Effect of Azotobacter and Pseudomonas with Mineral Fertilizer on Yield and Yield Components of Malt Barley (Hordeum vulgare L.)

The effect bio-fertilizer composed of Azotobacter and Pseudomonas spp. was investigated on yield and yield components of malt barley (H. vulgar L). The treatments which were laid at Bekoji, a potential malt barley district in the southeastern highlands of Ethiopia, Azotobacter + Pseudomonas + 46/20.2 N/P kg ha -1, Pseudomonas +46/20.2 N/P kg ha -1, 46/20.2 N/P kg ha -1, Azotobacter +46/20.2 N/P kg ha -1, Azotobacter + Pseudomonas, Azotobacter, Pseudomonas, C- (uninoculated and unfertilized) in randomized complete block design(RCBD) with three replications. Results indicated that application of the bio-fertilizer treatments alone resulted in the minimum performance of different agronomic parameters studied, while inorganic fertilizer application with dual inoculation with Azotobacter + Pseudomonas gives the highest and significant effect on grain yield, harvest index, biological yield, plant height, and thousand seed weight as compared to other treatments. But this trend was not observed in the number of spikes for that case the result was reversed. Hence, the study showed that the use of chemical fertilizer along with dual inoculation (Azotobacter +Pseudomonas) should be considered as a component of inputs especially in inorganic farming systems of malt barley production.

this consortium could provide growers with a sustainable approach to reduce synthetic fertilizer usage in wheat production (AS et al., 2019;Ashok K and Vijay SM, 2019;Satyanarayana T and Johri BN, 2012;Sayyed RZ et al., 2019).
In Ethiopia, only a few studies on tef root-associated microorganisms have been undertaken. Accordingly, the effects of PGPR on growth and yield of tef were evaluated by Delelegn W. and Fassil A (2011). Microbial inoculum of two Bacillus species (Bacillus megatherium and Bacillus mucilaginous) improved the growth of the plant as well as the nutritional assimilation of the plant (Saida A et al., 2015). However, information on little has been known about the use of bio-fertilizers on malt barley in our soils, and hence, the present study was undertaken to generate information on the effects of phosphate solubilizing and nitrogen-fixing microorganisms (Azotobacter sp. and Pseudomonas sp.) on yield and yield components of malt barley.

Description of the Study Area
The study area, Bekoji, is found in the Arsi zone of Oromia Regional State in the southeastern part of Ethiopia. It is located at an altitude of 2780 m.a.s.l. with a latitude of 7°32'N and a longitude of 39°15'E. The area has a longterm mean annual precipitation of 1020 mm and a mean maximum and minimum temperature of 18.6°C and 7.9°C, respectively. The soil texture of the research site is reddish clay with a pH of 5.4.

Planting and Agronomic Practices
Field experiments were carried out in the three successive years of growing seasons at Bekoji, in the Arsi zone where barley production has high potential. The experimental plot size was 10.4m2 which is 2.6m x 4m x 13 rows of 20 cm apart. The space between each plot was 50 cm. Di-ammonium phosphate (DAP) containing phosphorus and nitrogen at a ratio of 18N:46P was used as a source of inorganic fertilizer. malt barley variety (cv. Holker) was sown at a seed rate of 150 kg ha-1. To inoculate with biological fertilizers, the seeds were first sterilized with 70% ethanol for two minutes and cleaned five times with distilled water, and were mixed with a sugar solution to serve as a sticker. Then the seeds were inoculated with the powder of Pseudomonas and Azotobacter at a rate of 500 g ha-1. The bacteria were applied with a carrier of fine lignite powder capable of passing through 75-106 µm sieves. The inoculants contain a minimum of 108 (at manufacturing date) up to107 (15 days before the expiry date) of viable cells. The Barley seeds were sown on the experimental plots soon after inoculation with the bio-fertilizers. All agronomic practices were carried out as recommended for the area. At harvest, the data recorded were plant height (cm), number of grain per spike, 1000-grain weight (g), biological yield (kg ha-1), harvest index (grain yield/biological yield × 100), grain yield (kg ha-1). Data were analyzed with Proc GLM procedure, SAS Tukey Honest Significant Difference (HSD) test was used to separate treatment means at a probability level of 0.05 (SAS, 2009).

Number of Spikes
The combined analysis of variance over three years indicated that the number of spikes per m2 was significantly (p < 0.05) and positively influenced by inorganic fertilizer application and bio-fertilizer inoculation ( Table 3). The highest number of spikes per m2 was obtained from the application of Pseudomonas + 46/20.2 kg NP ha-1. The number of spikes per m2 was increased by integrating bio-fertilizer with chemical fertilizer. Application of Pseudomonas + 46/20.2 kg NP ha-1 increased the number of spikes per m2 by 28.6% and 33.1% compared to the control and Azotobacter alone, respectively. Inoculation of seeds with the bio-fertilizer (Pseudomonas or Azotobacter) treatment along with the application of chemical NP fertilizer increased the number of spikes per m2 by 24.2 % to 28.6% compared to non-inoculated plants (Table 3). These results were consistent with previous findings (Abedi et al., 2010;Kandil et al., 2011;Liu and Shi, 2013).

