Dec 20, 2013 - also reported to increase seed yield and protein content of soybean ... Isolasi dan Uji ... and protein level of medium vigor hot pepper seed.
World Applied Sciences Journal 26 (Natural Resources Research and Development in Sulawesi Indonesia): 31-36, 2013 ISSN 1818-4952 © IDOSI Publications, 2013 DOI: 10.5829/idosi.wasj.2013.26.nrrdsi.26006
Effectiveness of Bio-Invigoration Technologies on Seed Viability and Vigor of Cocoa (Theobroma cacao L.) Sutariati Gusti Ayu Kade, 2Kamaruzaman Jusoff, 1 Sadimantara I. Gusti Ray, 1Andi Khaeruni, 1Muhidin and 3Meisanti 1
Department of Agrotechnology, Faculty of Agriculture, Universitas Halu Oleo, Kendari Southeast Sulawesi 93212 Indonesia 2 Department of Forest Production, Faculty of Forestry, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 3 Department of Agriculture Economic, Faculty of Agriculture, Universitas Halu Oleo Kendari, Southeast Sulawesi 93212 Indonesia 1
Submitted: Sep 25, 2013; Accepted: Nov 27, 2013; Published: Dec 20, 2013
Abstract: The use a high vigor of source of plant material (seeds/seedlings) is an important requirement for increasing crop productivity, especially cultivated on marginal soil. The objective of this paper was to evaluate the effectiveness of bio-invigoration technologies in improving seed viability and vigor of cocoa. Laboratory test was arranged in split plot completely block design. The main plot consisted of two sources of cocoa seeds, i.e. Forastero and Trinitario, while the sub plot contained 10 seed bio-invigoration technique treatments, i.e. KNO3 1%, matriconditioning using ground brick, matriconditioning using ground burned-rice husk, rhizobacteria Bacillus sp. CKD061, rhizobacteria P. fluorescens PG01, matriconditioning using ground brick + Bacillus sp. CKD061, matriconditioning using ground burned-rice husk + Bacillus sp. CKD061, matriconditioning using ground brick + P. fluorescens PG01, matriconditioning using ground burned-rice husk + P. fluorescens PG01 and treated by sterile water as a control. Each treatment replicated three times, therefore, overall there were 60 experimental units. Data obtained were analized using analysis of variance and followed with Duncan’s Multiple Range Test. The seed viability and vigor were evaluated by measuring their germination rate, maximum growth potential, seed uniform, relative growth rate and seedling height. The result showed that bio-invigoration treatments were effective to increase seed viability and vigor of cocoa. Independently, the use of rhizobacteria Bacillus sp. CKD061 or P. fluorescens PG01 gives better results and effectively improve seed viability and vigor of Forastero and Trinitario. Based on this experiment, seed bio-invigoration using Bacillus sp. CKD061 or P. fluorescens PG01 could be recommended in improving viability and vigor of cocoa seed. Key words: Bio-invigoration
Cocoa
Rhizobacteria
INTRODUCTION
Seed viability
Vigor
Therefore the application of bio-invigoration techniques to improve seed vigor seeds/seedlings pre-planting, should be the standard treatment in crop cultivation. Seed invigoration treatment purpose to overcome the low productivity due to the use of low vigor of seed. Seed invigoration could be conduct by seed matriconditioning, to accelerate and uniformity of the growth and increase the percentage of germination and
The use a high vigor of source plant materials (seeds or seedlings) is an important requirement for increasing crop productivity when cultivated on marginal land. The use of low quality seeds will produce low vigor of seedlings, not uniform, less tolerant to biotic and abiotic stresses, which in turn lowers productivity.
Corresponding Author: G.A.K. Sutariati, Department of Agrotechnology, Faculty of Agriculture, Universitas Halu Oleo, Kendari Southeast Sulawesi 93212 Indonesia. Tel: +62401319169.
