Lyophilized preparation of root endophyte fungus Piriformospora indica with carbon-based nanomaterial (carbon dots) successfully colonizes the plant host, Cicer arietinum

Lyophilized preparation of root endophyte fungus Piriformospora indica with carbon-based nanomaterial (carbon dots) successfully colonizes the plant host, Cicer arietinum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Lyophilized preparation of root endophyte fungus Piriformospora indica with carbon-based nanomaterial (carbon dots) successfully colonizes the plant host, Cicer arietinum Bindu Yadav, Pallavi Mourya, Rajeshwar Pratap SIngh, Smriti Sri, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5241436/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Root endophyte fungus Piriformospora indica can be axenically cultivable, is easily obtained in pure cultures in the laboratory, and therefore, can be developed as a biofertilizer for bioaugmentation. In this study, an effort towards sustainable organic agriculture, we have made two completely eco-friendly, biogenic and biocompatible lyophilized formulations of P. indica , one, without carbon dots and second, with carbon dots. Scanning Electron Microscopy (SEM) observations, viability assays and colonization efficiency of both the formulations revealed that lyophilization does not in any way alter either the morphology, growth or colonization ability of the endophyte. The formulations were also tested for impact on growth of Cicer arietinum plants in experimental set up. The plants were analysed for changes in dry weight, shoot length, root length and branch numbers. While the dry weight increased by a maximum of 1.9-fold; average shoot length increased by 1.4-fold; average root length by 1.7-fold; and number of branches by 1.4-fold, when compared to plants grown without any P. indica . These increases were found to be statistically significant. We identify this work as a significant step towards optimizations and production of this formulation on a larger scale. We also perceive this attempt as commitment towards United Nations SDGs 2,3 and 13. Biological sciences/Microbiology Earth and environmental sciences/Environmental sciences Piriformospora indica lyophilization carbon dots Cicer arietinum SDGs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Piriformospora indica is a root endophyte which colonizes both monocots and dicots. The fungus was isolated from the rhizosphere of xerophytes in Rajasthan's Thar Desert in western part of India. 1 It helps the colonized plants alleviate a variety of abiotic and biotic stresses; has time and again been advocated variously as a plant probiotic, natural plant growth promoter, immune modulator, metabolic regulator, bio fertilizer, phytoremediator, antioxidant enhancer, bio-herbicide, bio-insecticide and bio-pesticide. It confers resistance against abiotic and biotic stresses, at the same time enhancing plant productivity. 2 , 3 , 4 , 5 , 6 , 7 It's role as bio-protector conferring systemic resistance to the plants against several toxins, heavy metals and pathogens is well documented. 8 , 9 , 10 , 11 The 25Mb genome of P. indica has been sequenced and annotated. 12 P. indica can be axenically cultivated and is easily obtained in a pure culture in the laboratory. Given so many credits to its being, it is possible that laboratory grown P. indica can be developed into a suitable formulation to be used as a biofertilizer for field applications. Lyophilization preserves the vitality as well as stability of microorganisms over long periods of time by reducing their moisture content through vacuum freeze-drying. 13 Lyophilization increases the shelf life of the microbial samples and make them convenient for transport. A lyophilized formulation of P. indica fungal biomass can ease its mixing in the farm soil but, the real challenge is survival of the lyophilized preparation in soil till the endophyte can find a suitable host. Agricultural nanotechnology is emerging to be a promising technique, yet, concerns about fate, transport, bioavailability, and toxicity of nanoparticles as well as inadequacy of the regulatory framework, limits the agricultural sector's willingness to adopt such technology. 14 , 15 However, the use of nanotechnology including biogenic synthesis of carbon-based nanomaterial including carbon dots (CDs), multiwalled carbon nanotubes, and biopolymers based fertilizers can be suitable alternatives to direct use of chemical fertilizers. 16 , 17 , 18 As Arbuscular Mycorrhiza Fungi (AMF) have already been used as biofertilizers 19 it makes good sense to use fungal microbes containing carbon dots in preparing bioinoculant formulations for field application as a step towards sustainable agriculture. CDs are proven biocompatible and biodegradable, highly stable particles for green synthesis. 20 These can be easily taken up by biological cell membranes and cause negligible damage to cell integrity. 21 Additionally, CDs exhibit excitation independent photoluminescence and are highly auto-fluorescent even when exposed to low UV light, thereby making them ideal in application with bioimaging and tracking. 22 Therefore, in the present study, we propose preparing biocompatible and eco-friendly lyophilized formulations of P. indica for field applications. In this study we describe two such formulations first, lyophilized fungal biomass not containing CDs (henceforth referred to as untreated fungal biomass) and second, lyophilized fungal biomass containing CDs (henceforth referred to as treated fungal biomass). The work done towards designing and testing these formulations includes synthesis of non-toxic, biocompatible carbon dots (CDs), internalization of the CDs in P. indica fungal biomass, lyophilization of the untreated and treated fungal biomass, viability testing of the untreated and treated fungal biomass in liquid broth as well as on agar plates and interaction of lyophilized preparations with the plant host Cicer arietinum (chickpea) to check colonization efficiency as well as impact on biomass of the plants. Results Characterization of CDs The UV absorption peaks at 360nm (Fig. 1 A ) because of the n-π* transition of C = O bonds. This n-π* is responsible for photoluminescence (PL). The PL emission spectra of CDs were recorded at different wavelengths from 310nm-410nm ( Fig. 1 B ) . The maximum emission of CDs was centred at 443 nm when excited at 360 nm. The functional groups of CDs were analysed using FTIR spectroscopy. IR peak at 3340 cm − 1 was attributed to the stretching vibration of NH 2 of L-cysteine. The bending vibration of N − H was observed at 1652, 1570, 1558, and 1542 cm − 1 . Strong absorption IR peaks observed at 1716, 1731, and 1635 cm − 1 were assigned to the C = O stretch. Absorption bands at 1223 cm − 1 were because of C − O−C stretch. The presence of sulfur in CDs was confirmed by the absorption peak at 2600 and 635 cm − 1 . The FTIR spectra confirmed that the CDs contained the aromatic ring of carbon and carboxylic acid, primarily amine and sulfur as the functional groups corresponding to CA and L-cysteine ( Fig. 1 C ) . The zwitterionic nature of CDs was confirmed from the zeta potential studies of CDs. The overall charge of CDs was found to be 89 mV. TEM images showed that CDs are uniformly distributed over the surface ( Fig. 2 A ) . The particles showed a narrow size distribution varying from 1.5nm − 4nm. The average particle size was found to be 2.5 ± 0.8 nm ( Fig. 2 B ) . CDs exhibited well resolved lattice fringes with d- spacing of 0.206 nm. This lattice spacing being the characteristic of graphitic carbon (100) facet in CDs, showed that CDs have crystallinity. However, we also observed that not all CDs were crystalline in nature. The selected area electron diffraction (SAED) pattern showed that most of the CDs were amorphous in nature. The amorphous nature of CDs was also observed in XRD. Internalization of CDs by P. indica The internalization of CDs by P. indica was confirmed by autofluorescence of CDs. We found uptake of CDs in P. indica was time and concentration-dependent. The maximum visible fluorescence was observed in fungal biomass treated with 400µg/ml CDs for 2h. ( Fig. 2 C ). We found that any increase in the concentration of CDs beyond 400µg/ml or time beyond 2h resulted in an actual decrease in autofluorescence. This could be because longer incubations or higher concentrations of the CDs likely results in a reverse flow from fungal cells back into the suspension. Our SEM data shows that CDs did not have any adverse effects on P. indica morphology because in both, the treated and untreated fungal biomass, the pear-shaped structure of fungal spores was found to be intact (Fig. 3 A &B) . Lyophilization of treated and untreated fungal biomass The treated as well as untreated fungal biomass was lyophilized to a fine pale-yellow powder under sterile conditions. ( Fig. 4 ) Multiple batches were run to generate aliquots of the powder. This lyophilized preparation was checked for viability on agar plates as well as in broth at different time points, immediately, one week, two weeks and one month after lyophilization. All the lyophilized preparations grew to maturity in 21 days just like the routine batch cultures of P. indica ( Fig. 5 ). Lyophilized P. indica successfully colonized plants We observed that while roots that were not exposed to P. indica (controls) did not show, as expected, presence of spores ( Fig. 6 A ) , colonization percentage in plant roots exposed to fresh P. indica was ~ 90% ( Fig. 6 B ) . Most encouragingly, the colonization efficiency of lyophilized P. indica ( untreated ) in plants exceeded 70% in 40 days ( Fig. 6 C ) . This set of plants did not reach any higher colonization percentages possibly because of the time lag lyophilized P. indica may have taken to grow from desiccated state into vegetative stage before further entering into sporulation. It was also observed that many of the root sections where treated biomass was used showed less colonization i.e., 75% ( Fig. 6 D ) . The reason could be the same as the premise taken for this study, that, CDs reduce the dependence of fungus on plant host for fixed C which in turn either delays its interaction or prolongs its vegetative stage in the host. Nonetheless, further in-depth studies at a molecular level are