Archives

Percent inhibition (PI) =(C-T)/C ×100
Where, C = the growth of pathogen (cm) in the absence of antagonistic isolate.
T = the growth of pathogen (cm) in the presence of antagonistic isolate.

Characterization of the potential actinomycete isolates
Selected isolates were characterized on the basis of morphological, biochemical and molecular characteristics using Burgey’s manual of Systematic Bacteriology (Williams et al., 1989).
Morphological characterization
Morphological characteristics of actinomycete isolates were recorded by two means after obtaining pure cultures on nutrient agar medium:

• Cultural characteristics of microorganisms viz. size, shape, elevation, margin, texture, opacity, and color were studied.
• Microscopic characteristics viz. size, shape, arrangement and Gram’s reaction were studied.

Molecular characterization
Three actinomycetes isolates were grown in Luria broth for 24 h, and genomic DNA was extracted by CTAB method. The integrity and concentration of purified DNA was determined by agarose gel electrophoresis. The total genomic DNA extracted was dissolved in sterile distilled water and stored at 4ºC. Primers for 16S rRNA genes were selected from standard scientific literature (Perez et al., 2007; & Mavrodi et al., 2001). Sequences of the primers used to amplify anddetect 16S rRNA genes are “27 F-AGAGTTTGATCCTGGCTCAG and 1492 R-GGTTACCTTGTTACGACTT”. The oligonucleotides were synthesized at MWG Bio-tech Pvt. Ltd.,Germany. Each pair of primers was highly specific and gave a PCR product of known size thatwas easily identified by electrophoresis on agarose gel.
DNA sequencing
Partial 16S rRNA gene sequencing was carried out for promising isolates’ and was performed using the ABI PRISM® BigDye™ Terminator cycle sequencing kit on the ABI PRISM 3100 genetic analyser. The 16S rRNA gene sequences were assembled using MEGA 4 software, compared with other strains using NCBI BLAST analysis for identification purpose and comparison of homologies of isolated strains with previously characterized strains.
Antifungal metabolite production by isolates
Production of fungal cell wall degrading enzymes
Amylase
Amylase production was determined by using 1% starch agar plates. The isolates were spot inoculated and incubated at 28±2ºC for 2 days. After incubation, flood the plates with iodine solution for 30 sec and pour of the excess iodine. Clear zone around the colonies indicates the presence of amylolytic activity.
Protease
Protease production was determined using milk casein agar. The isolates were spot inoculated on milk casein agar and incubated for 2 days at 28±2ºC. Proteolytic activities were identified by appearance of clear zone surrounding their growth.
Lipase
Lipolytic activity was determined by spot inoculated isolates on Tributyrin agar plates (Lawrence et al., 1967).
Cellulase
Cellulase activity was determined using cellulose agar plate and the isolates were spot inoculated on plate and after incubation, zone of clearance around colony indicates positive for cellulolytic activity.
Chitinase
Chitinase activity was done using colloidal chitin agar. The isolates were spot inoculated on plates. All the plates were incubated at 28±2ºC for 2 days. Isolates showing clear zone around the colony considered as chitinolytic activity.
Production of secondary metabolites
Salicylic acid production
For determination of salicylic production, the strain were grown in the succinate medium at 28±2ºC for 48 h. Cells were collected by centrifugation at 10,000 g for 5 minutes. One ml of cell free culture filtrate was acidified with 1N HCL to pH 2.0 and salicylic acid was extracted with equal volume of chloroform. Two ml of 2M FeCl3 was added to the pooled chloroform phases. The absorbance of purple ion salicylic acid complex developed in aqueous phase, was read at 527nm. A standard curve prepared with salicylic acid in succinate medium and quantify of salicylic acid produced was expressed as μg/ml.
Siderophore production
Catecholate nature
FeCl3Test – To 0.5 ml of cell free culture suspension was added from nutrient broth to 0.5 ml of 2% aqueous FeCl3 solution. Appearance of orange or red wine colour indicated the presence of Catecholate nature of siderophore.
Hydroxamate nature
Tetrazolium salt test – to a pinch of tetrazolium salt, was added 1-2 drops of 2 N NaOH and 0.1 ml of the test culture supernatant. Instant appearance of a red to dip red color indicated the presence of hydroxamate siderophores.
Volatile organic compound production
To determine antifungal role of bacterial volatiles, an assay was carried out in two compartmentalized petridish. One compartment of petridish was filled with sterile nutrient agar medium and another with PDA medium. The compartment containing nutrient agar medium was streaked with actinomycete strains, whereas compartment filled with PDA medium was inoculated with 3 mm plug of fungal pathogen previously grown on PDA plates. After inoculation the petri dishes were sealed tightly to avoid escape of volatiles and incubated at 28±2ºC in an incubator and the plates were observed for inhibition of fungal growth (mycelial or sporulation), if any. Control plates received fungal inoculation but was not inoculated with bacteria.
In vitro efficacy of selected isolates for biocontrol of fungal pathogens
The promising isolates were further tested for their efficacy for biocontrol of F.oxysporum and M. phaseolina in chickpea and soybean, respectively under in vitro conditions. The In vitro efficacy of isolates was tested on solid water agar (1 % agar) in tubes. Seeds weresurface sterilized by treatment with 0.5% HgCl2 for 30 sec (three times) followed by wash withsterilized distilled water.
One set of treatment received simultaneous inoculation of fungal pathogens and actinomycetes suspension (50 μl) keeping uninoculated controls. Another set receives sequential application, actinomycetes culture (50 μl) by seed treatment followed by fungal inoculation (50 μl) 5 days after inoculation of actinomycetes. The tubes were observed daily for disease development up to 15 days.

