Fungal pretreatment of lignocellulosic biomass for the production of plant hormone by Pichia fermentans under submerged conditions

The study was designed to evaluate the production of auxin by eukaryotic unicellular organism Pichia fermentans. Different media formulations were used for the production of indole-3-acetic acid (IAA) under broth and submerged conditions. Wheat straw-based production medium was formulated and optimized using statistical approach. The IAA production was significantly enhanced by nine folds, when the wheat straw was pretreated with Phanerochaete chrysosporium (150 µg/ml) as compared to untreated wheat straw (16.44 µg/ml). Partial purification of IAA was carried out by silica gel column chromatography and further confirmed by high-performance liquid chromatography. Exogenous application of crude and partially purified IAA positively influenced the Vigna radiata seedling growth. The number of lateral roots in the growing seedlings was significantly higher as compared to the control seeds. Thus, the present findings point towards an efficient production of plant hormone by yeast and white rot fungus using abundantly available wheat straw, which may lead to the development of cost-effective production of such metabolites and their further use in agricultural field to reduce the negative impact of chemical fertilizers.


Introduction
Worldwide increased food demands for the growing population have stressed the various agricultural systems to enhance their productivity and yield. The economic losses due to various biotic and abiotic stresses have affected the growth, and productivity of the numerous crops and, thus, the food supply. To deal with this problem several chemical and biological methods have been proposed. Among several methods, the application of auxin (indole-3-acetic acid) as a plant growth regulator is widely acceptable. Auxin is a plant hormone, which mainly includes indole acetic acid (IAA). Some microorganisms have IAA biosynthesis pathway, which converts tryptophan to IAA. IAA can be found in actively growing parts of plant such as apical meristem, young leaves and buds (Müller and Leyser 2011). IAA helps in stimulation of root initiation, cell differentiation, elongation and division, lateral root development, and gravitational responses (El-Tarabily 2008). IAA also acts as a signaling molecule for development of plant structure and overall growth (Teale et al. 2006).
Application of the synthetic indole-3-acetic acid (IAA) has been suggested but its high cost and less stability than synthetic auxin analogs such as 1-naphthaleneacetic acid (NAA) limit its practical use (Flasiński and Hac-Wydro 2014). The use of synthetic NAA is also toxic to both human and animal health (Jaishankar et al. 2014). This has raised concern with regard to the toxic contamination of the soil, air, natural water reservoirs and most importantly food crops. Application of eco-friendly technologies in the form of biosynthetic IAA could result as an alternative for synthetic IAA and NAA. Excessive production of IAA in plants is tough and time consuming compared to microorganisms. Therefore, it is of great interest to explore IAA production by microbes. Apart from its synthesis by plants, IAA is also produced by bacteria (Apine and Jadhav 2011), yeast, (Xin et al. 2009) and fungi (Ünyayar et al. 2000;Yürekli et al. 2003;Maor et al. 2004). Applications of IAA-producing yeasts, such as Candida valida, Rhodotorula glutinis, Trichosporon asahii, Lindera saturnus and Rhodotorula mucilaginosa, to promote plant growth have already been reported (Xin et al. 2009). However, information on the production of phytohormones using fermentation is sparse and little data on the IAA synthesized by fungi is known. Earlier, several white rot fungi such as Ganoderma lucidum, Lentinula edodes, Pleurotus ostreatus and Agaricus bisporus were found to have promising antioxidant properties to protect the cells from reactive oxygen species (ROS) (Jayakumar et al. 2009;Jaszek et al. 2014). However, physiological role of auxins in fungi remains unclear. One of the suggested roles for IAA produced by fungus is to mediate fungal-plant interaction thereby favoring the transfer of phytohormones from plant system to soil and vice versa (Fu et al. 2015). High concentrations of IAA can inhibit the hypersensitive response (Jouanneau et al. 1991) and may suppress expression of plant defense genes (Yamada et al. 1985;Shinshi et al. 1987), thus suggesting involvement of IAA in fungal phytopathogenicity as well.
Industrial production of IAA is gaining importance as it is widely used in both research and agriculture sectors. Production of secondary metabolites in microbes is faster, easily managed and cost can be minimized. Among different microbes, yeast belonging to Saccharomycetaceae can be a good choice for the production of such compounds as this group of microbes naturally occur in fruits and widely used in fermentation process and also have low risk of pathogenicity (Cousin et al. 2017). Recently, about seven different yeasts including Pichia have been found to be endophytic in nature (Ling et al. 2019). Pichia fermentans is widely present in nature and habitually found in fruits and juices, as well as being connected to humans and animals (Las Heras-Vazquez et al. 2003). Recently, this yeast species was also reported to possess exoelectrogenic property . The species has been successfully used for bio-control of brown rot of apple, but found to be pathogenic when applied to peach fruit (Giobbe et al. 2007). Besides this, P. fermentans is well accepted for the production of wines together with Saccharomyces cerevisiae (Kong et al. 2019). In view of the isolation of P. fermentans from various plant species, it seems likely that this species may have some significant role in plant growth promotion by producing plant growth regulating hormone.
In the industrial processes, production media plays a key role, which is responsible for its high cost. In the present study, an economic medium based on agricultural residues was formulated. Wheat straw as agricultural residue is obtained after harvesting of wheat grains, which has about 529 million tons of global production (Govumoni et al. 2013). Wheat straw contains cellulose (34-40%), hemicellulose (20-25%), and lignin (20%) (Carvalheiro et al. 2009). Among these components, lignin is the most resistant component of plant cell walls, which makes the cellulose like polymers inaccessible for microbial degradation or depolymerization. Few fungi are able to completely degrade lignin to carbon dioxide and thereby increase the accessibility of carbohydrate polymers present in plant cell walls. Selective degradation of lignin is carried out by white rot fungi (Sharma and Arora 2015). Among different white rot fungi, Phanerochaete chrysosporium is known for its selective degradation of lignin and have potential in wide range of biotechnological applications (Sharma et al. 2011). Abundantly available wheat straw can be pretreated using white rot fungi, which converted the complex plant biomass, i.e., cellulose into the easily fermentable sugars glucose (Sadh et al. 2018). P. fermentans may utilize this sugar and may produce IAA in the presence of tryptophan. Besides efficient pretreatment of wheat straw during primary fermentation, P. chrysosporium may also produce IAA in the presence of tryptophan (Chandra et al. 2019), which may further contribute to increase the IAA production during secondary fermentation.
To validate the root proliferating potential of the IAA produced by yeast and fungus, mung bean (Vigna radiata) was used as a model plant, since it is commonly cultivated in the most of the parts of South Asian subcontinent. Owing to its short growing period, these are commonly used for crop rotation to enhance the soil fertility as their roots have symbiotic association with rhizobia (Sharma et al. 2019b).
In the present study, P. chrysosporium pretreated wheat straw was used to produce IAA under submerged fermentation conditions by P. fermentans. After optimizing the production level, the product was isolated and purified using column chromatography and further confirmed by HPLC. IAA produced by Pichia fermentans and P. chrysosporium was exogenously used to evaluate its effect on plant growth.