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World Appl. Sci. J., 26 (Natural Resources Research and Development in Sulawesi Indonesia): 31-36, 2013
Effect of Seed Bio-Invigoration Treatments on Viability and Vigor of Cocoa Seed: The experiment was performed by Split Plot design with two factors in a two sources of cocoa seeds, i.e. Forastero and Trinitario, while the sub plot contained 10 seed bio-invigoration technique treatments, i.e. KNO3 1% (KNO3), rhizobacteria Bacillus sp. CKD061 (BC), rhizobacteria P. fluorescens PG01 (PG), matriconditioning using ground burned-rice husk (RM), matriconditioning using ground brick (MB), matriconditioning using ground burned-rice husk + Bacillus sp. CKD061 (RM-BC), matriconditioning using ground burned-rice husk + P. fluorescens PG01 (RM-PG), matriconditioning using ground brick + Bacillus sp. CKD061 (BM-BC), matriconditioning using ground brick + P. fluorescens PG01 (BM-PG) and treated by sterile water as a control (C). Each treatment were replicated three times with an overall total of 60 experimental units. The treated seeds were sowed on sterile burned rice husk placed in a plastic box (20 cm x 15 cm x10 cm). A total of 20 seeds were sowed per box and three boxes were prepared per treatment and stored in room temperature during 14 days. The seed viability and vigor were evaluated by measuring their germination rate, maximum growth potential, seed uniform, relative growth rate and seedling height.
seedling. This technique is shown effective in improving seed viability and vigor based on the results of previous studies [1-4]. Seed invigoration (matriconditioning) can be integrated with rhizobacteria, named bio-matriconditioning [5]. Integration seed invigoration technique with biological agents proved to increase seed vigor before planting and protecting seedlings grown from seed-borne as well as soil borne pathogen fungi [5-8]. The objective of this paper was to evaluate the effectiveness of bio-invigoration technologies in improving viability and vigor of cocoa seed. MATERIALS AND METHODS Preparation of Seed Invigoration Materials and Rhizobacteria Inoculum: Materials used for seed invigoration with solid material (matriconditioning) are ground burned-rice husk and ground brick in size 0.5 mm sieve passes. Rhizobacteria used in this study are Bacillus sp. CKD061 and Pseudomonas fluorescens PG01 (collection of Agronomy Laboratory, Universitas Haluoleo). During times active use Bacillus sp. CKD061 was routinely cultivated on agar plate of Tripthic Soy Agar (TSA) medium at the room temperature, while Pseudomonas fluorescens PG01 was cultivated on King’s B Medium (KBM). The TSA medium composition (g/l) are Tripthic Soy Broth (Difco) 30 g and agar 20 g, while KBM are peptone protease 20 g, K2HPO4 2.5 g, MgSO4.7H2O 6 g, glycerol 15 ml and agar 20 g. After 48 hours, the growing bacterium colony was suspended in sterile deinonized water till a population density of 109cfu/ml and ready for use in seed treatment [9].