RESULTS AND DISCUSSION

Isolation of actinomycetes
Total 10 soil samples were collected from different area of agriculture fields and dumping sites. Total 40 actinomycetes were isolated on Glycine Asparagine Agar media consisting of antifungal agent cyclohexamide (50μg/ml) by serial dilution method. Out of 40 actinomycetes, 28 cultures were isolated from agriculture fields while 12 cultures were obtained from dumping sites. The colonies showing typical characteristics of the actinomycetes like forming branched network of hyphae and sexual spores were selected, purified and stored at 4ºC temperature in refrigerator for further studies.
Screening of actinomycetes for antifungal activity
Screening of all the isolates for their anti-pathogenic activity against soil borne fungal pathogens viz. M. phaseolina and F. oxysporum was performed by dual culture method. All isolates under the test were screened by their potentiality to check the mycelial growth of soil borne fungal pathogens M. phaseolina and F. oxysporum. After 7 days of incubation the mycelial growth was measured and the inhibition of mycelial growth due to presence of antagonistic bacteria was recorded as percentage inhibition.
Data in Table 1 shows that isolate I 14 was found significantly superior as compared to other isolates showing maximum inhibition of mycelial growth i.e. 50% followed by isolate M 1 48% inhibition and isolate M 14 showing 44% of inhibition of M. phaseolina. Similarly Isolates M 1, M 14 and I 14 showed 48.89 %, 44.44 % and 35.56 % inhibition of mycelial growth of F. oxysporum, respectively (Figure 1).
Microorganisms have tendency of antagonism for their survival in specific niche. Phytopathogenic fungi are major threat for crop production which could be inhibited by the antagonistic microbes present in the same habitat which ultimately proved to be beneficial for plant health. Gopalakrishnan et al. (2011) isolated 137 actinomycetes cultures, from 25 different herbal vermicomposts, and screened out them on the basis of their antagonistic potential against F. oxysporum f. sp. ciceri by dual-culture assay.
Morphological characterization
Colonial characteristics of all the three isolates were studied by growing them on NA plate and observed after 72 h of incubation. All the isolates showed variable growth patterns on NA as narrated in Table 2. All the isolates were also found to produce spores and earthy odor. Further the isolates were subjected to the microscopic characterization through wet mount and Gram’s staining. Microscopic examination revealed that all the isolates were Gram positive appear as filamentous growth and produced spores which is the characteristic feature of the actinomycetes. Streptomycetes are gram-positive found predominantly in soil and decaying vegetation, mainly streptomycetes produce spores and are noted for their distinct “earthy” odour which results from production of a volatile metabolite, geosmin (Madigan & Martinko 2005).
Molecular characterization
16S rRNA gene sequencing and identification of promising isolates
16S rRNA partial gene sequence of ~ 1500 bp was carried out (with technical support of Eurofins genomics, Banglore) and the output data were stored in FASTA format. The output sequences were subjected for BLAST (Basic Local Alignment Search Tool) analysis to identify the cultures and to find out the nearest match of the cultures (http://www.ncbi.nlm.nih.gov/). The sequence analysis of partial 16S rRNA gene of isolate I 14, M 1 and M 14 have been deposited in NCBI, GenBank under accession numbers MN577354, MN577357 and MN577358 respectively. The analysis named isolate I 14 as Streptomyces sp. strain AAUBC I 14, Isolate M 1 as Streptomyces asenjonii AAUBC M 1 and Isolate M 14 as Streptomyces cavourensis AAUBC M 14 (Figure 2 to 4).
Molecular identification of the actinomycetes is done by the nucleic acid sequencing method. The sequence of 16S ribosomal DNA is being used by various researchers for accurate identification of the actinomycetes up to genus level. With help of the 16S rDNA sequences phylogenetic relationship of different actinomycetes can be confirmed by Jami et al. (2015).
Antifungal metabolite production by isolates
Production of fungal cell wall degrading enzymes
Metabolites like hydrolytic enzymes viz. amylase, protease, lipase, cellulose, and chitinase secreted by many microorganisms helps in degradation of the fungal cell wall and thereby restrict their growth. Moreover, secondary metabolites like siderophores, salicylic acid and volatile compounds also help to inhibit fungal growth and thereby limit the pathogen attack to the crops.
Present study shows that all selected isolates were capable of producing amylase, protease, lipase, cellulase, and chitinase enzymes. (Table 3) secretion of these enzymes by isolates can result in the suppression of plant pathogen activity.
Thus, they play a vital role in the biological control of many plant diseases by degrading the cell walls of phytopathogens. It affects fungal growth by its lytic action on cell walls, hyphal tips, and germ tubes (Kim et al., 2003; & Sayyed et al., 2015) and partial swelling in the hyphae and at the hyphal tip leading to hyphal curling or bursting of the hyphal tip (Someya et al., 2000).
Production of secondary metabolites
All three isolates were capable of produced salicylic acid in succinate media. M 14 showed the highest salicylic acid production i.e. 50 µg/ml followed by M 1 (34.04 µg/ml) and I 14 (10.00 µg/ml) (Table 4).
Salicylic acid, a phenolic compound, is getting attention as plant stress regulator and induced both local and systemic plant defence response. It plays an important role in the regulation of plant growth and inhibits the growth of pathogen.
