MATERIALS AND METHODS

Sample collection sites: The rhizosphere soil and root samples of Karanj (Pongamia pinnata (L.) Pierre) nursery plants were collected during February-May, 2015 in summer season from viz., Naldurg Andur, Jalkot, Osmanabad, Paranda and Kalamb for biomass production and arbuscular mycorrhizal fungal infection status. Ten different replications of rhizosphere soil and root samples were collected in separate ziplock polythene bags from each sites. Soil samples were used for isolation and identification of arbuscular mycorrhizal fungal spores and roots for assessment of percentage root colonization.

Treatments of AMF inoculums to Karanj: Inoculums of indigenous AMF maintained in greenhouse with Coleus (Plectranthus scutellarioides) as host (containing 270-300 spores/100 g soil) was applied 30 g per each plant after 15 days of seedlings of Pongamia pinnata. Seedlings without treatment of AMF are considered as control. The biomass and AMF infection data revealed after 90th days seedling (DAS). Seedlings were watered on alternate days and 10 mL of Hoagland solution twice in a week [Stock solution (1000 mL) (NH₄H₂PO₄, 1 mL, KNO₃, 6 mL, Ca(NO₃) 2-4 mL, MgSO₄ 2 mL), micro-nutrients solution (H₃BO₃, 2.86 g, MnCl₂ 1.81 g, ZnSO₄, 0.22 g, CuSO₄, 0.08 g, NaMoO₄, 0.02 g)] to each pot, after 90th days of treatment⁸. Biomass, presence of arbuscules, vesicles, hyphal and Dark Septate Endophyte (DSE) and types of AMF species were monitored; then the root colonization index, i.e., percentage of root length colonized by AMF and spore population in 100 g of soil were calculated.

Biomass production: Pongamia pinnata treated and untreated plants, fresh weight of shoot and roots data were recorded. Shoots and roots were separated and oven dried at 60°C for 48 h for the determination of dry mass after recording their lengths⁹. Leaf area was measured by disc method. Fifty leaf disc of known size was taken from randomly selected leaves of the plant, discs and remaining leaf blades were oven dried and leaf area was calculated by using equation¹⁰:

Area of total leaves (cm²) = No of disc from total leaves×Area of single disc

AMF spore density: One hundred grams of rhizospheric soil was dissolved in 1000 mL of water and decanted through a series of 355-35 μm sieves¹¹. Residues were filtered through Whatman filter paper No. 1 and all spores were counted under the stereo-zoom dissection microscope. Intact and healthy AM fungal spores were mounted in PVLG (Polyvinyl alcohol-lactoglycerol) with and without Melzer×s reagent for identification using keys⁵ and INVAM (http://www.invam.caf.wvu.edu). The AMF spores were identified up to species level.

Assessment of percentage root colonization: The roots were fixed in to 4% Formalin Aceto Alcohol (FAA). Fixed roots were washed to free FAA cleared in water. Roots are placed in glass vial with 10% KOH and stained in 0.05% trypan blue lactophenol to determine mycorrhizal colonization, these VAM fungal colonized segments studied. Then transferred the sieving on to a gridded petri plate and observed it under the binocular microscope 400X (Lawrence and Mayo LM-52-3521). The percent root colonization was measured by using the equataion¹²:

Statistical analysis: Statistical analysis of the experiments were performed by using the method described by Mungikar¹³.

RESULTS

Biomass production: The present study, 10 parameters of biomass productivity was studied in Pongamia pinnata (Table 1). In this study, six replications were used. In biomass production, height of stem (121 cm), width of stem (5.2 cm), length of root (54.8 cm), the number of leaflets was observed more in Kalamb site (74), dry weight of shoot (18.21 g), fresh weight of shoot was found more in Kalamb site (44.7 g) and leaf area (2233.57 cm²) were observed more in Kalamb sites. Fresh weight of root was increased in Osmanabad site (29.41 g) and less in Kalamb (21.6 g). After the AMF inoculums treatments promising data were observed almost in all study sites after 90th Days After Seedlings (DAS). Kalamb site was found good AMF inoculums source of soil.

