Inoculation of Pongamia pinnata with phosphate solubilizing FUNGI: effect on plant growth and transplantation establishment
Soumya Ranajan Nayak, Manas Ranjan Panigrahi and Nibha Gupta
Plant Pathology and Microbiology Division,Regional Plant Resource Centre, Bhubaneswar -751015 Odisha (India)
Pongamia pinnata is well known forest tree speices having medicinal and biofuel properties. It is slow growing and seed dormancy is major constraints behind its large scale propagation. Micorbial inoculations are known to enhance the plant growth and productivity on is application of host rhizosphere. In view, a pot culture experiment was carried out on Pongamia pinnata for which 30 days old seedlings were raised from freshly collected seeds and transplanted in the poly pots containing red laterite soil and subsequently supplemented with 7 days old liquid culture of six different pretested phosphate solubilising fungi.
The fungal inoculation was done trice with monthly interval upt 120 days of plant growth. Data recorded for plant height , biomass , leaf no and leaf area , other physiological growth parameters RGR, NAR, LAR , QI and R/S exhibited growth promoting activity of the three fungal inoculants viz., Aspergillus flavus, Penicillium implicatum and Fusarium oxysporum. However, Aspergillus flavus was found to enhance the plant growth and biomass significantly (P<0.05).
Key Words: Aspergillums, Forest, Penicillium, Pongamia pinnata, Transplantation, tree
Pongamia pinnatais non edible and medium sized tree legume endowed with biodiesel and medicinal properties. It is native to India, Malaysia, Australia and Indonesia1.The tree is generally considered to be saline and drought tolerant species and is able to grow in a range of suboptimal conditions including alkaline soils2. It is also useful as anti-inflammatory, antiplasmodial, anti- lipidperoxidative, anti-diarrhoeal, anti-ulcer, anti-hyperammonic and antioxidant activity 3 Pongamia can grow on low fertility soil due to its symbiotic association with Rhizobia. However, the seed dormancy is the major constraints towards large scale propogation of this tree species.
Bioinoculatns can be helpful in breaking the seed dormancy by producing various plant growth substances 4. In fact, microbial population in the rhizosphere soil contributes towards enhancement of plant growth through nutrient recycling and making them available for plants 5. Such types of bioinoculants also involve the phosphate solubilising fungi which play a vital role in biomineralization of bound phosphate present in rhizosphere soil and make it available to the host plants 6. In view to analyse the effect of some phosphate solubilising fungi on growth and development of Pongamia pinnataon transplantation conditions, a pot experiment was planned and the result obtained on growth performance in terms of plant height, biomass and physiological growth parameters are presented here.
Materials and methods
The study was carried out during May-September, 2015 in the experimental field of Regional Plant Resource Centre, Bhubaneswar, Odisha, India. The experiment was done in poly bags containing 5.5 kg red soil. Pot soil was fumigated with 1% formalin for 48 hrs prior to the experiment. The brownish to deep brown colored seeds were decapsulated and sown in pre-saturated poly bags. Seedlings of 30 days were transplanted into the poly bags. Six different phosphate solubilisng fungal isolates (confirmed earlier through plate culture test performed on Pikovskaya’ medium) identified as Rhizopus stolonifer , Aspergillus flavus, Penicillium implicatum, Fusarium oxysporum, Aspergillus kanagawaensis, Aspergillus brunneo were used for the experiment.
The seven day old culture developed in Sabouraud Dextrose broth at 30 °C were inoculated (@ 100 ml /poly bag ) separately & thrice with monthly interval. Uninoculated plants were considered as control and regular watering was done to keep the plants healthy. Final data was recorded on 140th day for Plant height, fresh and dry biomass, root shoot ratio, physiological growth parameter like relative growth rate, net assimilation rate, leaf area ratio, quality index, were calculated 7. Soil of pot culture experiment was also analysed for its physiochemical properties like pH, EC, OC, available N , P , K and Na And Fe8. All data were collected in five replications and subjected to statistical analysis for variance and significance.
Different pattern of plant growth and development was observed in different treatments as well as uninoculated control. ANOVA indicated significant effect of the treatments at P < 0.05. The data of shoot height, root length of seedlings in the nursery at both uninoculated control and treated transplanted seedlings are depicted the prominent and significant differences among the treatments (P<0.05, R2 0.4254 and 0.3118) and it was higher in plants treated with Aspergillus flavus and Fusarium oxysporum (Fig. 1, 2).
