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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)

 Abstract

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

Introduction

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.

Results

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.  

Discussion

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.

Acknowledgments

The financial assistance received from Forest and Environment Dept., Govt. of Odisha through State Plan 2015-16 is gratefully acknowledged.

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