Actinobacteria of arid and semi-arid soils: antagonism to fungal pathogens and plant growth promoting potential.
MATERIAL AND METHODS
Isolation of Actinobacteria
Soil samples were collected from the rhizosphere of Sorghum (Sorghum vulgare), Pearl millet (Pennisetum typhoideum), Pigeon pea (Cajanus cajan), Finger millet (Eleusine coracana) and Groundnut (Arachis hypogea) grown in arid region of Jaisalmer in Raj asthan; arid and semi arid regions of Belgaum, Hubli, Bijapur and Tumkur districts of Karnataka and Anantapur in Andhra Pradesh. Two soil samples from pristine forest in humid region of Karnataka (Kegdal, Vibuthi) were also taken. Four different media were used for isolation of Actinobacteria viz., actinomycetes isolation agar, starch casein agar, arginine glycerol salts medium and humic acid vitamin agar. Isolates were characterized by referring to the 9th edition of Bergey's manual of determinative bacteriology (Holt et al. 1994) and identified by standard methods (International Streptomyces project)
In vitro antagonism to fungal pathogens
The Actinobacterial strains were screened for the antagonism (biocontrol potential) to soil borne plant pathogens viz., Macrophomina phaseolina, Sclerotium rolfsii, Rhizoctonia solani and Fusarium oxysporum. The fungal cultures were obtained from the Directorate of Oilseeds Research, Hyderabad and Central Research Institute of Dryland Agriculture (CRIDA), Hyderabad, India. The mycelia plug of the pathogen (grown on PDA for two days) was cut and placed at the centre of fresh PDA plates using a cork borer. In the same PDA plate at 3 cm distance from the pathogen a plug of Actinobacteria (grown on oat meal agar for five days) was placed. This test was performed separately for all the Actinobacterial strains. A control was maintained without inoculation of Actinobacterial strains but grown only with pathogen. In vitro screening for antagonism was made in three replica plates. The inoculated plates were incubated at 30 [+ or -] 2[degrees]C for 7 days. Colony growth inhibition (%) was calculated by using the formula: C - T/C *100, where C is the colony growth (in mm) of pathogen in control, and T is the colony growth of pathogen in dual culture. All strains were tested in triplicate.
Growth Promoting Characteristics
The strains were tested for plant growth promoting attributes like Indole acetic acid (IAA) production (Gordon and Weber 1951); Gibberellic acid (GA) production (Paleg 1965); P solubilisation on Pikovskaya agar (Pikovskaya, 1948) followed by spectrophotometric quantisation of P solubilized in broth after 10 days growth at 28 [+ or -] 2[degrees]C in shake cultures at 125rpm; K solubilization on Aleksandrov agar (Hu et al. 2006) followed by flame photometric quantification of K solubilized in broth after 10 days growth at 28 [+ or -] 2[degrees]C in shake cultures at 125rpm; Alkaline phosphatase production (Tabatabai and Bremner, 1969); Ammonia production (Cappuccino and Sherman, 2005); Siderophore production (growth in chrome azurol S-CAS medium (Schwyn and Neilands, 1987); and chitinase production (Hamid et al. 2013) were determined.