Mycorrhizal study: Arbuscular Mycorrhizal Fungal (AMF) assessment of percentage root colonization and spore density was studied in Pongamia pinnata (Table 2). Spore density was found more in Andur site (446/100 g soil) and less than Jalkot site (63/100 g soil) followed by Andur site and Paranda site. After treatments of AMF inoculums considerably increased the AMF spore density per 100 g of soil in Paranda site i.e., 63 (334) followed by others. Among AMF spore, Acaulospora rehmii, Glomus macrocarpum, Glomus geosporum, Sclerocystis sp. were observed from all study sites (Fig. 1, 2). The AMF percentage of root colonization was increased in Kalamb site (91.66%), while less Paranda site (70.83%). The AMF root colonization types are Arbuscular (A), Vesicular (V), Hyphal (H), colonization and DSE was found in all sites.

DISCUSSION

In the literature, there are several reports in which AMF inoculations decreased plant biomass but this decrease has often been found to be transient and reversed later, being followed by a positive growth response^14,15. Results also suggested that the extent of growth depression or reduction in P. pinnata varied with inoculated AMF species, which can be related to the variations in carbon demand of different AMF species. It was suggested that such differences in growth response towards different AMF inoculants are directly related to the balance between benefits and costs of the symbioses¹⁶. It was examined the mycorrhization level of some important regional plants (Azadirachta indica, Dalbergia sissoo, Dendrocalamus strictus, J. curcas, Leucaena leucocephala, Madhuca latifolia and P. pinnata), which suggested that it was minimum in P. pinnata¹⁷. This could be due to the large seeds of P. pinnata and pointed out those plants with large seeds generally exhibit lower AMF densities. It was concluded that AMF inoculations should enhance biomass of P. pinnata only after depletion of metabolic reserves in its cotyledons and such mycorrhizal seedlings can be utilized for biofuel plantation^18,19.

Table 1:	Impact of AMF treatment for biomass production in Pongamia pinnata from different study sites (90 DAS)
Values means of three replications, values in parentheses are AMF treatment, N: Naldurg, A: Andur, J: Jalkot, O: Osmanabad, P: Paranda, K: Kalamb

Table 2:	Arbuscular mycorrhizal fungal status in Pongamia pinnata different sites (90 DAS)
Values represent three replications, Values in parentheses are AMF treatment, A: Arbuscular, V: Vesicular, H: Hyphal, DSE: Dark septate endophytes

Fig. 1(a-d):	Identified genera of AMF (400X), (a) Acaulospora rehmii, (b) Glomus macrocarpum, (c) Glomus geosporum and (d) Sclerocystis sp.

Fig. 2(a-d):	Types of AMF colonization (400X), (a) Vesicles, (b) Hyphal, (c) Arbuscules and (d) DSE and hyphal

It is relevant to mention that the possible synergistic effect would be the uptake by AM fungal hyphae and translocation into the plant of P released by PSB in soil^20-23. Recently, the results showed that the combined inoculation of both PSB, AMF and rock phosphate produced vigorous plant growth of tree seedlings for quick planting²⁴. It was reported that, Azadirachta indica, Pongamia pinnata, Leucaena leucocephala and Acacia catechu were most effective in catching mycorrhizae and can be used as the effective tool in rehabilitation of the degraded ecosystems²⁵.

CONCLUSION

It was concluded that AMF inoculations should enhance biomass of P. pinnata only after depletion of metabolic reserves in its cotyledons and such mycorrhizal seedlings can be utilized for biofuel plantation. It was also discussed that AMF inoculations when amended enhanced biomass and AMF status of P. pinnata after 90 DAS of treatment. The importance of AM fungi in nursery management and in revegetation efforts has become an integral part of all stages of afforestation programmes. When AMF are more colonized to plants then enhanced the biomass productivity.

SIGNIFICANCE STATEMENTS