Morpho Cultural and Biochemical Characterization of Different Isolates of Trichoderma species Isolated from Rhizospheric Soil of Cotton in Haryana

doi:10.21203/rs.3.rs-4353981/v1

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Morpho Cultural and Biochemical Characterization of Different Isolates of Trichoderma species Isolated from Rhizospheric Soil of Cotton in Haryana

https://doi.org/10.21203/rs.3.rs-4353981/v1

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Trichoderma biocontrol agents are proven to be effective agents against soil-borne diseases. Trichoderma species are vital for root growth and development, crop yield, resistance to abiotic stress, and nutrient uptake and utilisation in addition to preventing the spread of illnesses caused by soil-borne pathogens. Cell wall degrading enzymes, or CWDEs, are essential to mycoparasitic action because they hydrolyze pathogen cell walls, releasing the organism's contents in the process. The antifungal action of Trichoderma includes a great variety of lytic enzymes such as chitinase, proteases, cellulases, IAA production, siderophore production, Zn solubilisation and phosphate solubilisation etc. which play a key role in bicontrol activity of Trichoderma. Cultural and morphological studies of different isolates of Trichoderma were also studied and observed that maximum mycelial growth (53.67) was recorded by HST2, Whereas, least (36.33mm) was showed by HMT2 after 6 DAI. The colony colour of all the isolates varied from dark green, whitish green to light green. The reverse colony colour of native Trichoderma isolates varied from creamish white, colourless and colourless to yellowish and the edge of colony varied from wavy to smooth. The length of conidia varied from 2.69 to 4.66 µm. The longest conidium was observed in HFtT with a conidial length of 4.66 µm. Width of conidia of different isolates varied from 2.31 to 3.65 µm. The maximum width was observed in case of HFtT (3.65 µm).

Trichoderma

biocontrol

essential

mycoparasitic

colony

enzyme

Trichoderma has been known to have biocontrol properties since the 1920s. Certain species of Trichoderma have the ability to decrease illness and increase productivity. The ability of Trichoderma species to colonise and thrive alongside plant roots is known as rhizospheric competence ability. The ability of Trichoderma species to biocontrol a wide variety of soil-borne illnesses, such as Macrophomina, Pythium and Fusarium, is well documented. Trichoderma species are well known for their ability to increase the systemic resistance of plants, which aids in controlling the development of the plant as well as its susceptibility to disease. Trichoderma uses a range of tactics to fight phytopathogens, including as resource competition, mycoparasitism, antibiotic synthesis, secondary metabolite production, etc. Mycoparasitsm is the main biocontrol technique that Trichoderma employs to fight phytopathogens. The direct mycoparasitic activity of Trichoderma spp. is one of the major mechanisms proposed to explain their antagonistic activity against soil-borne plant-pathogenic fungi (Dennis & Webster, 1971; Elad et al., 1982; Lynch, 1987; Ridout et al., 1986). The fungus produces a number of enzymes that contribute significantly to antagonism, such as chitinase, glucanase, cellulase, xylanase and lipase, which also help to lessen plant infections. These enzymes attack and degrade the polysaccharide, chitin, and β-glucan chains that generate the rigidity of the cell wall (Mitchell & Alexander, 1963; Henis & Chet, 1975, Bull & Chesters, 1966, Reese & Mandels, 1959). Numerous studies have been conducted to better understand the chitinase, cellulase, xylanase and glucanase systems of Trichoderma spp. during growth on different carbon sources. A great deal of research has been done to understand how different Trichoderma species express chitinase and glucanase. What mechanism governs the creation of these enzymes is still unknown, though. To choose the most potent Trichoderma biocontrol strain, it is crucial to comprehend the mechanisms underlying the regulation of these enzymes. Due to their capacity to lower the prevalence of disease caused by plant pathogenic fungi, especially several common soil-borne diseases, Trichoderma spp. are frequently used as biocontrol agents in agriculture (Dubey SC 2007, Papavizas GC 1985). It has been suggested that the lytic activity of several strains of T. harzianum on cell walls of Sclerotium rolfsii, Rhizoctonia solani and Pythium aphanidermatum can be correlated with the degree of biological control of these pathogens in vivo ( Elad et al., 1982).The process of mycoparasitism causes Trichoderma to undergo morphological changes, including coiling and the creation of appressorium-like structures that allow the parasite to enter the host (McIntyre M, 2004). The ability of Trichoderma species to create enzymes that break down cell walls was examined in the current study.

