BIODIVERSITY OF PHOSPHATE SOLUBILIZING FUNGIISOLATED FROM AGRICULTURAL FIELDS OF MARATHWADA REGION

was by a standard protocol and analyzed on 0.8% agarose electrophoresis. Further, PCR was carried out to the 18S rRNA gene of the extracted genomic DNA of UK-2 using PCR primers (Forward Primer 5'-GAGTTTGATCCTGGCTCAG-3' and Reverse primer 5'-GAAAGGA GGTGATCCAGCC-3'). The genus of the strain was determined based on the sequence of 356 bp of the 18S rRNA gene. The obtained 18S rRNA gene sequences were assembled. All species Penicillium canescens,Penicillium sp, Penicillium , Curvularialunata , Fusarium oxysporum, Rhizopus sp, and Trichoderma spp, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Aspergillus awamori and Aspergillus terreus, Aspergillus sp., evaluated on Pikovskaya (PVK) selective media, they tested phosphate solubilization capacity, and some of them discovered excellent solubilizing properties.The successful isolates from the sample were evaluated for their plant promotion activities such as antagonism, phytohormone production, siderophores, and biopesticide

Fungi are the core components of soil microorganisms, accounting for more of the soil biomass than bacteria, depending on soil depth and nutritional requirements. In the current study, phosphate solubilizing fungi were isolated from the soil of Maharashtra's Marathwada area, and all fungal isolates were examined for their ability to solubilize phosphate.Only 11 fungal isolates out of a total of 40 were found to have P-solubilizing activity. After 48 hours of incubation, the fungal isolates Aspergillus niger (PQ9), Trichoderma spp (PQ36), and Penicillium spp (PQ19) demonstrated a considerable zone of solubilization with 34, 31 to 30 mm on selective agar medium. The potent phosphate solubilizing fungi were identified in 18SrRNA analysis.The study, therefore, proposed that these fungal species have strong phosphate solubilizing properties and can be used for excellent crop productivity as a biofertilizer.
Fungi have been demonstrated to have a better ability to solubilize insoluble phosphate than bacteria [4].Insoluble phosphorous species have been reported to be solubilized by soil fungi such as Aspergillus niger and Penicillium, which are the most prevalent fungi capable of solubilizing phosphate [5].Many soil researchers have performed research into phosphate solubilizing microorganisms [6,7], mangrove [8], and rhizosphere [9,10].From such exploration's different forms of phosphate solubilizing microorganisms have been successfully described. In recent decades, phosphate has been identified in a broad range of rhizosphere bacteria and fungi, including Bacillus subtilis, Bacillus cereus, Bacillus megaterium,Pseudomonas putida, Pseudomonas aeruginosa, Escherichia coli, Pantoeaagglomerans, Enterobacter aeruginosa,Penicillium, and Azotobacter chroococcum [11].
Fertilisers are now widely used in the Marathwada region in place of manures. As a result, crop production is increasing at a rapid rate, while soil quality and microbial diversity are declining. However, little is known about the diversity of phosphate-solubilizing fungus present in the rhizospheres in this region. The aim of this research was to look into the diversity of phosphate solubilization efficiency of isolated fungi from the Marathwada region that have the potential to promote plant growth in nutrient-deficient soils.

Materials And Methods:-Sample Collection:
A total of 160 rhizosphere soil samples were collected from various locations namely Aurangabad, Beed, Hingoli, Jalna, Latur, Nanded, Osmanabad, and Parbhani districts of Marathwada, Maharashtra (India). The study's main focus was on analyzing soil samples obtained from every tehsil in the Marathwada district.

Physicochemical analysis of Soil Samples:
Soil samples were collected in a sterilized bottle and thoroughly mixed on a clean cloth before being broken up with a wooden pestle and mortar and air-dried [15]. The air-dried samples were sieved into ten mesh sizes, placed in glass bottles, and labelled for analysis. After collection, a portion of each sample was immediately transferred to the laboratory and stored at 4 0 C for microbial analysis. Physicochemical parameters like pH, Electrical Conductivity (EC), Total Organic Carbon, Available Nitrogen (N), Available Phosphorus (P 2 O5), and available potassium (K 2 O) of soil samples were analyzed as per the methods recommended by APHA [16].

Primary Screening of Phosphate Solubilizing Fungi
Soil samples were prepared for microbial analysis, which included the isolation of phosphate solubilizing fungi on Pikovskaya's (PKV) agar medium supplemented with 25 g/mL chloramphenicol to prevent bacterial growth [17].The existence of a transparent halo zone around the fungal colony suggested that the fungus was capable of phosphate solubilization, and the zone's diameter was measured in millimetres.

Characterization of Phosphate SolubilizingFungi
After Screening of Phosphate Solubilizing all fungal isolates were transferred on Potato Dextrose Agar to accelerate the growth rate andthe production of enough conidia (Pandey et al., 2008).Isolates were compared to mycological identification keys and taxonomic descriptions to classify the isolated fungi to the genus level [17].such as color of the colonies both from the upper and lower side,surfaceappearance, and texture.Microscopy was also used to establish conidia, conidiophores, spore arrangement, and vegetative structures [18].For further analysis, the detected fungi were held on Potato Dextrose Agar (PDA) slant at 4 0 C.

Qualitative analysis of Phosphate solubilization
In the centre of Pikovskaya's plate, spot inoculated isolates with phosphate solubilizing capability were placed and incubated at 37 0 C. The diameter of the clearance zone was measured every 24 hours for up to 7 days.