Biological control of soil borne pathogens is often attributed to improved nutrition that boosts host defences or to direct inhibition of pathogen growth and activity. Amendment with certain abiotic factors (inducers) appears to stimulate the disease resistance by indirectly stimulating indigenous populations of microorganism that are beneficial to plant growth and antagonistic to pathogen.
Many studies have indicated that salicylic acid (SA) plays an important role in plant defence response against pathogen attack and is essential for the development of SAR (Ryals et al., 1996; & Yalpani et al., 1993). Ward et al. (1991) found that this increased resistance was correlated with accumulation of pathogenesis-related (PR) proteins, which are generally considered to be markers of SAR.
Exogenous application of SA or its functional analogs, such as 2, 6-dichloroisonicotonic acid and benzo- (1, 2, 3) - thiadiazole-7-carbothioic acid S-methyl ester (BTH), induces SAR in plants, resulting in resistance to certain pathogens (Ryals et al., 1996).
Production of siderophores
FeCl3 test for catecholate type of siderophore
During the present study, all isolates failed to produce catecholate type of siderphore. As started by Neilands (1981) absence of wine color proves absence of catecholate type of siderophore in sample.
Tetrazolium test for hydroxamate type of siderophore
The test is based on capacity of hydroxamic acids to reduce tetrazolium salt by hydrolysis of hydroxamate groups using strong alkali. The reduction and release of alkali shows a deep red color. Tetrazolium test was carried out to find out presence of hydroxamate type of siderophore. All isolates produced hydroxamate type of siderophore because of instant appearance of deep red color in tubes. Similar results have been reported by Dave and Dube (2000).
The antagonism depends on the amount of iron available in the medium, siderophore produced by a biocontrol agent and sensitivity of target pathogens. Siderophore producing rhizobacteria improve plant health at various levels: they improve iron nutrition, inhibit growth of other microorganisms with release of their antibiotic molecule and hinder the growth of pathogens by limiting the iron available for the pathogen, generally fungi, which are unable to absorb the iron siderophore complex.
This finding is in agreement with Nimnoi et al. (2010) who reported that Pseudonocardia halophobica isolated from roots of Aquilaria crassna exhibited high ability for siderophore production and produced 39.30mg/ml. Khamna et al. (2009) also reported that Streptomyces CMU-SK 126 isolated from Curcuma manggarhizospheric soil exhibited high ability for siderophore production and produced catechol type (16.19μg/ml) as well as hydroxamate type (54.9μg/ml) siderophores.
Production of volatile compounds
In addition to diffusible substances, bacteria emit a wide range of volatile compounds into the atmosphere. Over the last ten years, evidence has been accumulated that these volatiles are not only able to promote plant growth, but also strongly inhibit fungal growth. Production of volatile compounds by biocontrol agents is one of the mechanisms to bring about inhibition of fungal pathogens. To evaluate the role of volatile compounds produced by all the individual isolates for inhibition of phytopathogenic fungi, dual culture assay in two compartmentalized petridish was carried out. Here, due to compartments there was no possibility of direct contact of microorganism and pathogens and thereby inhibition which we observed may solely due to effect of volatile compounds produced by microbes.
Data indicated that, all the individual strains showed inhibition of M. phaseolina and F. oxysporum. Major impact is seen on the sporulation efficiency of the fungi as there wasreduction in fungal sporulation observed in the plates which received bacterial inoculation ascompared to uninoculated control indicating the impact of volatile compounds on sporulationefficiency of the fungi which in turn affects the proliferation of fungal pathogens by inhibitingits reproduction (Figure 5).
In vitro efficacy of selected isolates for biocontrol of fungal pathogens
Charcoal rot caused by the fungus, M. phaseolina, have emerged as serious concern for cultivation of soybean. M. phaseolina causes huge annual losses to the crop and can survives in the soil mainly as microsclerotia for 2 years or longer and; germinate repeatedly during the crop-growing season. The pathogen generally attacks the young plants of the soybean. The results of the study clearly indicate that all the isolates were capable of inhibiting the growth of M. phaseolina when inoculated simultaneously. The plants showed better root and shoot development as well as no disease symptoms of M. phaseolina infection up to 15 days (Figure 6A). The plants inoculated with isolates and fungi simultaneously were as healthy as the uninoculated control plants which do not received inoculation of the fungi. These results prove that all the isolate were capable of restricting growth of the M. phaseolina and thereby prevents disease outbreak in soybean plants.
Fusarium wilt caused by F. oxysporum is the major soil-borne fungus affecting chickpeas globally. Fusarium wilt epidemics can devastate crops and cause up to 100% loss in highly infested fields and under favourable conditions. The fungus can be transmitted by seed and may survive in plant debris in soil. It was demonstrated that the fungus chlamydospore was found free in soil, in the seed, in cotyledons and axis. Results of present study indicated that tubes receiving inoculation of isolates do not showed any disease symptoms up to 15 days after inoculation and plants were found healthy as compared to the tubes which received fungal inoculation alone (Figure 6B).
The overall results of the in vitro biocontrol studies indicated that, all the isolates were capable of restricting the disease development by M. phaseolina and F. oxysporum in soybean and chick pea, respectively.

