Journal of Experimental Biology and Agricultural Sciences, February - 2016; Volume – 4(1)
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ISSN No. 2320 – 8694
CHARACTERIZATION AND IMPACT OF MYCORRHIZA FUNGI ISOLATED FROM WEED PLANTS ON THE GROWTH AND YIELD OF MUSTARD PLANT (Brassica juncea L.) Halim1,*, Resman2 and Sarawa3 1
Specifications Weed Science, Department of Agrotechnology, Faculty of Agriculture, Halu Oleo University, Southeast Sulawesi, Indonesia, Specifications Soil Science, Department of Agrotechnology, Faculty of Agriculture, Halu Oleo University, Southeast Sulawesi, Indonesia Specifications Agronomy, Department of Agrotechnology, Faculty of Agriculture, Halu Oleo University, Southeast Sulawesi, Indonesia
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Received – October 01, 2015; Revision – November 02, 2015; Accepted – February 20, 2016 Available Online – February 20, 2016 DOI: http://dx.doi.org/10.18006/2016.4(1).85.91
KEYWORDS ABSTRACT Mycorrhiza fungi Grasses weed Mustard plant Ultisols
Present study was aim to determined the impact of mycorrhiza fungi isolated from grasses weed on the growth and yield of mustard in Ultisols. Study was conducted in net house located in Sindang Kasih Village, Dictrict West Ranomeeto; Regency South Konawe Province of Southeast Sulawesi in the month of June to September 2014. Mycorrhiza fungi infection observated on plants root done in the Laboratory of the Faculty of Forestry and Environmental Science, Halu Oleo University, Kendari, Indonesia. Study was conducted in completely randomized block design (CRBD) with five treatments, each treatment was replicated with 5 replications. The variables observed for results were characteristic of mycorrhiza fungi, plant height, number of leaves, leaf area, fresh plant weight, dry plants weight, shoot root ratio, percentage of mycorrhiza fungi infection to plant roots. Results of study revealed that the treatments contains mycorrhiza fungi propagules @ 100 g per polybag show superiority over all the tested treatments in improving plant growth characteristics and yield of mustard plant.
* Corresponding author E-mail:
[email protected] (Halim) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.
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1 Introduction Mustard (Brassica juncea L.) crop is one of the most important vegetable crops which play an important role in the improving living standards of farmers. Haryanto (2006) reported the nutritive value of this crop and suggested that each 100g of green mustard leaf lettuce contains 2.3g protein, 4.0g carbohydrates and 0.3g of fat. Furthermore, it is also a good of sources of vitamins and 100g of mustard leaves had 1.94mg of vitamin A, 102mg of vitamin C and 0.09mg of vitamin B. Furthermore, Margianto (2008) suggested that these nutrient and vitamins are essential for the human body. Annual production of this crop from Indonesia is still below its maximum potential. Various factors such as type of soil, farmer practices and low fertility are responsible for the low production of this crop in Indonesia. Statistical data of agricultural department suggested that average production of mustard plants in Ultisol in Southeast Sulawesi only 3.74 tons ha-1 (BPS, 2010). The dominant soil types in Southeast Sulawesi is Ultisols which is characterized by pH 5.77, 1.92% organic carbon, 0.17% Nitrogen, 12.75 ppm Phosphorus and 0.22 me100g-1 Potassium (Halim & Rembon, 2013; Halim et al., 2015). Various efforts based on principles of conservation and ecofriendly natural sources to improve soil fertility of this Ultisols were carried out. Use of mycorrhiza for improving soil fertility is a common practice for many crops. Halim (2013) reported that some kinds of grasses weeds were naturally infected by mycorrhiza fungi. Mycorrhiza isolated from these types of weed grasses had positive impact on the soil fertility. Similar types of results was also reported from the another research conducted by same authors (Halim et al., 2014). They also reported that roots of broad-leaved weeds, grasses weed and sedges weed are infected by mycorrhiza fungi.