Screening of yeast and white rot fungus for IAA production
Yeast and white rot fungus were screened for IAA production as per the method described earlier (Chandra et al. 2019). Flask containing 50 ml of 0.5% (w/v) malt extract were sterilized at 15 lbs/in. 2 for 15 min and aseptically amended with tryptophan (0.05% (w/v) (filter sterilized). The flasks were inoculated with P. fermentans (24 h old 1 ml of 1 OD 600 ) or P. chrysosporium (2 mycelial discs of 4-day-old culture), and incubated at 30 °C up to 10 days along with uninoculated control flask. Two ml aliquot was aseptically taken out from the flask at 1-day interval and centrifuged at 8000 rpm for 10 min. The supernatant was used for IAA estimation.

IAA production using wheat straw
Flasks containing 1 g wheat straw and 20 ml of 0.1% (w/v) malt extract were sterilized and amended with 0.05% (w/v) tryptophan. P. fermentans or P. chrysosporium were inoculated aseptically and incubated up to 10 days at 30 °C and processed as described in previous step.

Pretreatment of wheat straw
Wheat straw was pretreated with P. chrysosporium under submerged conditions (primary fermentation). Flask containing 2 g wheat straw and 20 ml of 0.1% malt extract (w/v) were inoculated with fungus and then incubated at 30 °C. One ml of the sample was taken out each day to estimate sugar content up to 10 days. A similar set was prepared and P. fermentans was inoculated with tryptophan in the flask on 5th day (as the primary fermentation produced significantly higher sugar on this day) and further incubated for 5 days (secondary fermentation). After incubation, the aliquot was tested for the presence of IAA and the contents of the flask were dried at 80 °C for 48 h to calculate the amount of wheat straw consumed, while the dried residual straw contained degraded straw along with fungal and yeast biomass.

