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Research Article Microbiology

International Journal of Pharma and Bio Sciences ISSN

0975-6299

PLANT PROMOTING ACTIVITIES OF FUNGAL ENDOPHYTES ASSOCIATED

WITH TOMATO ROOTS FROM CENTRAL HIMALAYA, INDIA AND THEIR

INTERACTION WITH PIRIFORMOSPORA INDICA

NEHA CHADHA, RAM PRASAD AND AJIT VARMA*

Amity Institute of Microbial Technology, Amity University of Uttar Pradesh,

Sector 125, Noida- 201303, India

ABSTRACT

Endophytic fungi promote plant growth through the production of IAA, siderophore and phosphate solubilization. Out of thirty fungal endophytes isolated from tomato roots grown in a stressed, cold climate of Central Himalayan hills of district Pithoragarh, Uttarakhand, India twelve showed distinct morphological and study was undertaken, to understand the plant growth promoting properties possessed by these endophytes residing under stress environment. All the fungi showed the zone of phosphate solubilization, seven were positive for siderophore, four for HCN and three-showed ammonia production. IAA production was found maximum in Fusarium fusarioides. All the endophytic fungi showed remarkable phosphate solubilizing activity up to eight days with a decrease on the 10th day with maximum reporting from Trichoderma peudokoningii. All the isolates showed maximum siderophore production at 21th day after the inoculation. These isolated root endophytic fungi possess plant growth promoting properties and thus have a potential to be used for biofertilizer. KEYWORDS: Root endophytic fungi, Central Himalayas, Phosphate solubilisation, IAA, siderophore, Piriformospora indica

*Corresponding author

AJIT VARMA

Amity Institute of Microbial Technology, Amity University of Uttar Pradesh,

Sector 125, Noida- 201303, India

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INTRODUCTION

Root endophytic fungi live asymptomatically within the roots of many plant species without causing any harm to the host. Every plant harbor at least one or more endophytic fungi in the universe 1, 2. Endophytic fungi are ubiquitous and highly diverse group in their geographical location. It has been documented that endophytes appear more in tropical than temperate zone 3, 4. They are also found in xerophytic 5 and aquatic environment 6. Fungal endophytic diversity in extreme environmental conditions is of great interest and little attention has been given to these microbes because of their slow growth rate and strenuous handling. Pandey and Palni (2007) have reported that the microbial population decreases with an increase in the altitude 7. Endophytic fungi interact mutualistically with the host plant by producing metabolites that induces resistance and protection against various pathogens. They protect plant in drought 8 as well as in cold climatic conditions. Endophytes are considered as an important source of bioactive components 9. Plant growth promoting fungi (PGPF) promotes plant growth through the production of enzymes, phosphate solubilization10, 11, siderophore production 12 and antagonism to phytopathogens 13. The identification of endophytic fungi through their ability to produce IAA, phosphate, siderophore may be useful in increasing the production of crop plants 14 or in suppression of diseases by pathogen or pests. The Uttarakhand Himalayas, India is extremely rich in flora and fauna. Pithoragarh, situated in the middle hill region of Central Himalaya’s hills has an average altitude of about 5,000 feet. Annual rainfall is 1200 mm approximately and temperature ranges from maximum to minimum i.e. 35°C (summer) to -2°C (winter). Tomato (family Solanaceae) is the most widely cultivated vegetable crop next to potato; they are rich source of nutrition and play an important role in scavenging free radicals from the body. Lycopene present in tomato has an excellent antioxidant activity to fight against anti-aging, cancer and heart diseases 15. Plant growth promoting fungi (PGPF) are non- pathogenic saprophytes and

act as a biocontrol agent against pest and soil pathogens 13. Root colonized with PGPF can lead to systemic resistance in distal parts of the plant 16, 17. PGPF are crop specific and their significance on plant is limited because of variation in climate and soil inconsistency 18,

19. The endophytic fungi residing in cold conditions, where soil is deficient in organic matter and low moisture content possessed plant growth promoting activities and these fungi can be used in increased production of plant in the same environment 20, 21, 22, 23. The present study revealed the plant growth promoting properties of fungal isolates from tomato roots residing under cold stress condition of the central Himalayas of India.