needed to understand the reason behind extensive hyphal growth before sporulation inside the host roots, when fungus is treated with CDs. Lyophilized P. indica enhances growth of the colonized plants We found that plants colonized with the fresh cultures of P. indica , lyophilized untreated P. indica and lyophilized treated P. indica showed morphologically better growth and biomass in comparison to the non-colonized plants (controls) ( Fig. 7 A-D ) . Growth parameters were analysed for dry weight, shoot length, root length and branch numbers. The dry weight of plants grown with lyophilized untreated fungal biomass as well as plants grown with fresh fungal cultures showed an equal increase of 2.5-fold when compared to plants grown without any P. indica (control). The dry weight of plants grown with lyophilized treated fungal biomass showed a 2.3-fold increase when compared to plants grown without any P. indica (control). All these differences were found to be statistically significant (p < 0.001) ( Fig. 8 A ). The average shoot length of plants grown with lyophilized untreated fungal biomass and those grown with fresh fungal culture increased by 2.1 and 2.3-fold, respectively, when compared to plants grown without any P. indica (control). These differences were found to be statistically significant (p < 0.001) Shoot length of those grown with lyophilized treated fungal biomass showed only a 2-fold increase over plants grown without any P. indica (control). This difference was also found to be statistically significant (p < 0.01) ( Fig. 8 B ). The average root length of plants grown with lyophilized untreated fungal biomass and those grown with fresh fungal culture increased by 1.7-fold and 1.9-fold, respectively, when compared to plants grown without any P. indica (control). These differences were found to be statistically significant (p < 0.01and p < 0.001, respectively). However, root length of those grown with lyophilized treated fungal did not show any statistically significant increase ( Fig. 8 C ). Number of branches in plants grown with lyophilized untreated fungal biomass and those grown with fresh fungal culture increased equally by 1.4-fold when compared to plants grown without any P. indica (control). These differences were found to be statistically significant (p < 0.001). However, number of branches in plants grown with lyophilized treated fungal did not show any statistically significant increase ( Fig. 8 D ). Discussion P. indica has great potential to be used as a biofertilizer in increasing biomass and productivity of crops when compared to other mycorrhiza including AMFs. Though found natively in soils, P. indica faces all the usual challenges presented by a complex ecosystem as soil, towards growth and colonization of its host. Therefore, to be able to put it to maximally beneficial use in agriculture, we suggest bio-augmentation of this endophyte in agricultural farmlands. However, field application requires a suitable formulation that is eco-friendly, sustainable, easy to add in the soil, can increase the shelf life of the fungus to give it sustained longevity in the absence of a host and can also be scaled up for commercial use. In the present study, we have successfully attempted to make such a working formulation by lyophilizing the fungal biomass harvested from laboratory grown pure cultures of P. indica . Further, to strengthen its stability, viability and efficiency after addition in the soil, we have integrated nano-sized, photostable, non-toxic and biocompatible CDs inside the fungal biomass before lyophilization. Indeed, as our results show, lyophilized P. indica is revivable in laboratory cultures and not only thrives just as well as the routine fresh batches, there is also no change in either the growth rate or morphology of the spores. Moreover, like fresh cultures, the lyophilized formulation is also able to colonize the chickpea plants efficiently and is comparable to it in its positive impact on plant growth and biomass. Further, while lyophilization with CDs inside the fungal biomass did show good revival and colonization, our results showed that it is the lyophilized preparation without CDs which has the desirable impact on plant growth and biomass. However, before making any conclusions it will be worthwhile to expand this study over a longer period of plant growth to also include the reproductive phase and fruiting. In a parallel, yet unpublished study done in our laboratory it has been shown that addition of axenically grown P. indica does not alter the microbial diversity of the soil, therefore giving a more promising prospect towards its bioformulation. This preliminary work, to the best of our knowledge also a first for lyophilization of P. indica , certainly entails optimization of shelf-life, survival, colonization efficiency and larger in-situ experimental cohorts before scaling-up the process for bio-augmentation on commercial scale. The larger aim is utilizing the potential of P. indica in sustainable organic agriculture to achieve food security, enhanced productivity and health of food crops in the face of climate change, as also realized in United Nations SDGs 2,3 and 13. Material and Methods Fungal culture P. indica culture was a gift from Prof. Ajit Varma, Amity University, Noida, UP, India. P. indica cultures was routinely maintained in both, KF culture medium broth (20 g L - 1 Glucose, 2 g L - 1 Peptone, 1 g L - 1 Yeast Extract, 1 g L - 1 Casamino Acid, 520 mg L - 1 KCl, 520mg L - 1 MgSO 4 7H 2 O, 1.52g L - 1 KH 2 PO 4 , 55mg L - 1 ZnSO 4 7H 2 O, 27mg L - 1 H 3 BO 3 , 12 mg L - 1 MnCl 2 4H 2 O, 4 mg L - 1 CoCl 2 6H 2 O, 4 mg L - 1 CuSO 4 5H 2 O, 16.2 mg L - 1 CaCl 2 , 16.2 mg L - 1 FeCl 3 , 0.5 mg L - 1 Biotin, 0.5 mg L - 1 Nicotinamide, 1 mg L - 1 Pyridoxal Phosphate, 1 mg L - 1 Aminobenzoic Acid, 2.5 mg L - 1 Riboflavin and 2% Agar; pH 6 0.5) as well as on agar plates. 23 P. indica was grown in liquid KF medium in a 250-ml culture flasks under constant shaking at 110 rpm, and at 30 0 C for 7–9 days in a metabolic shaker (Orbitek shaker, India). Synthesis and characterization of carbon dots (CDs) To synthesize CDs, citric acid (0.52M) and 20.8mM L-cysteine were dissolved in 10ml ultrapure water as described. 22 , 24 The clear solution was placed in a microwave for 3min at 700W. After allowing the yellow precipitate to cool at room temperature, 5ml ultrapure water was added to dissolve it, yielding a yellow color solution with the desired CDs. To remove impurities, formulation was dialyzed against pure water for three days using a dialysis membrane (Spectra/Por MWCO 1000Da). Finally, a solution containing CDs was oven-dried at 80°C, yielding 0.157g CDs. From this 1mg CDs were dispersed in 1ml ultrapure water before being used. The UV-Vis and fluorescence spectra of CDs were measured using a T90 + UV/vis spectrometer (PG Instruments Ltd, UK). The fluorescence behavior of CDs was examined using a Nikon real-time confocal laser scanning microscope (A1R). High-resolution Transmission Electron Microscopy (HRTEM, JEOL JEM-2200 FS, JAPAN) was used to characterize the size and crystallinity of CDs. Since the functional group in the CDs correspond to citric acid and L-cysteine, the Fourier Transform Infrared Spectroscopy (FTIR) confirmed that the CDs include the aromatic ring of carbon and carboxylic acid, mainly amine and sulfur. Internalization of CDs by P. indica P. indica being a root endophyte fungus is dependent on its plant host for fixed carbon, which implies, that without a host the endophyte will either have limited survival or may not survive altogether. Therefore, laboratory synthesized CDs, as a source of fixed carbon, were internalized in the lyophilized fungal biomass in a bid to ensure its survival in the field till the time it can find and colonize the plant host roots. P. indica fungal biomass was harvested from culture agar plates by flooding the plates with liquid KF broth and carefully scraping the fungus with a sterile glass spreader in a centrifuge tube. Small hyphal fragments were removed by centrifugation. The supernatant was discarded and pellet obtained ( untreated fungal biomass) was diluted with sterile distilled water. The number of spores was quantified under a light microscope using a hemacytometer (Neubauer Scientific, PA, USA). Concentration and time-dependent study was performed to check the uptake of CDs. To investigate at what concentration CDs uptake P. indica varying concentrations of CDs (1000, 800, 400, 200, 100, 50 and 25µg/ml) were added to the fungal biomass in separate wells and incubated for various time periods (0, 1, 2, 4 and 6h) ( treated fungal biomass). Autofluorescence in spores was visualized by Confocal Laser Scanning Microscope (Olympus FluoView ™ FV1000) at 40X excited by 405nm laser. Further, both, treated and untreated samples were viewed under Scanning Electron Microscope (SEM) (EVO 18 special edition, ZEISS) to check for any morphological changes post internalization of CDs. To do SEM, samples were rinsed with PBS buffer, placed in fixative (2.5% glutaraldehyde) for 4h, washed again with PBS buffer, and stored overnight at 4°C. Further, samples were dehydrated in an ascending series of 10% increments from 50–100% ethanol for 20min each before being kept for drying. Untreated fungal biomass served as control for making comparisons. A viability check was done to test the viability of fungal biomass after internalization of CDs. For this purpose, variously treated fungal biomass defined by different concentrations and time was grown on separate KF agar plates and liquid broth at 30 ± 2 0 C for 10 days. Fungal biomass incubated only with distilled water for 2h was used as control. Lyophilization of P. indica For this purpose, 100ml broth culture of both untreated and treated fungal biomass were separately sieved, washed using sterile distilled water and transferred to sterile lyophilization tubes. The tubes were kept frozen in liquid nitrogen for 2min. Tubes were uncapped and covered with sterile aluminium foil and loaded on the lyophilizer (Operon FDB 5503) at temperature and vacuum of -57 0 C and 10 − 3 Torr, respectively for 24h. The lyophilized powder was stored in batches of 10mg in sterile capped vials at 4 0 C. To test the viability of lyophilized preparations, 10mg each of untreated and treated lyophilized fungal biomass were inoculated separately on KF agar plates (incubated at 30˚C in an incubator) and in KF broth incubated at 30˚C, 111 rpm in a metabolic shaker (Orbitek shaker, India). Interaction of P . indica with plant To find out if lyophilization or the CDs have any effect on the ability of P. indica to interact with the host plant, four sets of pots were prepared (1) plants were grown without any P. indica (2) plants were grown only with fresh cultures of P. indica mixed with soil at final concentration of 0.1% (w/w) (3) plants were grown with lyophilized untreated fungal biomass mixed with soil at final concentration of 0.1% (w/w) ( 4 ) plants were grown with lyophilized treated fungal biomass mixed with soil at final concentration of 0.1% (w/w). Chickpea plant ( Cicer arietinum ) seeds were purchased from Indian Agriculture Research Institute, Pusa, New Delhi. To grow chickpea plants, seeds were surface sterilized. To do this, approx. 