Department of Biotechnology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India.
Received: 22-Feb-2022, Manuscript No. M-50159;Editor assigned: 25-Feb-2022, PreQC No. P-50159; Reviewed: 11-Mar-2022, QC No. Q-50159;Revised: 16-Mar-2022, Manuscript No. R-50159; Published: 23-March-2022, Invoice No. J-50159
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Six streptomyces sp ESS-4 (Streptomyces fradiae), DVS-11 (Streptomyces olivoviridis), KZSS-6 (Streptomyces koyangensis) NVS-10 (Streptomyces flavofuscus), TSS-3 (Streptomyces griseorubens), and TVS-2 (Streptomyces lunaelactis) were previously reported to antagonistic activity in fungal pathogens, PGP traits, green and field conditions in black gram. These isolates were characterized for their antibacterial activity for streak plate method, antibiotic resistances, and optimization of pH, salinity, temperature, carbon sources, and heavy metals. These streptomyces sp show inhibition to 11 antibiotics they produce the halo-zone produces 14 to 19 cm. This streptomyces sp grow in pH 3 to 13, salinity up to 0% to 12%, temperature up to 20-50, carbon sources for glucose, fructose, sucrose, and starch, and heavy metals tolerances. Out of six sp, KZSS-6 was better results. So KZSS-6 strain was further evaluated on the greenhouse and black gram field conditions. This streptomyces greenhouse condition they enhance the root length, yields enhanced when compared to the control. In greenhouse conditions black gram plant height 8 to 14 cm, root length 1 to 6 cm, the seeds 2 to 5cm, total plant weight is 0.20 to 0.60 mg, and total dry weight for plant 7 to 14 mg. In the field experiment, root height and weight (10 to 13 cm, 0.25 to 0.50 mg), shoot length and weight (3.10 to 3.50 cm), stem height and stem weight (22 to 32 cm, 4.0 to 4.25 gm), plant weight (5.50 to 7.50 gm), number of fruits weight (10, 100, and 1000) (0.4 to 0.50, 2.50 to 5.0 and 22 to 45 gm), and finally yield (11.5 to 29.50 tons)
Streptomyces koyangensis; antibacterial activity; optimization; greenhouse; field conditions
Agriculture is a major contributing factor to the global food supply. Now present circumstances agriculture was facing severe threats like loss of soil fertility, poor soil quality, climatic changes conditions, more use of pesticides. To overcome these challenges, eco-friendly approaches like bio-fertilizers. To enhance the agricultural outputs sustainably, novelty and eco-friendly strategies must be employed in agriculture, they reduced chemical pesticides. Plants, as non-locomotive organisms live. Pulses have contained are sources of proteins, micronutrients, and dietary fiber. Black gram plants, green gram plants have contained good proteins for 100 g of seeds the range of about 20.97 to 31.32 g and 25 to 30 g of protein, experts. India is one of the country largest pulses producers and consumers in the world. Preethi, R. Vigna mungo (L) hepper is commonly known as a black gram. One of the important Asiatic legumes crops these seeds contains a major protein and vitamins .
Streptomyces are filamentous gram (+) bacteria, belonging to the order of Actinomycetales, they are presented by more than 570. These actinomycetes were found in soil, marine water, vermicompost esperts, Streptomyces is to survive during the unfavourable environmental condition. Therefore, the streptomyces is more efficient against many other microorganisms present in rhizosphere soil . Recently streptomyces has been considered as a prospective bio control agent in agriculture, also to produces antibiotics and intense antagonistic activity through the production of various antifungal metabolites experts. When compared to other microorganisms, actinomycetes are inhibited by a wide range of plants as entophytes. Therefore, actinobacteria are efficient plant growth promoters, and potential to serve as an effective bio control agent experts. They are plays important role in plant growth-promoting, plant protection, and anti-microbial activity, also produce secondary metabolites for commercial interest, experts .
Earlier, we reported a set of six streptomyces strains were isolated from black gram soil and vermicompost samples . The strains are using this study namely DVS-11, ESS-4, KZSS-11, NVS-10, TSS-2, and TVS-2 which PGP traits and antagonistic activity of fusarium oxysporum in Black gram. Six streptomyces strains were effective control heavy metals, physiological properties and production of antibiotics . Then further green house and filed assay were conducted and results .
