Combine Inoculation Effect of Pink Pigmented Facultative Methylotrophs and B. Japonicum on Plant Growth and Yield of Soybean

A field experiment was conducted during kharif 2017-18 All growth and yield parameters of soybean as well as the population of Methylobacterium were influenced by the (S.T of Rhizobium +S.T of PPFMs +Foliar spray consortium of PPFMs + 100 % N) which was significantly superior over rest of all the treatments and it was at par with treatment (S.T of Rhizobium +S.T of PPFMs +Foliar spray consortium of PPFMs + 75 % N) during flowering and harvesting stage. This shows that the consortium of efficient strain of PPFMs could reduce N fertilizer application in field condition. Keyword: Methylobacterium, Pink pigmented facultative methylotrophs, Soybean. Research Article Cite this article: Govekar, Y.R., Navale, A.M., Deokar, C.D., & Surve, U.S. (2020). Combine Inoculation Effect of Pink Pigmented facultative Methylotrophs and B. Japonicum on Plant Growth and Yield of Soybean, Ind. J. Pure App. Biosci. 8(5), 348-355. doi: http://dx.doi.org/10.18782/2582-2845.8312 Govekar et al. Ind. J. Pure App. Biosci. (2020) 8(5), 348-355 ISSN: 2582 – 2845 Copyright © Sept.-Oct., 2020; IJPAB 349 In response, PPFMs produces plant growth promoting substance such as Indole acetic acid (IAA), Gibberlic acid (GA) etc which are known to stimulate plant growth (Ivanova et al., 2001 & Koenig et al., 2002); fix the atmospheric nitrogen (Sy et al., 2001); solubilise mineral phosphate (Jones et al. 2007); induce systemic resistance against plant pathogens (Madhaiyan et al., 2006) and chelation of inorganic compound such as iron. Due to these importance, the PPFMs have received a great deal of attention as bioinoculants for use in agriculture. Combined use of two or three beneficial microorganisms as inoculation have been found to perform better than single inoculations (Alagawadi & Gaur, 1988; Jisha & Alagawadi, 1996; & Prathibha et al., 1995). Therefore the present study was undertaken to study the effect of combined inoculation PPFMs (Methylobacterium) and B Japonicum on growth and yield of soybean. MATERIALS AND METHODS The PPFM consortium was prepared by using Sterilized Ammonium mineral salt (AMS) broth and base material such as Fe. EDTA, Arabinose, Glycerol, PVP and Trehalose and was inoculated with loopful of efficient isolates of Methylobacterium cultures and kept in temperature controlled shaker (150 rpm/min) at 30 0 C for 5 days. After attaining the full growth (10 -9 cfu/ml) it was diluted to1:100(1%) and sprayed @ 3.0 lit/ha after 45 days of sowing. The consortium of B.Japonicum was obtain from the Department of plant pathology and Agricultural microbiology, MPKV, Rahuri The treatments were as follows. Treatment No. Treatment details


INTRODUCTION
In a recent years, the pink pigmented facultative methylotrophs (PPFMs) is one of such a bacteria receiving more attention as a plant growth promoting bacteria. They are known to play an important role in increasing crop yields and land fertility. These are physiologically an interesting group of bacteria able to grow on methanol, methylamine as well as on a variety of C 2 , C 3 and C 4 compounds as sole sources of carbon and energy (Lidstrom, 1992), potentially dominating the phyllosphere population. They are commonly found in soil as well as on the surfaces of leaves, seeds and in the rhizosphere of a wide variety of plants (Holland & Polacco, 1994;Chanprame et al., 1996;Holland, 1997;& Shepelyakovskaya et al., 1999).
The association of PPFMs with plant possesses an associative symbiotic relationship in which PPFMs utilizes the methanol emitted from leaves of plants as sole carbon and energy source.
In response, PPFMs produces plant growth promoting substance such as Indole acetic acid (IAA), Gibberlic acid (GA) etc which are known to stimulate plant growth (Ivanova et al., 2001& Koenig et al., 2002; fix the atmospheric nitrogen (Sy et al., 2001); solubilise mineral phosphate (Jones et al. 2007); induce systemic resistance against plant pathogens (Madhaiyan et al., 2006) and chelation of inorganic compound such as iron. Due to these importance, the PPFMs have received a great deal of attention as bioinoculants for use in agriculture. Combined use of two or three beneficial microorganisms as inoculation have been found to perform better than single inoculations (Alagawadi & Gaur, 1988;Jisha & Alagawadi, 1996;& Prathibha et al., 1995). Therefore the present study was undertaken to study the effect of combined inoculation PPFMs (Methylobacterium) and B Japonicum on growth and yield of soybean.

MATERIALS AND METHODS
The PPFM consortium was prepared by using Sterilized Ammonium mineral salt (AMS) broth and base material such as Fe. EDTA, Arabinose, Glycerol, PVP and Trehalose and was inoculated with loopful of efficient isolates of Methylobacterium cultures and kept in temperature controlled shaker (150 rpm/min) at 30 0 C for 5 days. After attaining the full growth (10 -9 cfu/ml) it was diluted to1:100(1%) and sprayed @ 3.0 lit/ha after 45 days of sowing. The consortium of B.Japonicum was obtain from the Department of plant pathology and Agricultural microbiology, MPKV, Rahuri The treatments were as follows.