Ten efficient biocontrol strains Streptomyces A1, A2, A6, A10, A11, A17, A18, A30, A40 and A45 were screened for their ability to promote plant growth in soil microcosms under glass house conditions at Indian Institute of Soil Science, Bhopal. The experiment had five replications and laid out in completely randomized design. 330 ml paper cups were taken and filled with 300g soil (Vertic Eustochrept). The Actinobacterial strains were grown separately on starch casein broth for 7 days with shaking at 125 rpm. Farm yard manure (FYM) used as a carrier material was air dried for 3-4 days and passed through 0.2 mm sieve and sterilized three times by steam sterilization (121[degrees]C for 20 min) on successive days and then two times dry heat sterilization (160[degrees]C for 3 h each time). Forty milliliter of the culture broth was added to 100 gm FYM in a plastic pouch, mixed by hands and sealed. One gram of the FYM based inoculants was added to 10 ml of 1% carboxy methyl cellulose (CMC). Maize Seeds (var. JM-216) were surface sterilized by dipping them in 95% ethanol solution for 5 min and in 3% sodium hypochlorite (NaClO) solution for 5 minutes and subsequently washed in sterile distilled water for five times. Then 24 seeds of maize were transferred to the CMC culture suspension and kept overnight. The seeds were removed aseptically and air dried in a laminar air flow work station. The inoculants coated seeds were sown @ 4 seeds per cup. After germination, the plants were thinned to maintain three plants in each cup. After 15 days urea was applied as solution to the cups at 40 [micro]g N [g.sup.-1] soil. The cups were watered regularly with tap water (boiled for 30 min and cooled) to maintain optimum moisture. The plant height, number of leaves and plant dry mass was recorded at 4 weeks and 6 weeks growth stage (30 and 45 days after sowing). The same methodology was used for chickpea (JG-16) seed treatment. Observations like plant height recorded both at 30 and 60 days after sowing (DAS), nodule number and dry weight, dry weight of plants was recorded at 60 DAS.
RESULTS AND DISCUSSION
The soil samples obtained from different crop rhizosphere at different places of arid, semi arid and humid regions had a wide variation in pH from 6.0 to 9.0. The organic carbon content ranged from 0.12 to 3.95 per cent (Table 1). The Actinobacterial count of the soil samples on four different nutrient media employed for enumeration is given in Table 1. Higher populations were recorded on Humic acid vitamin agar (14.8 X [10.sup.4] cfu/g soil) while the lowest Actinobacterial population was recorded in Argnine glycerol salts medium (3.5 X [10.sup.4] cfu/g soil). Humic acid vitamin agar was the best medium for enumeration of Actinobacteria from all soils. These results agree with Hayakawa and Nonomura (1987) who showed that Humic acid vitamin (HV) agar was superior to the other media including colloidal chitin agar, glycerolarginine agar and starch-casein-nitrate agar for the isolation and enumeration of soil Actinobacteria. Similarly, Coombs and Franco (2003) observed that the nutrient poor media such as HV agar, TWYE (Tap water yeast extract) and YECH (Yeast extract- casein hydrolysate) agar are most effective for isolation of endophytic Actinobacteria from wheat root. When the average counts of Actinobacteria obtained from different type of soils grown on four different media were compared, the highest Actinobacterial population was observed in rhizosphere of crops sampled in black soils (Vertisols) which may be due to soil type and higher pH. Varalakshmi et al. (2010) who observed that black soils harboured more population of actinomycetes in the rhizosphere of fox tail millet as compared to red soil.
Forty one Actinobacterial isolates were obtained in total from all the soil. Based on morphological, biochemical and microscopic observations, these isolates were assigned to four genera (data not shown)--Streptomyces (26 no.), Nocardia (12), Micromonospora (1) and Saccharopolyspora (2). Highest number of isolates belonged to the genus Streptomyces (63%), followed by Nocardia (29%) while rest of the isolates belonged to Micromonospora (5%) and Saccharopolyspora (2%). Dominance of Streptomyces among the actinomycetes was also earlier reported by many research workers (Alexander, 1961; Gupta 1991; Gesheva and Gesheva 2000).
In this study, 41 Actinobacterial strains were tested for their in vitro antagonistic activity against four plant pathogenic fungi viz., Macrophomina phaseolina, Sclerotium rolfsii, Rhizoctonia solani and Fusarium oxysporum. Some of the Streptomyces strains inhibited all the four pathogens whereas Nocardia, Saccharopolyspora and Micromonospora strains inhibited all the pathogens except Fusarium oxysporum (Table 2). The strains from humid regions were more effective in inhibiting mycelial growth of Macrophomina phaseolina (inhibited mycelial growth by 62.8%) (Fig. 1) followed by arid and semi arid region strains. Arid and semi arid region strains (40.8 and 40.6%) are almost similar in inhibiting mycelia growth of Sclerotium rolfsii compared to humid region strains. Semi arid region strains (24.2%) are more effective in inhibiting the mycelia growth of Rhizoctonia solani followed by arid and humid region strains, whereas only arid region strains (44.5%) showed inhibition towards Fusarium oxysporum. This biocontrol potentiality can be attributed to the production of antimicrobial compounds including ammonia production. Khamna et al. (2009) observed biocontrol activity of 23 Streptomyces isolates against five phytopathogenic fungi viz., Alternaria brassicicola, Colletotrichum gloeosporioides, Fusarium oxysporum, Penicillium digitatum and Sclerotium rolfsii. Subhashini (2010) isolated 40 actinomycetes from tobacco soils of India and reported that 10 Streptomyces isolates showed high antagonistic activity against Pythium aphanidermatum under in vitro conditions.
