T.K. Radha1,2, D.L.N. Rao3 and K.R. Sreeramulu

1Indian Institute of Horticulture Research, Bengaluru-560 089, India.
2 Indian Institute of Soil Science, Nabi Bagh, Bhopal-462 038, India.
3University of Agricultural Sciences, GKVK, Bengaluru-560065, India.

Abstract

Biological control of plant pathogens is assuming increasing importance in organic and low input sustainable agriculture. Forty one Actinobacterial strains isolated from the arid, semi arid and humid regions of Rajasthan, Karnataka, Andhra Pradesh in India were tested for their antagonism to four plant pathogenic fungi viz., Macrophominaphaseolina, Sclerotiumrolfsii, Rhizoctoniasolaniand Fusariumoxysporum. The Streptomyces strains from humid regions were more effective in inhibiting mycelial growth of Macrophomina phaseolina(62.8%). Arid and semi arid region strains (40.8 and 40.6%) were almost similar in inhibiting mycelial growth of  Sclerotiumrolfsii compared to humid region strains. Semi arid region strains (24.2%) were more effective in inhibiting the mycelial growth of Rhizoctoniasolanifollowed by arid and humid region strains. However only the arid region strains (44.5%) showed inhibition towards Fusariumoxysporum. The strains with best antogonistic ability (10 no.s) also exhibited other plant growth promoting traits like production of Indole acetic acid (1.9 – 5.7 mg ml-1)and Gibberellic acid (24.1-41.4 mg ml-1). All the strains except Streptomyces A30 could solubilize P from inorganic tri-calcium phosphate. The amount of P solubilization ranged from 3.2 – 6.8%.  All the strains except Streptomyces A40 produced alkaline phosphatase which ranged from 60.0-160.9 µg p-nitrophenol/ml /h. Some strains produced ammonia whereas none of the strains produced HCN, siderophore or exhibited chitinase activity. In a glass house evaluation in soil microcosms, among the ten strains Streptomyces A6 was found best for promoting maize and Streptomyces A17 for chickpea growth.

Keywords: Actinobacteria, Streptomyces, Biocontrol, PGPR, dryland crops.

Introduction

Pathogenic micro-organisms affecting plant health are a major and chronic threat to food production worldwide. Use of pesticides to control the pathogens is an inevitable part of food production strategy but their unscientific usage in developing countries has often led to adverse effects on soil and human health.  Several negative effects like development of pathogen resistance and adverse influence on non-target populations have been reported.  Furthermore, the growing cost of pesticides and consumer demand for pesticide-free food has led to a search for substitutes for these products. There are also a number of fastidious diseases for which chemical solutions are few, ineffective or nonexistent. Biological control is thus considered as an alternative or a supplemental way of reducing the use of chemicals in agriculture. Some of the Actinobacteria naturally occurring in soils and on rhizosphere of various crops act as potential biocontrol agents.Actinobacteria possess several properties which help in plant growth promotion and as biocontrol agent in combating several plant diseases. They colonize plant root surfaces, exhibit antibiosis against plant root pathogens, synthesis of particular extracellular enzymes, hydrogen cyanide production and siderophore production (Tokala et al. 2002). They are able to tolerate unfavourable growth conditions by forming spores (Alexander 1977).  Actinobacteria, which have the ability to thrive under dry conditions, could be a valuable biocontrol resource to the poor farmers of dry land areas. Since Actinobacteria prefer dry to semi-dry soils, the crops grown in arid and semi-arid regions are likely to benefit by their inoculation. In the present study, Actinobacteria were isolated from arid, semi arid and also humid regions (for comparison) of western and peninsular India and screened for antagonism against some important fungal plant pathogens; their plant growth promoting attributes were also tested in vitro and in vivo on maize and chickpea in soil microcosms.