Optimization of conditions for IAA production
On the basis of results obtained from above experiments, three independent variables (distilled water, tryptophan and malt extract) were selected to enhance the production of auxin. The optimization of the selected variables was done as described previously by response surface methodology (RSM) using a Box-Behnken design (Box and Behnken 1960). The experimental design included 15 flasks with 3 center points. Each 100-ml conical flask contained 1 g of wheat straw, 0.1-0.5% (w/v) of malt extract and 15-50 ml of distilled water ( Table 1). The flasks were sterilized, inoculated with P. chrysosporium and incubated for 5 days (primary fermentation). Then, P. fermentans was inoculated aseptically along with appropriate concentration of tryptophan (0.1-1% (w/v)) ( Table 1) and again incubated for 5 days. The contents of the flasks were filtered and the filtrate was centrifuged at 8000 rpm for 10 min. The obtained supernatant was used for further analyses. Complete experiment was repeated and validated using optimized concentration of supplements. The mathematical relationship of response G (for each parameter) and independent variable X (X 1 , distilled water; X 2 , tryptophan; and X 3 , malt extract) was calculated by the quadratic model equation (Box and Behnken 1960): where G is the predicted response; β 0 , intercept; β 1 , β 2 , β 3 and, linear coefficients; β 11 , β 22 and β 33 , squared coefficients and β 12 , β 13 and β 23 interaction coefficients. MINITAB and statistical software package Design Expert ® version 8.0 (State ease, Inc., Minneapolis, USA) were used to find out the optimal working conditions and prepare response surface graphs. The predicted concentrations were used to confirm the maximum production of IAA.

Estimation of IAA
IAA was quantitatively estimated as per the method described earlier (Chandra et al. 2019). Briefly, 1 ml of the supernatant was mixed with the equal amount of Salkowski's reagent and the OD was read at 530 nm after 30 min of incubation. An uninoculated sample was used as control. The standard IAA was used to prepare a standard curve for quantitative comparison.

Estimation of released sugar content
Reducing sugar content was measured in pretreated wheat straw containing flasks as per the standard protocol using 3,5-dinitrosalicylic acid (DNSA) reagent (Miller 1959). Briefly, 1 ml of the supernatant was transferred into separate test tubes containing 2 ml of DNSA reagent (1 g DNSA, 30 g sodium potassium tartrate and 20 ml of 2 M sodium hydroxide to a final volume of 100 ml in distilled water); the test tubes were kept in a water bath for 10 min at 100 °C. After incubation the tubes were allowed to cool down at room temperature and the absorbance was recorded at 540 nm.

Purification of IAA
Partial purification of IAA from crude extract was carried out by silica gel column (22 × 5 cm) using the methanol:water (9:1) as a mobile phase. The flow rate was kept at 1 ml/min and the fractions (2 ml) were collected up to 50 fractions. Each fraction was tested for the presence of IAA using Salkowski's reagent. The positive fractions were pooled together and evaporated to dry in a rotary evaporator at 60 °C then solubilized in 2 ml of methanol. Presence of IAA was further confirmed by HPLC using C18 column (5 μm; 25 × 0.46 cm) with elution performed using the ratio 9:1 of methanol and water, containing 0.5% acetic acid with a flow rate of 0.5 ml/min and the detection was monitored at 220 nm at 40 °C.

Effect of crude and purified IAA on seedling and root development
Plant growth promotion and root proliferation ability of the crude and purified IAA was assessed in vitro in Petri plates. The Vigna radiata seeds were surface sterilized using 0.1% HgCl 2 followed by 4-5 repeated washings with sterilized distilled water. These seeds were treated with crude (150 µg/ml) or purified (100-200 µg/ml) IAA extract and incubated for 7 days at 30 °C, 1 ml sterilized water was added to each plate along with the control. Root length, seedling length and weight were measured after incubations.

Statistical analyses
Except RSM, all the results were represented as mean ± standard error (n = 20). Statistically significant difference was calculated using t test and one-way ANOVA as appropriate. Least significant difference was calculated to find out the statistical significant difference (P < 0.05)

IAA production by yeast and white rot fungus
P. fermentans and P. chrysosporium produced IAA in malt extract and wheat straw-based medium supplemented with 0.05% (w/v) tryptophan. P. fermentans and P. chrysosporium produced IAA in the range of 1.99-129.33 µg/ ml (Table 2). Maximum production of IAA was observed in medium (without wheat straw) inoculated with P. fermentans on the 6th day (129.33 ± 11.20 µg/ml). On the other hand, P. chrysosporium produced maximum IAA on the 10th day (51.49 ± 3.68 µg/ml) when inoculated in wheat straw-based medium.