Ten Actinobacterial strains showing best biocontrol ability were screened for plant growth promoting attributes and also tested for their influence on plant growth of maize (Table 4) and chickpea (Table 5) under glass house conditions. The Indole acetic acid (IAA) production of the strains under in vitro conditions ranged from 1.9-5.7 mg [ml.sup.-1], Gibberellic acid (GA) production ranged from 24.1-41.4 mg [ml.sup.-1]. Solans et al. (2009) found that three rhizoactinomycetes Streptomyces MM40, Actinoplanes ME3 and Micromonospora MM18 produced and released IAA, GA3, and zeatine in a defined media. All the strains except Streptomyces A30 could solubilize P from inorganic tri-calcium phosphate. The amount of P solubilzation ranged from 3.2-6.8%. Among the 10 strains 7 strains could solubilize K from muscovite mica which ranged from 3.7-6.3 mg [ml.sup.-1]. Some strains produced ammonia whereas none of the strains produced HCN, siderophore or exhibited chitinase activity. All the strains except Streptomyces A40 produced alkaline phosphatase which ranged from 60.0-160.9 [micro]g p-[nitrophenol.sup.-1][ml.sup.-h]
Franco-Correa et al. (2010) isolated 30 actinomycetes from the rhizosphere of Trifolium repens L. and found that 21 isolates exhibited phosphate solubilizing ability on Pikovskaya's medium, all the isolates produced acid phosphatase and only thirteen isolates produced alkaline phosphatase.
Results of glass house screening showed that Actinobacterial strains significantly differed in influencing plant growth attributes like plant height, number of leaves and total plant dry weight. Among all the strains the highest plant height (20.0 cm), number of leaves (8.9) and plant dry weight (1044 mg [plant.sup.-1]) was obtained in plants inoculated with Actinobacterial strain A6 (Streptomyces isolated from arid soils) at 45 DAS. Gopalakrishnan et al., (2013) evaluated five strains of Streptomyces on Sorghum under green house conditions and found that all the five strains had significantly enhanced plant growth parameters over un-inoculated control. In Chickpea, strain A17 (Streptomyces) showed highest plant height (30.5 cm) number of nodules (29.0) nodule dry weight (26.7 mg [plant.sup.-1]) and plant dry weight (936 mg [plant.sup.-1]). The increase in nodule numbers could be due to improvement of Rhizobium symbiosis caused by the plant growth promoting action of the Actinobacteria. Solans et al. (2009) co-inoculated actinomycetes with Siniorhizobium meliloti and found that nodulation and growth of Alfalfa (Medicago sativa) plants were significantly increased when compared to plants inoculated only with Siniorhizobium meliloti. The findings of this study is of much significance to crop production in utilizing efficient Actinobacterial strains like Streptomyces A6 for maize and Streptomyces A17 in chickpea cultivation to obtain higher crop yields. The identified strains have high potential for use particularly in dry land agriculture as bioinoculants for suppressing plant pathogens, solubilzation of P and K with the added advantage to crops in terms of improved crop growth and yields due to plant growth promoting hormones elaborated by the bacteria in crop rhizosphere.
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T.K. Radha (1,2), D.L.N. Rao (3) and K.R. Sreeramulu
(1) Indian Institute of Horticulture Research, Bengaluru-560 089, India.