Quantitative analysis of Phosphate solubilization
Pikovskaya's broth medium with Tricalcium phosphate (0.3g/100ml) was prepared and sterilised for quantitative analysis of phosphate; 1ml of each isolate was inoculated into the broth medium. The sterilised sample was then incubated for 5 days on a rotatory shaker at 370C, after which the culture broth was centrifuged for 30 minutes at 10,000rpm. As a control, uninoculated broth was used.Calorimetrically at 410nm with standard KH2PO4, the usable phosphorus was calculated.

Assessment of the biodiversity of isolates
The diversity of the isolates was madefrom the microscopic, cultural/morphological description of thefungal isolates. Thisdescription allowed highlighting thesimilarities and the differences between isolates.

Molecular identification of fungal isolates
The molecular techniques are being very much employed for the classification and characterization of various fungal species [19]. For species-level identification, genomic DNA of the potent phosphate solubilizing fungi was extracted 801 by a standard protocol and analyzed on 0.8% agarose electrophoresis. Further, PCR was carried out to amplify the 18S rRNA gene of the extracted genomic DNA of fungi using Gene Amp PCR with the forward and reverse primers. The genus of the strain was determined based on the sequence of 601bp of the 18S rRNA gene. The obtained 18S rRNA gene sequences were assembled and exploited for phylogenetic analysis. The 18S rRNA gene sequence-related taxa were acquired from the GenBank database at the National Center for Biotechnology Information. The report (sent online) includes NCBI-BLASTn (http://blast.ncbi.nlm.nih.gov) result for fungi showing closest VALID neighbor of the organism in the TYPE database along with percent similarity.

Results and Discussion:-
The pH values of the soil samples vary from 7.7 to 8.7, while the ideal pH range is 7.5 to 7.8, suggesting that the soil of Marathwada is slightly alkaline due to excessive evaporation of water in dry areas, which brings salts to the surface. Alkaline medium restricts plant growth, and pH may affect nutrient availability in the soil [20] (Table 1). This is most likely due to the fact that these soils were formed from basaltic parent material with a high concentration of basic cations. Padole and Mahajan found similar results [21].  Colonies began as white and evolved into a yellowish-green to light green colour.

Aspergillus fumigatus
3. PQ13, PQ40, PQ14 On potato dextrose agar, colonies were lime green in colour. The texture ranged from woolly to cottony to granular.
Phialides are born on cylinder branches and arranged in a brushlike head, septate distinct and bearing a cluster of branches.

Contribution of P-solubilizing fungi in Soil of Marathwada region
At different locations, the percentage contribution of different fungal species to the overall fungal population varied.Since they were described by a large number of species, the genus Aspergillus flavus contributed the most to all of the samples. The percentage contribution of fungal species resulted in significant differences. Penicillium sp ranked first of all P-solubilizers, contributing the highest percentage of 9.15 percent to the total fungal population. Aspergillus flavus contributed 3.26 percent of the total, Penicillium sp contributed 2.61 percent, and Trichoderma spp contributed 5.22 percent. Together, these species account for 24.81 percent of the total fungal population (Fig.2).

Fig 2:-Contribution of P-solubilizing fungi in Soil of Marathwada region
A total of 347 fungal isolates were isolated in this study, with 40 of them proving to be effective phosphate solubilizers. PQ13, PQ19, PQ36, PQ40, PQ67, PQ88, PQ135, PQ153, PQ207, PQ233, and PQ277, among the best 11 PSF isolates, developed a maximum zone of solubilization.PQ153 was found to be a potent phosphorus solubilizing bacterium, with a 42 mm zone of solubilization, relative to other isolates. Phosphorus deficiency is a natural phenomenon on soil all over the world, and it is one of the factors that limits crop production. Fertilizers containing phosphorus account for a substantial portion of agricultural production costs.

803
In the present study, genomic DNA was extracted by a standard protocol and analyzed on 0.8% agarose electrophoresis. Further, PCR was carried out to amplify the 18S rRNA gene of the extracted genomic DNA of UK-2 fungi using Gene Amp PCR with the forward and reverse primers (Forward Primer 5'-GAGTTTGATCCTGGCTCAG-3' and Reverse primer 5'-GAAAGGA GGTGATCCAGCC-3'). The genus of the strain was determined based on the sequence of 356 bp of the 18S rRNA gene. The obtained 18S rRNA gene sequences were assembled. All fungal species Penicillium canescens,Penicillium sp, Penicillium digitatum, , Curvularialunata, Fusarium oxysporum, Rhizopussp, and Trichodermaspp, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Aspergillus awamoriandAspergillus terreus, Aspergillus sp., evaluated on Pikovskaya (PVK) selective media, they tested phosphate solubilization capacity, and some of them discovered excellent phosphorus solubilizing properties.The successful isolates from the sample were evaluated for their plant growth promotion activities such as antagonism, phytohormone production, siderophores, and biopesticide function.

Conclusion:-
The biodiversity of fungal species from the rhizosphere soils of various locations in the Marathwada area was investigated in terms of morphological characterization and phosphate solubilizing potential in the current study. Aspergillus fumigates, Aspergillus niger, Aspergillus flavus, Penicillium spp., and Trichoderma spp. were evaluated in the study. These could be used to create inoculum, and the effects of inoculation on plant growth should be tested in vitro, in greenhouses, and in field trials.