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Introduction
Capsicum annuum L. (chilli) is an important spice crop of India which is cultivated in an area of 774.9 thousand hectare with productivity of 1.93 tonnes per hectare.1 The word “capsicum” is derived from Greek word “Kapsimo” which means “to bite”. Chilli is also known as bell pepper, pod pepper, hot pepper, red pepper, cayenne pepper, paparika and capsicum in different parts of the world. It is believed to be originated in Mexico as a wild crop around 7500 BC and was first introduced to India by Portuguese during 15th century, more particularly in Goa.2
Heavy losses in capsicum can occur due to fungi, viruses, nematodes and bacteria many of which survive in seed. Seed borne mycoflora reported in chilli includes, Aspergillus niger Tiegh., Aspergillus flavus Link, F. oxysporum, P. aphanidermatum, Colletotrichum sp., Fusarium moniliforme J. Sheld., Alternaria alternata (Fr.) Keissl., Penicillium sp., Cladosporium sp., Curvularia sp., Drecheslera sp., Macrophomina phaseolina, Rhizopus sp. and Mucor sp.3,4 Many of these pathogens were carried on seed either internally or externally and can result in reducing seed germination and seedling vigour which leads to poor yield.
Association of seed borne mycoflora with chilli seeds results in loss of seed viability and seedling mortality (post and pre emergence), discolouration of fruit and rotting of fruit. Fruit rot is of great concern as it declines the market value of fruit and the low seed quality may cause yield losses of upto 50 per cent.4 Dead chilli seedling in field serves as a potential source of inoculum for further spread of pathogen in field. To increase the production and productivity of chilli in terms of quality and quantity, farmer requires healthy seeds, with high germination and purity.
Hence, it is of vital importance that seeds should be treated before they are used in the field. This investigation was carried out to check the efficacy of bioagents on seed mycoflora of chilli.
Materials and methods
Collection of seed samples
Seeds of chilli variety GVC-111 was selected for research purpose and were collected from the Regional Horticultural Research Station (RHRS), Navsari Agricultural University, Navsari. Seeds were packed and preserved in polythene bags at room temperature for further investigation.
Identification of dominant seed mycoflor
flavus, A. niger, Alternaria sp., Colletotrichum sp., Penicillium sp., M. phaseolina, Curvularia sp., Fusarium sp., sterile septate and aseptate fungi were isolated from chilli seed variety GVC 111 by agar plate and standard blotter method during the experiment. Among those isolated fungi, A. niger, Colletotrichum sp. and Fusarium sp. were found to be more common and dominant fungi as it was noticed from almost all the seed samples.
Evaluation of bioagents as seed treatment on dominant seed mycoflora
The efficacy of bioagent seed treatment on inhibition of seed borne mycoflora through seed germination and seedling vigour was studied by paper towel method in vitro. Spore suspension of most dominant seed mycoflora (A. niger, Colletotrichum sp. and Fusarium sp.) recorded from seeds of chilli variety GVC 111 were prepared separately in sterilized distilled water from 10 days old cultures. By using an improved Double Neubaur Haemocytometer, concentration of spores was maintained to 5 X 104spores/ml in the suspension and it was then filtered with muslin cloth to separate the mycelium from spore suspension.5
Healthy seeds were surface sterilized with mercuric chloride (0.1%) and then washed three times with sterilized distilled water. Those seeds were soaked in the spore suspension of most dominated fungi for 8 hours separately. The soaked seeds then taken out from the solution and kept on blotter paper for drying. After that suspension of talc based formulation of respective bioagents @ 5g/kg for fungal bioagents (106cfu/g) and @ 6g/kg for bacterial bioagents (108cfu/g) was prepared (Table 1). Then mix the seeds in suspension and again wait for 8 hours. Soaked seeds were taken out from the suspension and dried in blotter paper. Treated seeds were placed equidistantly between two previously wetted germination papers. Seed soaked only in spore suspension of corresponding pathogen was kept as control. Three repetitions for each of the fungal species in each variety were kept. The paper towels were rolled without disturbing the position of the seed and labelled properly. The ends of paper towels were closed with rubber bands, kept in polythene bag and incubated in upright position. After 7 days of incubation period, they were observed and following parameters were measured and calculated.