Other inoculants Rhizopus stolonifer, A. brunneo, Penicillium implicatum exhibited growth promoting effect over control in transplanted seedlings as far as no. of root nodules, leaf area, and plant biomass is concerned (Fig.4, 5, 6). Significant differences for number of leaves did not reflected in all control and inoculated plants (Fig.3). Conversely, the treatment of Aspergillus flavus and Aspergillus brunneo enhanced the leaf area ( P<0.05, R2 0.9952 Fig. 5). Inoculation of Aspergillus flavus was found to be best in enhancing shoot fresh and dry biomass over other treatments as well as control. (P<0.05, R2 0.6091 and 0.5614). The inoculation of phosphate solublizing fungi Penicillium implicatum also yielded good root growth in transplanted seedlings as compared to non inoculated control plants ( Fig. 7 and 9, P<0.05, R2 0.8314 and 0.8107).
Similar differential response due to different inoculations were observed on physiological growth of Pongamia pinnata. It is evident that seedlings inoculated with Aspergillus flavus and Fusarium oxysporum showed better growth and biomass as compared to other treatments under transplantation conditions. Relative growth rate (RGR) and Net assimilation rate (NAR) was also changed due to the enhancement in dry biomass, stem height and leaf area as depicted in fig 10 and 11. With the inoculation of Aspergillus flavus and F.
oxysporum where growth of seedlings was maximum, relative growth rate (RGR) measured 0.088 & 0.066 d-1 , respectively. Inoculation of Aspergillus brunneo showed quite higher LAR ( leaf area ratio) as compared to the other treatments (Fig.11) . Data recorded on R/S presented in fig.12 exhibited the superior response of Aspergillus flavus, Penicillium implicatum and Fusarium oxysporum inoculations . Data recorded for the quality index of plants grown with selected fungal isolates in transplanted and conditions showed better quality and comparatively more growth than the uninoculated control.
The physiochemical properties of the pot culture experimental soil have also been measured. The pH of soil was observed in range of 5.12 -5.50. The soil treated with Aspergillus flavus and Fusarium oxysporum exhibited low pH i.e.5.12 and 5.20 respectively as comparared to control along with other treatments. The measured electrical conductivity was higher in these treatments in comparison to others.
The organic content and available nitrogen was lower in rhizosphere soil of the plants treated with Aspergillus flavus. In contrast, the available phosphorus was higher (53 kg/ha ) followed by Fusarium oxysporum ( as compared to uninoculated control in soil under this treatment. Both the above fungal treated soil were also found to be enriched with Na i. e. 60.3 and 59.8 mg/kg. The uninoculated control soil was found to be enriched with iron content i. e. 110 mg/kg which was lowered down upto 30.0-35.0 mg/kg in soils inoculated with Penicillium implicatum and Fusarium oxysporum, respectively.
In view to evaluate the effective plant growth promoting ability of phosphate solublising fungi on forest trees , present experiment was set up on Pongamia pinnata in pot culture condition which was supplemented with liquid cultures of phosphate solubilising fungi namely: Rhizopus stolonifer, Aspergillus flavus, Penicillium implicatum, Fusarium oxysporum, Aspergillus kanagawaensis, and Aspergillus brunneo. The different fungal strains used in the present study were having phosphate solubilising potential tested in plate culture method in laboratory earlier. Odee et. al (2002) recommended inoculation of liquid cultures to raise the healthy seedlings in the nursery conditions. This also helps in increasing fungal population in the rhizosphere and finally mineral solubilisation.
The costly affair of chemical fertilizers as well as limited supply of nutrient to the plants demands an alternative like mineral solubilising microbes. The enhancement in growth of Pongamia pinnata in pot culture conditions in the present experiment revealed the effect of fungal inoculation and their usefulness as bioinoculants for plant productivity. Experimental transplanted seedling of Pongamia pinnata supplied with different fungal cultures had exhibited good growth in terms of plant height and biomass. Under experimental conditions Aspergillus flavus was found to be very effective in increasing over all plant growth including shoot height, root length, number of branches, no. of leaves, leaf area and biomass. However, Fusarium oxysporum were also prominent in increasing plant height, leaf area, and biomass of the transplanted plants. The third fungal culture namely Penicillium implicatum was able to increase the root biomass in contrast to Aspergillus flavus that could be able to enhance the shoot biomass of experimental seedlings of P. pinnata.
