) and T. atroviride (29.46 and 33.83%) on 7^th and 14^th day after inoculation. B. subtilis (38.11 and 48.41%) and P. fluorescens (49.61 and 58.90%) found least effective. It was observed that among two treatments of inoculation, pre- inoculation treatment was found superior in managing the black mold rot severity as compared to post-inoculation.





Similar trend of results was recorded by Prajapati (2016). He reported that Trichoderma asperellum gave cent per cent reduction in black mould rot severity after 7^th and 0.33 per cent after 14^th day of inoculation, followed by T. harzianum, T. viride, T. virens, T. fasciculatum and T. atroviride against A. niger. Reddy (2012) reported that T. harzianum was found most effective in reducing the Penicillium rot severity by 4.50 and 10.00 per cent on 4^th and 8^th day after inoculation. Baig et al., (2012) reported that P. fluorescens and B. subtilis can effectively suppress the growth of A. niger up to 73 and 60 per cent respectively, in groundnut seeds. Bhuvneswari and Rao (2011) reported that the T. viride acts as a most potent bio-agent for controlling Aspergillus fruit rot of mango. Meena (2006) reported that T. viride and T. harzianum at 1 x 106 cfu/mL found very effective against Penicillium fruit rot of aonla. Bagwan (2003) recorded lowest Penicillium rot severity in citrus (P. digitatum and P. italicum) when the fruits were treated with Candida oleophila, Candida sp. and T. viride.
 
Effect of liquid culture filtrate (LCF) of Trichoderma spp. against A. niger
 
The lowest mycelial growth i.e. 28.33 mm and highest percent growth inhibition i.e. 68.52 was recorded in T. asperellum on 7^th day after incubation, followed by T. harzianum i.e. 45 mm and 50.00, was at par with T. atroviride and T. viride 47.41 and 45.93 per cent. While, minimum mycelial growth and lowest per cent growth inhibition was recorded in T. virens, was at par with T. fasciculatum. The results are presented in Table 3 and Plate 3.





The results of present investigations corroborate with the results obtained by Prajapati (2016). He recorded highest mycelial growth inhibition by LCF of T. harzianum followed by T. asperellum and T. viride i.e.88.14, 87.41 and 85.56 per cent after seven days of incubation against A. niger. Odebode (2006) reported that the culture filtrates of T. harzianum revealed highest per cent mycelial growth inhibition i.e. 45.60 against A. niger. Rajendran et al., (2010) studied inhibitory effect of culture filtrate (50% v/v) of T. viride on mycelium growth inhibition of A. niger, A. fumigatus and A. flavus and reported 64, 49 and 48 per cent mycelial growth inhibition, respectively in fruits and vegetables through poisoned food technique. Terna et al. (2013) evaluated antagonistic potential of culture filtrates of a soil isolate of T. viride in vitro, against three common post-harvest rot pathogens; A. niger, A. flavus and F. oxysporum. They observed that the synergistic combination of all three enzyme-induced culture filtrates (chitinase, protease and cellulase) of T. viride gave the highest radial growth inhibitions by A. niger (77.69%), A. flavus (67.89%) and F. oxysporum (46.95%), respectively.  Kataty and Emam (2012) found that, culture filtrate of T. harzianum (T3 and T24) greatly inhibited spore germination of the test post-harvest pathogenic fungi (Geotricum candidum, Penicillium steckii, Rhizopus sp., Fusarium sp. and Aspergillus sp.). Nirupama and Singh (2011) screened culture filtrates of ten isolates of Trichoderma spp. and evaluated the production of volatile and non-volatile inhibitors against the F. oxysporum f. sp. lycopersici, causing wilt in tomato. Culture filtrate of T. viride (TV19) at 15 percent concentration gave highest inhibition of pathogen growth followed by T. harzianum (TH7). The similar trend of results in growth inhibition of the pathogen was observed in volatile metabolites of 15-days old cultures of TV19 and TH7. Prajapati et al., (2015) showed that the LCF of T. harzianum and T. viride revealed complete inhibition of mycelial growth of Sclerotium rolfsii.

In pre-inoculation treatment, the LCF obtained from Trichoderma asperellum found superior in reducing the black mold rot severity i.e. 20.05 and 21.32 after 7^th and 14^th day after inoculation followed by T. harzianum and T. viride i.e. 20.96, 25.49 and 23.29, 30.14, respectively. T. virens and T. atroviride showed mediocre effect. While, the culture filtrate of T. fasciculatum found least effective in managing black mold rot severity i.e. 33.21 and 44.46 per cent as compared to control  after 7^th and 14^th days after inoculation, respectively. Similar trend of results was observed in post-inoculation treatments. The culture filtrate of T. asperellum was found effective in reducing the per cent black mold rot severity i.e. 27.43 and 28.60 after 7^th and 14^th days of inoculation followed by T. harzianum, T. viride. T. virens and T. atroviride gave mediocre effect. T. fasciculatum found least effective. The results obtained are presented in Table 4 and Plate 4.





The results of present investigations corroborate with the results obtained by Prajapati (2016) he reported that the liquid culture filtrates of Trichoderma asperellum, T. harzianum and T. viride found significantly superior showing no black mould rot severity followed by T. fasciculatum i.e. 5.66 and 9.67 per cent on 7^th and 14^th day after inoculation. Adebesin et al. (2009) showed that the culture filtrate of T. asperellum (NG-T161) inhibited the mycelial growth of F. oxysporum and Colletotrichum musae by 49.7 and 60.3 per cent, respectively at 50 per cent (v/v) concentrations. Patel (2009) reported that culture filtrate of T. harzianum at 1250 µl/ml gave complete mycelial growth inhibition of A. niger of groundnut while in dual culture it was 71.11 per cent. Mortuza and Ilag (1999) reported that conidia and culture filtrates of Trichoderma spp. effectively reduced the rotting in banana fruits when artificially inoculated with Lasiodiplodia theobromae.

Among all the antagonists significantly lowest mycelial growth with highest mycelial growth inhibition was recorded in T. asperellum followed by T. harzianum. Pre-inoculation treatment with, T. asperellum  found significantly superior in reducing the garlic black mould rot severity on 7^th and 14^th day after inoculation followed by T. harzianum, T. viride, T. atroviride  over control. Similar trend of results was observed in post-inoculation treatment. Culture filtrate of T. asperellum T. harzianum found effective in in vitro and in vivo.

The future of food production and environmental safety are in jeopardy due to issues in agriculture. The emergence of plant pathogenic microorganisms in a crop plantation caused a pandemic plant diseases phenomenon. The best solution to overcome this problem is the application of biological control by using Trichoderma spp. in agricultural products. Trichoderma spp. significantly suppress the growth of plant pathogenic microorganisms and regulate the rate of plant growth.