Table 1: Various bioagents treatment evaluated against the most dominant seed mycoflora of chilli in vitro.
| Treatment No. | Name of bioagents | Dose
(g/kg seeds) |
| T1 | Trichoderma viride (Navsari isolate) | 5 |
| T2 | Trichoderma harzianum (Navsari isolate) | 5 |
| T3 | Pseudomonas fluorescens (Navsari isolates) | 6 |
| T4 | Bacillus subtilis (Navsari isolates) | 6 |
| T5 | Trichoderma harzianum+ Pseudomonas fluorescens | 5+6 |
| T6 | Trichoderma viride+ Pseudomonas fluorescens | 5+6 |
| T7 | Pseudomonas fluorescens + Bacillus subtilis | 6+6 |
| T8 | Control (treated with respective pathogen only) | – |
|
(Fungal antagonists @ 106cfu/g and bacterial antagonists @ 108cfu/g) |
||
Germination percentage (%)
Count of both germinated and ungerminated seeds was taken from healthy and infected seeds. Seedling emergence from seed was considered as successful germination. The germination percentage was calculated as the ratio of number of normal seedlings to the total planted seeds i.e., sum of the abnormal, normal and ungerminated seeds.6
Seedling length (plumule and radical length)
Ten normal seedlings from all repetitions were randomly selected and total seedling length including plumule and radical length was measured. Lot with maximum seedling length was taken as vigorous.
Seedling fresh weight (plumule, radical and remaining seed fresh weight) (mg).
The samples which used for seedling length were also used for recording fresh seedling weight from each repetition separately on electronic weighing balance.
Seedling dry weight
The samples used for recording seedling fresh weight were kept in hot air oven at 600C for 20 minutes. Dry weight of each sample was taken with electronic weighing balance.
Seedling vigour index.
It is the combination of standard germination test with seedling length. Vigorous seed may be those who give high vigour index. Vigour index was calculated by using formula of Abdul-Baki.7
Vigour index (%) = Average seedling length on final count
(plumule + radical length (cm)) x per cent germination
Results and Discussion
The average per cent seed germination, seedling length, fresh weight, dry weight and vigour index were found to be increased in all treatments tested over control. The effect of bioagents as seed treatment on most dominant seed mycoflora (A. niger, Colletotrichum sp., Fusarium sp.) of chilli in var. GVC 111 are presented and discussed as under.
Aspergillus niger
Results presented in Table 2 revealed that average seed germination was significantly higher in seed treatment with T. harzianum+P. fluorescens (93.33%) as compared to other treatments and was statistically at par with P. fluorescens (90.00%). Next best significant group was B. subtilis (86.00%), T. harzianum (84.67%) and T. viride+P. fluorescens (82.00%), where all of them were at par with each other. Relatively lower seed germination was seen in P. fluorescens+B. subtilis (80.00%), followed by T. viride (64.67%). While lowest seed germination was found in in control (59.33%). Average seedling length was more in the seed treated with T. harzianum+P. fluorescens (3.02cm) and was found significantly superior over the rest of treatments. Next best in order was seed treatment with P. fluorescens (2.57cm), which was at par with B. subtilis (2.42cm) and T. harzianum (2.39cm). The length of germinated seeds was comparatively shorter in T. viride+P. fluorescens (2.32cm), followed by P. fluorescens+B. subtilis (2.01cm) and T. viride (1.62cm). The lowest seedling length was recorded in control (1.35cm).