Materials And Methods
Antibacterial activity for streak plate method
Antimicrobial activity was performed by cross streak technique. SCA plates were prepared, isolates were streak on the center of Petri plates then incubated at 28OC for 7days . Than the plates were streak on test organisms, at 90o then plates were incubation at 37OC for 12 hrs. The anti-bacterial activity was measured by inhibition of target bacteria and isolated actinomycetes Oskay M, Kumar PS, Sangkanu S. The human bacterial pathogen was used for test, Bacillus subtitles (MTCC No. 10407), Micrococcus luteus (MTCC-106), Staphylococcus aureus (MTCC No. 6908), Streptococcus mutans (MTCC No. 890). Gram negative bacteria Klebsiella pneumonia (MTCC No. 9024), Proteus vulgaris (MTCC No. 744), Pseudomonas aeruginosa (MTCC No. 1034), Saccharomyces cerevisiae (MTCC-251). All the bacterial cultures were procured from IMTECH, Chandigarh .
Antibiotic assay (or) Antibiotic resistances
The Potential isolates were tested for their antibiotic susceptibility and resistance activity against 11 different antibiotics such as Amoxicillin, Ampicillin, Cefpodoxime, Chloramphenicol, Ciprofloxacin, Neomycin, Novobiocin, Penicillin, Rifampicin, Tetracycline, Vancomycin . Muller Hinton Agar media was prepared, autoclaved at 121°C for 15 minutes at 15 lbs pressure, cooled and poured into Petri plates. After solidification, 3 days old Actinomycetes culture was swabbed and followed by placing the antibiotic disc aseptically. The plates were incubated at 28±2°C for four to five days. The results were noted as the zone of inhibition in mm .
The selected actinomycetes against the stress, by the addition of heavy metals to its growth environment. PBS buffer prepared and adjust the pH to 6.8. Heavy metals are using the Cuso4 (copper (II) sulfate, Znso4 (Zink sulfate), (CH3 coo) 2Pb.3h2O(Lead acetate tri hydrate), CH 3 COONH 4 (ammonium ethanoate), K4 so4 (Potassium sulfate), and CH3 COONA (sodium acetate), Heavy metals salts are prepared in different concentration in 10mm, 50mm, 100mm, 500mm, and 1000mm were dissolved PBS buffer . To prepare the starch casein agar, centre of plate well in 12mm. On each plate six actinomycetes stains are streaks, incubated at 28±2°C for 7 days .
To calculated the % of growth=Complete inoculated length of streak –inhibited length/Complete inoculated length × 100
Effects of carbon source
The capacity of carbon sources utilization by isolated Actinomycetes was tested against four different carbon sources such as glucose, fructose, sucrose, and starch . All the four carbon sources separately sterilized by membrane filtration and 1% carbon source were added to basalmineral salt agar media. The selected Actinomycetes were streaked on basal mineral salt agar media and incubated at 28 ± 2°C for five days . The plates were observed for the growth of Actinomycetes and noted as the scale of 0-3 as follows; 0=no growth, 1=slight growth, 2=moderate growth, and 3=good growth .
Effect of Salinity
The Actinomycetes strains were streaked on Bennett`s agar (Himedia Laboratories, Mumbai, India), amended with different concentrations of NaCl (0-12%, at 2% intervals) and incubation, at 28 ± 2°C for five days Gopalakrishnan et al., 2012 . After incubation the plates were observed for growth of Actinomycetes. Based upon the growth the scale was given as 0-3 as follows; 0=no growth, 1=slight growth, 2=moderate growth, and 3=good growth .
Effect of pH
The selected Actinomycetes were streaked on Bennett`s agar (Himedia Laboratories, Mumbai, India), adjusted with pH ranging from 5, 7, 9, and 11, incubated at 28 ± 2°C for five days. After incubation, the plates were observed for the growth of Actinomycetes . Based upon growth the scale was given 0-3 as follow; 0=no growth, 1= slight growth, 2=moderate growth, and 3=good growth .
Effect of temperature
The selected Actinomycetes were streaked on Bennett’s agar (Himedia Laboratories, Mumbai, India) and incubated at different temperatures of 20°C, 30°C,40°C, and 50°C for five days . After incubation, the plates were observed for the growth of Actinomycetes. Based upon growth the scale was given 0-3 as follow; 0=no growth, 1= slight growth, 2=moderate growth, and 3= good growth .
