ISOLATION, CHARACTERIZATION AND EVALUATION OF PLANT GROWTH PROMOTING RHIZOBACTERIA FROM TOMATO RHIZOSPHERE FROM DIFFERENT PARTS OF ETHIOPIA.

Abstract

Rhizobacteria inhabit and colonize the plant root-zone and play a significant role in soil quality, health, biodiversity, and productivity. Rhizobacteria that possess beneficial traits in plant growth-promotion and disease protection are called plant growth-promoting rhizobacteria (PGPR). Phosphate solubilizing bacteria (PSB) are the known plant growthpromoters with their multidimensional benefits in plant nutrient access, stress alleviation, phytohormone production, and broad host-range interaction, among others. They improve plants’ physiological processes to grow well and produce quality yield. Therefore, exploring and developing potential or competent plant growth-promoter is required to improve soil fertility, farm production, and yield quality with a sustainable and eco-friendly approach. A huge diversity of soil microbes capable of P-solubilization and other plant growth-promoting attributes have been reported so far and remain to be explored. However, local isolation, strain identification, and host-specific evaluation are helpful for better farm production, indigenous competition, and sustainable environmental health. Multistage screening procedures (lab screening, greenhouse trials, and field evaluation) help to select efficient and competent strains. Each stage of evaluation possibly confers the different dimensional attributes of the candidate strain and then empirically strengthens the screening technique. This project aimed to screen efficient PSB strains and develop competent biofertilizers and plant growth-promotors with multidimensional benefits possibly applicable for tomato and other crop production at Koka, Meki, and Ziway Zuria. The project started by assessing the overall tomato production system, agrochemicals, and biofertilizer practices in these specific sites using randomly selected smallholder farmers. Accordingly, smallholder farmers in the study sites produced different crops, including tomatoes, in both seasons under repeated agrochemical practices. Sample soils were collected from different sites, including the tomato rhizosphere at Koka, Meki, and Ziway Zuria, as well as the natural forest soil from Wendogenet and Yirgalem Zuria. Then the experiment proceeded in the laboratory by screening potential PSB strains from collected soil samples using selective media and other preliminary screening techniques. The top 10 PSB isolates were selected and then further evaluated under different conditions. Their P dissolving ability was evaluated in liquid medium from different sources and the amount of dissolved P concentration quantified, plant growth-promoting traits verified, representative biochemical tests conducted, molecular characters (sequence of the 16S rDNA and IGS region between 16S and 23S) analysed and taxonomy identified, antagonistic effects, N2-fixing ability, symbiotic interactions, and host viii range were evaluated accordingly. PSB and possible P-substrates dual inoculation impact on tomato growth and yield were evaluated both at a greenhouse and open field levels, while a host range symbiotic interaction trial was conducted at field conditions using 5 different crops (maize, wheat, faba bean, kidney bean, and onion). From each experimental unit, various parameters were analysed against each PSB strain. During screening in the lab for instance, upon 8 days of incubation on PVK agar plate, Mk-1-25 and K-10-41 strains recorded a higher solubilization index (3.1 and 3.0, respectively), while 5 days of incubation in PVK broth resulted in a growing medium pH change where K-10-41 and K-10-27 significantly lowered the pH to 4.02 and 4.12, respectively. Inoculation in modified PVK broth with different P-substrates (iron phosphate, aluminium phosphate, and bone meal) and incubation for 10 days resulted in a substantial medium pH change and dissolved P. Accordingly, strains from the Koka site dissolved the highest overall mean P concentrations (260.83, 260.38, and 241.91 µg/ml by K-1-29, K-10-41, and K-10-27, respectively) and lower medium pH (4.93 and 4.95 by K-10-27 and K-10-41, respectively). Likewise, Z-12-20 was isolated from Ziway Zuria and found to be a competent strain in dissolving an average of 223.52 µg/ml P and reducing the pH to 4.98 upon 10 days of incubation. Among Psubstrates, the highest dissolved P (253.46 µg/ml) was obtained from TCP; nevertheless, the rest of the substrates recorded comparable concentrations. Furthermore, these potential 10 PSB isolates were characterized (morpho-biochemically and molecularly) and taxonomically identified. As a result, they placed to Bacillus, Priestia and Burkholderia species (i.e., K-1- 29, Mk-20-7, and Mk-20-20 belong to Priestia megaterium, while K-10-27 and Z-12-20 belong to Bacillus subtilis; Mk-1-25 and Z-13-4 Bacillus halotolerance; K-10-41 Bacillus velezensis, Mk-13-16 belongs to Bacillus amyloliquefaciens, and Z-1-16 belongs to Burkholderia cenocepacia). These strains are highly studied and repeatedly cited by different scholars for their plant growth-promoting and biocontrol roles. To evaluate their symbiotic effectiveness and plant assay responses, the current PSB strains were transferred and inoculated to tomato under greenhouse conditions. So as to improve their interaction and responses, all of them were co-inoculated with six possible P sources (TCP, BM, FeP, AlP, DAP fertilizer, and compost) independently. Accordingly, strains inoculation significantly improved plant growth parameters: shortened tomato germination, shoot length (highest 108.19, 106.43, and 101.81 cm by Z-12-20, Mk-1-25, and K-10-41), leaf parameters (substantially improved by Mk-1-25 and K-1-29), branch and node number, floral parameters (number, cluster, bud), fruit parameters (number, weight, marketability) (on average 6.1 and 5.95 fruits per plant were harvested from K-1-29 and K-10-27, respectively, ix 158.7 and 149.2 g fruits collected from Mk-20-20 and Z-1-16 inoculation, respectively), root length, root fresh and dry weight substantially were promoted by Z-12-20, shoot fresh and dry weight were significantly enhanced by Mk-1-25 and Z-12-20. Moreover, the overall highest phosphate-based symbiotic effectiveness was recorded by Mk-1-25 (PBSE%=176), followed by Z-12-20 and K-1-29 (PBSE%=144). Among the added P substrates, compost induced the symbiotic interaction, which then resulted in enhanced tomato vegetative growth, fruit number (6.03), and biomass, while AlP and AmP promoted fruit weight (collected on average of 156.73 and 149.61 g, respectively). Having these encouraging and positive responses/results from laboratory and greenhouse trials, the strains were further evaluated at open field conditions using tomato and other host crops. This is because, the in vitro solubilization and beneficial response of the strains might not be appropriately reflected and related to their effects at field level. Similar to greenhouse experiment, tomato-field trial was conducted under dual-inoculation of PSB and possible external P substrates (BM, compost, DAP fertilizer, and a 50% rate mixture of DAP and compost). This synergetic inoculation significantly improved the tomato’s growth, development, and yield parameters over uninoculated (control) group. For instance, elevated average shoot length (67.2 cm recorded by Mk-20-7), branch and leaf development (Z-12-20 and Mk-1-25), floral development (K-1- 29, Mk-1-25, Mk-20-20, and K-10-41), and fruit parameters (average highest total fruit number: 21.87, 21.82, 21.31, and 20.69 by K-10-41, K-10-27, K-1-29, and Mk-20-20, respectively) and highest fruit weight: 2821.6, 2793.3, and 2780.53 g harvested by K-10-41, K-1-29, and Mk-20-7, inoculation respectively). From the collected total fruits, a greater average number of marketable fruits were obtained from the inoculation of Mk-20-7 (10.44), K-10-41 (10.42), and K-1-29 (10.0 fruits per plant). However, because of strong biological competitors (birds, porcupines, and hyena), blossom end rot and early harvesting, a substantial number of unmarketable fruits were collected from the inoculation of K-10-27 (12.64) and Mk-13-16 (11.44). Compost application improved tomato-bacterial symbiotic interaction and significantly promoted tomato early vegetative growth, while a 50% rate mixture of DAP and compost demonstrated substantial responses at late growth period and resulted in highest tomato shoot length (67.39 cm) and fruit yield (on average 63.06 fruits with a gross weight of 2617.39 g). Similarly, the addition of bone meal enhanced fruit yield (total fruit number (62.82), quality (larger fruit with 10 cm length), and marketability (9.67 healthy fruits per plant)). To strengthen the screening processes and to promote farm practices, the candidate strains were inoculated to different crops to check their symbiotic interaction/response and growth promotion efficiencies at open field. Accordingly, maize, x wheat, onion, faba, and kidney bean were inoculated, and agronomy parameters were evaluated. Because of biological competitors’ attacks, grain yield was excluded from the analysis for most of these crops. Among the 10 PSB inoculants, Mk-20-20, Z-12-20, and Z13-4 significantly induced maize shoot length, whereas leaf number, cob number, and shoot fresh and dry weight were enhanced by inoculation of Z-12-20 and Z-13-4. Similarly, wheat inoculation with Z-1-16 promoted shoot length, tiller, and leaf development; Z-12-20 enhanced wheat flower development; and Mk-20-7 improved wheat biomass yield. Onion inoculation with Mk-20-20 increased shoot length (53.45 cm), shoot fresh weight (44.11 g), Mk-1-25 improved shoot length, leaf number, shoot fresh weight, bulb length, and Z-12-20 induced bulb diameter and bulb weight, while K-10-41 (which was one of the top promotors to tomato and legumes) showed the poorest interaction and limited response against onion. Inoculation of Mk-1-25, Z-1-16, Z-12-20, K-10-41, and K-1-29 promoted faba bean and kidney bean growth and biomass yield. Generally, hierarchical procedures (laboratory-tofield work) were conducted to screen these potential biological growth-promotors. In conclusion, 10 competent PSB stains were selected and evaluated under various conditions including plant beneficial traits, substrate, and host crop preference. Consequently, the current strains have shown positive and encouraging plant-benefiting traits and responses on tomato and other crops especially, K-1-29, K-10-27, K-10-41, Z-12-20, Mk-20-7, and Mk-1- 25, can be developed and recommended as biotechnological farm-inputs as potential biofertilizers and PGPR. Besides screening efficient strains, it is critical to apply other supplementals (possible P-substrates) for fruitful symbiotic effectiveness and improved production. Moreover, the future perspective should focus on conducting site-specific field trials, exploring more potential strains, and studying the detailed molecular and physiological symbiotic interactions and responses of the strains to develop competent bioinoculants that are cheap, affordable, ecofriendly, and sustain production especially for smallholder farmers.