Variation in growth performance of plants inoculated with the different fungal cultures may be due to differences in their phosphate solubilising potential. The experiment was set on the transplanted seedlings of P. pinnata. The experimental plants were surviving and growing well even after 140 days without any further addition of chemical fertilizers indicate the its role towards seedlings establishment after transfer and subsequent growth (Nenwani et. al, 2010; Maliha et. al, 2004, Hossain et. al, 2007) .
Many fungi like Penicillium and Aspergillus etc. were reported as phosphate solubilisers as well as evaluated for the plant growth and productivity (Dash et al., 2013; Vibha et al., 2014). Present study also confirm this findings as inoculation of Aspergillus flavus and Penicillium implicatum into the rhizosphere of P. pinnata seedlings were found to be effective in enhancing and improving plant growth. The carbon assimilation and allocation may be reflected in trait of LAR as well as growth variation may be due RGR and NAR (Krishnan and Satakappam, 2009). Significant differences in plant’s physiological parameters like relative growth rate, net assimilation rate and leaf area ratio was observed in inoculated and uninoculated seedlings.
Phosphorus is one of the essential macronutrient required in large amounts by the plants P and are generally added to soil in fertilizers. Plant utilizes 0.1% of phosphorus present in soil and rest is rapidly fixed as insoluble forms. Beneficial microbes in rhizosphere are receiving greater attention, as they can solubilize inorganic phosphate into soluble form. Mostly, Aspergillus and Penicillium spp. are most common fungi endowed with phosphate solubilising potential.
It is important to note that present study also revealed the growth enhancing properties due to inoculation of fungi especially Aspergillus flavus and Penicillium implicatum to the rhizosphere of Pongamia pinnata in experimental conditions. Availability of more amount of P content in the soil treated with these fungi indicated the good solubilizing capacity. Significant improvement in N, P and K availability in soil was recorded with some variations among treatments. Increase in nitrogen content in the soil in the presence of fungal inoculations may be due to the cellular decay and metabolite production of plants as well rhizosphere microbial mass .
It has been reported that besides P, phosphate solubilisng microbes produces considerable amount of Nitrogen and other growth promoting metabolites in the rhizosphere (Kucey ,1989). The growth promoting effect of fungal inoculations may be due these factors also. The increase in P content in soil may be due to the production of organic acid by these fungal inoculants as reduction in soil pH could also be observed in the experimental soil. This result reflects that reduction in iron content in the rhizosphere soil. It may be due to the higher production of iron chelators other than the organic acids ( Dori et al., 1990; Manulis et al., 1987).
The usefulness of fungal inoculations in the present study has also been corroborated with reports on superior response of Aspergillus niger and Penicillium chrysogenum for better growth and biomass production of Dalbergia sissoo over control (Dash et al., 2013). The transplanted plants of Pongamia pinnata showed substantial increase in plant height , total biomass, leaf area when experimented with different fungal inoculation over control suggesting usefulness of these fungi for the establishment of Pongamia pinnata seedlings in transplantation field.
A elaborative experiments towards impact of different edaphic and environmental factors like alkaline, saline and drought stress condition on the performance of fungal inoculations as well plant growth is required to reach any fruitful conclusion as Pongamia pinnata is generally known as saline and drought tolerant species ( Chaukiyal et al., 2000). However, data recorded through present study will be helpful in development of bioinoculants of Aspergillus flavus, Penicillium implicatum and Fusarium oxysporum for the mass scale propagation of P. pinnata for commercial use as this tree species is a good source of medicinal compounds and biofuel production.
The financial assistance received from Forest and Environment Dept., Govt. of Odisha through State Plan 2015-16 is gratefully acknowledged.
- Arote SR, Yeole PG (2010). Pongamia pinnata L: A comprehensive review. International Journal PharmTech Research 2:2283–2290.
- Basak UC, Gupta N, Rautaray S & Das P (2004). Effects of salinity on the growth of mangrove seedlings. Journal of Tropical Forest Science 16:437-439.
- Biswas B, Scott PT, Gresshoff PM (2011). Tree legumes as feedstock for sustainable biofuel production: opportunities and challenges. Journal Plant Physiology 168:1877–1884.
- Chaukiyal SP, Sheel SK, Pokhriyal TC (2000). Effects of seasonal variation and nitrogen treatments on nodulation and nitrogen ﬁxation behaviour in Pongamia pinnata. Journal Tropical Forest Sciences 12:357–368.