Evaluation of actinobacterial PGP activity under greenhouse condition
Six strains were possessed in greenhouse condition by using mass cultivating pot experiments. Six strains were assayed with sterile and non-sterile soils in the presences of FOC.
Mass culturing of streptomyces spps fermentation broth
Six strains were inoculated into starch casein agar medium and incubated at 28 ± 2oC for 3 to 5. Agar blocks were prepared, 1 sq cm of agar block from the periphery of actively growing colony was cut and inoculated in 100 ml of ISP-2 broth and additionally 10% maltose, 4% glucose and 4% yeast extract, adjusted with pH 7.2. The media was prepared and inoculated the selected actinomycetes. The medium was transfer to in each flask 100 ml and to take 250ml flask, the flasks were incubated 28 ± 2°c in rotary shaker with stirring at 170 rev min. To prepare ISP-2 broth additionally added 4% yeast malt extract, transfer to the 48hr log phage actinobacterial broth culture added. The inoculated flask was placed on incubator rotary shaker for 4-5 days. On the fifth day, 001 ml of FOC spores stock prepared as earlier was added to the culture medium .
Pot filling the study
Each pot was filling with 1 kg soil (sterile and non-sterile soils using this study). Pot filling with sterile soil in aseptically, and inoculated with fungal spores for which each 1 kg of sterile soil was mixed with 50 ml of FOC spores’ stock (concentration 10-6 spores per 1 gram of soil).
Seed sowing: Black gram seeds were procured from commercial markets in GUNTUR. The seeds were selected by appearance, surface sterilization (with aqueous sodium hypochlorite—NaOCl, 05% v/v, 2 min) and washed with sterile distilled water three times . One (or) two seeds were sown per pot thus giving a total of 10 seeds (five replication pots) for each treatment in study .
Growing of seedlings and plant growth analysis: In green house temperature was maintain 27°C and humidity 55%. In treatment pot were arranged in a Completely Randomized Design (CRD) five replicates. The pot was maintaining 65% moisture level, and watered uniformly every day. Treatment post was label, and maintained with succent distances . Antagonistic treatments allowed growing until 15 to 30 days.
Measurement of plant growth parameters: The black gram plants were grown in 15 to 30 days, measure the root length, plant height, the plants were uprooted, fresh biomass, and disease index . In black gram root length was measure from the base of the shoot to the tip of the primary root and shoot length was measured from the base of the shoot to tip of primary leaf. Seed germination was calculated in percent by using the formula .
Percent of seed germination=[(number of greminated seeds )/(total number of seed sown) ]×100
Evaluation of Streptomyces for PGP potential on black gram under field conditions
Field experiment was conducted in 2018-2019 (rabi and kharif season) 16.3763° N, 80.5277° E Nagarjuna university Andhra Pradesh, India. Experiment was conducted with 4 types of veritas, like Poush, PU-31, TBG-104, Q-31 soda (90 to 120 days) these seeds were collected form Acharya N G Ranga Agricultural University, Guntur. which normally yields 1.2–1.5 t?ha-1. The experiment was laid out in a randomized complete block design with 3 replicates and subplot sizes of 10 m×7.5 m . The Streptomyces spp were grown on starch casein broth at 28 °C for 5 days. The control contained no Streptomyces strains. The 10-14-day old single seedlings (black gram) were uprooted from the green house, black gram plant roots were dipped in the respective Streptomyces spp. broth (containing 10-8 CFU•mL−1) for 50 min and transplanted at site soil, the plants were planted in row-to-row with spacing of 25 cm and a plant-to-plant spacing of 25 cm. Broth culture (1000 mL 10-8) was applied one in 15 days until flowering stage along with irrigation. The black gram crop was harvested manually on 2018 and 2019 and observed for plant height, hole plant weight, stem height, stem width, stem weight, internodes number of fruits, leaf width, leaf weight, shoot length, shoot height, root height, root weight, number of root nodules, root nodules weight, one fruit weight, 10 seeds weight, 100 seeds weight, 1000 seeds weight.
percentage Yeild (%) tonnes=(square foot×number of fruits×seeds weight)/(100×one hectare land)
The antagonistic activity was conducted for the completely randomized design with three replicates in three fungal pathogens viz., physiological properties like Temperature, pH salinity, carbon source utilization, and antibiotic production. The data were subjected to Analysis of Variance (ANOVA) and the mean values were compared at a 5% Least Significant Difference (5% LSD) and Coefficient of Variation (% CV).