Germination Rate (GR), was calculated based on the formula: [10] GR =
Σ NS at observation 1 + Σ NS observation 2
X 100%
Σ seeds planted
NS = Percentage of normal seedlings, Observation 1 = 7 days after planting (dap). Observation 2 = 14 dap. Maximum Growth Potential, observed by calculated the number of seed germinated in the lates observation (14 dap) [10]. Growth Uniform, was measured based on presentage of normally seedling germination at 10 dap [10]. Relative Growth Rate (RG-r), depicting seed vigor, is the ratio of growth rate(RG) to maximum growth rate (RG-m). The maximum growth rate itself was obtained from the assumption that at the first observation, normal seedlings had reached 100%. RG was calculated based on the accumulation of daily growth rate: [10]
Seed Bio-Invigoration Treatments: Cocoa seeds were peeled (testa), washed with sterile water and wipe with a paper towel to dry before treated. The seeds were air dried in the laminar air flow cabinet for one hour. Seed bio-matriconditioning were conducted by entering 60 g seed into a plastic container which were mixed with 20 mL of rhizobacteria suspension and added 60 g of ground burned-rice husk or ground brick (as the matriconditioning media). Plastic container was then covered by sealed with plastic. To avoid the occurrence anaerobic conditions, three small holes were made on the plastic cover by using a needle and placed at room temperature for eight hours after treatment. The seeds that have been treated were cleaned from material media then air-dried for two hours and ready to used. 32
World Appl. Sci. J., 26 (Natural Resources Research and Development in Sulawesi Indonesia): 31-36, 2013 Table 1: The effect of bio-invigoration treatments and seed source on growth rate of cocoa seed Seed Source Seed bio-invigoration
RG-r for cocoa seeds was calculated by using the following formula:
RG-r =
RG x 100% 14.28
---------------------------------------
Rate of seed
Forastero
bio-invigoration
Trinitario
Control
26.67eB
46.67dA
36.67
KNO3
75.00bA
76.67bA
75.83
BC
95.00aA
90.00aA
92.50
PG
91.67aA
91.67aA
91.67
RM
73.33bA
61.67cB
67.50
BM
81.67bA
78.33bA
80.00
RM-BC
80.00bA
61.67cB
70.83
RM-PG
71.67bcA
65.00cB
68.33
BM-BC
78.33bA
80.00bA
79.17
BM-PG
76.67bA
75.00bA
75.83
Rate of seed source
75.00
72.67
Note: Means in the same column suffixed with different lower case letters or means in the
Seedling height at 14 dap, was measured from root neck (medium surface) to the highest growing point.
same row suffixed with different capital letters are different at 5% levels of significance according to DMRT. BC (Bacillus sp. CKD061), PG (P. fluorescens PG01), RM (ground burned-rice husk matrix), BM (ground brick matrix)
RESULTS AND DISCUSSION
Table 2:
The effect of bio-invigoration treatments dan seed source on maximum growth potential of cocoa seed
Effect of Bio-Invigoration Treatments on Viability and Vigor of Cocoa Seed: Compared to the control, bio-invigoration technique that integrated with rhizobacteria treatments were significantly increased growth rate of cocoa seed. Almost of bio-invigoration treatments showed better seed viability than control in both seed sources. Among the treatments were used, seed bio-invigoration with Bacillus sp. CKD061 or P. fluorescens PG01 without matriconditioning media showed best performance. The Forastero seed showed higher growth rate than Trinitario (Table 1). The experiments showed that maximum growth potential of cocoa seed treated with rhizobacteria significantly different to the control, both bio-invigoration using Bacillus sp. CKD061 or P. fluorescens PG01 treatments, showed the highest maximum growth potential were 100% in Forastero and in Trinitario seed. Both of treatments significantly different to the control and other treatments. In generally, Trinitario seed showed that higher maximum growth potential than Forastero seed (Table 2). Both seed matriconditioning and biomatriconditioning using rhizobacteria treatments showed not significantly different on growth uniform of cocoa seed. However, bio-invigoration treatmens using rhizobacteria Bacillus sp. CKD06 or P. fluorescens PG01 suspensions effectively increased growth uniform compared to the control and other treatments. In generally, Trinitario seed showed better performance on growth uniform than Forastero (Table 3).