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The soil has been taken from the study area field, it cleared from debris such as twigs, roots, leaves and small rocks. Cleared soil sifted into a polybag with a weight of 10kg soil and recommended dose basic NPK fertilizer along with organic manure was added to each polybag. The mustard seed sown for 7 days in media seedbed mixture of rice husk, soil, sand, with the volume ratio 1: 0.5:1 was transferred to the polybags. Mycorrhiza fungi isolated from the roots of Imperata cylindrica that had previously been propagated on maize (Halim, 2012) were transferred to the each polybags. 2.3 Observation of Variables Isolated mycorrhiza fungi were characterized with the standard identification key for mycorrhiza. Various growth parameters such as plant height, number of leaves and leaf area were measured on the intervals of each seven days which start from the 7th days after planting and continue upto 28 days after planting (DAP). The total leaf area was measured by using the formula proposed by Sitompul & Guritno (1995): L= p x l x k x j Note: L= leaf area, P = length of leaf, l = plant fresh weight (g plant-1), k = the coefficient of leaf area (0.78), j = number of leaves. The fresh weight of the plants (g plant-1) was measured after harvest while the dry weight of the plant (g plant -1) was measured after harvesting drying plant in oven at a temperature of 80 0C for 48 hours. Further, Ratio leaf area was measured (cm2 g-1) by the using formula proposed by Sumarsono (2010). Ratio leaf area = L/ W Whereas L = leaf area (cm2), W = dry weight the plant (g)
2 Materials and Methods 2.1 Study area and Experimental setup Present study was conducted from June to September 2014 in net house, village Sindang Kasih, District West Ranomeeto, South Konawe Regency, Southeast Sulawesi Province and Laboratory of the Faculty of Forestry and Environmental Science, Halu Oleo University Kendari, Indonesia. Plant were grown in polybag (40 cm x 50cm) and study was conducted in completely randomized block design (RCD) with five treatments i.e. without mycorrhiza fungi propagules (M0), mycorrhiza fungi propagules@ 25 g per polybag (M1), mycorrhiza fungi propagules@ 50 g per polybag (M2), mycorrhiza fungi propagules @ 75 g per polybag (M3) and mycorrhiza fungi propagules@ 100 g per polybag (M4), each treatment was replicated with 5 replications. 2.2 Preparation of planting media
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Shoot root ratio of the mustard plant was measured by using the formula proposed by Gardner et al. (1991). Shoot root ratio = shoot dry weight/ root dry weight 2.4 Percentage of the mycorrhiza fungi infection The Observations were carried out using a dissecting microscope at a magnification of 40X. Furthermore, mycorrhiza fungi infection was calculated by using the formula proposed by Brian & Schults (1980).
Where IP= the percentage of mycorrhiza fungi infection; r1= the number of root infected examples and r2= the number of root not infected examples
Characterization and impact of mycorrhiza fungi isolated from weed plants on the growth and yield of mustard plant (Brassica juncea L.)
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Table 1 Effect of mycorrhiza fungi on the average height of mustard (cm) at the age 7-14, 14-21 and 21-28 DAP. Treatment
Average plant height 7-14DAP 14-21DAP 21-28DAP without mycorrhiza fungi (M0) 3.17d 6.20c 8.26d d b 25 g mycorrhiza fungi (M1) 4.20 8.70 10.35c c b 50 g mycorrhiza fungi (M2) 5.58 9.45 11.47bc b ab 75 g mycorrhiza fungi (M3) 7.06 10.90 12.40ab a a 100 g mycorrhiza fungi (M4) 8.89 12.60 13.47a SEM value 1.04 1.19 2.78 2 1.34 2.19 1.60 DRMT 0.05% 3 1.41 2.30 1.68 4 1.45 2.37 1.74 5 1.48 2.42 1.77 Here, DAP = day after planting, SEM = standard error mean, the numbers followed by the same superscript letters in the same column are not significantly differ on DRMT 0.05% 2.5 Data Analysis Data of each variable were observed were analyzed by variance of analysis. If the F count is greater than the F table, then continued with Duncan Range Multiple Test (DRMT) at 0.05% confidence level.