Plant Height
Plant height was significantly (p <0.05) and positively affected by nitrogen, phosphorus, and bio-fertilizer application as shown in Table 3. The highest plant height (109.2 cm) was obtained from the addition of dual inoculants (Azotobacter + Pseudomonas) with inorganic fertilizer +46/20.2 NP kg ha -1). The next good treatments (Pseudomonas + 46/20.2 NP; NP alone and Azotobacter +NP) were not significantly different from the top performer. The 5th treatment (Azotobacter + pseudomonas) had an intermediate plant height (96.7cm) which is Journal of Natural Sciences Research www.iiste.org ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online) Vol.12, No.11, 2021 27 significantly lower than the four top performances. The last three treatments including negative control did not much to plant height increase (Table 3). Previous studies have also reported similar findings (Zorita and Canigia, 2009;Daneshmand et al., 2012;Namvar et al., 2012;Liu and Shi, 2013).

Grain Yield
Analysis of variance for individual and combined years showed that the mean grain yield of malt barley was significantly (p < 0.05) affected by applying bio-fertilizers ( Table 2). The highest (3686.0 kg ha-1) and lowest (1924 kg h-1) grain yields were obtained by applying dual bio-fertilizer inoculants along with NP fertilizer, and from the control, respectively. This showed a remarkable 91.6 % of yield increase ( Table 2). The inoculants (Azotobacter and Pseudomonas) are effective only when combined with NP fertilizers. Comparing Pseudomonas +NP and Azotobacter +NP treatments, the first is significantly better than the latter in increasing both grain and biomass yields ( Table 2). The combination of the two inoculants along with inorganic NP resulted in superior grain and biomass yield increments. Similar results were reported on the effects of N fertilizer (Kizilkaya, 2008;Wortman et al., 2011;Scursoni et al., 2012;Getachew et al., 2014) and bio-fertilizer (Saini et al., 2004;Sary et al., 2009;Namvar et al., 2012) on grain yield of different crops. The increase in grain yield in the inoculated plants could be attributable to the exudation of plant growth regulators (PGRs), such as auxins, gibberellin, and cytokinin by Azotobacter sp., in addition to the increase in nutrient availability (Vessey, 2003). Piccinin et al. (2013) showed that the combined application of chemical NP fertilizers and bio-fertilizer inoculation improved the grain yield of malt barley. Generally, nano bio-fertilizer application increased crop growth and improved yield and yield components in barley (Seyed M. et al., 2013) and extended the growing period in wheat (Mahmoud M. et al., 2014).

Biomass Yield
Results revealed that treatment effects for either biofertilizers or uninoculated were significant for both grain yield and total biomass yields of malt barley for each year (Table 1). In the combined analysis, application of Azotobacter + Pseudomonas + 46/20.2 N/P kg ha-1 increased total biomass by 35.8 %, compared to the Azotobacter treatments alone, and 50.1% increase to sole Pseudomonas inoculation similarly, Azotobacter + Pseudomonas inoculated plants with NP fertilizer resulted in higher total biomass (9388 kg ha -1) than the control with 5469 kg ha-1. Bio-fertilizer inoculation with NP fertilizer increased straw yield by 60.7 % as compared to the control ( Table  2). Several workers have reported similar effects of N fertilizer on total biomass of different crops (Kizilkaya, 2008;Wortman et al., 2011;Scursoni et al., 2012;Getachew et al., 2014) and bio-fertilizer (Saini et al., 2004;Sary et al., 2009;Namvar et al., 2012), Piccinin et al. (2013) also indicated that total biomass of malt barley was improved when malt barley plants were grown with a combination of NP fertilizer and bio-fertilizer inoculation.

CONCLUSIONS
The results of this study indicated that the combined use of organic and inorganic fertilizers improved yield and yield components of malting barley. The results indicated that the application of Azotobacter and Pseudomonas with chemical fertilizer (nitrogen and phosphorus) has shown a significant influence on yield, and yield components of malt barley. Thus, it seems that inoculating barley with bio-fertilizers (Azotobacter and Pseudomonas) caused the developing root system activity, and increasing plant access ability to nutrients in addition to producing hormones stimulating growth, it also seems that these bio-fertilizers had best and probably synergistic effects on each other which finally led to improving growth traits of barley. So the study results indicated that the application of suitable bio-fertilizers could be efficient in increasing yield, improving growth traits of barley. Thus, we suggested that the combined application of organic and inorganic fertilizers could improve the productivity of malt barley.

ACKNOWLEDGEMENTS
The authors would like to thank the Ethiopian Institute of Agricultural Research (EIAR) and Kulumsa Agricultural Research Center (KARC) for providing all the necessary facilities and supports during the execution of this study. Our heartfelt thanks also go to the soil microbiology research team of Holetta Agricultural Research Center, Our utmost gratitude to Dr. Asfaw Hailemariam and his team at the National Soil Testing Center for the preparation and provision of Azotobacter biological fertilizers. And we would like to take this opportunity to express our