Seed Source Seed bio-invigoration
---------------------------------------
Rate of seed
Forastero
Trinitario
bio-invigoration 81.67
Control
71.67cB
91.67bA
KNO3
91.67bB
100.00aA
95.83
BC
100.00aA
100.00aA
100.00 100.00
PG
100.00aA
100.00aA
RM
96.67abA
100.00aA
98.33
BM
96.67abA
100.00aA
98.33
RM-BC
93.33abA
95.00abA
94.17
RM-PG
86.67bB
95.00abA
90.83
BM-BC
91.67bB
100.00aA
95.83
BM-PG
95.00abA
100.00aA
97.50
Rate of seed source
92.33
98.17
Note: Means in the same column suffixed with different lower case letters or means in the same row suffixed with different capital letters are different at 5% levels of significance according to DMRT. BC (Bacillus sp. CKD061), PG (P. fluorescens PG01), RM (ground burned-rice husk matrix), BM (ground brick matrix) Table 3:
The effect of bio-invigoration treatments and seed source on growth uniform of cocoa seed Seed Source --------------------------------------
Rate of seed
Seed bio-invigoration
Forastero
bio-invigoration
Control
6.67jB
25.00iA
15.83
KNO3
33.33hB
58.33dA
45.83
Trinitario
BC
75.00aA
66.67bA
70.83
PG
73.33aA
65.00bcA
69.17
RM
46.67fA
51.67eA
49.17
BM
35.00hB
61.67cdA
48.33
RM-BC
41.67gB
50.00efA
45.83
RM-PG
36.67hB
48.33efA
42.50
BM-BC
40.00gB
60.00dA
50.00 48.33
BM-PG
33.33hB
63.33bcdA
Rate of seed source
42.17
55.00
Note: Means in the same column suffixed with different lower case letters or means in the same row suffixed with different capital letters are different at 5% levels of significance according to DMRT. BC (Bacillus sp. CKD061), PG (P. fluorescens PG01), RM (ground burned-rice husk matrix), BM (ground brick matrix)
33
World Appl. Sci. J., 26 (Natural Resources Research and Development in Sulawesi Indonesia): 31-36, 2013 Table 4:
less effective in improving the viability and vigor in both seed source, Forastero or Trinitario. In-effective technology is thought to be caused by the characteristics of the cocoa seeds are relatively vulnerable to external factors flogging. This is consistent with the results of the assessment [11], that cocoa seeds had experienced peeling testa (outer shell), highly susceptible to cellular damage because there is no protector, so it should be conserved properly. In addition, the characteristics matriconditioning media that have high water holding capacity thought to affect the moisture content of cocoa seeds are very susceptible to decreased water content (recalcitrant seeds). This condition can lead to seed stressed that implies decreased seed viability and vigor. In general, all of the viability and vigor seed variables were observed with the suspension use of Bacillus sp. CKD061 and P. fluorescens PG01 gave better results compared to other treatments in both of the seed sources, Forastero or Trinitario. The improvement of cocoa seed viability and vigor obtained with the use of Bacillus sp. CKD061 or P. fluorescens PG01 was mainly due to the ability of both rhizobacteria in generating growth hormones IAA and phosphate solubling. Bacillus sp. CKD061 produce IAA 346,97 ppm [12], whereas P. fluorescens PG01 100 ppm [13]. Some previous researchers have also reported that the role of Bacillus spp. and P. fluorescens as plant growth-promoting rhizobacteria (PGPR) were closely related to the ability to synthesize growth hormones, fixing nitrogen or phosphate solubling. Bacillus spp. capable fixing nitrogen and phosphate dissolving [14], synthezing IAA [13, 15], gibberellins [16] and cytokinins [17]. Similarly, P. fluorescens was capable of producing IAA [18], gibberellins and cytokinins [19], nitrogen fixation [20] and dissolved phosphate [21]. Other researchers also reported hat the main contribution of rhizobacteria Bacillus spp. or P. fluorescens in association with plants is to increase the availability of growth hormone that serves as IAA stimulate plant growth and increase the availability of plant nutrients such as phosphate for plant growth and development [22]. The use of solvent rhizobacteria phosphate could substitute some or all of the needs of phosphate fertilizer plant that is able to give positive results on plant growth. Dissolution of phosphate is caused by bacteria that produce enzymes that can decide phosphate phosphatase bound by organic compounds into forms that are available so that the availability of adequate plant phosphate [23]. Seed inoculation with rhizobacteria was also reported to increase seed yield and protein content of soybean seeds compared with control [24].