white or brownish yellow. Spores are located on the terminal gametangium located on the undifferentiated hyphae in a sporocarp (Halim, 2012). Mostly these spores are formed on the external hyphae near the root zone. 3.2 Effect of mycorrhizal application on the growth attributes
3 Result
3.2.1 Plant Height
3.1 Characterization of Mycorrhiza Fungi
The effect mycorrhizal fungi on average plant height are represented in table 1; results of study revealed that application of mycorrhizal fungi increase the height of mustard plants. This improvement in the plant height is continued upto the 21 DAP; after this plants are not showing much improvement in height and no significant difference was reported between 21 and 28 DAP. Highest dose of mycorrhiza (100g) shows superiority over the other treatments and 7 DAP is was 8.89cm which reached 13.4cm at 28DAP.
Two species of mycorrhiza fungi viz Gigaspora sp. and Glomus sp. were isolated from the selected weed species. (Halim, 2009), among these Gigaspora sp. was identified by the presence of a single brown color terminal spore. These spores have globular or spherical shape with more than one layer. A complementary tool in the form of bulbous suspensor was also reported for this species. While the Glomus sp. are characterized by the presence of single or bunch of ripe hyaline
Gigaspora sp.
Glomus sp.
Figure 1 Spore form of Gigaspora sp and Glomus sp. _________________________________________________________
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Table 2 Effect of mycorrhizal fungi on the average number of mustard plant leaves at the age of 7- 28 DAP. Treatment without mycorrhiza fungi (M0) 25 g mycorrhiza fungi (M1) 50 g mycorrhiza fungi (M2) 75 g mycorrhiza fungi (M3) 100 g mycorrhiza fungi (M4) SEM value 2 3 4 5 Here, DAP = day after planting, SEM = standard error are not significantly differ on DRMT 0.05%. DRMT 0.05%
Average Leaves Number 7DAP 14DAP 21DAP 28DAP 3.00c 4.40b 5.40c 7.20c bc b bc 3.60 4.60 5.80 7.80bc ab b a 3.40 4.60 7.20 9.20a ab ab ab 3.80 5.00 6.80 8.60ab a a a 4.00 5.60 7.20 9.20a 0.16 0.24 0.62 0.52 0.52 0.64 1.03 0.95 0.55 0.67 1.09 0.99 0.57 0.69 1.12 1.02 0.58 0.71 1.14 1.05 mean, the numbers followed by the same superscript letters in the same column
3.2.2 Number of Leaves The effect mycorrhiza fungi to the average number of leaves of mustard plants shown in Table 2. The trends of leaves numbers are similar to plant height and it increased with the increasing of day of planting and dose of mycorrhiza fungi. The highest average number of leaves per mustard plant at age of 7 DAP was 4.00 and it was reported in the treatment M4; this number reached 9.20 per plant in the same treatment at the interval of 28DAP. At 28DAP, treatment M2 is also showing similar leaf number and it is significantly not differ from the M4.
application and days. Highest total leaf area was reported from the treatment containing 100g mycorrhiza at 28DAP. 3.3 Fresh and dry weight of mustard Plant
3.2.3 Total Leaf Area
The effect mycorrhiza fungi to the average fresh and dry weight the plants are calculated on the harvesting of plants. Average fresh and dry weights are represented in Table 4. The value of fresh and dry weight increased with the increasing the dose of mycorrhiza fungi application, lowest fresh and dry weight was reported from the treatment without mycorrhiza application (M0) while the highest plant was reported from the highest dose of mycorrhiza fungi application (M4).
Total leaf area was calculated by the method given by Sitompul & Guritno (1995). The effect mycorrhiza fungi to the total leaf area of mustard plants shown in Table 3. Total leaf area also increased with the increasing the dose of mycorrhizal
The treatment M4 is showing 37.35 and 51.58% higher fresh and dry weight respectively as compared to the treatment without mycorrhiza. Treatment M3 and M4 is not showing any significant difference (DMRT = 0.05%).