Isolation, purification and multiplication of native Trichoderma isolates

Collection of samples

Four cardinal sites inside the rhizosphere of cotton plants were used to gather soil samples from various cotton-growing regions in Haryana. Using a soil auger that was pushed into the soil to a depth of around 10–15 cm and then pulled out, soil samples were gathered from a depth of 10–15 cm at horizontal spacing of 100–120 cm. The dirt core was pushed out of the tubular augur using a steel handle, and it was then extracted using a sterilized knife or blade. About 500 gram of the soil core sample were removed, put into polyethylene bags, sealed with wire or lids, and labelled with the locations of the sample collection sites and the date of sample collection. As quickly as feasible, collected soil samples were transferred to the lab and kept at 4°C until needed. The soil samples underwent proper mixing and drying. For the purpose of isolating Trichoderma from soil, one gram of the soil was obtained and serially diluted.

Isolation of native Trichoderma from soil samples

The isolated Trichoderma was obtained from the gathered soil samples using the serial dilution procedure. Prior to serial dilution, the obtained samples were uniformly mixed, finely crushed, and dried in the shade. PDA medium served as the isolation medium. Using glass marking pencils, test tubes were designated as 10^− 1, 10^− 2, 10^− 3 and up to 10^− 8. 10 millilitres of distilled water were added to the first test tube, and 9 millilitres to the other test tubes. Non-absorbent cotton plugs were placed into test tubes, and they were autoclaved for 20 minutes at a temperature of 121.6˚C and 15 psi of pressure. One gram of representative soil was added to the first test tube, which contained 10 ml of sterilized water, to create the initial dilution in Test tube 10^− 1 once it had cooled. To achieve a uniform dispersion of the soil sample, the contents were mixed by rolling the test tubes back and forth between palms of hands for five minutes. One millilitre of the suspension from the first dilution was taken while the object was moving and put into the test tube marked 10^− 2 along with nine millilitres of sterilized water using a brand-new, sterilized pipette. Up until the original sample was diluted to a 10^− 8 ratio, the same process was repeated. Each and every time pipettes were used, they were sterile. One millilitre of each dilution's suspension was put in the centre of the sterilized water blanks using a sterile pipette. Following the final step, 1 ml of the dilution suspension was added to each PDA containing petri plate. Glass marking pencils were used to mark the plates. The entire process took place in a laminar air flow environment with sterilised conditions. For five to seven days, these plates were kept in a BOD incubator at 26 ± 1˚C. Every day, the plates were checked to see if Trichoderma spp. had grown.

In order to perform a preliminary screening for Trichoderma species, the macroscopic and microscopic characteristics of the fungal colonies were examined. For macroscopic screening, the colonies growth rate and colours were evaluated, while slides were made for microscopic screening. Each isolate's mycelium was removed from the PDA plate, spread out onto a sanitized slide, mounted with a drop of water, covered with a cover slip, and examined under a light microscope at 10X and 40X magnifications. We looked at the sizes and forms of the colonies' conidia.

Purification of Trichoderma isolates

For purification, a bit of mycelium was cut with the help of sterilized cork borer and transferred to sterilized potato dextrose agar (PDA) plate. The plates were incubated at 25˚C for 5–7 days in BOD incubator. Later the bit of mycelium was transferred on PDA slants and pure culture was obtained by hyphal tip method (Rangaswami, 1972). Within 5–7 days pure culture was obtained and it was stored at 4˚C for further studies.

Cultural and morphological characterization of native Trichoderma isolates

The purpose of this experiment was to investigate potential morphological differences between various native Trichoderma isolates. For this, 15 millilitres of sterilized PDA were added to petri dishes, which were then inoculated with a 5-mm mycelial disc that had been cut from the edge of the colony that was actively growing using a cork borer. The plates were then incubated for five days at 25˚C in a BOD incubator. Characters from the colony were noted. A loopful culture of Trichoderma isolates was collected from a five-day-old culture and placed on glass slides mounted with a drop of water and covered with a coverslip for the mycelial, conidial, and phialid features. The slides were examined under a microscope with magnifications of 10X and 40X, and notes were made on the observations.

