Pink pigmented facultative methylotrophs (PPFMs) from black pepper (Piper nigrum L.) for drought stress mitigation and plant growth promotion

Abstract

The study entitled “Pink Pigmented Facultative Methylotrophs (PPFMs) from black pepper (Piper nigrum L.) for drought stress mitigation and plant growth promotion" was carried out during the period from 2021-2023 in the Department of Agricultural Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram, with the objective to isolate, screen and characterize Pink Pigmented Facultative Methylotrophs (PPFMs) for plant growth promotion and abiotic stress mitigation with special emphasis on drought management in black pepper (Piper nigrum L.) under Kerala conditions. The study comprised an in vitro screening experiment and a pot culture experiment with black pepper variety Karimunda. A total of 60 PPFM cultures were isolated from phyllosphere and rhizosphere by leaf imprint method and serial dilution and plating method on Ammonium Mineral Salt (AMS) agar medium supplemented with 0.5% methanol and 10 µg cycloheximide. These isolates were tentatively identified as PPFMs based on the characteristic pink pigmented colonies. Characterization of the isolates by colony morphology, gram reaction and biochemical tests were carried out. 30 PPFM isolates were selected for in vitro screening of PPFM isolates for plant growth promotion. IAA production by the different isolates of methylotrophs showed wide variations ranging from 10.02 to 51.25 µg mL-1 culture filtrate (without tryptophan) and from 10.06 to 60.00 µg mL-1 culture filtrate (with tryptophan). Maximum IAA production of 51.25 µg mL-1 and 60.00 µg mL-1 culture filtrate (without and with tryptophan, respectively) was recorded by PNPPFM 59. GA production by different isolates were ranging from 29.19 to 1.81 µg mL-1. Maximum GA production of 29.19 µg mL-1 was recorded by PNPPFM 60. Extracellular ammonia production by the different isolates of methylotrophs ranged from 1.43 to 7.07 µmol mL-1. Maximum extracellular ammonia production of 7.07 µmol mL-1 was recorded by PNPPFM 59. ACC deaminase activity production by different isolates were ranging from 0.89 to 13.94 µg mL-1. Maximum ACC deaminase activity production of 13.94 µg mL-1 was recorded by PNPPFM 60. For the assessment of drought tolerance potential, these isolates were grown in different concentrations of PEG 6000 viz. 0%, 5%, 10%, 20%, 30% and 40%. All the cultures were able to tolerate up to 30% concentration of PEG 6000. Index ranking was done to select the better performing isolates for the subsequent experiments. Consequently PNPPFM 60, PNPPFM 59 and PNPPFM 44 were selected for the pot culture experiment. The pot culture experiment was undertaken with black pepper cuttings to study the effect of PPFM isolates on plant growth promotion and drought tolerance. The experiment was laid out in CRD with 7 treatments and three replications. The treatments comprised four PPFM isolates (PNPPFM 60, PNPPFM 59, PNPPFM 44 and Ref. culture PPFM 38) and three control treatments (sterile water, uninoculated media and absolute control). These treatments were given as cutting dipping and foliar spray at 45, 75 and 90 DAP. The study revealed that PPFM isolates had significant effect on growth and establishment of black pepper cuttings. Maximum number of leaves (4.66), shoot length (28.30 cm), root length (17.70 cm), number of roots (14.66 and 138.33, primary and secondary roots respectively), fresh weight of roots (1.01 g), dry weight of roots (0.4 mg), fresh weight of newly emerged shoots and leaves (10.74 g) and dry weight of newly emerged shoots and leaves (2.96 g) were recorded with PNPPFM 60 and least was recorded with either one or more of controls. Cell membrane integrity and relative water content were highest with PNPPFM 60 on 4th as well as on 14th day. Highest chlorophyll stability index on 14th day was recorded with PNPPFM 60. Proline content (78.67 µg g-1 tissues), super oxide dismutase activity (0.45 activity g-1 min-1), catalase activity (18.48 activity g-1 tissues), peroxidase activity (47.18 activity g-1 min-1) and malondialdehyde (0.54 µmol g-1 tissues) were highest with PNPPFM 60. Considering the major drought tolerance parameters such as proline content, SOD, catalase, peroxidase and malondialdehyde, PNPPFM 60 was the best among the PNPPFM isolates tested in the pot culture experiment. The present study revealed that the isolate PNPPFM 60 (cutting dip – 1% PPFM culture broth and foliar spray – 1% culture broth at 45, 75 and 90 DAT) were effective in improving the growth parameters and drought tolerance in black pepper.