(2) Indian Institute of Soil Science, Nabi Bagh, Bhopal-462 038, India.
(3) University of Agricultural Sciences, GKVK, Bengaluru-560065, India.
(Received: 11 March 2017; accepted: 04 May 2017)
* To whom all correspondence should be addressed.
Caption: Fig. 1. Biocontrol ability of Actinobacteria against fungal plant pathogens (dual culture) MP: Macrophomina phaseolina; SR: Sclerotium rolfsii; RS: Rhizoctonia solani; FO: Fusarium oxysporum; H: Humid; SA: Semi arid; A: Arid
Caption: Fig. 2. Antagonism of Actinobacteria against fungal plant pathogens a) Macrophomina phaseolina b) Sclerotium rolfsii c) Rhizoctonia solani d) Fusarium oxysporum
Table 1. Enumeration of Actinobacteria (x [10.sup.4]cfu-g soil [+ or -] SEm) in different growtli media * Climatic Soil order Place and Origin region state Arid Alfisol Tumkur (Karnataka) Finger millet Ananthpur (Andhra Pradesh) Groundnut Aridisol Jaisalmer (Rajasthan) Sam Pearl millet (Rajasthan) Desert Semi-arid Verti sol Hubli, Bijapur Sorghum, (Karnataka) Pigeon pea, Pearl millet Alfisol Gadag (Karnataka) Sorghum Elumid Lateritic Uttarakannada (Karnataka) Forest Verti sol Uttarakannada Forest (Karnataka) Belgaum Sorghum Average (Karnataka) Climatic Soil order PH OC AIA region (%) Arid Alfisol 6.3 0.36 5.2 [+ or -] 0.12 Aridisol 9.0 0.12 6.7 [+ or -] 0.16 Semi-arid Verti sol 8.6 0.66 8.5 [+ or -] 0.17 Alfisol 8.4 0.65 5.2 [+ or -] 0.17 Elumid Lateritic 6.0 3.95 7.5 [+ or -] 0.14 Verti sol 6.2 0.81 8.2 [+ or -] 0.15 Average 7.4 1.1 6.88 [+ or -] 0.15 Climatic Soil order SCA HVA region Arid Alfisol 4.5 [+ or -] 0.18 11.3 [+ or -] 0.25 Aridisol 6.3 [+ or -] 0.23 5.8 [+ or -] 0.23 Semi-arid Verti sol 7.2 [+ or -] 0.26 31.7 [+ or -] 0.30 Alfisol 4.6 [+ or -] 0.08 10.0 [+ or -] 0.32 Elumid Lateritic 7.2 [+ or -] 0.23 9.0 [+ or -] 0.26 Verti sol 7.7 [+ or -] 0.26 21.2 [+ or -] 0.48 Average 6.25 [+ or -] 0.20 14.83 [+ or -] 0.30 Climatic Soil order AGS region Arid Alfisol 2.6 [+ or -] 0.05 Aridisol 5 [+ or -] 0.01 Semi-arid Verti sol 4.2 [+ or -] 0.17 Alfisol 2.8 [+ or -] 0.17 Elumid Lateritic 3.0 [+ or -] 0.31 Verti sol 3.5 [+ or -] 0.22 Average 3.5 [+ or -] 0.15 * AIA: Actinomycetes Isolation agar, SCA: Starch casein agar, HVA: Humic acid vitamin agar, AGS: Arginine glycerol salts medium. OC: organic carbon Table 2. Biocontrol potential of different genera of Actinobacteria against fungal plant Pathogens Genera Number of strains Number of strains inhibited MP inhibited SR Streptomyces (26 no's) 20 20 Nocardia (12 no's) 7 7 Saccharoployspora (2 no's) 2 2 Micromonospora (1 no) 1 1 Genera Number of strains Number of strains inhibited RS inhibited FO Streptomyces (26 no's) 18 2 Nocardia (12 no's) 6 0 Saccharoployspora (2 no's) 1 0 Micromonospora (1 no) 1 0 * MP: Macrophomina phaseolina, SR: Sclerotium rolfsii, RS: Rhizoctonia solani, FO: Fusarium oxysporum Table 3. Plant growth promoting attributes of Actinobacteria (Streptomyces spp.) Isolates IAA GA % Pi released (mg [ml.sup.