Biochemical characterization of native Trichoderma isolates

Hydrogen Cyanide production assay

The HCN production was measured using the Bakker and Schippers (1987) methodology. Microorganisms carried out the cyanogenesis of glycine, which resulted in the creation of HCN. Due to its volatile nature, HCN changed colour from deep yellow to orange to dark brown when it reacted with picric acid in the presence of Na₂Co₃. Filter paper dipped in 0.5 percent picric acid solution in 0.2 percent (w/v) Na₂Co₃ changed colour when natural Trichoderma isolates producing HCN was added. The filter paper was then placed in test tubes and cultured for 72 hours at 28˚C. The change in the colour of strips from deep yellow to brown were considered as positive test and no change in colour indicated negative results.

Indole acetic acid production assay

A modified version of the Brick et al. (1991) procedure was used to measure IAA production. For 72 hours at 25˚C, fungal cultures of native Trichoderma isolates were cultivated on potato dextrose broth with one gm/lt of L-tryptophan as a precursor. Following incubation, the native Trichoderma isolate cultures were centrifuged for 30 minutes at 3000 rpm to collect the supernatant. Four millilitres of Salkowski reagent (50 millilitres: 35% perchloric acid, 1 millilitre 0.5% M FeCl₃) were added after two drops of orthophosphoric acid were added to two millilitres of supernatant. The solution's growth of a pink hue suggested the synthesis of IAA.

Siderophore production

Siderophore production was detected by CAS (Chrome azurol S) assay (modified method of Schwyn and Neilands, 1987).

Solutions

Chrome azurol S (CAS) agar medium

Dye solution (Solution A)

10 ml of an iron (III) solution comprising 1 mM FeCl3.6H2O in 10 mM HCl was combined with 50 ml of dye solution containing 60.5 mg CAS that had been produced in distilled water. A solution containing 72.9 mg of hexadecyltrimethylammoniumbromide (HDTMAC) in 40 millilitres of distilled water was gradually supplemented with the mixture. Ten millilitres of the resulting dark blue liquid were used for a 100 millilitre autoclave, which was run for ten minutes at 15 pounds of pressure.

Solution B

A base medium was produced with 30.42 g of Pipes buffer, 15 g of agar agar, 2 percent K₂HPO₄ (10 ml), 10 percent NaCl (2 ml), 5 percent Na₂MoO₄ (0.1 ml), deferrated 1M sucrose (3 ml), deferrated 1M CaCl₂ (0.4 ml), and deferrated 1M MgSO₄.7H₂O (0.8 ml) in 800 ml of distilled water. 50% (w/w) NaOH was added to the medium to raise its pH to 6.8, and it was then autoclaved for 20 minutes at 15 pounds of pressure.

Following a cooling process to 50ºC, 30 millilitres of a 10% mannitol solution were added as a carbon source. Next, solution A, the dye solution, was added, carefully mixing it to prevent the development of air bubbles. Sterilized petri dishes were filled with the liquefied substance. Each log phase developed culture was inoculated with five microliters (µl) on siderophore plates, which were then incubated for three to four days at 28 + 2ºC. The blue ferric dye complex decolorized, creating yellow halo zones around the cultures, indicating the presence of iron chelator (siderophore).

Cellulase Production

Cellulolytic activity of native Trichoderma isolates was assessed on carboxy methyl cellulose (CMC) agar plates. On CMC agar plates, freshly developing bacterial cultures were spot injected, then allowed to grow at 28 ± 2°C for 48 hours. After that, the plates were flooded with a 0.1% aqueous solution of congo red for 15–20 minutes, and the surface was cleaned with 1 M NaCl. A distinct zone encircling the colony showed the generation of cellulase.

Pectolytic Activity

The composition of the Pectinase Screening Agar Medium (PSAM) was as follows (g/L): 2g of NaNO₃, 0.5g of KCl, 0.5g of MgSO₄, 1g of K₂HPO₄, 0.5g of trypton, 20g of agar, and 10g of pectin. PSAM was added to petri dishes and inoculated with the medium mentioned above. A clear zone encircling the injected culture suggested pectolytic activity.

