From Piriformospora indica to Rootonic : A review

Piriformospora indica (Hymenomycetes, Basidiomycota) is a cultivable endophyte that colonizes roots and has been extensively studied. P. indica has multifunctional activities like plant growth promoter, biofertilizer, immune-modulator, bioherbicide, phytoremediator, etc. Growth promotional characteristics of P. indica have been studied in enormous number of plants and majority of them have shown highly significant outcomes. Certain secondary metabolites from the fungus are reasons behind such promising outputs. Effect of P. indica has been studied on more than 150 plants. Promising outputs of laboraatory experiments and small field trials indicated the need for its mass cultivation and usage. For field trials, a formulation “Rootonic” was prepared by mixing P. indica biomass in magnesium sulphite. The quantity of formulation (Rootonic) to be used per acre of land for maximum productivity has also been standardized for about 50 plants. P. indica has proved to be highly beneficial endophyte with high efficacy in field. This article is a review on our journey from P. indica to “Rootonic”.


INTRODUCTION
Piriformospora indica was discovered by Prof Dr. Ajit Varma and his colleagues in Thar Desert of Western India in 1992 from the root system of several xerophytic plants (Varma et al., 1999;Verma et al., 1998).It belongs to Hymenomycetes, Basidiomycota.A new family Sebacinaceae and new order Sebacinales Glomeromycota was created for this fungus due to its unique features (Weiß et al., 2004;Qiang et al., 2011).This is a very unique symbiotic fungus which not only promotes plant growth but also has other multifunctional activities such as plant growth promoter, bio-protectant, bio-pesticide, helps in enhanced flowering and fruiting etc.Its properties have been patented in Germany (European Patent Office, Muenchen, Germany, Patent No. 97121440.8-2105, Nov. 1998) dating back to 1997.P. indica is deposited at the Deutsche Sammlung fu¨r Mikroorganismen und Zellkulturen, Braunschweig, Germany (DSM 11827).It promotes plant growth, increases the resistance of colonized plants against fungal pathogens and increases their stress tolerance (Harman 2011;Varma et al., 2012a).
Colonization by P. indica increases nutrient uptake, allows plants to survive under water, temperature and salt-stresses, confers (systemic) resistance to toxins, heavy metal ions and pathogenic organisms and stimulates growth and seed production.The valuable secondary metabolites excreted by P. indica influence early seed germination, better plant productivity, early flowering, etc. Use of P. indica to increase desiccation tolerance in higher plants has been studied by Varma and his colleagues (2012c) and significant increase tolerance was achieved.
Genome wide study revealed that its genome is assembled into 1,884 scaffolds containing 2,359 contigs with an average read coverage of 22 and a genome size of 24.97 Mb.The estimated DNA content of P. indica nuclei ranges from 15.3 to 21.3 Mb.To assess the genome completeness of P. indica a blast search was performed with highly conserved core genes present in higher eukaryotes (Zuccaro et al., 2009).P. indica can be stably transformed by random genomic integration of foreign DNA and that it possesses a relatively small genome as compared to other members of the Basidiomycota (Zuccaro et al., 2011).
P. indica is a model organism used in mycorrhizal research, and its research outputs has been published in highly recognized journals like Nature, PNAS, Plos Pathogen, JBC, Plant Physiology, Molecular Plant Pathology, etc. Extensive research on this organism has brought it to an appreciable state and made its field trials and marketing possible.Objective of this review is to give a general view of journey of P. indica from laboratory to field and finally towards industrialization.

CULTIVATION AND MORPHOLOGICAL CHARACTERISTICS
Simple morphological features of P. indica contain hyphae and pear shaped large spores (Figure 2).It can be cultivated on basic defined medium at an optimum temperature of 25°C ± 2, pH 6.8.Maximum biomass production was obtained after 7 days incubation at 120 rpm (Varma et al., 2013).

FUNCTIONAL CHARACTERISTICS OF P. INDICA
Plant growth promotion P. indica promotes growth of plants of forestry, horticulture and agriculture importance.Numerous plants have been tested for the effect of P. indica on their growth and interestingly majority of them have shown beneficial effect.Few results of its effect on sugarcane, Pinus and potato are shown in Figure 3.It is important to note that in addition to enhancement of plant growth, the fungus also helps in enhancement of active ingredients in plants.In the case of Ratoon crop of sugarcane it was seen that plants not associated with P. indica turned yellow due to iron deficiency, whereas plants subjected to P. indica treatment remained green, indicating that the fungus also helps in iron transport.Almost 39% enhancement in iron content and 16% increase in sugar content were recorded in P. indica treated plants (Table 1).Noticeable increase in plant size and tuber size was observed in the case of Pinus and potato, respectively.