MATERIALS AND METHODS (i) Sample collection and Isolation Tomato plants with the roots were randomly collected from Defence Institute of Bio-Energy Resources (DIBER) field station Pithoragarh district of Uttrakhand India (29°35’N 80°13’E) in the month of September 2012. The whole plant was kept in sterile polythene bags and transported to laboratory for further study. The roots were surface sterilized with 70% ethanol followed by 4% sodium hypochlorite with subsequent washing with distilled water. The roots were crushed with phosphate buffer saline solution pH 7 in mortar pestel and spread on Potato dextrose agar (PDA) plates. Distinct colonies were picked from the mother plate for isolation and were sub cultured on fresh PDA plates. (ii) Plant growth promoting characteristics

of fungal root endophytes The fungal root endophytes were analyzed for their plant growth promoting characteristics viz., production of IAA, ammonia, siderophores, HCN and their ability to solublize phosphates. (iii)Qualitative estimation of phosphate

solubilization on solid medium 24 The phosphate solubilizing activity of fungal isolates were tested on Pikovskaya’s solid agar medium with following composition; (Glucose 10g, Ca3(PO4)2 5g, (NH4)2SO4 0.5g,

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NaCl 0.2g, MgSO4.7H20 0.1g, KCl 0.2g, Yeast extract 0.5g, MnSO4.H2O 0.002g, FeSO4.7H2O 0.002g and Agar 15g in one litre of medium) with bromo phenol blue at a concentration of 0.003%. After incubation of seven days at 28°C, the formation of clear zone around the fungal hyphae indicates the ability of fungus to solubilize inorganic phosphorous. (iv)Quantitative estimation of phosphate

solubilization in liquid medium 25 Fungal disc was inoculated in Pikovskaya’s broth and culture was filter out using whatman filter paper 1 after two, four, six, eight and ten day after inoculation. 1ml supernatant was taken and 10 ml chloromolybdic acid to 40 ml distilled water was added. After this, 1ml chlorostanous acid was added to the above solution and distilled water was added to make up the volume to 50ml. The absorbance of the resulting blue color was measured at 600nm. The concentration of soluble phosphate was calculated by the standard curve of KH2PO4. (v) Quantitative determination of Indole

Acetic Acid (IAA) production 26 For indole acetic acid production, isolates were grown in 25ml Czapek medium (pH 6.5) with and without tryptophan. After 7 days of incubation, 1ml culture filtrate, 2ml Salkowski reagent (0.5M FeCl3 in 35% perchloric acid) was added and incubated for 20 minutes in dark. The reading was taken at 530 nm using UV spectrophotometer (UV 1800 Schimadzu). The concentration of indol was determined using IAA standard curve. (vi)HCN production 27 Fungal isolates were placed on potato dextrose agar medium amended with 4.4g glycine/l. Whatman filter paper no.1 soaked in 2% sodium carbonate in 0.5% picric acid solution was placed at the top of the plate. Plates were sealed with parafilm and incubated for HCN production for 7 days at

28ºC. Discoloration of the filter paper from yellow to orange brown after incubation was considered as positive result for cyanide. (vii) Qualitative estimation of Siderophore

production 28 Detection of siderophore production was carried on CAS medium with some modification. The oranges zone around the fungal colony was an indicative for siderophore production. (viii) Quantitative estimation of

Siderophore production 29 For Quantitative estimation, FeCl3 test was done on culture filtrate. Fungal endophytes were grown on Fries basal medium. The mycelium was separated by whatmann filter paper 1. The culture filtrate was harvested at 4-day interval for a period of 28 days. 1-5 ml of ferric chloride solution (3 %) was added to 1 ml of culture filtrate and absorbance was taken at 260nm. ` (ix)Ammonia production 30 Fungal disc were inoculated in 10 ml potato dextrose broth and incubated at 30°C for 7 days in a rotatory shaker. After incubation, 0.5 ml Nessler’s reagent was added to each tube. The development of yellow to brown color indicated a positive reaction for ammonia production (x) Interaction of root endophytic fungi

with Piriformospora indica 31 The effect of root endophytic fungi on Piriformospora indica was performed on Yeast Maltose Peptone (YMP) agar plates by the dual culture method. Disc of P. indica and fungal isolates (5 mm diameter) were placed on the same YMP agar media, 6 cm from each other. The plates were incubated at 25°C for 7 days. Radius of P. indica and other fungal isolates were recorded. All the experiments were done thrice with three replicates.