Zn Solubilization

Using a modified Bunt and Rovira medium (glucose 10 gm, (NH₄)₂SO₄ 1.0 gm, KCL 0.2 gm, K₂HPO₄ 0.1 gm, MgSO₄. 7H₂O 0.2 gm, ZnO 1 gm, and Agar 15 gm) supplemented with 0.1% zinc oxide, all isolates were tested for their capacity to solubilize zinc. On medium plates containing zinc oxide, approximately 3 µl of log phase developed isolate cultures were spotted, and the plates were incubated at 28°C for 6 days. The creation of the solubilization zone served as a proxy for the zinc solubilization activity.

Phosphate solubilization

The solubility of phosphate on Pikovskaya agar (10 g glucose, 5 g Ca₃(PO₄)₂, 0.5 g (NH₄)₂SO₄, 0.2 g KCl, 0.1 g MgSO₄. 7H₂O, 0.5 g yeast extract, 25 mg MnSO₄, 25 mg FeSO₄, and 20 g agar-agar in 1 L, pH -7) was tested independently for each of the native Trichoderma isolates. After being incubated at the proper temperature, cultures that produce a transparent halo zone around each colony are tested as PSM.

Ammonia Production

The ability of native Trichoderma isolates to produce ammonia in peptone water was investigated. In each tube, 10 ml of peptone water was used to inoculate freshly formed cultures, which were then incubated for 48 hours at 28°C. Each tube received 0.5 ml of Nessler's reagent. A positive test result for ammonia production was the development of a brown to yellow hue.

Mycelial growth of native Trichoderma isolates

A total of twelve Trichoderma isolates were recovered from soil samples in the following locations: Bhiwani, Charkhi Dadri, Faridabad, Fatehabad, Gurugram, Jhajjar, Mahendergarh, Rewari, and Sirsa. Each isolate was given a unique code, such as HBhT, HCdT, HFrT, HFtT, HGT, HJT, HMT, HRT, or HST. Plate 1

After incubating the native Trichoderma isolates for six days, the maximum mycelial growth (53.67%) was detected by HST2, which is statistically significant greater than the mycelial growth of the other native Trichoderma isolates. However, as Table 2 illustrates, HMT2 displayed the least amount (36.33mm), which is comparable to all other native Trichoderma isolates save HST2.

Morphology and measurement of mycelial and conidia characters of Trichoderma isolates

The most crucial factors in characterising an individual are their morphological traits, which make it simple to distinguish different native Trichoderma isolates. Table 3 makes clear that there were hue variations in the colonies, ranging from light green to pale green to dark green. HRT, HGT, HST2, HBhT, HMT1, and T13 isolates showed a dark green colony colour; HST1, HRkT, HFrT, HMT2, and T13 isolates showed a whitish green colony colour; and HCdT, HFtT, and HJT isolates showed a light green colony colour. Native Trichoderma isolates ranged in colour from creamish white to colourless to yellowish in their reverse colony colour. The reverse colony's cream-colored whitish appearance was noted in the isolates of HRT, HGT, HST2, and HBhT. The reverse colony colour was found to be colourless in HST1, HCdT, HFtT, HJT, HMT2, and T13 isolates, and colourless to yellowish in HRkT, HFrT, and HMT1 isolates (Table 3 Plate 1).

The edge of colony could be smooth or wavy. As shown in Table 3, the wavy colony edge was found in the isolates of HRT, HGT, HST2, HRkT, HFrT, HMT2, and T13, while the smooth colony edge was found in the isolates of HST1, HCdT, HFtT, HJT, HBhT, and HMT1.The colony's edge could be smooth or wavy. As shown in Table 3, the wavy colony edge was found in the isolates of HRT, HGT, HST2, HRkT, HFrT, HMT2, and T13, while the smooth colony edge was found in the isolates of HST1, HCdT, HFtT, HJT, HBhT, and HMT1. The colony's edge could be smooth or wavy. As shown in Table 3, the wavy colony edge was found in the isolates of HRT, HGT, HST2, HRkT, HFrT, HMT2, and T13, while the smooth colony edge was found in the isolates of HST1, HCdT, HFtT, HJT, HBhT, and HMT1.

The colony's edge could be smooth or wavy. As shown in Table 3, the wavy colony edge was found in the isolates of HRT, HGT, HST2, HRkT, HFrT, HMT2, and T13, while the smooth colony edge was found in the isolates of HST1, HCdT, HFtT, HJT, HBhT, and HMT1.

The data of Table 4 also revealed that width of mycelia of native isolates of Trichoderma varied from 2.46 to 4.23 µm. The maximum width was recorded in HFtT (4.23 µm) followed by HFrT (4.21µm) whereas, the minimum width of 2.46 µm was recorded in T13.

