Actinobacteria of Arid and Semi-arid Soils: Antagonism to Fungal Pathogens and Plant Growth Promoting Potential

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., Macrophomina phaseolina, Sclerotium rolfsii, Rhizoctonia solani and Fusarium oxysporum. 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 Sclerotium rolfsii compared to humid region strains. Semi arid region strains (24.2%) were more effective in inhibiting the mycelial growth of Rhizoctonia solani followed by arid and humid region strains. However only the arid region strains (44.5%) showed inhibition towards Fusarium oxysporum. 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.

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 semidry 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.

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 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 Actinobacteria viz., actinomycetes isolation agar, starch casein agar, arginine glycerol salts medium and humic acid vitamin agar. Isolates were characterized by referring to the 9 th 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 ± 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.

Plant Bioassay
Te n e ff i c i e n t b i o c o n t r o l s t r a i n s 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°C for 20 min) on successive days and then two times dry heat sterilization (160°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.

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 (14.8X10 4 cfu/g soil) while the lowest Actinobacterial population was recorded in Argnine glycerol salts medium (3.5 X 10 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.
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)      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)  Micromonospora MM18 produced and released IAA, GA 3 , 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 -1 ml -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 -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 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.