Medium formulation using pretreated wheat straw
Wheat straw, the basic component of the medium, was pretreated by P. chrysosporium to release sugar in the medium which may be utilized by P. fermentans for IAA production. P. chrysosporium released a significant amount of sugar from the 2nd day onwards, which increased with time and was maximum (0.89 mg/ml) on the 9th day (Fig. 1). However, it could release five times higher sugar (0.747 mg/ml) from wheat straw after 5 days of incubation (primary fermentation) as compared to uninoculated control (0.152 mg/ml). Thus, the yeast was inoculated on the 5th day after primary fermentation. As a result, IAA production by P. fermentans was significantly enhanced (P < 0.05) by six times after secondary fermentation, when wheat straw was pretreated with P. chrysosporium (128 ± 4.43 µg/ml) as compared to untreated wheat straw (16 ± 0.33 µg/ml). About 20% (w/w) wheat straw was consumed during the 10 days of fermentation process (Fig. 2).

Optimization of the medium using RSM
An enhancement in the IAA production was obtained during RSM experimental design. Based on Eq.
(1) multiple regression analysis method was used to analyze the data obtained from Box-Behnken design, which were statistically significant. It was verified by F values and the analysis of variance by fitting the data of all independent observations in response surface quadratic model. R 2 value for IAA produced was 95.42%, which showed the appropriateness of the model in the present experiments. For IAA production one linear ( X 2 ) and one squared ( X 2 2 ) effect was significant. The plot of predicted versus actual IAA produced (Fig. 3a) demonstrated a high positive correlation between the predicted and actual results. Maximum IAA production (152.36 µg/ml) was obtained at minimum malt extract concentration, maximum tryptophan concentration and 32.5 ml distilled water (Fig. 3b). The optimized values of different factors were validated by executing the experiment and repeating the same with distilled water 32.5 ml, tryptophan 1% and malt extract 0.1% which gave significant IAA produce (150 ± 2.5 µg/ml). Regression coefficients value significance is inversely proportional to the P-values and bears a direct effect with the magnitude of t-value. Hence from Table 3, it can be observed that the linear effect of tryptophan was the most significant (P-value = 0.0003). Quadratic effect was more significant for tryptophan (P-value = 0.0730) followed by malt extract and distilled water. The interaction of tryptophan and malt extract was more significant than that of other factors. The fitness of the model was determined by the coefficient of determination R 2 . A model having an R 2 value higher than 0.9 was considered as very high correlation between experimental value and predicted value from the model (Chen et al., 2009). The R 2 value in this model was found to be 0.9542, which depicts that 95.42% of the total variation that occurred in the response value could be explained by the model and the remaining 4.58% is not explained Table 2 Production of IAA (µg/ml) at different days by P. fermentans and P. chrysosporium Similar superscripts (a, b, c, d) show no statistically significant difference (P > 0.05) within each row, while similar superscripts (p, q, r s, t, u, v, w) show no statistically significant difference (P > 0.05) within each column  . 2 Production of IAA (µg/ml) by P. fermentans and wheat straw (WS) consumed (% (w/w)) with and without fungal pretreatment after secondary fermentation (* or ** represents statistical significant difference (P < 0.05)) by the model. The ANOVA for the response surface quadratic model is shown in Table 3; the model was highly significant with the P value 0.0074.

Purification of IAA
Fraction number 5 to 15 obtained from column chromatography showed the presence of IAA. These fractions were pooled, and concentrated to 2 ml. HPLC detected the peak at 6.77 min when standard IAA was run (0.1 mg/ml).
A peak comparable to standard IAA confirmed the presence of IAA in the methanolic extract (Fig. 4).

Effect of crude and partially purified IAA on seedling and root development
An increase in lateral root count and weight of seedlings as compared to control was observed upon inoculation of seeds with crude and purified IAA (Table 4). All seeds used in the experiments germinated under the experimental conditions. The treated seeds with crude and partially purified IAA showed statistically significant (P < 0.05) increase in the lateral root count, which increased from 5.35 to 14.1 and 17.7, respectively (Fig. 5).