Treatment
No.  In present study, significant differences were observed between various treatments by applications of PPFMs and Rhizobium consortium under graded levels of nitrogenous fertilizers on all growth attributing parameters viz, plant height, root and shoot length, fresh and dry weight of shoot and root, number of effective and non-effective nodules, of soybean during both flowering and harvesting stage. The phyllosphere as well as rhizosphere PPFMs population along with no. of pods per plant, stover yield and grain yield also shows similar trend. All the above growth parameters along with PPFMs population and the no. of pods per plant, stover and grain yield of soybean influenced by the treatment T 10 (S.T of Rhizobium +S.T of PPFMs +Foliar spray consortium of PPFMs + 100 % N) which was significantly superior over rest of all the treatments and it was at par with treatment T 9 (S.T of Rhizobium +S.T of PPFMs +Foliar spray consortium of PPFMs + 75 % N) in respect of their influence on all growth attributing character at flowering and harvesting stage. It is also found that the treatment T 7 (S.T of Rhizobium +S.T of PPFMS+ Foliar spray consortium of PPFMs) recorded all the above parameters which was significantly higher over T 2 (Seed treatment of Rhizobium) (86.75) treatment. The superiority of T 10 treatment is due the high dose of nitrogenous fertilizer and combine beneficial effect of both Rhizobium and PPFMs. The increase in growth parameters is due to the as per increase in graded dose nitrogenous fertilizer as reported by Ntambo et al. (2017). The Krushnanjali (2017) reported that combine effect of Rhizobium along with increase in graded doses of nitrogenous fertilizer results in increase in the growth parameters of the plant. The significant improvement found in the growth parameter such as height of the plant, development of root and shoot, and nodulation and yield parameters of the crop in the treatment inoculated with Rhizobium and PPFMs. This is due to combine beneficial effect of these two organisms. Rao and Dhir, (1993) reported that the presence of Rhizobia in the legume rhizosphere influencing the legume roots to release plant growth and promoting substances which, in turn might have enhanced the growth of Methylobacterium in situ and synergistic effect might have occurred in treatments. The development of root and shoot is due to the action of plant growth promoting substances such as IAA and GA produced by the PPFMs. This has been supported by Suresh Reddy, 2002 who reported that these growth promoters allows a balanced growth of shoot and root system. When roots become more extensive due to the action of IAA, then the cytokinins of the plant signals the shoot system to form more branches.
The results also supported by some workers such as Senthilkumar, 2003;Madhaiyan et al., 2009. They reported that the plant growth promoters such as IAA and GA also enhanced root growth enabling the plants to absorb more nutrient in soybean ultimately resulting in better growth, dry matter production. The maximum nodule number may be attributed to the presence of flavonoid compounds taken up by the roots of soybean plants. This has been supported by Subba Rao who reported that the flavonoid compounds secreted by the roots of soybean plants helps to release naringenin, genistein and diadzein (iso flavones).So these flavonoid molecules by legumes can either induce or block the transcription of nod genes in rhizobium and Bradyrhizobium resulting in increased nodulation as well as nitrogen fixation . Similar results were shown by Radha (2007) and Meenakshi (2009) Radha et al. (2009) reported that the combined inoculation of Methylobacterium sp with Rhizobium sp .significantly increased plant growth parameters such as root and shoot development, number of leaves, nodulation, compared with individual inoculation of Rhizobium sp or uninoculated control. While, Meenakshi (2008) reported significantly higher number of leaves, shoot dry weight and root dry weight and nodulation was recorded in the treatments that received Bradyrhizobium and PPFM spray than Bradyrhizobium alone and uninoculated control. In case of phyllosphere and rhizosphere PPFM population. The results are mainly due to the growth hormone production by Methylobacterium sp. especially high cytokinin production in apical plant tissues and rhizosphere soil. The another reason behind that the foliar spraying PPFMs which significantly influenced the PPFMs population in the foliar region such as phyllosphere. of rice crop as reported by Holland, (1997b).
However, the phyllosphere region of the soybean recorded the highest PPFMs load compared to soybean rhizosphere. The increased population on the leaves is due to the fact that they utilize the gaseous methanol. This has been supported by Nemecek-marshal et al. 1995 andDaniel et al. (2006). They reported that the PPFMs population utilize gaseous methanol emitted by the stomata of the leaves of the plants during leaf expansion by pectin demethylation as carbon and energy source and promote the growth of their host through the release of metabolites. The increase in yield parameters in the treatment containing 100% N is due to as per graded dose nitrogenous fertilizer reported by Krushnajali (2017). The Krushnajali (2017) and Ntambo et al 2017 both worker reported that increase in graded doses of nitrogenous fertilizer results in increase in the yield parameters of the plant. The superior performance of the soybean inoculated with PPFMs and Rhizobium isolates may be due to the cumulative effect of increased plant growth substances, enhanced nutrient uptake, nitrogen fixation and control of plant pathogen by sidrophore production resulting in higher yield of soybean crop.
This shows that the combination of Rhizobium and PPFMs along with 75%N enhances the all growth parameters, as well as yield of soybean crop concluding that there is a possibility of saving fertilizer nitrogen to an extent of 25% to soybean crop. It is also found that the PPFMs consortium in combination with Rhizobium without any nitrogen dose shows significant improvement in growth attributing characters and yield of soybean as compared to the Rhizobium alone indicating the importance of dose of PPFMs consortium along with Rhizobium as compared to Rhizobium alone.
Associative effect of rhizobium and phosphate soluiblizing bacteria on the yield and nutrient uptake of