Table 3 Effect of mycorrhiza fungi on the total leaf area of mustard plants at the age 7 - 28 DAP. Treatment
Total leaf area (cm) 7DAP 14 DAP 21 DAP 28 DAP without mycorrhiza fungi (M0) 21.36c 77.75d 341.69d 776.88d c c cd 25 g mycorrhiza fungi (M1) 24.52 128.11 462.99 995.38c bc c bc 50 g mycorrhiza fungi (M2) 27.01 142.46 582.07 1266.39b 75 g mycorrhiza fungi (M3) 32.72b 228.74b 711.03ab 1364.30ab 100 g mycorrhiza fungi (M4) 42.86a 288.55a 812.68a 1532.68a SEM value 0.87 0.24 1.25 20.5 2 6.22 42.10 143.50 198.60 DMRT 0.05% 3 6.53 44.19 150.60 208.50 4 6.73 45.52 155.20 214.80 5 6.86 46.45 158.30 219.10 Here DAP = day after planting, SEM = standard error mean, the numbers followed by the same superscript letters in the same column are not significantly differ on DRMT 0.05%.
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Table 4 Effect of the mycorrhiza application of the average fresh and dry weight (g plant ) of the mustard plant. Treatment Fresh weight (g DMRT Dry weight (g DMRT 0.05% -1 -1 plant plant without mycorrhiza fungi (M0) 71.39c ) 2(0.05%) = 18.47 3.99c ) 2 = 0.86 25 g mycorrhiza fungi (M1) 81.70bc 3 = 19.39 5.96b 3 = 0.90 50 g mycorrhiza fungi (M2) 96.32ab 4 = 19.97 6.41b 4 = 0.93 75 g mycorrhiza fungi (M3) 107.00a 5 = 20.38 7.62a 5 = 0.95 100 g mycorrhiza fungi (M4) 113.95a 8.24a SEM value 5.39 0.43 Here DAP = day after planting, SEM = standard error mean, the numbers followed by the same superscript letters in the same column are not significantly differ on DRMT 0.05%. Table 5 Average shoot root ratio of the mustard plant under the influence of mycorrhiza application. Treatment Shoot root ratio DRMT 0.05% without mycorrhiza fungi (M0) 2.01c 2 = 0.64 25 g mycorrhiza fungi (M1) 2.58c 3 = 0.67 50 g mycorrhiza fungi (M2) 3.24b 4 = 0.69 75 g mycorrhiza fungi (M3) 3.61b 5 = 0.71 100 g mycorrhiza fungi (M4) 4.39a SEM value 0.24 Here DAP = day after planting, SEM = standard error mean, the numbers followed by the same superscript letters in the same column are not significantly differ on DRMT 0.05%.
3.4 Shoot Root Ratio The effect mycorrhiza fungi to the average shoot root ratio of the mustard plant shown in Table 5. The trends are similar to the growth parameters and highest shoot root ratio was obtained from the treatment containing 100g mycorrhiza culture, and it was 52.21 percent higher than the polybag without mycorrhiza. With the increasing dose of mycorrhiza, shoot root ration also increased and a significant difference was reported between all the tested doses.
3.5 Percentage of mycorrhiza colonization The average percentage of mycorrhiza fungi colonization in the mustard plant roots are listed in Table 10. Highest dose of mycorrhiza (M4) shows the highest colonization (42%) and this was followed by the treatment M3, M2 and M1 respectively. Highest colonization provides higher nutrition to the mustard plant and because of this plant shows superiority in all the studied attributes. Discussions The results of research showed that the application of mycorrhiza fungi significantly affected all the variables of mustard plants. The possible reason of this was the availability of sufficient nutrients which favor the plant growth. According to the Simarmata & Herdiani (2004) biological fertilizers such
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as mycorrhiza fungi can increase the availability of nutrients for plants in marginal land of Indonesia, which in turn increased crop production. Findings of present study are in the agreement with the findings of these authors. Further, Marschner & Dell (1994) stated that the infection of mycorrhiza fungi change the growth and activity of plant roots by the formation of mycelia on the external surface which caused increase in the absorption of nutrients and water. These higher nutrients increase the plant height, number of leaves and plant weight. Similar type of growth with respect to plants height, number of leaves and leaf area of plants was obtained by Mayerni & Hervani (2008). These researchers reported that mycorrhizal infection increases the metabolism of plant growth which could mainly take place in vegetative phase. Husin (1997) reported that by changing plant metabolic activities, mycorrhiza fungi influence the production of growth hormones such as auxin and gibberellins. Among these auxin prevent the aging of plant roots, so in this condition roots can function longer and absorption of nutrients will also higher. While the giberelin performing the function of enlargement and stimulate the cell division. The ability of mycorrhiza fungi in absorption of phosphate is not only determined by the fungal colonies in the roots and development in the soil, but also determined by ability of external hyphae. In fact, the percentage mycorrhiza fungi infections on plant roots are not always comparable to effect on crop yields. Mycorrhiza fungi infection and the effect decreases with increasing phosphate available in the soil.
