Isolation and screening of freshly isolated bradyrhizobium and rhizobium spp. for multi-host plant growth promotion.

Abstract

Biological nitrogen fixation (BNF) is an essential part of sustainable agriculture. Inorganic nitrogenous fertilizers are extensively used in modern, high input agriculture. However, they are expensive, and poorly absorbed by plants so much of the nitrogen that is applied is lost due to leaching, which results in soil acidification and ground water pollution. In contrast, rhizobia have the capacity to fix atmospheric nitrogen effectively, resulting in the uptake and assimilation of nitrogen into plants without the harmful environmental and related application costs. This study aimed to isolate, screen and identify freshly isolated Bradyrhizobium spp. and Rhizobium spp. for multi-host plant growth promotion. Freshly isolated rhizobial isolates were obtained from the nodules on roots of soybean (Glycine max (L.) Merrill), cowpea (Vigna unguiculata (L.) Walp.), pigeonpea (Cajanus cajan (L.) Millsp.) and groundnut (Arachis hypogaea (L.) Kohler). Thirty-three isolates were isolated and stored in 40% glycerol solution at -80°C. Two commercial multi-host soybean strains, XS21 and WB74, to be used as controls, were obtained from the Agricultural Research Council, Mpumalanga, South Africa, in subsequent trials. The development of visual growth scales of each of the test crops [soybean, cowpea, dolichos (Lablab purpureus (L.) Sweet) and dry bean (Phaseolus vulgaris (L.) var. Gadra)], were developed in order to study the dynamics of nitrogen fluxes that occurred in the growth cycles of each of the crops. The total chlorophyll and leaf nitrogen contents were determined, based on the chlorophyll and leaf nitrogen content at specific growth stages, for each crop, at multiple levels of nitrogen fertilization. A linear correlation was observed between the total chlorophyll content and that of the leaf nitrogen content of each test crop except for soybean. However, due to physiological crop damage due to red spider mite (Tetranychus urticae Koch.), powdery mildew (Erysiphe polygoni D.C.) and mealy bug (Pseudococcus filamentosus Guen.) infestations, that caused yellowing leaves, there was no linear correlation between chlorophyll content and leaf nitrogen content for soybean. There were significant differences in total chlorophyll content for dolichos and dry bean crops and for total leaf nitrogen content between the soybean, cowpea and dry bean crops. The thirty-three freshly isolated rhizobial strains and the two commercial multi-host soybean strains (WB74 and XS21) were tested in vitro with for three secondary plant growth promoting traits: inorganic phosphate solubilization, siderophore production and indole-3- acetic acid production. Eight freshly isolated rhizobial isolates tested positive for all three traits: Isolates CT1-6, Ukulinga I1-1, 93015 T1-1, Royes I3-3, Royes I1-2, CI1-G, 00040I2 and 87051 I2-1. Neither of the commercial controls performed well for these traits. The 31 freshly isolated rhizobial strains and the two commercial strains were screened in vivo, compared with the two fertilizer controls (0% optimum nitrogen and 100% optimum nitrogen). Infection and nodulation were determined to occur at the V2 and V3 growth stages, depending on the rhizobial strain. Peak chlorophyll readings were noted at the V3 and R1 growth stages and thereafter, exponential declines in chlorophyll contents occurred at the R3, R6 and R7 growth stages. The best performing rhizobial isolates on soybean were: Ukulinga I1-1, Royes I1-2 and 93051 T1-1; on cowpea: Royes I1-2, 87091 C1-2, CCI3 and Ukulinga I1-1; on dolichos, CI2 and Royes I1-2; and dry bean, Isolate SCI performed nearly as well as the 100% Nitrogen Control for chlorophyll content, and XS21 performed nearly as well as the 100% Nitrogen Control for dry biomass. The best 17 rhizobial isolates and the 2 commercial controls were screened with two fertilizer controls (0% optimum nitrogen and 100% optimum nitrogen) for nodulation, total nodule number and nodules containing leghaemoglobin. On soybean only four isolates produced nodules, and only one nodule contained leghaemoglobin. On cowpea 11 of the rhizobial isolates tested produced nodules, with nearly every nodule containing leghaemoglobin. On dolichos, 18 rhizobial isolates produced nodules, half of which contained leghaemoglobin. On dry bean, 9 rhizobial isolates produced nodules, and less than half of the nodules contained leghaemoglobin. Subsequently, the freshly isolated rhizobial strains that performed best were used in combination with potassium silicate (KSi), to determine their combined effects on chlorophyll content and dry biomass. A full factorial trial was not conducted due to lack of glasshouse space. The 17 rhizobial isolates, 2 commercial rhizobial strains and 4 fertilizer controls (0% optimum nitrogen, 100% optimum nitrogen, 0% optimum nitrogen + KSi and 100% optimum nitrogen + KSi) were tested. The total chlorophyll content and dry biomass was measured for each crop and analyzed using ANOVA, or Kruskal-Wallis analysis. The soybean plants treated with XS21 + KSi, and Royes I3-3 + KSi had higher chlorophyll contents than the 100% Nitrogen Control. Plants treated with CI2 + KSi, and Royes I3-3 + KSi had higher dry weights than the 100% Nitrogen Control. Twelve of the rhizobial isolates tested on cowpea plants produced more chlorophyll than the 100% Nitrogen Control. Nine of those rhizobial isolates had higher chlorophyll contents than the plants treated with 100% Nitrogen + KSi Control. All of the rhizobial isolates tested on cowpea plants had higher dry weights than the plants treated with 0% nitrogen, 0% nitrogen + KSi and the 100% nitrogen. Sixteen of the nineteen rhizobial isolates tested on dolichos had higher chlorophyll contents than the plants treated with 0% N + KSi and 0% nitrogen. The plants treated with Isolates 87051 I3-1 + KSi had a higher chlorophyll content than the 100% Nitrogen Control. Eighteen of the rhizobial isolates tested on dry bean had higher dry weights than the 0% Nitrogen Control + KSi. Only Isolate CI2 + KSi on dolichos resulted in a dry weight comparable to both the 100% Nitrogen Control and 100% Nitrogen + KSi Control. None of the rhizobial isolates tested on dry bean plants caused chlorophyll contents comparable to both the 100% Nitrogen and 100% Nitrogen + KSi Controls. Fourteen rhizobial isolates were identified using 16S DNA sequencing. Nine of these belonged to the genus Bradyrhizobium and the remaining five were identified as Achromobacter xylosoxidans Strain zc1, Pseudomonas moraviensis Strain IARI-HHS1-33 and Pseudomonas sp. LC182, Pseudomonas chlororaphis Strain UFB2 and Bacillus acidiceler Strain TSSAS2-2.