Discussion
Plant-associated yeast might be a good source of plant hormone as confirmed by the experimental results. As reported earlier, Pichia spartinae also influenced the IAA content in the plant (Nakamura et al. 1991). IAA production by yeast has been reported earlier (Pandi et al. 2019), while P. fermentans is being reported for the first time. In the present study, P. fermentans produced high amount of IAA and using fungal pretreated wheat straw positively influenced its production. Pichia guilliermondii and Hanseniaspora uvarum produced less than 25 μg/ml when inoculated in yeast extractdextrose based medium after 7 days of incubation   (Basha and Ramanujam 2015). Also, Williopsis saturnus an endophytic yeast in maize roots was found to be capable of producing IAA (22.51 µg/ml) in vitro in glucose-peptone broth (GPB) medium (Nassar et al. 2005).
In the present study a much higher amount of IAA (129.33 µg/ml) was produced by P. fermentans under submerged conditions after 6 days of incubation. However, some phylloplane yeast strains Rhodosporidium fluviale demonstrated efficient IAA (565 mg/L) production (Limtong et al. 2014) almost comparable to P. fermentans as demonstrated in the present study. Similarly, R. paludigenum also produced a sufficient level of IAA (25.4 mg/L/h) under fed-batch fermentation (Nutaratat et al. 2016). Besides IAA production, several rhizosphere yeast strains have also demonstrated plant growth promoting properties, e.g., Sporobolomyces roseus (Perondi et al. 1996), Rhodotorula sp. (Abd El-Hafez and Shehata 2001), Candida valida, Rhodotorula glutinis and Trichosporon asahii (El-Tarabily 2004) have been reported to promote the growth of wheat, tomato and sugar beet, respectively. Two different pathways for auxin biosynthetic have been proposed for plants: production via indole-3-glycerophosphate and tryptophan-dependent pathways. It differs in the intermediates as: indole-3-pyruvate (IPA) (Woodward and Bartel 2005), tryptamine (TAM), indole-3-acetaldoxime (IAOx) (Zhao et al. 2002), and indole-3-acetamide (IAM) (Pollmann et al. 2009). Another way to convert indole-3-acetonitrile (IAN) into IAA has also been suggested (Mano and Nemoto 2012). Approximately 37.7% of the investigated yeast strains collected from the phyllosphere of various plant species in Thailand produce IAA (Limtong and Koowadjanakul 2012). Three endophytic yeasts isolated from Populus tree also produced IAA when incubated with tryptophan. It suggests that IAA production is common in some yeasts and tryptophan is a major IAA precursor.
White rot species are widely used in biotechnological and biochemical applications, i.e., delignification or bioremediation. However, their application is limited to the production of plant growth promoting factors. Plant growth regulators, e.g., abscisic acid production were reported by some white rot fungi (Crocoll et al. 1991). Besides abscisic acid, Funalia trogii and Trametes versicolor also produced cytokinins, gibberellic acid and indole  acetic acid by using olive oil mill and alcohol factory wastewaters as substrate for fermentation (Yürekli et al. 1999). Numerous fungi have demonstrated IAA production, but their biosynthetic pathway and enzyme involved have not been documented (Sukumar et al. 2013). However, researchers have confirmed the presence of IAM and IPA pathways in Colletotrichum gloeosporioides f. sp. aeschynomene. It also revealed that IAM pathway to be a major pathway used this fungus to produce IAA (Robinson et al. 1998). Gas chromatography analyses confirmed the presence of metabolic intermediates of IPA, IAM, and TRA pathways in the culture filtrates of Fusarium delphinoides (Kulkarni et al. 2013). Production of indole-3-acetic acid by Colletotrichum gloeosporioides f. sp. Aeschynomene was strictly dependent on external tryptophan (Maor et al. 2004). Trichoderma atroviride also produced high level of IAA (6.2, 9.8 and 38.55 µg/ml) in the presence of 200 µg/ml tryptophan, tryptamine and tryptophol, respectively (Gravel et al. 2007). Thus, IAA can be produced when the external tryptophan is available to the fungus. Most of these studies have used costly medium to produce IAA. Earlier, immobilized culture of P. chrysosporium has also been used for the IAA production. The fungi produced maximum auxin on the 18th day (76 µg/ml) (Ünyayar et al. 2000), while the same strain Phanerochaete chrysosporium ME446 demonstrated 0.198 µg IAA/ml in broth medium (Ünyayar 2002). Recently, another wood-degrading basidiomycetes Pleurotus pulmonarius was reported to convert 10 mM tryptophan to approximately 15 μg/ml IAA using, cellulose as a sole carbon source (Pham et al. 2019). Three white rot fungi