Value addition in spices and plants of pharmaceutical importance
Effect of P. indica has been studied on large number of spices and plants of medicinal importance.To name few are Curcuma longa, Spilanthus calva, Artemisia annua, Tridax procumbens, Abrus precatoriu, Bacopa monnieri, Coleus forskohlii, Adhatoda vasica, Withania somnifera, Chlorophytum tuberosum, Foeniculum vulgare, Linum album, Podophyllum sp., etc (Das et al., 2012).The organism has shown significant increase in concentration of active ingredients like curcumin, artemisnin, podophyllotoxin and bacoside leading to value addition to the plant.
Interaction of P. indica with C. longa resulted in approximately 21, 19 and 13% increase in essential oil, Curcumin and rhizome yield.Field trials showed that increase in rhizome yield after treatment with P. indica would benefit a farmer with Rs. 16,000/ (US $ 280.00) per hectare of land.It is also probable that healthy and shiny rhizomes would fetch better price.Increase in plant size, secondary metabolite release and increase ability to fight against infections was observed upon interaction of P. indica with A. annua, B. monnieri, etc (Figure 4).
The tissue culture results obtained were evaluated for field trial and similar promising outputs were obtained.Field trial of A. annua was done in Central India.Improved plant growth and 1.6 fold increased concentration of active ingredient artemisinin was observed.In the case of B. monnieri 3.5 fold increase in bacoside concentration was observed.
Interaction of Ephedra ciliata (used for treatment of hay fever, asthma etc.) with P. indica also led to enhanced growth of treated plants (Varma et al., 2013).

P. indica as bioprotectant, rejuvenate fruiting, promoting early flowering
Experimental data suggests that P. indica suppresses the growth of a large number of pathogens like Geaumannomyces graminis, Alternaria sp., Colletotrichum falcatum, Fusarium oxysporum, Fusarium udum, Rhizoctonia bataticola, R. solani, Sclerotinum rolfsii, Verticillium sp. and many more (Dolatabadi et al., 2011;Ghahfarokhy et al., 2011).Field trials on Lagenaria siceraria and Tagetes sp.showed that interaction with P. indica suppressed the infestation by plant pathogens including viruses (Figure 5).P. indica protecting a Tagetes sp. from mite infection has also been recorded.Rigorous fruiting was observed in kinnow (Citrus reticulata) plant after interaction with P. indica with a limitation that this fruiting was time dependant and seen only at the early stage.P. indica also possess unique characteristics of inducing early flowering.This property is seen in case of plants viz., tobacco, Coleus, Brassica, etc.. Orchids are well known ornamental plants and their cultivation is expensive.Another limiting factor to its growth is that among millions of tiny seeds, unfortunately 99% do not germinate and transform into fully grown and mature plants unless they establish contact with mycorrhiza.Its interaction with P. indica has shown increased seed germination.P. indica has also shown early seed generation and enhanced plant growth in highly valuable plants like Jatropha curcas and Populus deltoides (Kaldorf et al., 2005).

Unique features of P. indica
P. indica shows striking unique features upon interaction with various plants.Field trials to investigate activity of the fungus have been conducted in North, Central and in extreme cold deserts (Ladakh-Leh) of India.P. indica mixed with frozen Leh soil (temperature -30°C) showed significant results where all the fifteen seeds of Cichorium endivia germinated within 12-25 days (Singh and Varma,

2000)
. The germinated plantlets when transferred to macro-plots attained full growth and imparted better productivity than the control.In contrast, not a single seed broke dormancy in absence of the fungus was noticed.

Liquid fertilizer from P. indica
Culture filtrate (liquid fertilizer) of P. indica also acts as an excellent plant growth promoter.In order to prepare culture filtrate, P. indica was grown in broth and for 10 days followed by removal of biomass.Culture filtrate helped in early seed generation and early flowering as well (Figure 6).It was then interpreted that the fungus secretes some secondary metabolites that works as fertilizers (Bagde et al., 2010a(Bagde et al., , b, 2011)).