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Table10 The average percentage of mycorrhiza fungi infection in the mustard plant roots. Treatment Percentage mycorrhiza fungi colonization DRMT 0.05% without mycorrhiza fungi (M0) 0e 2 = 4.93 25 g mycorrhiza fungi (M1) 10 d 3 = 5.18 50 g mycorrhiza fungi (M2) 18 c 4 = 5.33 75 g mycorrhiza fungi (M3) 26 b 5 = 5.44 100 g mycorrhiza fungi (M4) 42 a SEM vaue 21.64 Here DAP = day after planting, SEM = standard error mean, the numbers followed by the same superscript letters in the same column are not significantly differ on DRMT 0.05%.
Further, Turk et al. (2006) also confirmed that mycorrhiza can improve the availability of Phosphorus in soil that experienced scarcity of Phosphorus. Phosphorus uptakes in plants affect the physiological and morphological conditions of the plant which led to increase the production of energy in the plant body and fresh and dry weight of plant increases (Nuhamara, 1994). These observations confirmed the finding of present study where higher fresh and dry plant weight was obtained by the application of mycorrhiza. The shoot root ratio described the patterns of plants growth as a resultant of plant responses to the environment. Though, shoot root ratio determined by genetic factors but it is also strongly influenced by environmental factors such as soil and climate. Sutedjo & Kartasapoetra (1997) suggested that if one factor has stronger influence than any other factor, the factor will be closed off from each of the factors that have different properties and real work to support the production of plant. The roots which have greater absorption area will have a chance to absorb more nutrients, therefore the plants associated with mycorrhiza fungi will able to improve its capacity to absorb nutrients and water. In addition, these plant has 2-4 times higher metabolic rate as compared to the plants that do not colonized by mycorrhiza fungi (Sieverding, 1991). Similarly, Rasouli-Sadaghiani et al. (2010) reported that the higher dose of mycorrhiza fungi increased the uptake of several nutrients. Results of this study confirmed that the higher dose of mycorrhiza fungi in the planting hole caused higher colonization of mycorrhiza fungi in plant roots. Acknowledgements The author would like to thank to the Ministry of National Education, Republic of Indonesia for the financial assistance through the scheme of National Priorities Research Grant Master Plan for the Acceleration and Expansion of Indonesian Economic Development 2011-2025 in 2014. The author also thank to the Rector of Halu Oleo University and the Chairman of the Research Institute of Halu Oleo University for providing us moral support and space carry out this study.