-1]) (mg [ml.sup.-1]) at 10 days A1 5.1 34.8 6.0 A2 1.9 24.3 4.8 A6 5.7 35.3 6.5 A10 5.6 33.0 6.02 A11 3.9 24.1 4.9 A17 5.42 41.4 6.8 A18 -- -- 3.2 A30 3.3 29.5 -- A40 2.5 -- 5.6 A45 3.4 28.6 4.3 Isolates Alkaline K solubilization Ammonia Phosphatase ([micro]g (mg [ml.sup.-1]) production p-nitrophenol/ml /h) at 10 days A1 83.6 6.1 + A2 60.0 3.7 + A6 141.6 6.3 + A10 106.9 5.9 + A11 78.6 4.0 - A17 160.9 6.3 + A18 79.2 3.9 - A30 74.4 -- - A40 -- -- - A45 62.3 -- + * All strains negative for HCN production, siderophore production and chitinase activity. Table 4. Effect of Actinobacteria (Streptomyces spp.)on Maize (Var. JM-216) at different growth stages Isolates Plant height (cm Plant height (cm [plant.sup.-1]) 30 [plant.sup.-1]) 45 DAS DAS A1 14.4 19.2 A2 12.6 16.8 A6 15.0 20.0 A10 13.1 17.5 A11 14.1 18.9 A17 12.8 17.0 A18 12.9 17.2 A30 13.5 18.0 A40 11.7 15.6 A45 15.7 21.0 Control 10.5 14.0 S.Em [+ or -] 0.15 0.20 CD @ 1% 0.48 0.63 Isolates No.of leaves No.of leaves ([plant.sup.-1]) 30 ([plant.sup.-1]) 45 DAS DAS A1 6.3 8.4 A2 6.0 8.0 A6 6.7 8.9 A10 6.3 8.4 A11 6.0 8.0 A17 6.0 8.0 A18 5.0 6.7 A30 5.0 6.7 A40 5.7 7.6 A45 5.0 6.7 Control 4.0 5.3 S.Em [+ or -] NS NS CD @ 1% Isolates Total DM (mg [plant.sup.-1]) 45 DAS A1 560 A2 554 A6 1044 A10 485 A11 467 A17 493 A18 364 A30 551 A40 456 A45 417 Control 380 S.Em [+ or -] 2.5 CD @ 1% 8.0 Table 5. Effect of Actinobacteria (Streptomyces spp.) on Chickpea var (JG-16) at different growth stages Isolates Plant height Plant height (cm (cm [plant.sup.-1]) [plant.sup.-1]) 60 DAS 30 DAS A1 21.8 28.9 A2 20.2 28.2 A6 21.7 29.0 A10 17.2 30.8 A11 24.2 27.5 A17 20.3 30.5 A18 21.5 29.6 A30 19.2 28.7 A40 21.2 29.8 A45 21.8 30.2 Control 11.3 27.0 S.Em [+ or -] 0.10 0.36 CD @ 1% 0.31 1.08 Isolates Nodule number Nodule dry weight (No.[plant.sup.-1]) (mg [plant.sup.-1]) 60 DAS 60 DAS A1 23 13.3 A2 29 23.3 A6 22 16.7 A10 29 23.7 A11 28 11.7 A17 29 26.7 A18 23 16.7 A30 21 16.7 A40 21 20.0 A45 21 11.7 Control 19 10.3 S.Em [+ or -] 1.0 0.2 CD @ 1% 3.0 0.6 Isolates Total Dw (g [plant.sup.-1]) 60 DAS A1 797 A2 861 A6 808 A10 896 A11 863 A17 936 A18 848 A30 793 A40 860 A45 736 Control 661 S.Em [+ or -] 3.2 CD @ 1% 9.6
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|Author:||Radha, T.K.; Rao, D.L.N.; Sreeramulu, K.R.|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Jun 1, 2017|
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