Mechanisms behind the unique action of P. indica
The fungal interactions are characterized by increase in efficiency of nutrient uptake from soil due to better hyphal penetration as compared to thicker root hairs.Plants deliver phosphor assimilates to fungus and during mycorrhizal associations; plants acquire phosphates from extensive network of extra radical hyphae.Interaction of P. indica with plant alters pathway for nitrogen metabolism, thereby helping plants to absorb more nitrogen.This phenolmenon gives higher resistance to water deficiency and makes plants drought tolerant.Enhanced growth of plants under mycorrhizal condition amplifies its starch requirement.This starch is obtained from deposition in root amyloplasts.Thus, it is interpreted that one of the major starch degrading enzymes, the glucan-water dikinase is activated by P. indica (Iris et al., 2010).
Uptake and transportation of important macronutrients like iron, zinc, manganese, copper, etc. are also regulated by the fungus.Along with this, beneficial phytohormones are synthesized by plants associated with P. indica.The cumulative effect of macro-micro-nutrients and phytohormones regulates plant metabolism leading to value addition, early flowering, plant growth promotion, etc. Massive proliferation of useful rhizospheric microorganisms sustains soil fertility (Varma et al., 2013).

STEP TOWARDS COMMERCIALIZATION
Laboratory scale data and field trials have evidently concluded that P. indica can be extensively used to increase plant growth quantitatively and qualitatively (Sahay andVarma, 1999, 2000;Rai et al., 2001).Main motive behind such huge previous experimental subject was further applying it in normal fields so that it is available to all.For making this target come true, a step towards commercialization was taken.Fungal biomass is mass cultivated in fermentor (Bagde et al., 2010b).These fungal biomass formulated with magnesium sulphite (carrier) was prepared (Figure 7).Most effective formulation was standardized to 2% (w/w).Moisture content and colony forming unit was maintained at 20% and 10 9 , respectively.This formulation is named "Rootonic".Seed treatment was done by mixing Rootonic to seed and incubating it under shade overnight.Protocol for seed treatment has been given as Figure 8.
Quantity of formulation required for seed treatment has been standardized for large number of plants.Details are included in Table 2.

Preliminary studies with nanoparticles
Nanotechnology has significant benefits on food and agriculture system.Preliminary work on interaction of nanoparticle embedded P. indica biomass with Broccoli has shown better growth promotional property as compared to the control (P.indica without nano material); as tested in our laboratory (Suman et al., 2010).

CONCLUSION
P. indica is a rewarding organism with its huge and distinguished properties.Colonization by P. indica increases nutrient uptake, allows plants to survive in drought, salt-stress and temperature stress.Excellent plant growth promotion, growth at extremes of climate and bio-protecting capability of the organisms has paved way for its varied field applications.Large field trials at various locations in India showed beneficial effects of P. indica on plant growth and development.Promising outputs of field trials showed that it should be used at large scale so that common farmers are benefited and finally countries economy is at profit.Increase in productivity of certain crop upon interaction with P. indica will increase total land usage.Enhanced field usage of the microorganisms requires its mass production.Field trials of the same are done by formulating biomass with powder and inoculating the mixture into root of plants.The formulation is termed "Rootonic".The journey from P. indica to Rootonic is exciting and very fulfilling.Large scale production and application of the product is still under process and we are looking forward to its commercialization soon.
Table 2. Quantity of formulation required for the seed treatment.(c.f.Varma et al., 2013).

Figure 3 .
Figure 3. (A) Effect of P. indica on sugarcane (Saccharum officinarum) cultivated in field at Punjab.(B) Growth promotion of Cyprus plants (pinus) upon interaction with P. indica grown on rocky sand.(C) Increases size and improves texture of potato plants upon interaction with P. indica, cultivated in field at Punjab.(c.f.Varma et al., 2013).

Figure 5 .
Figure 5. (1) Field trial on interaction of P. indica with Lagenaria siceraria showing how it suppresses the infestation by plant pathogens including viruses (upper); a magnified view of the same (lower).(2) Interaction of P. indica with plant of Tagetes sp.Note: Control plant with folded leaf due to mite infestation; no such symptoms were observed on treated plants (c.f.Varma et al., 2013).

Figure 7 .
Figure 7. Steps for the preparation of the formulation (c.f.Varma et al., 2013).
* °Bx (degree Brix) is the sugar content of an aqueous solution.One degree Brix is 1 g of sucrose in 100 g of solution.