 

Material and Methods

Isolation of Actinobacteria

Soil samples were collected from the rhizosphere of Sorghum (Sorghum vulgare), Pearl millet (Pennisetumtyphoideum), Pigeon pea (Cajanuscajan), Finger millet (Eleusinecoracana) and Groundnut (Arachis hypogea) grown in arid region of Jaisalmer in Rajasthan; 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 Actinobacteriaviz.,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)

 

Table 1.Enumeration of Actinobacteria (x104cfu/g soil ±SEm) in different growth media*

Climatic region Soil order Place and state Origin pH OC (%) AIA SCA HVA AGS
Arid Alfisol Tumkur (Karnataka)

 

 

Ananthpur (Andhra Pradesh)

Finger millet

 

Groundnut

 

6.3

 

0.36

 

5.2±0.12

 

4.5±0.18

 

11.3±0.25

 

2.6±0.05

Aridisol Jaisalmer (Rajasthan)

 

Sam (Rajasthan)

Pearl millet

 

Desert

 

9.0

 

0.12

 

6.7±0.16

 

6.3±0.23

 

5.8±0.23

 

5±0.01

Semi-arid Vertisol Hubli, Bijapur (Karnataka) Sorghum, Pigeon pea, Pearl millet  

8.6

 

0.66

 

8.5±0.17

 

7.2±0.26

 

31.7±0.30

 

4.2±0.17

Alfisol Gadag (Karnataka) Sorghum 8.4 0.65 5.2±0.17 4.6±0.08 10.0±0.32 2.8±0.17
Humid Lateritic Uttarakannada  (Karnataka) Forest 6.0 3.95 7.5±0.14 7.2±0.23 9.0±0.26 3.0±0.31
Vertisol Uttarakannada  (Karnataka)

 

Belgaum (Karnataka)

Forest

 

Sorghum

 

6.2

 

0.81

 

8.2±0.15

 

7.7±0.26

 

21.2±0.48

 

3.5±0.22

Average 7.4 1.1 6.88±0.15 6.25±0.20 14.83±0.30 3.51±0.15

*AIA: Actinomycetes Isolation agar, SCA: Starch casein agar, HVA: Humic acid vitamin agar, AGS: Arginine glycerol salts medium.

OC: organic carbon

 

In vitroantagonism to fungal pathogens

The Actinobacterial strains were screened for the antagonism (biocontrol potential) to soil borne plant pathogens viz., Macrophominaphaseolina, Sclerotiumrolfsii, Rhizoctoniasolani and Fusariumoxysporum. 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 ± 2 ºC 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.

 

Table 2.Biocontrol potential of different genera of Actinobacteria against fungal plant

Pathogens

Genera Number of strains inhibited MP Number of strains inhibited SR Number of strains inhibited RS Number of strains inhibited FO
Streptomyces  (26 no’s) 20 20 18 2
Nocardia (12 no’s) 7 7 6 0
Saccharoployspora (2 no’s) 2 2 1 0
Micromonospora (1 no) 1 1 1 0

* MP: Macrophominaphaseolina, SR:Sclerotiumrolfsii, RS:Rhizoctoniasolani, FO:Fusariumoxysporum

 

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 quantification of P solubilized in broth after 10 days growth at 28±20 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±20 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.

 

Table 3.Plant growth promoting attributes of Actinobacteria(Streptomyces spp.)

Isolates IAA

(mg ml-1)

GA

(mg ml-1)

% Pi released  at 10 days Alkaline Phosphatase

(µg p-nitrophenol/ml /h)

K solubilization (mg ml-1) at 10 days Ammonia production
A1 5.1 34.8 6.0 83.6 6.1 +
A2 1.9 24.3 4.8 60.0 3.7 +
A6 5.7 35.3 6.5 141.6 6.3 +
A10 5.6 33.0 6.02 106.9 5.9 +
A11 3.9 24.1 4.9 78.6 4.0
A17 5.42 41.4 6.8 160.9 6.3 +
A18 3.2 79.2 3.9
A30 3.3 29.5 74.4
A40 2.5 5.6
A45 3.4 28.6 4.3 62.3 +

*All strains negative for HCN production, siderophore production and chitinase activity.

 

Plant Bioassay

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 (VerticEustochrept). 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 (1210 C for 20 min) on successive days and then two times dry heat sterilization (1600 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 µg N g-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.

 

Table 4:Effect of Actinobacteria(Streptomyces spp.)on Maize (Var.  JM-216) at different growthstages

Isolates Plant height

(cm plant-1)

30 DAS

Plant height

(cm plant-1)

45 DAS

No.of leaves

(plant-1)

30 DAS

No.of leaves

(plant-1)

45 DAS

Total DM

(mg plant-1)