Pleurotus ostreatus, Phanerochaete chrysosporium and Trametes versicolor produced IAA when plant biomass (Jatropha seed cake) was used as a substrate. Among these fungi, P. chrysosporium produced maximum IAA in Jatropha seed cake-based medium, which took about 15 days for the production, while the other fungi followed P. chrysosporium (Bose et al. 2013). Phlebia species and P. chrysosporium have also been reported to produce maximum IAA (31-20 μg/ ml) in complex yeast extract glucose broth (Chandra et al. 2019). Among different white rot fungi, P. chrysosporium is a well-known wood-degrading fungus, which has already been reported to treat wheat straw for various industrial applications (Sharma and Arora 2015). This fungus can degrade lignin, cellulose and hemicelluloses to release a sufficient amount of sugar. Secondly, the IAA production ability of this fungus along with lignocellulosic biomass degradation made P. chrysosporium the preferable choice for wheat straw pretreatment. Pretreatment of complex lignocellulosic biomass may enhance the efficiency of the fermentation process to be used for the production of various secondary metabolites by the microorganisms. Among various pretreatment methods of lignocellulose, the biological method using microorganisms are better in terms of economic and environmental friendly. The co-culture (simultaneous inoculation) of bacteria and/ or fungi in bio-processing seems to be highly useful in the breakdown of complex biopolymers because of their high enzyme activity (Sharma et al. 2019a).
Level of IAA produced by P. fermentans under broth and submerged conditions decreased gradually after reaching threshold. This might be due the fact that high IAA concentrations substantially reduced yeast growth (Sun et al. 2014). Previous studies have similarly indicated that IAA inhibits the growth of fungi (Prusty et al. 2004;Kulkarni et al. 2013). Similarly, the fungal strain, P. chrysosporium, was able to grow and produce IAA in the presence of tryptophan up to 10th day of incubation thereafter the IAA production decreased sharply. Besides yeast and fungi, it was observed that IAA production increased gradually from 2nd to 8th day, and decreased thereafter in the case of Pseudomonas putida (Bharucha et al. 2013).
IAA production is regulated by several factors like strain, concentration of precursor, media components, growth stage, etc. (Jasim et al. 2014). Therefore, the medium formulation needs to be optimized to get maximum production under the experimental conditions. In industrial production, downstream processing and the quality of the product is one of the important parameters considered. During HPLC analysis, an adjacent peak was also observed during the analysis, which was also seen in the previous studies. This peak might belong to some related indole compounds (indole lactic acid) as described during the IAA production by fungus Colletotrichum acutatum (Chung et al. 2003).
Shoot length of V. radiata was enhanced during the treatment of fungal strain Trichoderma viride, however, no much change was observed in case of root length (Kumar et al. 2017). As demonstrated earlier, IAA enhanced the seedling length at lower concentrations, while promoted lateral root growth and weight at higher concentrations (Malik and Sindhu 2011). The present study also confirmed the typical behavior of IAA, which enhanced the number of lateral roots and weight of seedlings. Thus, it could be concluded that the effect of exogenous IAA is concentration dependent, where lower concentrations can promote root growth, whereas higher concentrations can inhibit the same. Endogenous IAA produced by plants has a limited amount and is not used directly by plants, while IAA obtained from fungus may be applied as biological fertilizers.

Conclusion
Pichia fermentans and Phanerochaete chrysosporium produced IAA independently. The findings also highlighted the importance of pretreatment of wheat straw by P. chrysosporium, for better IAA production. Exogenous treatment of IAA demonstrated the potential of partially purified compound to increase the number of lateral roots in Vigna radiata seedlings and subsequent plant growth. Thus, the study provides an application of agricultural residue (wheat straw) to be used sued as substrate for the production of plant growth regulating hormone under submerged conditions. Abbreviations IAA: Indole-3acetic acid; HPLC: High performance liquid chromatography; MTCC : Microbial type culture collection; OD: Optical density; DNSA: 3,5-Dinitrosalicylic acid; ANOVA: Analysis of variance; IPA: Indole-3-pyruvate; TAM: Tryptamine; IAOx: Indole-3-acetaldoxime; IAM: Indole-3-acetamide; IAN: Indole-3-acetonitrile.