Bio control activities of native Trichoderma isolates

Potency of native Trichoderma isolates for production of HCN and IAA

HGT, HCdT, HST1, and HRkT isolates demonstrated positive findings for HCN generation, while the remaining isolates demonstrated negative results for HCN production, according to Table 5 data. All of the isolates showed positive results for IAA production; however, isolate HST2 produced the most IAA (21.78 µg/ml), followed by isolates HST1 (21.48 µg/ml) and HBhT (16.57 µg/ml). T13 produced the least amount of IAA (11.48 µg/ml).

The isolates HGT, HST2, HFtT, HMT2, and T13 all produced positive cellulase results, while the remaining isolates produced negative cellulase results. The isolates HST2, HFtT, HMT2, and T13 displayed positive pectolytic activity results, while the remaining isolates displayed negative pectolytic activity data. The isolates HRT, HGT, HST2, HMT2, and T13 produced ammonia in positive amounts, while the isolates remaining in the sample produced ammonia in negative amounts.

Some isolates, including HRT, HGT, HST1, HST2, and HFtT, produced siderophores in positive results, while the other isolates produced siderophores in negative results. The zinc and phosphate solubilization activities yielded positive findings for isolates HRT, HST2, HFtT, and T13, while the remaining isolates yielded negative results for both activities (Table 5, Plate 2).

Table 1

Details of native *Trichoderma* isolates collected from different districts of Haryana
Sr. No.	District	Latitude	Longitude	Nomenclature of isolate
1.	Bhiwani	28.79	76.13	HBhT
2.	Charkhi Dadri	28.59	76.26	HCdT
3.	Faridabad	28.39	77.31	CFrT
4.	Fatehabad	29.51	75.45	HFtT
5	Gurugram	28.45	77.02	HGT
6.	Jhajjar	28.60	76.65	HJT
7.	Mahendergarh	28.26	76.15	HMT1
8.	Mahendergarh	28.27	76.15	HMT2
9.	Rewari	28.18	76.61	HRT
10.	Rohtak	28.89	76.60	HRkT
11.	Sirsa	29.55	75.00	HST1
12.	Sirsa	29.53	75.02	HST2

Table 2

Radial growth of native *Trichoderma* isolates
Sr. No.	Name of Isolates	6 DAI (in mm)
1.	HBhT	37.00* (37.43)**
2.	HCdT	42.67 (40.75)
3.	HFrT	40.00(39.20)
4.	HFtT	36.67 (37.24)
5.	HGT	42.00 (40.37)
6.	HJT	39.33 (38.82)
7.	HMT1	38.33 (38.22)
8.	HMT2	36.33 (37.02)
9.	HRT	41.33 (39.98)
10.	HRkT	39.33 (38.82)
11.	HST1	40.33 (39.40)
12.	HST2	53.67 (47.09)
13.	T13	40.33 (39.40)
	CD (p = 0.05)	5.81
	SE(m) ±	1.98
*Mean of four replications
** Values in parenthesis are angularly transformed

Table 3

Morphological characters of native isolates of *Trichoderma* collected from different districts of Haryana
Isolate	Colony Colour	Reverse colony colour	Colony Edge	Mycelia colour
HBhT	Dark Green	Creamish white	Wavy	Watery white
HCdT	Light Green	Colourless	Wavy	Watery white
HFrT	Whitish Green	Colourless to Yellowish	Smooth	Watery white
HFtT	Light Green	Colourless	Smooth	Watery white
HGT	Dark Green	Creamish White	Wavy	Watery white
HJT	Light Green	Colouless	Wavy	Watery white
HMT1	Dark Green	Colourless to yellowish	Wavy	Watery white
HMT2	Whitish Green	Colourless	Smooth	Watery white
HRT	Dark Green	Creamish white	Smooth	Watery white
HRkT	Whitish Green	Colourless to yellowish	Smooth	Watery white
HST1	Whitish Green	Colourless	Smooth	Watery white
HST2	Dark Green	Creamish white	Wavy	Watery white
T13	Dark Green	Creamish white	Smooth	Watery white