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Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. References BPS (2010) Survei Pertanian. Biro Pusat Statistik. Jakarta. Brian.PPWC, Schultz RC (1980) Procedures and Equipment for Staining Large Number of Plant Roots for Endomycorrhizal Assay. Cam I.Microbial. Gardner PF, Pearce RB, Roger L, Mithcell (1991) Fisiologi Tanaman Budidaya.Terjemahan Herawati Susilo. Penerbit UIPress. Jakarta. Halim (2009) Peran mikoriza indigenous gulma Imperata cylindrica (L.) Beauv dan Eupatorium odorata (L.) terhadap kompetisi gulma dan tanaman jagung. Disertasi program doktor Universitas Padjadjaran Bandung. Halim (2012) Peran mikoriza indigen terhadap indeks kompetisi antara tanaman jagung (Zea mays L.) dengan gulma Ageratum conyzoides. Berkala Penelitian Agronomi 1:86-92. Halim (2013) Identification of indigenous mycorrhiza fungi of weed in The Biosciences Park Area of Halu Oleo University. Proceedings of the 8th International Confrence on Innovation and Collaboration towards ASEAN community. ISBN 978602-8161-57-2.17-19. Halim, Rembon FS (2013) Peningkatan produksi tanaman jagung berbasis bioteknologi Mikoriza Indigenous Gulma. Laporan hasil penelitian MP3EI Dikti. Lembaga Penelitian Universitas Halu Oleo Kendari. Halim, Rembon FS, Kandari AM, Resman, Sani A (2014) Characteristics of indigenous mycorrhiza of weeds on marginal dry land in South Konawe, Indonesia. Agriculture, Forestry and Fisheries 3:459-463. doi: 10.11648/j.aff.20140306.14.
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Halim, Arma MJ, Rembon FS, Resman (2015) Impact of mycorrhiza fungi from grassland rhizosphere and liquid organic fertilizer to the growth and yield of sweet corn on Ultisols in South Konawe, Indonesia. Agriculture, Forestry and Fisheries 4:209-215. doi: 10.11648/j.aff.20150405.12
Rasouli-Sadaghiani M, Hassani A, Barin M, Danesh YR, Sefidkon F (2010) Effects of arbuscular mycorrhizal (AM) fungi on growth, essential oil production and nutriends uptake in basil. Journal of Medicinal Plant Research 4:2222-2228. doi: 10.5897/JMPR10.337.
Haryanto (2006) Sawi dan Selada. Edisi revisi. Penebar Swadaya. Jakarta.
Sieverding E (1991) Vesicular-Arbuscular Mychoryza Management in Tropical Agrosystems, Deutshe Gesellicaft Fur Tehnische. Germany.
Husin EF (1997) Respon beberapa jenis tanaman terhadap mikoriza vesikula arbuskular dan pupuk fosfat pada tanah Ultisol. Prosiding pemanfaatan cendawan mikoriza untuk meningkatkan produksi tanaman pada lahan marjinal. Hal.4-8. Asosiasi Mikoriza Indonesia. Universitas Jambi. Margianto E (2008) Swadaya. Jakarta.
Budidaya
tanaman sawi. Penebar
Marschner H, Dell B (1994) Nutrient Uptake in Mycorrhiza Symbiosis. Plant and Soil 159:89-102. doi: 10.1007/BF00000098. Mayerni R, Hervani D (2008) Pengaruh Jamur Arbuskula terhadap Pertumbuhan Tanaman Selasih (Ocimum sanctum L.). Jurnal Akta Agrosia 11:7-12. Nuhamara ST (1994) Peranan mikoriza untuk reklamasi lahan kritis. Program Pelatihan Biologi dan Bioteknologi Mikoriza. Penerbit Tarsito. Bandung.
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Simarmata T, Herdiani E (2004) Efek pemberian inokulum CMA dan pupuk kandang terhadap P tersedia, retensi P dalam tanah dan hasil tanaman bawang merah (Allium Acanolicum L.) hlm 20-40 di dalam prosiding: Pemanfaatan Cendawan Mikoriza untuk meningkatkan Produksi Tanaman pada Lahan Marjinal. Asosiasi Mikoriza Indonesia. Universitas Jambi. Sitompul SM, Guritno B (1995) Analisis pertumbuhan tanaman. Gadjah Mada University Press. Jogyakarta. Sumarsono (2010) Analisis kuantitatif pertumbuhan tanaman kedelai (Soy beans). Fakultas Peternakan Universitas Diponegoro. Semarang. Sutedjo M, Kartasapoetra M (1997) Pupuk dan pemupukan. Rineka Cipta Jakarta. Turk MA, Assaf TA, Hameed KM, Al-Tawaha AM (2006) Significance of Micorrhyzae. World Journal of Agricultural Sciences 2: 16-20.