50 seeds were soaked in 100ml sterile distilled water for 1h. Thereafter, they were washed in consecutive steps serially with 10% SDS, 20ml 90% ethanol (for 45sec), 50ml sterile distilled water (washed thrice), 50ml 2% (v/v) NaOCl (for 2min), 50ml sterile distilled water (washed thrice), 50ml 70% ethanol (for 45sec) and finally five times with 50ml of sterile distilled water. Germinated seedlings were placed in pots filled with a mixture of sterile sand and soil in the ratio of 3:1 (garden soil from the Jawaharlal Nehru University campus and acid washed riverbed sand). Plants were supplied with half strength modified Hoagland solution (5mM KNO 3 , 5mM Ca(NO) 2 , 2Mm MgSO 4 , 10 M KH 2 PO 4 , 10 M MgCl 2 , 4M ZnSO 4 , 1M CaSO 4 , 1M NaMoO 4 , and 50 M H 3 BO 3 ) weekly. All the pots were grown for 40 days in a Controlled Environment Chamber Facility (CECF) maintained at daily temperature of 29°±2°C. Plants were grown under controlled conditions in a greenhouse with an 8-h light:16-h-dark photoperiod (light intensity of 1,000 lux, using high-intensity fluorescent light) at a temperature cycle of 25C/18C with a relative humidity 60–70%.They were watered as needed. Histochemical analysis Histochemical analysis of the roots of plants from all sets was done to check colonization. Root sections of ~ 1cm were placed in 2ml centrifugation tubes. They were incubated in 1ml 10% KOH solution at 60°C for 15min to soften the plant material and thereafter neutralized in 1N HCL for 5min. The samples were then washed thrice with sterile distilled water and stained in 1ml 0.02% trypan blue at 60°C for 15 min. Samples were de-stained with 1ml 50% lactophenol for 15–60 min. and mounted on a slide, under coverslip for observation with light microscope. Percentage colonization for the inoculated plants was calculated using the following formula as described previously. 8 $$\:\varvec{\%}\:\varvec{C}\varvec{o}\varvec{l}\varvec{o}\varvec{n}\varvec{i}\varvec{z}\varvec{a}\varvec{t}\varvec{i}\varvec{o}\varvec{n}=\frac{\varvec{r}\varvec{o}\varvec{o}\varvec{t}\:\varvec{s}\varvec{e}\varvec{c}\varvec{t}\varvec{i}\varvec{o}\varvec{n}\:\varvec{w}\varvec{i}\varvec{t}\varvec{h}\:\varvec{P}.\varvec{i}\varvec{n}\varvec{d}\varvec{i}\varvec{c}\varvec{a}}{\varvec{T}\varvec{o}\varvec{t}\varvec{a}\varvec{l}\:\varvec{r}\varvec{o}\varvec{o}\varvec{t}\:\varvec{s}\varvec{e}\varvec{c}\varvec{t}\varvec{i}\varvec{o}\varvec{n}\varvec{s}\:\varvec{o}\varvec{b}\varvec{s}\varvec{e}\varvec{r}\varvec{v}\varvec{e}\varvec{d}}\times\:100$$ Determination of Biomass Well-grown chickpea plants from all the four sets were harvested for analyses after 40 days of growth. The pots were tapped lightly all across the outside to loosen the soil inside. Scraper was used to scrape the soil sticking on the inside walls. Each pot was held and gently turned upside down to remove the intact plant with its soil. The bulk soil around the roots was removed very carefully with hands so as to not damage the roots of the plant. The plants were washed gently multiple times to remove residual traces of soil sticking to the root. Washed plants were stretched lengthwise on blotting sheets to remove excess water. Plants from all the four sets were kept distinct on separate blotting sheets. Dry weight, number of branches, shoot and root lengths of plants from each of these four sets were recorded. To measure the dry weight, harvested plants were washed with water, and oven dried at 80°C for 72 h. 10 Statistical analysis GraphPad Prism was used to conduct the unpaired t test to check the significance. Declarations Author Contribution MD and AKJ initiated the project. BY , SS, RPS and PM have performed all the experiments. MS is written by MD. AKJ, BY and PS. Acknowledgments MD gratefully acknowledges DST-PURSE and UGC-UPE-II, New Delhi, India, for financial help. BY is thankful to JNU for providing research fellowship and to AIRF, JNU for SEM analysis, confocal and fluorescence microscopy. The authors are very thankful to Shalini Sinha for her help with statistical analysis. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request" References Verma, S., Varma, A., Rexer, K.H., Hassel, A., Kost, G., Sarbhoy, A., Bisen, P., Bütehorn, B., & Franken, P. (1998). Piriformospora indica , gen. et sp. nov., a new root-colonizing fungus. Mycologia , 90(5), 896-903. Waller, F., Achatz, B., Baltruschat, H., Fodor, J., Becker, K., Fischer, M., Heier, T., Hückelhoven, R., Neumann, C., von Wettstein, D., Franken, P., & Kogel, K. H. (2005). The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proceedings of the National Academy of Sciences , 102(38), 13386-13391 Franken, P. (2012). The plant strengthening root endophyte Piriformospora indica : potential application and the biology behind. Applied Microbiology Biotechnology , 96 (6), 1455-1464. Ansari, M. W., Trivedi, D. K., Sahoo, R. K., Gill, S. S., & Tuteja, N. (2013). A critical review on fungi mediated plant responses with special emphasis to Piriformospora indica on improved production and protection of crops. Plant Physiology Biochemistry , 70 , 403-410. Prasad, R., Kamal, S., Sharma, P. K., Oelmüller, R., & Varma, A. (2013). Root endophyte Piriformospora indica DSM 11827 alters plant morphology, enhances biomass and antioxidant activity of medicinal plant Bacopa monniera . Journal of basic Microbiology , 53 (12), 1016-1024. Jogawat, A., Vadassery, J., Verma, N., Oelmüller, R., Dua, M., Nevo, E., & Johri, A. K. (2016). PiHOG1, a stress regulator MAP kinase from the root endophyte fungus Piriformospora indica , confers salinity stress tolerance in rice plants. Scientific Reports , 6(1), 1-15. Gill, S.S., Gill, R., Trivedi, D.K., Anjum, N.A., Sharma, K.K., Ansari, M.W., Ansari, A.A., Johri, A.K., Prasad, R., Pereira, E., Varma, A. & Tuteja, N. (2016). Piriformospora indica : potential and significance in plant stress tolerance. Frontiers in Microbiology , 7 , 332. Kumar, M., Yadav, V., Tuteja, N., & Johri, A. K. (2009). Antioxidant enzyme activities in maize plants colonized with Piriformospora indica . Microbiology , 155(3), 780-790. Narayan, O.P., Verma, N., Singh, A.K., Oelmüller, R., Kumar, M., Prasad, D., Kapoor, R., Dua, M., & Johri, A.K. (2017). Antioxidant enzymes in chickpea colonized by Piriformospora indica participate in defense against the pathogen Botrytis cinerea . Scientific Reports , 7(1), 1-11. Narayan, O. P., Verma, N., Jogawat, A., Dua, M., & Johri, A. K. (2021). Sulfur transfer from the endophytic fungus Serendipita indica improves maize growth and requires the sulfate transporter SiSulT. The Plant Cell , 33 (4), 1268-1285. Verma, N., Narayan, O. P., Prasad, D., Jogawat, A., Panwar, S. L., Dua, M., & Johri, A. K. (2022). Functional characterization of a high‐affinity iron transporter (PiFTR) from the endophytic fungus Piriformospora indica and its role in plant growth and development. Environmental Microbiology , 24(2), 689-706. Zuccaro, A., Lahrmann, U., Güldener, U., Langen, G., Pfiffi, S., Biedenkopf, D., Wong, P., Samans, B., Grimm, C., Basiewicz, M., Murat, C., Martin, F., & Kogel, K. H. (2011). Endophytic life strategies decoded by genome and transcriptome analyses of the mutualistic root symbiont Piriformospora indica . PLoS Pathogens , 7(10), e1002290. Prakash, O., Nimonkar, Y., & Shouche, Y. S. (2013). Practice and prospects of microbial preservation. FEMS Microbiology Letters , 339 (1), 1-9. Mishra, S., Keswani, C., Abhilash, P. C., Fraceto, L. F., & Singh, H. B. (2017). Integrated approach of agri-nanotechnology: challenges and future trends. Frontiers in Plant Science , 8 , 471. He, X., Deng, H., & Hwang, H. M. (2019). The current application of nanotechnology in food and agriculture. Journal of Dood Drug Analysis , 27 (1), 1-21. Phadke, C., Mewada, A., Dharmatti, R., Thakur, M., Pandey, S., & Sharon, M. (2015). Biogenic synthesis of fluorescent carbon dots at ambient temperature using Azadirachta indica (Neem) gum. Journal of Fluorescence , 25 (4), 1103-1107. Kasibabu, B. S. B., D’souza, S. L., Jha, S., & Kailasa, S. K. (2015). Imaging of bacterial and fungal cells using fluorescent carbon dots prepared from carica papaya juice. Journal of Fluorescence , 25 (4), 803-810. Singhal, R., & Kalra, V. (2017). Cobalt nanoparticle‐embedded porous carbon nanofibers with inherent N‐and F‐doping as binder‐free bifunctional catalysts for oxygen reduction and evolution reactions. ChemPhysChem , 18 (2), 223-229. Basiru, S., Mwanza, H. P., & Hijri, M. (2021). Analysis of arbuscular mycorrhizal fungal inoculant benchmarks. Microorganisms . 2021; 9: 81. Maholiya, A., Ranjan, P., Khan, R., Murali, S., Nainwal, R. C., Chauhan, P. S., Sathish, N., Chaurasia, J. P. & Srivastava, A.K. (2023). An insight into the role of carbon dots in the agriculture system: a review. Environmental Science: Nano , 10(4), 959-995. Janus, Ł., Piątkowski, M., Radwan-Pragłowska, J., Bogdał, D., Matysek, D. (2019). Chitosan-Based Carbon Quantum Dots for Biomedical Applications: Synthesis and Characterization. Nanomaterials (Basel) , 16;9(2):274. Sri, S., Kumar, R., Panda, A. K., & Solanki, P. R. (2018). Highly biocompatible, fluorescence, and zwitterionic carbon dots as a novel approach for bioimaging applications in cancerous cells. ACS Applied Materials & Interfaces , 10 (44), 37835-37845. Hill, T. W., & Kafer, E. (2001). Improved protocols for Aspergillus minimal medium: trace element and minimal medium salt stock solutions. Fungal Genetics Reports , 48 (1), 20-21. Liu, Q., Zhang, N., Shi, H., Ji, W., Guo, X., Yuan, W., & Hu, Q. (2018). One-step microwave synthesis of carbon dots for highly sensitive and selective detection of copper ions in aqueous solution, New Journal of Chemistry , 42 , 3097-3101. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5241436","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":376082363,"identity":"6358f1de-cd66-4597-b4f7-fb1acfc509b8","order_by":0,"name":"Bindu Yadav","email":"","orcid":"","institution":"Jawaharlal Nehru University","correspondingAuthor":false,"prefix":"","firstName":"Bindu","middleName":"","lastName":"Yadav","suffix":""},{"id":376082365,"identity":"aa8097ff-abb6-4335-88a4-0c9c94ed2aca","order_by":1,"name":"Pallavi Mourya","email":"","orcid":"","institution":"Jawaharlal Nehru University","correspondingAuthor":false,"prefix":"","firstName":"Pallavi","middleName":"","lastName":"Mourya","suffix":""},{"id":376082366,"identity":"0734a4d1-86e5-4b69-80d0-a925b65584ae","order_by":2,"name":"Rajeshwar Pratap SIngh","email":"","orcid":"","institution":"Jawaharlal Nehru University","correspondingAuthor":false,"prefix":"","firstName":"Rajeshwar","middleName":"Pratap","lastName":"SIngh","suffix":""},{"id":376082367,"identity":"ea523c7b-2dd9-4cb0-8daa-f8e0d257dd86","order_by":3,"name":"Smriti Sri","email":"","orcid":"","institution":"Jawaharlal Nehru University","correspondingAuthor":false,"prefix":"","firstName":"Smriti","middleName":"","lastName":"Sri","suffix":""},{"id":376082368,"identity":"5e5ced2c-f57d-4d0b-be23-32bb22967574","order_by":4,"name":"Pratima Solanki","email":"","orcid":"","institution":"Jawaharlal Nehru University","correspondingAuthor":false,"prefix":"","firstName":"Pratima","middleName":"","lastName":"Solanki","suffix":""},{"id":376082369,"identity":"c2ac621f-ebda-43c2-8b48-fc4b4caffb38","order_by":5,"name":"Atul Kumar Johri","email":"","orcid":"","institution":"Jawaharlal Nehru University","correspondingAuthor":false,"prefix":"","firstName":"Atul","middleName":"Kumar","lastName":"Johri","suffix":""},{"id":376082370,"identity":"59afbe4e-ea8c-477b-92b1-6cb2128eaa18","order_by":6,"name":"Meenakshi Dua","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYNCCAwwJbPKPDz4Gc5iZG4jUwpCWbMzAYADUwkikFgaGHDNpsBYGAlp025uPPfhxpi6Pj+GAWXVBxZ9o/naglh8V23BqMTtzLN2w58bhYjbGhrTbM84Y5M44zNjA2HPmNm4tN3LMJHg+HEhsY2Y4dpu3zSC3AaiFmbENj5b7779J/vlQl9jGxthWDNIyn6CWGzxs0jw3mBPbeJjZmEFaNhDUcibNTFrmzOHENgk2ZmmeM8a5G4FaDuL1y/HDzyTfHKtLnD+D/+Nnngq53HnnDx988KMCtxbs4ACJ6kfBKBgFo2AUoAEAx7xcTTCtOt4AAAAASUVORK5CYII=","orcid":"","institution":"Jawaharlal Nehru University","correspondingAuthor":true,"prefix":"","firstName":"Meenakshi","middleName":"","lastName":"Dua","suffix":""}],"badges":[],"createdAt":"2024-10-10 17:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5241436/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5241436/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68904172,"identity":"6edf9566-363c-4f1c-abda-18f00a98f57f","added_by":"auto","created_at":"2024-11-13 10:22:12","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":112237,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of Carbon Dots.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5241436/v1/dd6a31c922d9d59d480c90f7.jpeg"},{"id":68904173,"identity":"4cf910d0-f728-4f22-82b8-70b3b3b55d40","added_by":"auto","created_at":"2024-11-13 10:22:12","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":176101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) TEM\u003c/strong\u003e imaging of CDs\u003cstrong\u003e (b) Size distribution of CDs.\u003c/strong\u003e \u003cstrong\u003e(c) Autofluorescence in spores as visualized by CLSM after treating \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. indica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e with varying concentration of CDs (i) \u003c/strong\u003e200µg/ml CDs for 2h \u003cstrong\u003e(ii) \u003c/strong\u003e400µg/ml CDs for 2h \u003cstrong\u003e(iii) \u003c/strong\u003e600µg/ml CDs for 2h \u003cstrong\u003e(iv) \u003c/strong\u003e800µg/ml CDs for 2h.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5241436/v1/bb15fb44be0a55bb67e8314e.jpeg"},{"id":68906124,"identity":"c3d9ef24-24e5-46f7-9347-99f6fd7e34bc","added_by":"auto","created_at":"2024-11-13 10:46:12","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":172169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM of (A)\u003c/strong\u003e lyophilized\u003cem\u003e P. indica\u003c/em\u003e (\u003cem\u003euntreated \u003c/em\u003efungal biomass) (\u003cstrong\u003eB)\u003c/strong\u003elyophilized\u003cem\u003e P. indica \u003c/em\u003ewith 400µg/ml CDs for 2h (\u003cem\u003etreated\u003c/em\u003e fungal biomass).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5241436/v1/9ca68eea9bd02ab304f6b080.jpeg"},{"id":68904966,"identity":"2ddba43e-1ef4-4785-9670-b451a3c6afd1","added_by":"auto","created_at":"2024-11-13 10:30:12","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":140020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLyophilization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. indica\u003c/strong\u003e\u003c/em\u003e. Pale yellow powder in multiple aliquots.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5241436/v1/2c04d7b9f746f7710ca8c487.jpeg"},{"id":68905210,"identity":"707f7595-8a78-4703-a20d-acd531245af6","added_by":"auto","created_at":"2024-11-13 10:38:12","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":167924,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViability assay of lyophilized \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. indica \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eunder laboratory condition after two weeks of lyophilization. (A) \u003c/strong\u003eculture growing from lyophilized \u003cem\u003eP. indica \u003c/em\u003eon KF medium agar plates on left panel and in KF broth on right panel\u003cstrong\u003e (B) \u003c/strong\u003eculture growing from lyophilized \u003cem\u003eP. indica \u003c/em\u003etreated\u003cem\u003e \u003c/em\u003ewith\u003cem\u003e \u003c/em\u003e(400µg/ml CDs for 2h) on KF medium agar plates on left panel and in KF broth on right panel\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5241436/v1/dda9e9166b6fa2e34a921ec6.jpeg"},{"id":68904174,"identity":"fce02dca-6ea2-4ab6-b435-7f3fb3aa6f37","added_by":"auto","created_at":"2024-11-13 10:22:12","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":169636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColonization analysis in the roots of chickpea plants (A)\u003c/strong\u003e plants grown without any \u003cem\u003eP. indica\u003c/em\u003e \u003cstrong\u003e(B)\u003c/strong\u003e plants colonized with fresh \u003cem\u003eP. indica\u003c/em\u003e \u003cstrong\u003e(C)\u003c/strong\u003e lyophilized\u003cem\u003e P. indica\u003c/em\u003e (\u003cem\u003euntreated \u003c/em\u003efungal biomass)\u003cstrong\u003e (D) \u003c/strong\u003elyophilized\u003cem\u003e P. indica \u003c/em\u003ewith 400µg/ml CDs for 2h (\u003cem\u003etreated\u003c/em\u003e fungal biomass).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5241436/v1/fed34565bccfa13ff8cbf108.jpeg"},{"id":68904969,"identity":"9d7514d4-67c8-4df4-814e-0e0df4a09088","added_by":"auto","created_at":"2024-11-13 10:30:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":614225,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth and Biomass analyses.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e plants grown without any \u003cem\u003eP. indica\u003c/em\u003e \u003cstrong\u003e(B)\u003c/strong\u003e plants grown only with fresh cultures of \u003cem\u003eP. indica \u003c/em\u003emixed with soil at final concentration of 0.1% (w/w) \u003cstrong\u003e(C)\u003c/strong\u003e plants grown with lyophilized \u003cem\u003euntreated \u003c/em\u003efungal biomass mixed with soil at final concentration of 0.1% \u003cstrong\u003e(D)\u003c/strong\u003e plants grown with lyophilized \u003cem\u003etreated \u003c/em\u003efungal biomassmixed with soil at final concentration of 0.1% (w/w).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5241436/v1/175cdaa855bf84ba77a4fb03.png"},{"id":68904177,"identity":"a79e9516-3204-43bd-8497-38240dd55762","added_by":"auto","created_at":"2024-11-13 10:22:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":100716,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlant growth and development analyses \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003e(A)\u003c/strong\u003e Dry weight \u003cstrong\u003e(B)\u003c/strong\u003e Shoot length \u003cstrong\u003e(C)\u003c/strong\u003e Root length and \u003cstrong\u003e(D)\u003c/strong\u003e Number of branches. Data are shown as mean ± SEM (n=3 biological replicates and three technical replicates). The statistically significant differences are marked with asterix *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. Unpaired t-test was conducted using Prism Software version 8 (GraphPad).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5241436/v1/4501c811788c4acd2b14dd81.png"},{"id":81037948,"identity":"89aba401-fdf4-4f7d-8412-c5df0d95d64e","added_by":"auto","created_at":"2025-04-21 12:46:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2735792,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5241436/v1/73c8505a-9e10-45fd-8f72-04eacf85b60a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lyophilized preparation of root endophyte fungus Piriformospora indica with carbon-based nanomaterial (carbon dots) successfully colonizes the plant host, Cicer arietinum","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003ePiriformospora indica\u003c/em\u003e is a root endophyte which colonizes both monocots and dicots. The fungus was isolated from the rhizosphere of xerophytes in Rajasthan's Thar Desert in western part of India.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e It helps the colonized plants alleviate a variety of abiotic and biotic stresses; has time and again been advocated variously as a plant probiotic, natural plant growth promoter, immune modulator, metabolic regulator, bio fertilizer, phytoremediator, antioxidant enhancer, bio-herbicide, bio-insecticide and bio-pesticide. It confers resistance against abiotic and biotic stresses, at the same time enhancing plant productivity.