- Chopade VV, Tankar AN, Pande VV, Tekade AR, Gowekar NM, Bhandari SR, Khandake SN (2008). Pongamia pinnata: Phytochemical constituents, traditional uses and pharmacological properties. A review International Journal Green Pharmacy 2: 72-5.
- Dash S, Mohapatra AK & Gupta N (2013). Growth response of Dalbergia sissoo to mineral solubilizing bacteria and fungi in nursery conditions. Tropical Ecology 54(1):109-115.
- Dori S, Solel Z, Kashman Y and Barash I (1990). Characterization of hydroxamate siderophores and siderophore mediated iron uptake in Gaeumannomyces graminis tritici. Physiological and Molecular Plant Pathology 37:97-106.
- Gupta N, Sabat J, Parida R & Kerkatta P (2007). Solubilization of tricalcium phosphate and rock phosphate by microbes isolated from chromite, iron and manganese mine soils. Acta Botanica Croatica 66:197-204.
- Hossain M, Sultana F, Kubota M, Koyama H and Hyakumachi M (2007). The plant growth-promoting fungus Penicillium simplicissimum GP17-2 induces resistance in Arabidopsis thaliana by activation of multiple defence signals. Plant Cell Physiology 48: 1724-1736.
- Jiang Q, Yen SH, Stiller J, Edwards D, Scott PT, Gresshoff PM (2012). Genetic, biochemical, and morphological diversity of the legume biofuel tree Pongamia pinnata. Journal Plant Genome Science 1:54–68.
- Krishnan VM and Satakoppan VN (2009). Evaluation of growth parameters AGR, RGR and NAR of Soyabean [Glycine max Merr.] under Cd (II) stress. International Journal of Plant Science 4(2):449-453.
- Kucey RMN, Janzen HH and Legget ME (1989). Microbial mediated increase in plant available phosphorus. Advances in Agronomy 42:199-228.
- Leopold AC & Kriedemann PE (1975). Plant Growth and Development. McGraw-Hill Book Co., New York.
- Maliha R, Samina K, Najma A, Sadia A and Farooq L (2004). Organic acids production and phosphate solubilization by phosphate solubilizing microorganisms under in vitro conditions. Pakistan Journal of Biological Sciences 7:187–196.
- Manulis, S., Kashman Y and Barash I (1987). Identification of siderophores and siderophore-mediated uptake of iron in Stemphylium botryosum. Phytochemistry 26:1317- 1320.
- Nenwani V, Doshi P, Saha T and Rajkumar S (2010). Isolation and characterization of a fungal isolate for phosphate solubilization and plant growth promoting activity. Journal of Yeast and Fungal Research 1(1):009-014.
- Noggle GR & Fritz GJ (1986). Introductory Plant Physiology. Prentice Hall of India Private Ltd., New Delhi.
- Odee DW, Indieka SA, Lesueur D (2002). Evaluation of inoculation procedures for Calliandra calothyrsus grown in tree nurseries. Biology and Fertility of Soils. 36(2): 124-128.
- Roesti D, Gaur R, Johri BN, Imfeld G, Sharma S, Kawaljeet K, Aragno M (2006) Plant growth stage, fertiliser management and bio-inoculation of arbuscular mycorrhizal fungi and plant growth promoting rhizobacteria affect the rhizobacterial community structure in rain-fed wheat ﬁelds. Soil Biology and Biochemistry 38(5):1111-1120,
- Scott PT, Pregelj L, Chen N, Hadler JS, Djordjevic MA, Gresshoff PM (2008) Pongamia pinnata: an untapped resource for the biofuels industry of the future. Bioenergy Research 1:2–11.
- Smith SE, Read DJ (1997). Mycorrhizal Symbiosis. Academic Press, London.
- Tandon HLS (1999). Methods of analysis of soils, plants, waters and fertilisers. Publ. Fertiliser development and consultation organization, New Delhi , India . PP 13-35.
- Vibha, Geeta kumari and Nidhi (2014). Impact of phosphate solubilizing fungi on the soil nutrient status and yield mungbean (Vigna radiate L) crop. Annual Agriculture Research New Series 35(2):136-143.
- Weller DM, Raaijmakers JM, Gardener BBM, Thomashow LS (2002) Microbial populations responsible for speciﬁc soil suppressiveness to plant pathogens. Annual Review of Phytopathology 40:309–348.