Anti-bacterial activity for bacterial pathogens
Six isolates were screened against human bacterial pathogens. The percentage of activity was only 44 (41.50%) isolates showed good antimicrobial activity against pathogens. The percentage of activity to each pathogen is listed below: Bacillus subtitles (18%) Micrococcus luteus (14%), Staphylococcus aureus (18%), Streptococcus mutans (20%). Gram negative bacteria Klebsiella pneumonia (28%), Proteus vulgaris (16%), Pseudomonas aeruginosa (18%), Saccharomyces cerevisiae (16%), Table 1, Figure 1.
Effect on antibiotics
The six antagonistic isolates were able to resist all 11 antibiotics. The zone of inhibition range is 14mm-20mm. DVS-11 showed maximum resistance to Amoxiclav, Ampicillin, Ciprofloxacin, and Vancomycin, ESS-4 showed maximum resistance to Cefpodoxime and Tetracycline, TVS-2 showed maximum resistance to Chloramphenicol and Rifampicin, NVS-10 showed maximum resistance to Neomycin and Penicillin and KZSS-6 showed maximum resistance to Novobiocin . The results are represented in the Figure 2 and Table 2.
Table 1. Antibacterial activity in streak plate technique
|S.No||Isolate cultural||Bacillus subtitles||Micrococcus luteus||Staphylococcus aureus||Streptococcus mutans|
|2||DVS-11||++||[Indexing at]||++||[Indexing at]|
|3||KZSS-6||[Indexing at]||++||[Indexing at]||-|
|4||NVS-10||+||[Indexing at]||+||[Indexing at]|
|5||TVS-2||[Indexing at]||+||++||[Indexing at]|
|Klebsiella pneumonia||Klebsiella pneumonia||Pseudomonas aeruginosa||Saccharomyces cerevisiae|
|[Indexing at]||++||+||[Indexing at]|
|++||+||[Indexing at]||[Indexing at]|
The bacterial pathogens are showed the percentage of antibacterial activity Bacillus subtitles (18%) Micrococcus luteus (14%), Staphylococcus aureus (18%), Streptococcus mutans (20%). Gram negative bacteria Klebsiella pneumonia (28%), Proteus vulgaris (16%), Pseudomonas aeruginosa (18%), Saccharomyces cerevisiae (16%).
Figure 1. Antibacteria activity for bacterail straians.
Figure 2. Antibiotic activity and metals tolerance
Table 2. Antibiotic assays for most promising isolates
Am= Amoxyclav; AMP= AMPICILLIN; CF= Cefpodoxime; CP= Chloraphnicol; CIP= Ciproflaxacin;NE= Neomycin; NB= Novobiocin; P= Penicillin; RF= Rifampicin; TR= Tetracycline; VC= Vencomycin;SE =Standard Error; SED= Standard Errors of Differences of Means LSD=(5% Level)East signi?cant difference; CV=Coef?cient of Variance.
Heavy metals tolerances
Heavy metals tolerances Cuso4, Znso4, C4H12O7Pb, and CH3COONH4 are shown KZSS-6, ESS-4, NVS-10, DVS-11 and TSS-3 tolerance levels . Cuso4 metal tolerances rang is of five isolates 4 -32 out of five KZSS-6 are more activity, Znso4 metal tolerances rang is 11-32 more activity in ESS-4, C4H12O7Pb metal tolerances rang is of five isolates 3-26 more activity in TSS-3, CH3COONH4 metal tolerances rang is 2-14 more activity in KZSS-6. Out of six isolates only TSS-3 where’s none they grew at four metals (Cuso4, Znso4, C4H12O7Pb, and CH3COONH4). The results are represented in the Table 3, 4, Figure 3.
Table 3. Heavy Metals Tolerances Cuso4
|S.no||Cuso4 10mm||Cuso4 50mm||Cuso4 100mm||Cuso4 500mm||Cuso41000mm|
SE=Standard Error; SED= Standard Errors of Differences of means;
CV=coef?cient of variance.