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e It's role as bio-protector conferring systemic resistance to the plants against several toxins, heavy metals and pathogens is well documented.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e The 25Mb genome of \u003cem\u003eP. indica\u003c/em\u003e has been sequenced and annotated.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eP. indica\u003c/em\u003e can be axenically cultivated and is easily obtained in a pure culture in the laboratory. Given so many credits to its being, it is possible that laboratory grown \u003cem\u003eP. indica\u003c/em\u003e can be developed into a suitable formulation to be used as a biofertilizer for field applications.\u003c/p\u003e \u003cp\u003eLyophilization preserves the vitality as well as stability of microorganisms over long periods of time by reducing their moisture content through vacuum freeze-drying.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Lyophilization increases the shelf life of the microbial samples and make them convenient for transport. A lyophilized formulation of \u003cem\u003eP. indica\u003c/em\u003e fungal biomass can ease its mixing in the farm soil but, the real challenge is survival of the lyophilized preparation in soil till the endophyte can find a suitable host.\u003c/p\u003e \u003cp\u003eAgricultural nanotechnology is emerging to be a promising technique, yet, concerns about fate, transport, bioavailability, and toxicity of nanoparticles as well as inadequacy of the regulatory framework, limits the agricultural sector's willingness to adopt such technology.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e However, the use of nanotechnology including biogenic synthesis of carbon-based nanomaterial including carbon dots (CDs), multiwalled carbon nanotubes, and biopolymers based fertilizers can be suitable alternatives to direct use of chemical fertilizers.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e As Arbuscular Mycorrhiza Fungi (AMF) have already been used as biofertilizers\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e it makes good sense to use fungal microbes containing carbon dots in preparing bioinoculant formulations for field application as a step towards sustainable agriculture. CDs are proven biocompatible and biodegradable, highly stable particles for green synthesis.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e These can be easily taken up by biological cell membranes and cause negligible damage to cell integrity.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Additionally, CDs exhibit excitation independent photoluminescence and are highly auto-fluorescent even when exposed to low UV light, thereby making them ideal in application with bioimaging and tracking.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Therefore, in the present study, we propose preparing biocompatible and eco-friendly lyophilized formulations of \u003cem\u003eP. indica\u003c/em\u003e for field applications. In this study we describe two such formulations first, lyophilized fungal biomass not containing CDs (henceforth referred to as \u003cem\u003euntreated\u003c/em\u003e fungal biomass) and second, lyophilized fungal biomass containing CDs (henceforth referred to as \u003cem\u003etreated\u003c/em\u003e fungal biomass). The work done towards designing and testing these formulations includes synthesis of non-toxic, biocompatible carbon dots (CDs), internalization of the CDs in \u003cem\u003eP. indica\u003c/em\u003e fungal biomass, lyophilization of the \u003cem\u003euntreated\u003c/em\u003e and \u003cem\u003etreated\u003c/em\u003e fungal biomass, viability testing of the \u003cem\u003euntreated\u003c/em\u003e and \u003cem\u003etreated\u003c/em\u003e fungal biomass in liquid broth as well as on agar plates and interaction of lyophilized preparations with the plant host \u003cem\u003eCicer arietinum\u003c/em\u003e (chickpea) to check colonization efficiency as well as impact on biomass of the plants.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of CDs\u003c/h2\u003e \u003cp\u003eThe UV absorption peaks at 360nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e because of the n-π* transition of C\u0026thinsp;=\u0026thinsp;O bonds. This n-π* is responsible for photoluminescence (PL). The PL emission spectra of CDs were recorded at different wavelengths from 310nm-410nm \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The maximum emission of CDs was centred at 443 nm when excited at 360 nm. The functional groups of CDs were analysed using FTIR spectroscopy. IR peak at 3340 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was attributed to the stretching vibration of NH\u003csub\u003e2\u003c/sub\u003e of L-cysteine. The bending vibration of N\u0026thinsp;\u0026minus;\u0026thinsp;H was observed at 1652, 1570, 1558, and 1542 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Strong absorption IR peaks observed at 1716, 1731, and 1635 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were assigned to the C\u0026thinsp;=\u0026thinsp;O stretch. Absorption bands at 1223 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were because of C\u0026thinsp;\u0026minus;\u0026thinsp;O\u0026minus;C stretch. The presence of sulfur in CDs was confirmed by the absorption peak at 2600 and 635 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The FTIR spectra confirmed that the CDs contained the aromatic ring of carbon and carboxylic acid, primarily amine and sulfur as the functional groups corresponding to CA and L-cysteine \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. The zwitterionic nature of CDs was confirmed from the zeta potential studies of CDs. The overall charge of CDs was found to be 89 mV.\u003c/p\u003e \u003cp\u003eTEM images showed that CDs are uniformly distributed over the surface \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The particles showed a narrow size distribution varying from 1.5nm \u0026minus;\u0026thinsp;4nm. The average particle size was found to be 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 nm \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. CDs exhibited well resolved lattice fringes with d- spacing of 0.206 nm. This lattice spacing being the characteristic of graphitic carbon (100) facet in CDs, showed that CDs have crystallinity. However, we also observed that not all CDs were crystalline in nature. The selected area electron diffraction (SAED) pattern showed that most of the CDs were amorphous in nature. The amorphous nature of CDs was also observed in XRD.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInternalization of CDs by\u003c/b\u003e \u003cb\u003eP. indica\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe internalization of CDs by \u003cem\u003eP. indica\u003c/em\u003e was confirmed by autofluorescence of CDs. We found uptake of CDs in \u003cem\u003eP. indica\u003c/em\u003e was time and concentration-dependent. The maximum visible fluorescence was observed in fungal biomass \u003cem\u003etreated\u003c/em\u003e with 400\u0026micro;g/ml CDs for 2h. \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e We found that any increase in the concentration of CDs beyond 400\u0026micro;g/ml or time beyond 2h resulted in an actual decrease in autofluorescence. This could be because longer incubations or higher concentrations of the CDs likely results in a reverse flow from fungal cells back into the suspension.\u003c/p\u003e \u003cp\u003eOur SEM data shows that CDs did not have any adverse effects on \u003cem\u003eP. indica\u003c/em\u003e morphology because in both, the \u003cem\u003etreated\u003c/em\u003e and \u003cem\u003euntreated\u003c/em\u003e fungal biomass, the pear-shaped structure of fungal spores was found to be intact (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e\u0026amp;B)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLyophilization of\u003c/b\u003e \u003cb\u003etreated\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003euntreated\u003c/b\u003e \u003cb\u003efungal biomass\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003etreated\u003c/em\u003e as well as \u003cem\u003euntreated\u003c/em\u003e fungal biomass was lyophilized to a fine pale-yellow powder under sterile conditions. \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e Multiple batches were run to generate aliquots of the powder. This lyophilized preparation was checked for viability on agar plates as well as in broth at different time points, immediately, one week, two weeks and one month after lyophilization. All the lyophilized preparations grew to maturity in 21 days just like the routine batch cultures of \u003cem\u003eP. indica\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eLyophilized\u003c/b\u003e \u003cb\u003eP. indica\u003c/b\u003e \u003cb\u003esuccessfully colonized plants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe observed that while roots that were not exposed to \u003cem\u003eP. indica\u003c/em\u003e (controls) did not show, as expected, presence of spores \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, colonization percentage in plant roots exposed to fresh \u003cem\u003eP. indica\u003c/em\u003e was ~\u0026thinsp;90% \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Most encouragingly, the colonization efficiency of lyophilized \u003cem\u003eP. indica\u003c/em\u003e (\u003cem\u003euntreated\u003c/em\u003e) in plants exceeded 70% in 40 days \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. This set of plants did not reach any higher colonization percentages possibly because of the time lag lyophilized \u003cem\u003eP. indica\u003c/em\u003e may have taken to grow from desiccated state into vegetative stage before further entering into sporulation. It was also observed that many of the root sections where \u003cem\u003etreated\u003c/em\u003e biomass was used showed less colonization i.e., 75% \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. The reason could be the same as the premise taken for this study, that, CDs reduce the dependence of fungus on plant host for fixed C which in turn either delays its interaction or prolongs its vegetative stage in the host. Nonetheless, further in-depth studies at a molecular level are needed to understand the reason behind extensive hyphal growth before sporulation inside the host roots, when fungus is treated with CDs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLyophilized\u003c/b\u003e \u003cb\u003eP. indica\u003c/b\u003e \u003cb\u003eenhances growth of the colonized plants\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe found that plants colonized with the fresh cultures of \u003cem\u003eP. indica\u003c/em\u003e, lyophilized \u003cem\u003euntreated P. indica\u003c/em\u003e and lyophilized \u003cem\u003etreated P. indica\u003c/em\u003e showed morphologically better growth and biomass in comparison to the non-colonized plants (controls) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D\u003cb\u003e)\u003c/b\u003e. Growth parameters were analysed for dry weight, shoot length, root length and branch numbers.