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Table 1 Plant growth promoting attributes of endophytic fungi isolated from tomato roots

Root endophytic fungi Phosphate

solubilization Siderophore HCN Ammonia

Chaetomium globosum ++ ++ + - Fusarium oxysporum + + - - Fusarium semitectum + - - - Fusarium solani + - - - Fusarium fusarioides ++ - + - Fusarium moniliforme + - - + Mucor sp + + + - Aspergillus niger gr. ++ + - + Aspergillus sp + - - - Aspergillus versicolor ++ ++ + - Mucor hiemalis + + - + Trichoderma peudokoningii rifai

+ ++ - -

(++ maximum intensity; + minimum intensity; - no observation)

Figure 1 Fungal endophytes shows clear zone on (a) Pikovskaya’s medium with bromophenol blue,

(b) siderophore production and (c) HCN production test

Figure 2 Interaction of Piriformospra indica with a) Fusarium oxysporum, b) Aspergillus niger, c) Mucor sp., d) Chaetomium globosum, e) Aspergillus versicolor, f) Mucor hiemalis, g) Trichoderma peudokoningii rifai. One disc of each root endophytic fungi was placed on left side and P. indica disc on right side of YMP medium.

a) b) c)

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Graph 1 Quantitative estimation of phosphate solubilization (µg ml-1) for five days

i.e. two, four, six, eight and ten days after inoculation

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Graph 2 Comparison of indole acetic acid (µg ml-1) by root fungal endophytes

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Graph 3 Siderophore production by fungal root endophytes at different stages of growth

Graph 4 Effect of different fungal isolates on the radial growth of Piriformospora indica

RESULTS AND DISCUSSION

Endophytic fungi are the diverse group of organism that live in symbiotic association with plants and may produce beneficial substances for host 32, 33. Ascomycota and basidiomycota are common fungal endophytes, which colonizes the roots of the plants both intracellularly and intercellularly 34. The association between fungal endophytes and host plant varies from negative to positive or neutral and depends on host performance or host tissue nutrient concentrations 34, 35 and varies between different fungal taxa and strains 36. The present study emphasis us to understand the plant growth promoting properties possessed by fungal root endophytes residing under low temperature

environment. Out of thirty root endophytic fungi, twelve showed distinct morphology. These were identified as Chaetomium globosum, Fusarium oxysporum, Fusarium semitectum, Fusarium solani, Fusarium fusarioides, Fusarium moniliforme, Mucor sp, Aspergillus niger gr, Aspergillus sp, Aspergillus versicolor, Mucor hiemalis, Trichoderma peudokoningii rifai. The root fungal isolates mostly belonged to the genera Fusarium, Aspergillus, Mucor and Trichoderma. Fusarium and Trichoderma have been reported as endophytes also in leaves and stems 37. Table 1 shows the PGPF traits of twelve distinct fungal root endophytes. All the endophytes produced IAA production and

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solubilize phosphate. Seven were found positive for siderophore, four for HCN and three for ammonia production. Nahas (1996) reported that among the microbial group, fungi are more efficient in solubilizing phosphate than bacteria 38. All the endophytic fungi exhibited clear zone of phosphate solubilization around the fungal growth in Pikovskaya’s medium supplemented with tri calcium phosphate (Fig 1a). The quantitative estimation of phosphate solubilization for 5 days revealed a similar trend in all the endophytic fungi. The phosphate solubilization increased from day 2 to day 8 and then decreases slowly. The highest range was observed in T. peudokoningii (37.45±2.78 to 64.32±2.87) µg/ml followed by C. globosum (33.62±5.92 to 69.32±3.21) µg/ml, F. semitectum (32.64±1.89 to 57.63±2.11) µg/ml, A. versicolor (31.63±2.02 to 63.72±2.36) µg/ml (Graph 1). A similar finding has been reported on Pseudomonas striata 39 and on two different Penicillium sp 40, where they observed a gradual increase in the available P level in the medium up to a certain period and then available P level drops off. The mechanism behind the solubilization of phosphate could be the production of organic acids and a drop in pH 41, 42,43. P solubilization occurs when the media is supplemented with a simple carbon source along with phosphate solubilizing fungi. This also results in the production of various organic acids and other substances 44. All twelve endophytic fungi were screened for IAA production with tryptophan (5mg/ml) and the intensity of red color varied among all the fungal endophytes (Graph 2). F. fusarioides (49.21±3.5)µg/ml showed maximum IAA production followed by Aspergillus sp. (35.08±2.34) µg/ml and M. hiemalis (28.52±1.83) µg/ml. The other showed IAA comparatively in lesser quantity – C. globosum (26.78±1.11) µg/ml, T. peudokoningii (24.12±1.6) µg/ml, A. versicolor (20.85±1.43) µg/ml and F. moniliforme (20.54±1.1) µg/ml. C. globosum and other fungi produce IAA production through the IAA biosynthesis pathway. All the fungal endophytes were subjected to siderophore production. Seven showed siderophore production on on solid CAS agar media (Fig 1b). It was seen that the maximum intensity of siderophore production was shown by C.