Table 4. Heavy Metals Tolerances Znso4
|s.no||Znso4 10mm||Znso4 50mm||Znso4 100mm||Znso4 500mm||Znso4 1000mm|
SE =standard error; SED= Standard errors of differences of means;CV=coef?cient of variance.
Figure 3. Carbon sources, Nacl, pH and temperature.
Table 5. Heavy Metals Tolerances C4H12O7Pb
|s.no||C4H12O7Pb 10mm||C4H12O7Pb||C4H12O7Pb 100 mm||C4H12O7Pb 500||C4H12O7Pb 1000mm|
SE=standard error; SED=Standard errors of differences of means; CV=coef?cient of variance.
Figure 4. Green house and field trials conditions.
Effects of temperature pH, and Salinity
All the six promising isolates were able to grow in temperature up to 20oC to 40oC, expect 50oC (KZSS-6 tolerates up to 30oC), six isolates were grown in pH range between 5 to 13, expect (5, 13) out of six isolates TVS-2 up to range 9. Six isolates were grown in various NaCl concentration up to 0 to 12%, six isolates are performed (KZSS-6 up to 12%) The results are represented in the Table 6, 7, and 8.
Table 6. Heavy Metals Tolerances CH3COONH4
|s.no||CH3COONH4 10MM||CH3COONH4 50MM||CH3COONH4 100MM||CH3COONH4 500MM||CH3COONH4 1000MM|
SE=standard error; SED=Standard errors of differences of means, CV=coef?cient of variance.
Table 7. Carbon sources
|Name of isolates||Fructose||Glucose||Sucrose||Starch|
SE=standard error; LSD=least signi?cant difference; CV=coef?cient of variance.**statistically significant at 0.01 (P values), [Google scholar]Statistically signi?cant at 0.001.
Table 8. Effects of salinity
|Name of the isolates||0% Nacl||0.02||0.04||0.06||0.08||0.1||0.12|
Responses of the six Actinomycetes to salinity were record as follows, 0=no growth; 1=slight growth; 2=moderate growth; 3=good growth.SE =standard error; LSD=least signi?cant difference, CV=Coef?cient of Variance.**statistically significant at 0.01 (P values)[Google scholar]Statistically signi?cant at 0.001.
Greenhouse and MS medium growth factors
Four verities of seeds were growing green house and medium conditions. Plant height (cm) 7.50 to 14.30 ranges, root length (cm) 1.80 to 6.50 range, height seeds (cm) 2.80 to 5.7 range, leaf length 1.6 to 4.33 range, total plant weight (gm) 0.19 to 0.48 range and finally dry plant weight (mg) 7.60 to 14.50 range. Results are represented in the table(9-10).
Table 9. effect of pH and Temperature
|Name of the Isolates||20 oC||30 oC||40 oC||50 oC||pH-5||pH-7||pH-9||pH-11||pH-13|
Responses of the six Actinomycetes to pH and temperature were record as follows, 0= no growth; 1=slight growth; 2=moderate growth; 3=good growth.SE=standard error; LSD=least signi?cant difference; CV=coef?cient of variance.**statistically significant at 0.01 (P values)[Google scholar]Statistically signi?cant at 0.001.
Table 10. Normal greenhouse condition
|Name the variety seeds||Plant Hight (cm)||Root length (cm)||Hight of the seed (cm)||Leaf length (cm)||Total plant weight (mg)||Total dry weight for plant (mg)|
SE =standard error; SED= Standard errors of differences of means, CV = coef?cient of variance.
Evaluations filed condition
Under field condition, the KZSS-6 significantly enhanced plant height (cm plant-1) leaf, shoot, and root hight, leaf, shoot and root length, grains weight, 1000 grains weight, and grain yield percentage. grain was enhanced by 9-12%, and stover yield 15 to 30%. shoot length (mm-2)10 to 25%, plant height (cm plant-1) 15 to 30%, and root length (cm plant-1) were also enhancing, 12 to 24% respectively. The results are represented in the table 11-13.