Table 1: Screening of isolates against plant pathogenic fungi

Sr. No.	Isolates	% growth inhibition of test pathogenic fungi
		M. phasoelina	F.  oxysporum
1	I1	38.00	-
2	I2	36.00	-
3	I3	-	-
4	I4	-	-
5	I5	-	-
6	I6	34.00	17.78
7	I7	-	-
8	I8	-	31.11
9	I9	38.00	13.33
10	I10	42.00	-
11	I11	-	-
12	I12	-	-
13	I13	-	33.33
14	I14	50.00	35.56
15	I15	-	-
16	I16	-	-
17	I17		-
18	I18	-	-
19	M1	48.00	48.89
20	M2	-	-
21	M3	-	28.89
22	M4	36.00	35.56
23	M5	40.00	33.33
24	M6	42.00	-
25	M7	40.00	-
26	M8	36.00	31.11
27	M9	-	-
28	M10	-	26.67
29	M11	-	-
30	M12	-	-
31	M13	40.00	33.33
32	M14	44.00	44.44
33	M15	-	-
34	M16	-	-
35	M17	-	-
36	M18	-	-
37	M19	38.00	-
38	M20	36.00	-
39	M21	40.00	-
40	M22	36.00	-

Table 2: Colonial characterization of isolates on Nutrient agar plates

Table 3: Enzyme activity of isolates

Sr.No.	Isolates	Amylase	Protease	Lipase	Cellulase	Pectinase	Chitinase
1	I14	+	+	+	+	+	+
2	M1	+	+	+	+	+	+
3	M14	+	+	+	+	+	+

 

Table 4: Production of salicylic acid by isolates

 

CONCLUSION

In this study, the selected actinomycetes, Streptomyces sp., Streptomyces asenjonii and Streptomyces cavourensis were showed good antagonistic property in suppressing the mycelial growth of both fungi M. phaseolina and F. oxysporum. Hence, further investigation with these potential isolates may help to control charcoal rot in soybean and Fusarium wiltin chickpea caused by M. phaseolina and F. oxysporum respectively. Thus, it can be concluded from results of present investigation, actinomycetes isolates belonging to the genus Streptomyces can be explored as potential source of novel biological control agent.

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