\u003c/p\u003e \u003cp\u003eThe dry weight of plants grown with lyophilized \u003cem\u003euntreated\u003c/em\u003e fungal biomass as well as plants grown with fresh fungal cultures showed an equal increase of 2.5-fold when compared to plants grown without any \u003cem\u003eP. indica\u003c/em\u003e (control). The dry weight of plants grown with lyophilized \u003cem\u003etreated\u003c/em\u003e fungal biomass showed a 2.3-fold increase when compared to plants grown without any \u003cem\u003eP. indica\u003c/em\u003e (control). All these differences were found to be statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe average shoot length of plants grown with lyophilized \u003cem\u003euntreated\u003c/em\u003e fungal biomass and those grown with fresh fungal culture increased by 2.1 and 2.3-fold, respectively, when compared to plants grown without any \u003cem\u003eP. indica\u003c/em\u003e (control). These differences were found to be statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) Shoot length of those grown with lyophilized \u003cem\u003etreated\u003c/em\u003e fungal biomass showed only a 2-fold increase over plants grown without any \u003cem\u003eP. indica\u003c/em\u003e (control). This difference was also found to be statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe average root length of plants grown with lyophilized \u003cem\u003euntreated\u003c/em\u003e fungal biomass and those grown with fresh fungal culture increased by 1.7-fold and 1.9-fold, respectively, when compared to plants grown without any \u003cem\u003eP. indica\u003c/em\u003e (control). These differences were found to be statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). However, root length of those grown with lyophilized \u003cem\u003etreated\u003c/em\u003e fungal did not show any statistically significant increase \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNumber of branches in plants grown with lyophilized \u003cem\u003euntreated\u003c/em\u003e fungal biomass and those grown with fresh fungal culture increased equally by 1.4-fold when compared to plants grown without any \u003cem\u003eP. indica\u003c/em\u003e (control). These differences were found to be statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, number of branches in plants grown with lyophilized \u003cem\u003etreated\u003c/em\u003e fungal did not show any statistically significant increase \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003eD\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eP. indica\u003c/em\u003e has great potential to be used as a biofertilizer in increasing biomass and productivity of crops when compared to other mycorrhiza including AMFs. Though found natively in soils, \u003cem\u003eP. indica\u003c/em\u003e faces all the usual challenges presented by a complex ecosystem as soil, towards growth and colonization of its host. Therefore, to be able to put it to maximally beneficial use in agriculture, we suggest bio-augmentation of this endophyte in agricultural farmlands. However, field application requires a suitable formulation that is eco-friendly, sustainable, easy to add in the soil, can increase the shelf life of the fungus to give it sustained longevity in the absence of a host and can also be scaled up for commercial use. In the present study, we have successfully attempted to make such a working formulation by lyophilizing the fungal biomass harvested from laboratory grown pure cultures of \u003cem\u003eP. indica\u003c/em\u003e. Further, to strengthen its stability, viability and efficiency after addition in the soil, we have integrated nano-sized, photostable, non-toxic and biocompatible CDs inside the fungal biomass before lyophilization. Indeed, as our results show, lyophilized \u003cem\u003eP. indica\u003c/em\u003e is revivable in laboratory cultures and not only thrives just as well as the routine fresh batches, there is also no change in either the growth rate or morphology of the spores. Moreover, like fresh cultures, the lyophilized formulation is also able to colonize the chickpea plants efficiently and is comparable to it in its positive impact on plant growth and biomass. Further, while lyophilization with CDs inside the fungal biomass did show good revival and colonization, our results showed that it is the lyophilized preparation without CDs which has the desirable impact on plant growth and biomass. However, before making any conclusions it will be worthwhile to expand this study over a longer period of plant growth to also include the reproductive phase and fruiting. In a parallel, yet unpublished study done in our laboratory it has been shown that addition of axenically grown \u003cem\u003eP. indica\u003c/em\u003e does not alter the microbial diversity of the soil, therefore giving a more promising prospect towards its bioformulation. This preliminary work, to the best of our knowledge also a first for lyophilization of \u003cem\u003eP. indica\u003c/em\u003e, certainly entails optimization of shelf-life, survival, colonization efficiency and larger \u003cem\u003ein-situ\u003c/em\u003e experimental cohorts before scaling-up the process for bio-augmentation on commercial scale. The larger aim is utilizing the potential of \u003cem\u003eP. indica\u003c/em\u003e in sustainable organic agriculture to achieve food security, enhanced productivity and health of food crops in the face of climate change, as also realized in United Nations SDGs 2,3 and 13.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFungal culture\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. indica\u003c/em\u003e culture was a gift from Prof. Ajit Varma, Amity University, Noida, UP, India. \u003cem\u003eP. indica\u003c/em\u003e cultures was routinely maintained in both, KF culture medium broth (20 g L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Glucose, 2 g L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Peptone, 1 g L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Yeast Extract, 1 g L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Casamino Acid, 520 mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e KCl, 520mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e MgSO\u003csub\u003e4\u003c/sub\u003e7H\u003csub\u003e2\u003c/sub\u003eO, 1.52g L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 55mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e ZnSO\u003csub\u003e4\u003c/sub\u003e7H\u003csub\u003e2\u003c/sub\u003eO, 27mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 12 mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e MnCl\u003csub\u003e2\u003c/sub\u003e4H\u003csub\u003e2\u003c/sub\u003eO, 4 mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e CoCl\u003csub\u003e2\u003c/sub\u003e6H\u003csub\u003e2\u003c/sub\u003eO, 4 mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e CuSO\u003csub\u003e4\u003c/sub\u003e5H\u003csub\u003e2\u003c/sub\u003eO, 16.2 mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e CaCl\u003csub\u003e2\u003c/sub\u003e, 16.2 mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e FeCl\u003csub\u003e3\u003c/sub\u003e, 0.5 mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Biotin, 0.5 mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Nicotinamide, 1 mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Pyridoxal Phosphate, 1 mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Aminobenzoic Acid, 2.5 mg L\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Riboflavin and 2% Agar; pH 6 0.5) as well as on agar plates.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eP. indica\u003c/em\u003e was grown in liquid KF medium in a 250-ml culture flasks under constant shaking at 110 rpm, and at 30 \u003csup\u003e0\u003c/sup\u003eC for 7\u0026ndash;9 days in a metabolic shaker (Orbitek shaker, India).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSynthesis and characterization of carbon dots (CDs)\u003c/h3\u003e\n\u003cp\u003eTo synthesize CDs, citric acid (0.52M) and 20.8mM L-cysteine were dissolved in 10ml ultrapure water as described.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e The clear solution was placed in a microwave for 3min at 700W. After allowing the yellow precipitate to cool at room temperature, 5ml ultrapure water was added to dissolve it, yielding a yellow color solution with the desired CDs. To remove impurities, formulation was dialyzed against pure water for three days using a dialysis membrane (Spectra/Por MWCO 1000Da). Finally, a solution containing CDs was oven-dried at 80\u0026deg;C, yielding 0.157g CDs. From this 1mg CDs were dispersed in 1ml ultrapure water before being used. The UV-Vis and fluorescence spectra of CDs were measured using a T90\u0026thinsp;+\u0026thinsp;UV/vis spectrometer (PG Instruments Ltd, UK). The fluorescence behavior of CDs was examined using a Nikon real-time confocal laser scanning microscope (A1R). High-resolution Transmission Electron Microscopy (HRTEM, JEOL JEM-2200 FS, JAPAN) was used to characterize the size and crystallinity of CDs. Since the functional group in the CDs correspond to citric acid and L-cysteine, the Fourier Transform Infrared Spectroscopy (FTIR) confirmed that the CDs include the aromatic ring of carbon and carboxylic acid, mainly amine and sulfur.