Table 2: Effect of bioagents on seedling growth parameters of chilli seeds pretreated with A. niger
| Tr. No. | Treatment | Dose
(g/kg seeds) |
Seed
germination (%) |
Length of seedling (cm) | Fresh weight (mg) | Dry weight (mg) | Seedling vigour index |
| T1 | T. viride | 5 | 53.51*
(64.67) |
7.30
(1.62) |
3.62
(0.40) |
1.44
(0.06) |
104.53 |
| T2 | T. harzianum | 5 | 67.03
(84.67) |
8.88
(2.39) |
4.74
(0.68) |
1.92
(0.11) |
201.72 |
| T3 | P. fluorescens | 6 | 71.59
(90.00) |
9.21
(2.57) |
5.39
(0.88) |
2.26
(0.16) |
231.10 |
| T4 | B. subtilis | 6 | 68.03
(86.00) |
8.94
(2.42) |
5.29
(0.85) |
2.21
(0.15) |
207.73 |
| T5 | T. harzianum+P. fluorescens | 5+6 | 75.25
(93.33) |
10.01
(3.02) |
5.69
(0.98) |
2.60
(0.21) |
282.29 |
| T6 | T. viride+P. fluorescens | 5+6 | 64.89
(82.00) |
8.75
(2.32) |
4.68
(0.67) |
1.74
(0.09) |
189.87 |
| T7 | P. fluorescens +B. subtilis | 6+6 | 63.48
(80.00) |
8.15
(2.01) |
4.19
(0.53) |
1.65
(0.08) |
161.08 |
| T8 | Control (treated with A. niger only) | – | 50.36
(59.33) |
6.66
(1.35) |
3.38
(0.35) |
1.23
(0.05) |
80.20 |
| S.Em.± | 1.39 | 0.14 | 0.10 | 0.04 | 6.09 | ||
| C.D. at 5% | 4.16 | 0.42 | 0.29 | 0.12 | 18.26 | ||
| C.V. % | 3.74 | 2.83 | 3.64 | 3.67 | 5.79 | ||
| *Figures outside the parentheses indicate arc sine transformation value Figures in parentheses indicate original values
(Fungal antagonists @ 106cfu/g and bacterial antagonists @ 108cfu/g) |
|||||||
Average seedling fresh weight was found maximum in the seeds treated with T. harzianum+P. fluorescens (0.98mg) and was significantly higher over rest of the treatments. Next best was P. fluorescens (0.88mg), but was at par with B. subtilis (0.85mg) T. harzianum (0.68mg) and T. viride+P. fluorescens (0.67mg). The least weight was observed in the control (0.35mg). Higher average seedling dry weight was found in seeds treated with T. harzianum+P. fluorescens (0.21mg), followed by seed treatment with P. fluorescens (0.16mg), but both were at par with B. subtilis (0.15mg). Next best significant group was T. harzianum (0.11mg) followed by T. viride+P. fluorescens (0.09mg) and P. fluorescens+B. subtilis (0.08mg). The least weight was observed in control (0.05mg).
Average seedling vigour index was found significantly higher in seeds treated with T. harzianum+P. fluorescens (282.29) as compared to other treatments. Next best was seed treatment with P. fluorescens (231.10) followed by B. subtilis (207.73), which in turn were at par with T. harzianum (201.72) and T. viride+P. fluorescens (189.87). Relatively lower vigour index was observed in seed treatment with P. fluorescens+B. subtilis (161.08) and T. viride (104.53). The lowest vigour index was found in the control (80.20).
Colletotrichum sp.
Results presented in Table 3 revealed that average seed germination was significantly higher in seed treatment with T. harzianum+P. fluorescens (88.00%) when compared to all other treatments. Next best was P. fluorescens+B. subtilis (81.33%) and was statistically at par with T. viride+P. fluorescens (80.67%) followed by B. subtilis (75.33%). Comparatively lower seed germination was found in T. harzianum (51.33%), which was found statistically at par with T. viride (48.00%). While lowest seed germination was found in control (41.33%). Average seedling length was significantly the highest in seed treatment with combination of T. harzianum+P. fluorescens (2.27cm). However, the next best treatment was P. fluorescens+B. subtilis (2.03cm), which was at par with T. viride+P. fluorescens (1.98cm). Significantly the lowest seedling length was recorded in control (0.60cm).
Table 3: Effect of bioagents on seedling growth parameters of chilli seeds pretreated with Colletotrichum sp.