45 DAS

A1 14.4 19.2 6.3 8.4 560
A2 12.6 16.8 6.0 8.0 554
A6 15.0 20.0 6.7 8.9 1044
A10 13.1 17.5 6.3 8.4 485
A11 14.1 18.9 6.0 8.0 467
A17 12.8 17.0 6.0 8.0 493
A18 12.9 17.2 5.0 6.7 364
A30 13.5 18.0 5.0 6.7 551
A40 11.7 15.6 5.7 7.6 456
A45 15.7 21.0 5.0 6.7 417
Control 10.5 14.0 4.0 5.3 380
S.Em ± 0.15 0.20 NS NS 2.5
CD @ 1% 0.48 0.63 8.0

 

 

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 (14.8X104cfu/g soil) while the lowest Actinobacterial population was recorded in Argnine glycerol salts medium (3.5 X 104cfu/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, glycerol-arginine 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 endophyticActinobacteria 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; Gupta1991; Gesheva and Gesheva 2000).

In this study, 41 Actinobacterial strains were tested for their in vitro antagonistic activity against four plant pathogenic fungi viz., Macrophominaphaseolina, Sclerotiumrolfsii, RhizoctoniasolaniandFusariumoxysporum. Some of the Streptomyces strainsinhibitedall the four pathogens whereas Nocardia, Saccharopolyspora and Micromonospora strains inhibited all the pathogens except Fusariumoxysporum(Table 2). The strains from humid regions were more effective in inhibiting mycelial growth of Macrophominaphaseolina (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 Sclerotiumrolfsii compared to humid region strains. Semi arid region strains (24.2%) are more effective in inhibiting the mycelia growth of Rhizoctoniasolanifollowed by arid and humid region strains, whereas only arid region strains (44.5%) showed inhibition towards Fusariumoxysporum. This biocontrol potentiality can be attributed to the production of antimicrobial compounds including ammonia production. Khamnaet al. (2009) observed biocontrol activity of 23 Streptomyces isolates against five phytopathogenic fungi viz., Alternariabrassicicola, Colletotrichumgloeosporioides, Fusariumoxysporum, Penicilliumdigitatum and Sclerotiumrolfsii. Subhashini (2010) isolated 40 actinomycetes from tobacco soils of India and reported that 10 Streptomyces isolates showed high antagonistic activity against Pythiumaphanidermatumunder 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-1,Gibberellic acid (GA) production ranged from24.1-41.4 mg ml-1. Solans et al. (2009) found that three rhizoactinomycetesStreptomycesMM40, ActinoplanesME3 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-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 µg p-nitrophenol-1ml -h.

Franco-Correa et al. (2010) isolated 30 actinomycetes from the rhizosphere of TrifoliumrepensL. 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.

 

Table 5.Effect of Actinobacteria(Streptomyces spp.) on Chickpea var (JG-16) at different growth stages

Isolates Plant height

(cm plant-1)

30 DAS

Plant height

(cm plant-1)

60 DAS

Nodule number

(No.plant-1)

60 DAS

Nodule dry weight

(mg  plant-1)

60 DAS

Total Dw

(g plant-1)

60 DAS

A1 21.8 28.9 23 13.3 797
A2 20.2 28.2 29 23.3 861
A6 21.7 29.0 22 16.7 808
A10 17.2 30.8 29 23.7 896
A11 24.2 27.5 28 11.7 863
A17 20.3 30.5 29 26.7 936
A18 21.5 29.6 23 16.7 848
A30 19.2 28.7 21 16.7 793
A40 21.2 29.8 21 20.0 860
A45 21.8 30.2 21 11.7 736
Control 11.3 27.0 19 10.3 661
S.Em ± 0.10 0.36 1.0 0.2 3.2
CD @ 1% 0.31 1.08 3.0 0.6 9.6

 

 

 

 

 

 

 

 

 

 

Fig 1.Biocontrol ability of Actinobacteria against fungal plant pathogens (dual culture)

MP: Macrophominaphaseolina; SR: Sclerotiumrolfsii; RS:Rhizoctoniasolani;FO:Fusariumoxysporum

H: Humid; SA: Semi arid; A: Ari

 

 

 

 

 

 

 

 

 

 

 

Fig. 2Antagonism of Actinobacteria against fungal plant pathogens a) Macrophominaphaseolina b) Sclerotiumrolfsii c) Rhizoctoniasolani d) Fusariumoxysporum

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 -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-1) and plant dry weight (936 mg plant -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 Siniorhizobiummeliloti and found that nodulation and growth of Alfalfa (Medicagosativa) plants were significantly increased when compared to plants inoculated only with Siniorhizobiummeliloti. 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, solubilzationof  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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