globosum, A. vericolor and T. peudokoningii followed by F. oxysporum, Mucor sp, A. niger and M. hiemalis. However, F. semitectum, F. solani, F. fusarioides, F. moniliforme and Aspergillus sp did not produce siderophores. The quantitative estimation of siderophore production through FeCl3 test in Fries basal medium revealed that all the isolates showed maximum siderophore production at 21th day of the inoculation and the concentration decreases afterwards (Graph 3). The maximum concentration was seen in T. peudokoningii followed by C. globosum and A. versicolor. However, the siderophore production was found almost negligible in all Fusarium species. Similar findings were also reported in F. oxysporum 45. The rhizospheric organisms produce siderophores through several factors, one of which can be high iron concentrations in the soil 46. A. niger does not produce siderophore but is a good producer of citric and other organic acids that are thought to play a role in the reaction with CAS reagent 47. Siderophore production by A. niger and A. flavus in both solid and broth media is well supported by many workers. All the isolates were checked for HCN and ammonia production. Four showed postive results for HCN production and three for ammonia production. C. globosum, F. fusarioides, Mucor sp. and A. versicolor were found positive for HCN production and resulted in the filter paper color change from yellow to brown after incubation (Fig 1c). M. hiemalis, A. niger and F. moniliforme showed positive results for ammonia production and resulted in the development of a yellow to brown color onto reaction with Nesselers reagent. (Table 1) Chaetomium is a large genera belonging to family Chaetomiaceae (Ascomycota). It is a source of important bioactive secondary metabolites. More than 200 compounds have been reported from this genus 48. T. harzianum t – 22 and its cell-free culture filtrates solubilizes insoluble rock phosphate containing calcium phosphate and micronutrients like Mn and Zn49. For this reason, it has been hypothesized that the fungus can be applied as growth enhancement on various crops. In field plots, the beneficial action of F. oxysporum on AM fungi resulted in an increase in the growth and nutrient concentration during the early stage of

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root colonization in pea plants. This has been hypothesized by many workers 50, 51. Microscopic examinations of dual culture assay between fungal endophytes and P. indica shows an alteration in the mycelium of P. indica where it was in contact with the fungal endophytes (Fig 2). The interaction of fungal root endophytes with a known root endophyte, P. indica revealed that none of the isolated endophyte showed mutual interaction and the radial growth of P. indica incorporated with endophytes was drastically reduced (Graph 4). The experiment indicated that P. indica interacts with a diverse group of soil fungi and its interaction varied from the negative to positive association.

CONCLUSION Plant growth promoting fungi (PGPF) are closely associated with plant roots and have been reported in increasing the biomass and yield as well as modulate the nutrient uptake and protect the host plant from pathogen attack. The growth activity of PGPF is due to its colonization with the roots and concerting the inorganic minerals into organic forms that are rapidly taken up by the plants. This study

sheds light on the potential ability of some root endophytic fungi isolated from extreme cold conditions of Central Himalayas as plant growth promoters. However, further studies are required to establish the exact contribution by root endophytic fungi as PGPF organisms and as growth promoters. The geographical location represents a unique ecological niche for the isolation of root endophytic fungi from the tomato roots habituating that particular area. The beneficial microbes along with the crop evolve and adapts to the prevailing climatic conditions. The analysis of endophytic fungi of plants roots is an important step in the selection of new isolates and their survival under stress conditions.

ACKNOWLEDGEMENT

The work was supported by the Defence Institute of Bioenergy Resources, DRDO, Haldwani. We are also thankful to Dr. S.K. Singh from Agarkar Research Institute, Fungal Identification Service, Mycology and Plant Pathology Group Agharkar Research Institute, G, G Agarkar Road, Pune for identifying the fungal isolates.

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