| Tr. No. | Treatment | Dose
(g/kg seeds) |
Seed
germination (%) |
Length of seedling (cm) | Fresh weight (mg) | Dry weight (mg) | Seedling vigour index |
| T1 | T. viride | 5 | 43.83*
(48.00) |
6.18
(1.16) |
3.31
(0.33) |
1.36
(0.06) |
55.63 |
| T2 | T. harzianum | 5 | 45.74
(51.33) |
7.18
(1.56) |
3.49
(0.37) |
1.78
(0.10) |
80.10 |
| T3 | P. fluorescens | 6 | 58.90
(73.33) |
7.44
(1.68) |
3.55
(0.38) |
2.14
(0.14) |
122.85 |
| T4 | B. subtilis | 6 | 60.22
(75.33) |
7.54
(1.72) |
3.84
(0.45) |
2.24
(0.15) |
129.77 |
| T5 | T. harzianum+P. fluorescens | 5+6 | 69.74
(88.00) |
8.65
(2.27) |
4.59
(0.64) |
3.01
(0.28) |
199.37 |
| T6 | T. viride+P. fluorescens | 5+6 | 63.93
(80.67) |
8.08
(1.98) |
4.16
(0.53) |
2.29
(0.16) |
159.73 |
| T7 | P. fluorescens+B. subtilis | 6+6 | 64.40
(81.33) |
8.19
(2.03) |
4.40
(0.59) |
2.81
(0.24) |
165.47 |
| T8 | Control (treated with Colletotrichum sp. only) | – | 39.97
(41.33) |
4.44
(0.60) |
2.70
(0.22) |
1.19
(0.04) |
24.97 |
| S.Em.± | 1.16 | 0.10 | 0.08 | 0.06 | 3.31 | ||
| C.D. at 5% | 3.47 | 0.29 | 0.25 | 0.17 | 9.91 | ||
| C.V. % | 3.59 | 2.30 | 3.77 | 4.71 | 4.88 | ||
| *Figures outside the parentheses indicate arc sine transformation values Figures in parentheses indicate original values
(Fungal antagonists @ 106cfu/g and bacterial antagonists @ 108cfu/g) |
|||||||
Average seedling fresh weight was found significantly higher in seed treatment with combination of T. harzianum+P. fluorescens (0.64mg), which was statistically at par with P. fluorescens+B. subtilis (0.59mg). Next best significant group includes T. viride+P. fluorescens (0.53mg) followed by B. subtilis (0.45mg). Least weight was observed in the control (0.22mg). Average seedling dry weight was significantly higher in seed treatment with T. harzianum+P. fluorescens (0.28mg), followed by P. fluorescens+B. subtilis (0.24mg). Next best significant group includes T. viride+P. fluorescens (0.16mg), B. subtilis (0.15mg) and P. fluorescens (0.14mg). Least weight was observed in the control (0.04mg).
Average vigour index was found significantly higher in seeds treated with T. harzianum+P. fluorescens (199.37) as compared to others. Next best in order of merit was P. fluorescens+B. subtilis (165.47), which was at par with T. viride+P. fluorescens (159.73). Lower vigour index was observed in T. harzianum (80.10) followed by T. viride (55.63). The lowest vigour index was found in the control (24.97).
Fusarium sp.
Results presented in Table 4 revealed that average seed germination was significantly higher in seeds treated with P. fluorescens+B. subtilis (94.00%) and was at par with T. viride+P. fluorescens (93.33%). Next best significant group was T. harzianum+P. fluorescens (89.33%) with P. fluorescens (86.00%). The lowest seed germination was found in control (50.96%). Average seedling length was maximum in seeds treated with P. fluorescens+B. subtilis (2.93cm) and was at par with T. viride+P. fluorescens (2.76cm), followed T. harzianum+P. fluorescens (2.34cm). Next best significant group was P. fluorescens (1.97cm) with T. viride (1.86cm). The lowest seedling length was recorded in control (1.15cm).
Table 4: Effect of bioagents on seedling growth parameters of chilli seeds pretreated with Fusarium sp.
| Tr. No. | Treatment | Dose
(g/kgseeds) |
Seed
germination (%) |
Length of seedling
(cm) |
Fresh weight (mg) | Dry weight (mg) | Seedling vigour index |
| T1 | T. viride | 5 | 63.93*
(80.67) |
7.83
(1.86) |
3.67
(0.41) |
1.71
(0.09) |
149.62 |
| T2 | T. harzianum | 5 | 58.47
(72.67) |
6.19
(1.17) |
2.33
(0.18) |
1.32
(0.05) |
84.61 |
| T3 | P. fluorescens | 6 | 68.03
(86.00) |
8.06
(1.97) |
4.05
(0.50) |
2.16
(0.14) |
169.43 |
| T4 | B. subtilis | 6 | 60.27
(75.33) |
6.81
(1.41) |
3.19
(0.31) |
1.48
(0.07) |
106.14 |
| T5 | T. harzianum+P. fluorescens | 5+6 | 71.02
(89.33) |
8.79
(2.34) |
4.78
(0.70) |
2.60
(0.21) |
209.16 |
| T6 | T. viride+P. fluorescens | 5+6 | 75.12
(93.33) |
9.56
(2.76) |
5.64
(0.97) |
2.82
(0.24) |
258.05 |
| T7 | P. fluorescens +B. subtilis | 6+6 | 75.92
(94.00) |
9.86
(2.93) |
6.20
(1.17) |
3.24
(0.32) |
275.77 |
| T8 | Control (treated with