Table 4

Conidial and mycelial characters of native isolates of *Trichoderma*
Name of Isolate	Mycelial Width	Width of Conidia	Length of Conidia
HBhT	3.32* (10.49)**	2.89 (9.78)	3.36 (10.56)
HCdT	3.87 (11.34)	3.02 (9.99)	4.12 (11.70)
HFrT	4.21 (11.84)	3.1 (10.12)	4.54 (12.30)
HFtT	4.23 (11.86)	3.65 (10.96)	4.66 (12.46)
HGT	3.22 (10.32)	3.07 (10.08)	3.46 (10.72)
HJT	2.85 (9.72)	2.42 (8.93)	3.08 (10.11)
HMT1	2.56 (9.21)	2.31 (8.73)	2.85 (9.71)
HMT2	4.15 (11.75)	2.67 (9.41)	4.23 (11.86)
HRT	2.72 (9.47)	2.0 (8.12)	3.05 (10.05)
HRkT	3.08 (10.10)	2.58 (9.24)	3.26 (10.40)
HST1	3.86 (11.32)	3.08 (10.11)	4.37 (12.07)
HST2	4.22 (11.84)	2.88 (9.77)	4.46 (12.19)
T13	2.46 (9.01)	2.35 (8.77)	2.69 (9.44)
CD (p = 0.05)	0.35	0.58	0.27
SE (m) ±	0.12	0.20	0.09
*Mean of three replications
**Values in parenthesis are angularly transformed

Table 5

Bio control activities of native *Trichoderma* isolates
Isolate	HCN Production	IAA Production	Cellulase Activity	Pectolytic Activity	Ammonia Production	Siderophore Production	Zn Solubilisation	Phosphate Solubilisation
HBhT	–	(16.57)	–	–	–	–	–	–
HCdT	+	(16.13)	–	–	–	–	–	–
HFrT	–	(15.35)	–	–	–	–	–	–
HFtT	–	(17.22)	+	+	–	+	+	+
HGT	+	(12.74)	+	–	+	+	–	–
HJT	–	(12.35)	–	–	–	–	–	–
HMT1	–	(12.35)	–	–	–	–	–	–
HMT2	–	(16.26)	+	+	+	–	–	–
HRT	–	(13.78)	–	–	+	+	+	+
HRkT	+	(11.57)	–	–	–	–	–	–
HST1	–	(21.48)	–	–	–	+	–	–
HST2	+	(21.78)	+	+	+	+	+	+
T13	–	(11.48)	+	+	+	–	₊	+

The Trichoderma-plant-pathogen interaction is a very dynamic and multi-dependent system. An in-depth understanding of Trichoderma's processes towards plants and pathogens might greatly boost their efficacy. Trichoderma uses a variety of intricate direct and indirect biocontrol mechanisms to protect itself from abiotic pressures, such as unfavourable environmental conditions, and biotic stresses, such as a wide range of harmful microorganisms (fungi, bacteria, insects, and nematodes).. In the coming years, the methods of enhancing the effectiveness and reliability of Trichoderma preparations will be improved through treatments increasing the competitiveness of Trichoderma in the rhizosphere and rhizoplane. This can be achieved through strong and stable colonization of these zones and the combination with microorganisms supporting the positive effects of Trichoderma, the so-called supporting strains (Massart et al. 2015)

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The article contains the ethical approval and consent to participation.

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The manuscript has been read properly by all the authors and has been sent to anywhere for publication.

Avalability of data and materials

Data and material methods has been added.

Conflict of Interest

No conflict of interest.

Funding

No source of Funding.

Authors’Contribution

PV, NKY designed the study and prepared the manuscript. PV, P and LY analyzed the data. All authors interpreted the data, critically revised the manuscript for important intellectual contents and approved the final version.

Acknowledgements

The authors express their sincere thanks and gratitude to the Head of Department of Plant Pathology, CCSHAU, Hisar and Regional Director, RRS, Bawal for extending research facilities and support for conducting the present study

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Morpho Cultural and Biochemical Characterization of Different Isolates of Trichoderma species Isolated from Rhizospheric Soil of Cotton in Haryana

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Abstract

Figures

Background

Methods

Collection of samples

Hydrogen Cyanide production assay

Indole acetic acid production assay

Siderophore production

Solutions

Chrome azurol S (CAS) agar medium

Cellulase Production

Pectolytic Activity

Zn Solubilization

Phosphate solubilization

Ammonia Production

Results and Discussion

Conclusion

Declarations

References

Status:

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