The effect of bio-invigoration treatments and seed source on relative growth rate of cocoa seed Seed Source --------------------------------------
Rate of seed
Seed bio-invigoration
Forastero
bio-invigoration
Control
9.93
14.15
12.04g
KNO3
13.70
18.77
16.24c
BC
23.64
24.44
24.04a
PG
20.33
23.92
22.13b
RM
14.71
12.90
13.81ef
BM
15.48
14.78
15.13d
RM-BC
12.41
14.22
13.32f
RM-PG
13.38
15.35
14.36ef
BM-BC
15.15
16.73
15.94cd
BM-PG
15.85
13.01
14.43e
Rate of seed source
15.46
16.83
Trinitario
Note: Means in the same column suffixed with different lower case letters are different at 5% levels of significance according to DMRT. BC (Bacillus sp. CKD061), PG (P. fluorescens PG01), RM (ground burned-rice husk matrix), BM (ground brick matrix) Table 5:
The effect of bio-invigoration treatments and seed source on cocoa seedling height Seed Source ---------------------------------------
Rate of seed
Seed bio-invigoration
Forastero
Trinitario
bio-invigoration
Control
10.00dA
9.33dA
9.67
KNO3
14.78bA
12.22cA
13.50
BC
17.17aA
17.44aA
17.31
PG
16.94aA
18.11aA
17.53
RM
12.56cB
16.78aA
14.67
BM
12.17cA
9.50dA
10.83
RM-BC
12.00cA
13.33cA
12.67
RM-PG
16.28bA
16.72aA
16.50
BM-BC
13.50cB
16.00abA
14.75
BM-PG
13.22cB
16.56aA
14.89
Rate of seed source
13.86
14.60
Note: Means in the same column suffixed with different lower case letters or means in the same row suffixed with different capital letters are different at 5% levels of significance according to DMRT. BC (Bacillus sp. CKD061), PG (P. fluorescens PG01), RM (ground burned-rice husk matrix), BM (ground brick matrix)
Interaction between seed source and bio-invigoration seed treatment did not influence relative growth rate of cocoa seeds. A significant effect occurs only in the bio-invigoration seed treatment. Among the seed treatments tested, the bio-invigoration using Bacillus sp. CKD061 treatment provide seed relative growth rate percentage higher and significantly different compared to the control and other treatments (Table 4). The interaction between seed source and bioinvigoration seed treatments effectively increased the height of cocoa seedling. Seed treatment using rhizobacteria Bacillus sp. CKD061 or P. fluorescens PG01 showed high performance and seedling height. It was significantly different compared to the control and other seed treatments in both seed sources (Table 5). The results showed that the bio-invigoration seed technique uses matriconditioning media (ground burnedrice husk or ground brick) either independently or integrated with rhizobacteria (bio-matriconditioning), are 34
World Appl. Sci. J., 26 (Natural Resources Research and Development in Sulawesi Indonesia): 31-36, 2013
CONCLUSION
6.
The seed bio-invigoration technique with rhizobacteria could increase the viability and vigor of cocoa seeds. Utilization rhizobactera Bacillus sp. CKD061 or P. fluorescens PG01 showed better results and effectively improved the viability and vigor of cocoa seeds compared to control and other treatments in both Forastero and Trinitario seeds. Bio-invigoration treatment with Bacillus sp. CKD061 or P. fluorescens PG01 could be recomended in improving viability and vigor of cocoa seed. Future experiments are recommended to determine the effectiveness of bio-invigoration using rhizobacteria in improving growth and yield of cocoa on marginal land in the field.
7.
8.
9.
ACKNOWLEDGEMENTS The authors extend their gratitude to the Directorate General of Higher Education, Ministry of Education and Culture the Republic of Indonesia for providing research grant under MP3I program in the fiscal year 2012 to support this study.
10.
11.
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