Fusarium sp. only) |
– | 50.96
(60.33) |
6.16
(1.15) |
2.45
(0.17) |
1.19
(0.04) |
69.65 |
| S.Em.± | 1.47 | 0.12 | 0.11 | 0.06 | 6.05 | ||
| C.D. at 5% | 4.40 | 0.36 | 0.34 | 0.19 | 18.13 | ||
| C.V. % | 3.88 | 2.64 | 4.85 | 5.19 | 6.33 | ||
|
*Figures outside the parentheses indicate arc sine transformation values Figures in parentheses indicate original values (Fungal antagonists @ 106cfu/g and bacterial antagonists @ 108cfu/g) |
|||||||
Average seedling fresh weight was maximum in seed treatment with P. fluorescens+B. subtilis (1.17mg), followed by T. viride+P. fluorescens (0.97mg) and T. harzianum+P. fluorescens (0.70mg). Next best significant group possessed P. fluorescens (0.50mg), T. viride (0.41mg) and B. subtilis (0.31mg). Relatively lower vigour index was noticed in T. harzianum (0.18mg). The least was observed in the control (0.17mg). Average seedling dry weight was higher in seed treatment with P. fluorescens+B. subtilis (0.32mg), followed by T. viride+P. fluorescens (0.24mg) and T. harzianum+P. fluorescens (0.21mg). Least weight was found in control (0.04mg).
Average vigour index was found significantly higher in seeds treated with P. fluorescens+B. subtilis (275.77) and was at par with T. viride+P. fluorescens (258.05). Next best significant group in order of merit was T. harzianum+P. fluorescens (209.16), P. fluorescens (169.43) and T. viride (149.62). Comparatively lower seedling vigour index was noticed in B. subtilis (106.14) followed by T. harzianum (84.61). The lowest vigour index was found in the control (69.65).
On an overall the treatments were found significantly superior over control in increasing seed health parameter. Seeds treated with T. harzianum+P. fluorescens (5+6g/kg seeds) and P. fluorescens (6g/kg seeds) found effective when seeds were pretreated with A. niger, while T. harzianum+P. fluorescens (5+6g/kg seeds). Similarly P. fluorescens+B. subtilis (6+6g/kg seeds) found effective in seeds pretreated with Colletotrichum sp. and P. fluorescens+B. subtilis (6+6g/kg seeds) and T. viride+P. fluorescens (5+6g/kg seeds) found effective in seeds pretreated with Fusarium sp.
Machenahalli8 reported that seeds treated with T. harzianum+P. fluorescens @5+5g/kg seeds showed lower infection (14.89 %) with higher vigour index (930.74) which was followed by P. fluorescens (10.0g/kg) with 14.94 per cent infection and vigour index of 915.27. Reddy9 showed that seeds dressed with T. viride (10g/kg seed) and T. viride (5g/kg seed)+P. fluorescens (5g/kg seed) decreased the seed mycoflora infection in chilli by 81.8 per cent compared to untreated seed. The results were more or less in agreement with earlier works of seeds treated with bioagents on germination of seed and other health parameters of seed. The difference in results can be due to difference in dose of bioagents and varieties used for investigation.
From the present study it can be concluded that P. fluorescens+B. subtilis @ 6+6g/kg seeds and T. harzianum+P. fluorescens @ 6+6g/kg seeds were effective in controlling the growth of seed infecting fungi in chilli variety GVC 111.
Acknowledgement
I would like to thank my family members for their support and encouragement in completing my M.Sc. work. The research work can’t be fulfilled without the support of my guide and friends. So, I am taking this opportunity to thank all those helped me at the time of research. I am also thankful to the editor of the journal for publishing my article in this journal.
Conflict of Interest
There are no conflict of interest.
Funding source
The funds used during my experiment was provided by Department of Plant Pathology, Navsari Agricultural University, Gujarat.
References
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CrossRef - Machenahalli, S., Nargund, V. B. and Hegde, R. V. Management of fruit rot causing seed borne fungal pathogens in chilli. The Bioscan, 9: 403-406.
- Reddy, P., Jakhar, Y. N. and Dahiya, O. S. (2017). Influence of plant oils and bio- fungicides on seed mycoflora of chilli (Colletotrichum capsici). International Journal of Pure and Applied Bioscience, 5(6): 1544-1549.
CrossRef