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInternalization of CDs by\u003c/b\u003e \u003cb\u003eP. indica\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. indica\u003c/em\u003e being a root endophyte fungus is dependent on its plant host for fixed carbon, which implies, that without a host the endophyte will either have limited survival or may not survive altogether. Therefore, laboratory synthesized CDs, as a source of fixed carbon, were internalized in the lyophilized fungal biomass in a bid to ensure its survival in the field till the time it can find and colonize the plant host roots. \u003cem\u003eP. indica\u003c/em\u003e fungal biomass was harvested from culture agar plates by flooding the plates with liquid KF broth and carefully scraping the fungus with a sterile glass spreader in a centrifuge tube. Small hyphal fragments were removed by centrifugation. The supernatant was discarded and pellet obtained (\u003cem\u003euntreated\u003c/em\u003e fungal biomass) was diluted with sterile distilled water. The number of spores was quantified under a light microscope using a hemacytometer (Neubauer Scientific, PA, USA). Concentration and time-dependent study was performed to check the uptake of CDs. To investigate at what concentration CDs uptake \u003cem\u003eP. indica\u003c/em\u003e varying concentrations of CDs (1000, 800, 400, 200, 100, 50 and 25\u0026micro;g/ml) were added to the fungal biomass in separate wells and incubated for various time periods (0, 1, 2, 4 and 6h) (\u003cem\u003etreated\u003c/em\u003e fungal biomass). Autofluorescence in spores was visualized by Confocal Laser Scanning Microscope (Olympus FluoView\u003csup\u003e\u0026trade;\u003c/sup\u003e FV1000) at 40X excited by 405nm laser. Further, both, \u003cem\u003etreated\u003c/em\u003e and \u003cem\u003euntreated\u003c/em\u003e samples were viewed under Scanning Electron Microscope (SEM) (EVO 18 special edition, ZEISS) to check for any morphological changes post internalization of CDs. To do SEM, samples were rinsed with PBS buffer, placed in fixative (2.5% glutaraldehyde) for 4h, washed again with PBS buffer, and stored overnight at 4\u0026deg;C. Further, samples were dehydrated in an ascending series of 10% increments from 50\u0026ndash;100% ethanol for 20min each before being kept for drying. \u003cem\u003eUntreated\u003c/em\u003e fungal biomass served as control for making comparisons.\u003c/p\u003e \u003cp\u003eA viability check was done to test the viability of fungal biomass after internalization of CDs. For this purpose, variously \u003cem\u003etreated\u003c/em\u003e fungal biomass defined by different concentrations and time was grown on separate KF agar plates and liquid broth at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003e0\u003c/sup\u003eC for 10 days. Fungal biomass incubated only with distilled water for 2h was used as control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLyophilization of\u003c/b\u003e \u003cb\u003eP. indica\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor this purpose, 100ml broth culture of both \u003cem\u003euntreated\u003c/em\u003e and \u003cem\u003etreated\u003c/em\u003e fungal biomass were separately sieved, washed using sterile distilled water and transferred to sterile lyophilization tubes. The tubes were kept frozen in liquid nitrogen for 2min. Tubes were uncapped and covered with sterile aluminium foil and loaded on the lyophilizer (Operon FDB 5503) at temperature and vacuum of -57\u003csup\u003e0\u003c/sup\u003eC and 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e Torr, respectively for 24h. The lyophilized powder was stored in batches of 10mg in sterile capped vials at 4\u003csup\u003e0\u003c/sup\u003eC. To test the viability of lyophilized preparations, 10mg each of \u003cem\u003euntreated\u003c/em\u003e and \u003cem\u003etreated\u003c/em\u003e lyophilized fungal biomass were inoculated separately on KF agar plates (incubated at 30˚C in an incubator) and in KF broth incubated at 30˚C, 111 rpm in a metabolic shaker (Orbitek shaker, India).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInteraction of\u003c/b\u003e \u003cb\u003eP\u003c/b\u003e. \u003cb\u003eindica\u003c/b\u003e \u003cb\u003ewith plant\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo find out if lyophilization or the CDs have any effect on the ability of \u003cem\u003eP. indica\u003c/em\u003e to interact with the host plant, four sets of pots were prepared \u003cb\u003e(1)\u003c/b\u003e plants were grown without any \u003cem\u003eP. indica\u003c/em\u003e \u003cb\u003e(2)\u003c/b\u003e plants were grown only with fresh cultures of \u003cem\u003eP. indica\u003c/em\u003e mixed with soil at final concentration of 0.1% (w/w) \u003cb\u003e(3)\u003c/b\u003e plants were grown with lyophilized \u003cem\u003euntreated\u003c/em\u003e fungal biomass mixed with soil at final concentration of 0.1% (w/w) (\u003cb\u003e4\u003c/b\u003e) plants were grown with lyophilized \u003cem\u003etreated\u003c/em\u003e fungal biomass mixed with soil at final concentration of 0.1% (w/w).\u003c/p\u003e \u003cp\u003eChickpea plant (\u003cem\u003eCicer arietinum\u003c/em\u003e) seeds were purchased from Indian Agriculture Research Institute, Pusa, New Delhi. To grow chickpea plants, seeds were surface sterilized. To do this, approx. 50 seeds were soaked in 100ml sterile distilled water for 1h. Thereafter, they were washed in consecutive steps serially with 10% SDS, 20ml 90% ethanol (for 45sec), 50ml sterile distilled water (washed thrice), 50ml 2% (v/v) NaOCl (for 2min), 50ml sterile distilled water (washed thrice), 50ml 70% ethanol (for 45sec) and finally five times with 50ml of sterile distilled water. Germinated seedlings were placed in pots filled with a mixture of sterile sand and soil in the ratio of 3:1 (garden soil from the Jawaharlal Nehru University campus and acid washed riverbed sand). Plants were supplied with half strength modified Hoagland solution (5mM KNO\u003csub\u003e3\u003c/sub\u003e, 5mM Ca(NO)\u003csub\u003e2\u003c/sub\u003e, 2Mm MgSO\u003csub\u003e4\u003c/sub\u003e, 10 M KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 10 M MgCl\u003csub\u003e2\u003c/sub\u003e, 4M ZnSO\u003csub\u003e4\u003c/sub\u003e, 1M CaSO\u003csub\u003e4\u003c/sub\u003e, 1M NaMoO\u003csub\u003e4\u003c/sub\u003e, and 50 M H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e) weekly. All the pots were grown for 40 days in a Controlled Environment Chamber Facility (CECF) maintained at daily temperature of 29\u0026deg;\u0026plusmn;2\u0026deg;C. Plants were grown under controlled conditions in a greenhouse with an 8-h light:16-h-dark photoperiod (light intensity of 1,000 lux, using high-intensity fluorescent light) at a temperature cycle of 25C/18C with a relative humidity 60\u0026ndash;70%.They were watered as needed.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistochemical analysis\u003c/h2\u003e \u003cp\u003eHistochemical analysis of the roots of plants from all sets was done to check colonization. Root sections of ~\u0026thinsp;1cm were placed in 2ml centrifugation tubes. They were incubated in 1ml 10% KOH solution at 60\u0026deg;C for 15min to soften the plant material and thereafter neutralized in 1N HCL for 5min. The samples were then washed thrice with sterile distilled water and stained in 1ml 0.02% trypan blue at 60\u0026deg;C for 15 min. Samples were de-stained with 1ml 50% lactophenol for 15\u0026ndash;60 min. and mounted on a slide, under coverslip for observation with light microscope. Percentage colonization for the inoculated plants was calculated using the following formula as described previously.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\varvec{\\%}\\:\\varvec{C}\\varvec{o}\\varvec{l}\\varvec{o}\\varvec{n}\\varvec{i}\\varvec{z}\\varvec{a}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}=\\frac{\\varvec{r}\\varvec{o}\\varvec{o}\\varvec{t}\\:\\varvec{s}\\varvec{e}\\varvec{c}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}\\:\\varvec{w}\\varvec{i}\\varvec{t}\\varvec{h}\\:\\varvec{P}.\\varvec{i}\\varvec{n}\\varvec{d}\\varvec{i}\\varvec{c}\\varvec{a}}{\\varvec{T}\\varvec{o}\\varvec{t}\\varvec{a}\\varvec{l}\\:\\varvec{r}\\varvec{o}\\varvec{o}\\varvec{t}\\:\\varvec{s}\\varvec{e}\\varvec{c}\\varvec{t}\\varvec{i}\\varvec{o}\\varvec{n}\\varvec{s}\\:\\varvec{o}\\varvec{b}\\varvec{s}\\varvec{e}\\varvec{r}\\varvec{v}\\varvec{e}\\varvec{d}}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of Biomass\u003c/h3\u003e\n\u003cp\u003eWell-grown chickpea plants from all the four sets were harvested for analyses after 40 days of growth. The pots were tapped lightly all across the outside to loosen the soil inside. Scraper was used to scrape the soil sticking on the inside walls. Each pot was held and gently turned upside down to remove the intact plant with its soil. The bulk soil around the roots was removed very carefully with hands so as to not damage the roots of the plant. The plants were washed gently multiple times to remove residual traces of soil sticking to the root. Washed plants were stretched lengthwise on blotting sheets to remove excess water. Plants from all the four sets were kept distinct on separate blotting sheets. Dry weight, number of branches, shoot and root lengths of plants from each of these four sets were recorded. To measure the dry weight, harvested plants were washed with water, and oven dried at 80\u0026deg;C for 72 h.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism was used to conduct the unpaired t test to check the significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMD and AKJ initiated the project. BY , SS, RPS and PM have performed all the experiments. MS is written by MD. AKJ, BY and PS.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eMD gratefully acknowledges DST-PURSE and UGC-UPE-II, New Delhi, India, for financial help. BY is thankful to JNU for providing research fellowship and to AIRF, JNU for SEM analysis, confocal and fluorescence microscopy. The authors are very thankful to Shalini Sinha for her help with statistical analysis.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request\"\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eVerma, S., Varma, A., Rexer, K.H., Hassel, A., Kost, G., Sarbhoy, A., Bisen, P., B\u0026uuml;tehorn, B., \u0026amp; Franken, P. (1998). \u003cem\u003ePiriformospora indica\u003c/em\u003e, gen. et sp. nov., a new root-colonizing fungus. \u003cem\u003eMycologia\u003c/em\u003e, 90(5), 896-903.\u003c/li\u003e\n \u003cli\u003eWaller, F., Achatz, B., Baltruschat, H., Fodor, J., Becker, K., Fischer, M., Heier, T., H\u0026uuml;ckelhoven, R., Neumann, C., von Wettstein, D., Franken, P., \u0026amp; Kogel, K. H. (2005). The endophytic fungus \u003cem\u003ePiriformospora indica\u003c/em\u003e reprograms barley to salt-stress tolerance, disease resistance, and higher yield. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e, 102(38), 13386-13391\u003c/li\u003e\n \u003cli\u003eFranken, P. (2012). The plant strengthening root endophyte \u003cem\u003ePiriformospora indica\u003c/em\u003e: potential application and the biology behind. \u003cem\u003eApplied Microbiology Biotechnology\u003c/em\u003e, \u003cem\u003e96\u003c/em\u003e(6), 1455-1464.\u003c/li\u003e\n \u003cli\u003eAnsari, M. W., Trivedi, D. K., Sahoo, R. K., Gill, S. S., \u0026amp; Tuteja, N. (2013). A critical review on fungi mediated plant responses with special emphasis to \u003cem\u003ePiriformospora indica\u003c/em\u003e on improved production and protection of crops. \u003cem\u003ePlant Physiology Biochemistry\u003c/em\u003e, \u003cem\u003e70\u003c/em\u003e, 403-410.\u003c/li\u003e\n \u003cli\u003ePrasad, R., Kamal, S., Sharma, P. K., Oelm\u0026uuml;ller, R., \u0026amp; Varma, A. (2013).\u0026nbsp;Root endophyte \u003cem\u003ePiriformospora indica\u003c/em\u003e DSM 11827 alters plant morphology, enhances biomass and antioxidant activity of medicinal plant \u003cem\u003eBacopa monniera\u003c/em\u003e. \u003cem\u003eJournal of basic Microbiology\u003c/em\u003e, \u003cem\u003e53\u003c/em\u003e(12), 1016-1024.\u003c/li\u003e\n \u003cli\u003eJogawat, A., Vadassery, J., Verma, N., Oelm\u0026uuml;ller, R., Dua, M., Nevo, E., \u0026amp; Johri, A. K. (2016). PiHOG1, a stress regulator MAP kinase from the root endophyte fungus \u003cem\u003ePiriformospora indica\u003c/em\u003e, confers salinity stress tolerance in rice plants. \u003cem\u003eScientific Reports\u003c/em\u003e, 6(1), 1-15.\u003c/li\u003e\n \u003cli\u003eGill, S.S., Gill, R., Trivedi, D.K., Anjum, N.A., Sharma, K.K., Ansari, M.W., Ansari, A.A., Johri, A.K., Prasad, R., Pereira, E., Varma, A. \u0026amp; Tuteja, N. (2016). \u003cem\u003ePiriformospora indica\u003c/em\u003e: potential and significance in plant stress tolerance. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e, 332.\u003c/li\u003e\n \u003cli\u003eKumar, M., Yadav, V., Tuteja, N., \u0026amp; Johri, A. K. (2009). Antioxidant enzyme activities in maize plants colonized with \u003cem\u003ePiriformospora indica\u003c/em\u003e. \u003cem\u003eMicrobiology\u003c/em\u003e, 155(3), 780-790.\u003c/li\u003e\n \u003cli\u003eNarayan, O.P., Verma, N., Singh, A.K., Oelm\u0026uuml;ller, R., Kumar, M., Prasad, D., Kapoor, R., Dua, M., \u0026amp; Johri, A.K. (2017). Antioxidant enzymes in chickpea colonized by \u003cem\u003ePiriformospora indica\u003c/em\u003e participate in defense against the pathogen \u003cem\u003eBotrytis cinerea\u003c/em\u003e. \u003cem\u003eScientific Reports\u003c/em\u003e, 7(1), 1-11.\u003c/li\u003e\n \u003cli\u003eNarayan, O. P., Verma, N., Jogawat, A., Dua, M., \u0026amp; Johri, A. K. (2021). Sulfur transfer from the endophytic fungus \u003cem\u003eSerendipita indica\u003c/em\u003e improves maize growth and requires the sulfate transporter SiSulT. \u003cem\u003eThe Plant Cell\u003c/em\u003e, \u003cem\u003e33\u003c/em\u003e(4), 1268-1285.\u003c/li\u003e\n \u003cli\u003eVerma, N., Narayan, O. P., Prasad, D., Jogawat, A., Panwar, S. L., Dua, M., \u0026amp; Johri, A. K. (2022). Functional characterization of a high‐affinity iron transporter (PiFTR) from the endophytic fungus \u003cem\u003ePiriformospora indica\u003c/em\u003e and its role in plant growth and development. \u003cem\u003eEnvironmental Microbiology\u003c/em\u003e, 24(2), 689-706.\u003c/li\u003e\n \u003cli\u003eZuccaro, A., Lahrmann, U., G\u0026uuml;ldener, U., Langen, G., Pfiffi, S., Biedenkopf, D., Wong, P., Samans, B., Grimm, C., Basiewicz, M., Murat, C., Martin, F., \u0026amp; Kogel, K. H. (2011). Endophytic life strategies decoded by genome and transcriptome analyses of the mutualistic root symbiont \u003cem\u003ePiriformospora indica\u003c/em\u003e. \u003cem\u003ePLoS Pathogens\u003c/em\u003e, 7(10), e1002290.\u003c/li\u003e\n \u003cli\u003ePrakash, O., Nimonkar, Y., \u0026amp; Shouche, Y. S. (2013). Practice and prospects of microbial preservation. \u003cem\u003eFEMS Microbiology Letters\u003c/em\u003e, \u003cem\u003e339\u003c/em\u003e(1), 1-9.\u003c/li\u003e\n \u003cli\u003eMishra, S., Keswani, C., Abhilash, P. C., Fraceto, L. F., \u0026amp; Singh, H. B. (2017). Integrated approach of agri-nanotechnology: challenges and future trends. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e, 471.\u003c/li\u003e\n \u003cli\u003eHe, X., Deng, H., \u0026amp; Hwang, H. M. (2019). The current application of nanotechnology in food and agriculture. \u003cem\u003eJournal of Dood \u0026nbsp;Drug Analysis\u003c/em\u003e, \u003cem\u003e27\u003c/em\u003e(1), 1-21.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePhadke, C., Mewada, A., Dharmatti, R., Thakur, M., Pandey, S., \u0026amp; Sharon, M. (2015). Biogenic synthesis of fluorescent carbon dots at ambient temperature using \u003cem\u003eAzadirachta indica\u003c/em\u003e (Neem) gum. \u003cem\u003eJournal of Fluorescence\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e(4), 1103-1107.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKasibabu, B. S. B., D\u0026rsquo;souza, S. L., Jha, S., \u0026amp; Kailasa, S. K. (2015). Imaging of bacterial and fungal cells using fluorescent carbon dots prepared from carica papaya juice. \u003cem\u003eJournal of Fluorescence\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e(4), 803-810.\u003c/li\u003e\n \u003cli\u003eSinghal, R., \u0026amp; Kalra, V. (2017). Cobalt nanoparticle‐embedded porous carbon nanofibers with inherent N‐and F‐doping as binder‐free bifunctional catalysts for oxygen reduction and evolution reactions. \u003cem\u003eChemPhysChem\u003c/em\u003e, \u003cem\u003e18\u003c/em\u003e(2), 223-229.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBasiru, S., Mwanza, H. P., \u0026amp; Hijri, M. (2021).\u0026nbsp;Analysis of arbuscular mycorrhizal fungal inoculant benchmarks. \u003cem\u003eMicroorganisms\u003c/em\u003e. 2021; 9: 81.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMaholiya, A., Ranjan, P., Khan, R., Murali, S., Nainwal, R. C., Chauhan, P. S., Sathish, N., Chaurasia, J. P. \u0026amp; Srivastava, A.K. (2023). An insight into the role of carbon dots in the agriculture system: a review. \u003cem\u003eEnvironmental Science: Nano\u003c/em\u003e, 10(4), 959-995.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"21\"\u003e\n \u003cli\u003eJanus, Ł., Piątkowski, M., Radwan-Pragłowska, J., Bogdał, D., Matysek, D. (2019). Chitosan-Based Carbon Quantum Dots for Biomedical Applications: Synthesis and Characterization. \u003cem\u003eNanomaterials (Basel)\u003c/em\u003e, 16;9(2):274.\u003c/li\u003e\n\u003c/ol\u003e\n\u003col start=\"22\"\u003e\n \u003cli\u003eSri, S., Kumar, R., Panda, A. K., \u0026amp; Solanki, P. R. (2018). Highly biocompatible, fluorescence, and zwitterionic carbon dots as a novel approach for bioimaging applications in cancerous cells. \u003cem\u003eACS Applied Materials \u0026amp; Interfaces\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(44), 37835-37845.\u003c/li\u003e\n \u003cli\u003eHill, T. W., \u0026amp; Kafer, E. (2001). Improved protocols for Aspergillus minimal medium: trace element and minimal medium salt stock solutions. \u003cem\u003eFungal Genetics Reports\u003c/em\u003e, \u003cem\u003e48\u003c/em\u003e(1), 20-21.\u003c/li\u003e\n \u003cli\u003eLiu, Q., Zhang, N., Shi, H., Ji, W., Guo, X., Yuan, W., \u0026amp; Hu, Q. (2018). One-step microwave synthesis of carbon dots for highly sensitive and selective detection of copper ions in aqueous solution, \u003cem\u003eNew Journal of Chemistry\u003c/em\u003e, 42 , 3097-3101.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Piriformospora indica, lyophilization, carbon dots, Cicer arietinum, SDGs","lastPublishedDoi":"10.21203/rs.3.rs-5241436/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5241436/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRoot endophyte fungus \u003cem\u003ePiriformospora indica\u003c/em\u003e can be axenically cultivable, is easily obtained in pure cultures in the laboratory, and therefore, can be developed as a biofertilizer for bioaugmentation. In this study, an effort towards sustainable organic agriculture, we have made two completely eco-friendly, biogenic and biocompatible lyophilized formulations of \u003cem\u003eP. indica\u003c/em\u003e, one, without carbon dots and second, with carbon dots. Scanning Electron Microscopy (SEM) observations, viability assays and colonization efficiency of both the formulations revealed that lyophilization does not in any way alter either the morphology, growth or colonization ability of the endophyte. The formulations were also tested for impact on growth of \u003cem\u003eCicer arietinum\u003c/em\u003e plants in experimental set up. The plants were analysed for changes in dry weight, shoot length, root length and branch numbers. While the dry weight increased by a maximum of 1.9-fold; average shoot length increased by 1.4-fold; average root length by 1.7-fold; and number of branches by 1.4-fold, when compared to plants grown without any \u003cem\u003eP. indica\u003c/em\u003e. These increases were found to be statistically significant. We identify this work as a significant step towards optimizations and production of this formulation on a larger scale. We also perceive this attempt as commitment towards United Nations SDGs 2,3 and 13.\u003c/p\u003e","manuscriptTitle":"Lyophilized preparation of root endophyte fungus Piriformospora indica with carbon-based nanomaterial (carbon dots) successfully colonizes the plant host, Cicer arietinum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-13 10:22:07","doi":"10.21203/rs.3.rs-5241436/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"239d1013-8dd7-4a35-89cf-bf74c9fdf701","owner":[],"postedDate":"November 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40039403,"name":"Biological sciences/Microbiology"},{"id":40039404,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2025-04-21T12:38:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-13 10:22:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5241436","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5241436","identity":"rs-5241436","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}