Table 11. 14 days after greenhouse conditions
|Name the variety seeds||Plant Hight (cm)||Root length (cm)||Hight of the seed (cm)||Leaf length (cm)||Total plant weight (mg)||Total dry weight for plant (mg)|
Table 12. Harvesting period for black gram crop
|S.No||Root lenght (cm plant -1)||root weight (g m -2)||shoot height (cm plant -1)||shoot length (cm plant -1)||stem height (cm plant -1)||stem weight (g m -2)||stem width (cm plant -1)||leaf width (cm plant -1)||leaf weight (g m -2)||Internodes (no’s)|
Table 13. Harvesting period for black gram crop
|S.No||plant height( cm plant -1)||hole plant wight( g m -2)||number of fruits (no)||number of root nodules (no)||root nodules weight(g m -2)||one fruit weight( g m -2)||10 grains weight( g m -2)||100 grains weight( g m -2)||1000 grains weight( g m -2)||Yield % in control (tones)||Yield % in filed (tones)|
Responses of the KZSS-6 to field trials were record, with four types of black gram seeds as follows. SE=standard error; %CV=coef?cient of variance.
Antimicrobial activity of 54 actinomycetes isolates and 3 isolates showed antibacterial activity against E.coli, S. aureus, Bacillus subtilis, P. aeruginosa, Eterobacter aeruginosa, Salmonella typhi, Salmonella typhimurium and Vibrio c olerae Sengupta et al. 2015. Five Streptomyces which exhibited antibacterial effects against clinical isolates of methicillin-susceptible S. aureus and S. typhi Satheeja and Jebakumar 2011. 78 isolates 23 isolates showed antimicrobial activity against B subtilis, B cereus, S aureus and E coli.
Salinity stress, soil pH, and temperature was causes a major threat for plant growth and crop yield. Streptomyces spororaveus RDS28 growth condition was 31°C and pH of 7.5, then medium contains carbon source like glucose. Streptomyces thermolilacinus and Streptomyces werreansis showed significant antagonistic activities against some important gram negative and positive pathogenic bacteria. Streptomyces rimosus subsp antibiotic production including, growth at different pH values, different temperatures, different carbon sources.
Streptomyces rochei M78 isolate were observed using starch casein broth (SCB) as the best production medium, at initial pH 7.0 Starch and casein+yeast extract +peptone appeared to be the best carbon and nitrogen sources respectively and C:N ratio of 4:1 after 72 hr of incubation for optimal production of antibacterial metabolites Hayder NH, Mahmood MS 2016. Six isolates were found to have tolerance to wide range of physiological conditions including pH 11, salinity at 10% and temperature at 40oC and grown in different environmental conditions. Streptomyces werraensis the optimization studies for pH as 8.0, salinity (5%), starch and ammonium sulphate as suitable carbon and nitrogen sources respectively .
Streptomyces spectabilis, Streptomyces purpurascens, Streptomyces coeruleorubidus and Strepto-myces lavendofoliae. Streptomyces spectabilis were observed to be starch and casein as the carbon and nitrogen sources, pH 7, temperature 30°C, streptomyces spectabilis, cellobiose and peptone as the carbon and nitrogen sources, on the 5 th day at pH 5 at 30°C. Streptomyces coeruleorubidus, maximal production resulted on the sixth day at pH 6 and temperature of 35°C, 2015. The results revealed nine strains of Streptomyces Of them two (PS1 and PS28) isolates exhibited high activity against pathogenic bacteria. The optimum growth conditions were pH 7.5, temperature at 30°C.
Rhizobacteria, the two bacterial growing black gram plants in greenhouse conditions to improve growth, to exhibited maximum growth potential and growth-promoting attributes were evaluated. SA8 with kinetin (10μM) inoculated in black gram plants exhibited improved water relation, under salt stress. The artificial inoculation in wheat plant green house, to growth in both adult stage and seedling stages. Therefore, the inoculation in the field condition selecting resistance to wheat sharp eyespot.
In the present work six streptomyces sp was antimicrobial activity against the microbial pathogens in eight pathogenic bacteria its shows (30 to 40%) activity. Antibiotics assay to performed, the zone of inhibition of the six streptomyces sp was (10 to 30 mm). Carbon utilization to performed, the six streptomyces sp were utilized four carbons, high utilization was starch. These six isolates showed tolerance to a wide range of physiological conditions including pH 11, 40°C temperatures and 12% salinity in above information, and then choose the KZSS-6 (Streptomyces koyangensis) is more active than other isolates. In greenhouse and field conditions to understand the natural interaction with other soil native microflora, and host fungal pathogens.