Laboratory of Mycopathology and Microbial Technology, Centre of Advanced Study (CAS) in Botany,
Institute of Science, Banaras Hindu University, Varanasi, Uttar  Pradesh, India.

Corresponding author email: kumaridivyanshubhu@gmail.com

Article Publishing History

INTRODUCTION

Barley (Hordeum  vulgare L.) is a member of the Poaceae grass family, ranking fourth  among cereals behind maize, wheat and rice with respect to its worldwide production. It is  the fast growing  annual crop, grows in winter season (Ghanbari et al. 2012). Barley is a  diploid species with a high degree of inbreeding (Self pollinating) capability. It  has a low  chromosome number (2n=14) with a large genome size, can be easily cultivated and adapt to  different climatic and environmental conditions; and easily cross-bredable. Because of its  adaptability and  robustness, barley is grown in over 100 countries worldwide (Harwood 2019). Barley crops were used in the brewing  industry, as animal feed, and as healthy food options for human consumption. They were also used as a cover crop to increase soil  fertility (Ghanbari et al. 2012).

The  inclusion of barley in the diet provides several  health benefits, including  lowers blood  cholesterol levels, increased fibre intake, and a good source of beta-glucan, the highest  amount of beta-glucan was  found  in barley (Behall et al. 2004; Harwood 2019).

The  amount  of  grain  produced  is  insufficient  to  meet  the  food  demand  of  India’s  uncontrolled  growing  population;  thus, to  increase  crop  productivity  and  yield  to  feed the  growing  population,  synthetic  fertilizers  and  pesticides  were  incorporated  into farming  without  regard  for  the  negative  and  hazardous  impact  on  the  environment, food  chain, and  human  health.  The uncontrolled  use  of  chemical  fertilizers  in  agriculture is  presently  the  topic  of  debate  due  to  environmental  concern  and  fear  for  living being  health (Turan  et  al.  2010). The  synthetic  chemical  fertilizers  are  the  inorganic fertilizers  rich  in  major  nutrients  NPK (nitrogen,  phosphorus, potassium)  in  huge amount  and  do  not  incorporate  organic  manures  consequently  results  in  deterioration of  soil  quality  and  its  fertility (Choudhry  2005; Harwood 2019).

Plant growth  promoting rhizobacteria (PGPR) were the healthy and cost effective strategy  to enhance the crop productivity. Application of PGPR as biofertilizer are the most effective approach to enhance the sustainable agricultural systems (Sharma 2003). PGPR are  soilborne bacteria  that aggressively colonize the rhizospheric region of plants or when  applied to  the  seeds  or  crops  enhances  the  growth and yield  of plants (Kaymak 2011).  Incorporation of PGPR instead of chemical fertilizers are known to improve grain yield  through supply of plant nutrients may help to sustain and protect environmental  health (O’Connell 1992). Inoculation of phytomicrobiome members in agriculture for crop  productivity  is  a  sustainable  and  cost-effective approach  to disease control  and  artificial, chemical,  synthetic  supplements  could  reduce  the  negative  effects associated  with  the  excessive use of chemical fertilisers and pesticides (Antar et al. 2021b). These  phytmicrobiomes  have  been  used  as  an  effective  strategy  to  reduce  biotic  and  abiotic  stresses  that  could  improve  crop productivity  (Khan et al. 2020; Antar et al. 2021b).

Lignin is the  second richest biopolymer of high molecular weight having complex phenolic structure and called as major structural  component  of plant cell (Nayak et al. 2020). Lignin deposition in the cell wall is the important step during stem development, provides  strength  to stem which is interlinked to  barley  growth, agronomic traits and hence affects yield (Jones et al. 2001; Begović et al. 2015). Lignin deposition also play  crucial role  in  water and mineral transport, activates defence mechanism  against  biotic and abiotic stresses, provides rigidity and mechanical strength  to the  tissues through  thickening  of  plant  cell  wall  and  development  of secondary  growth, helps in plant tissue/organ growth  and  development,  imparts culm lodging resistance and many more in favour of plant metabolism  (Jayamohan  and  Kumudini  2011;  Liu  et  al.  2018).

It  was found  that  lignin  deposition in monocot plant such as barley is not very much intensive and thus have not that much intense secondary  growth  in comparision to dicot plants, in which the secondary cell wall is made  up of 20% of lignin (Vogel  2008). Lignin deposition  protects  the monocot plants such as barley from poor culm strength which is also termed as culm lodging (Hai et al. 2005; Ma 2009). Moreover, lignin deposition  generate resistance against culm or stalk bending (reduction in culm height), imparts strength to the barley culm which have direct impact  on  grain  yield as bending of culm or reduction in culm height leads to shorter plant height which consequently reduces the grain yield (Lalić et  al. 2005; Bonawitz and Chapple 2013).

It was reported that PGPR treated plants can enhances the lignification in plants (Jha  2019). Increased lignin deposition were found in Azospirillum  brasilense treated strawberry plant which also provide resistance against charcoal rot disease (Viejobueno et al. 2021) Maximum lignin  deposition  was found in vascular bundle of chickpea plant inoculated by fluorescent Pseudomonas and Rhizobium PGPR strains (Singh et  al. 2013; Viejobueno  et  al.  2021).

PGPR application as biofertilizer in plant is a sustainable approach to improving crop production. Employment of PGPR can be introduced to achieve  the pupose of achieving the sustainable and resilient agricultural production system without application of additional chemical fertilization. In the view  of above background information the present investigation was aimed to evaluate the efficacy of PGPR isolates; Pseudomonas punonensis (R1),  Pseudomonas plecoglossicida (R4), Pseudomonas aeruginosa (R2), Alcaligenes faecalis (DBHU5) and their consortium on yield and yield attributing  agronomic parameters such as plant height, leaf surface area, number of fertile tillers, spike length, grains per spike, 1000 grain weight, grain yield,  straw  yield, total biomass and harvest index % of barley plant, further to investigate the effect of these PGPR strains on lignin deposition  at  the  vascular bundle, cell wall of stem section of barley.

MATERIAL  AND  METHODS

In order to investigate the impact of PGPR on yield and yield components of barley (Hordeum vulgare L.) in the field condition, the barley variety PL-  426 were sown during the Rabi season of 2018-19 at  Botanical Garden of  Banaras Hindu University, Uttar Pradesh, India. The  soil of experimental  plot was fertile, alluvial loam and is characterized as type of Indo-Gangatic  plains. Rabi season is the winter season in the northern India where crop  is shown in the month of November-December and harvested in March-April of the subsequent year.

The experimental design was laid out in split randomise block design plot with date of sowing on 1^st December 2018, having 2 conditions that are PGPR treated and control (without any treatment of PGPR and chemical fertilizer) having 6 treatments, with 3 replications of each treatment. Seeds were sown in 6m by 3m total area (1m by 1m each  plot) having 6 rows and 3 columns,a total of 18 plots. Row to row distance were 20 cm. Seeds were inoculated by PGPR strains by dipping the seeds for 5 hours into the bacterial broth prior to sowing in the field. Maximum precautions were taken to avoid any contamination and mixing of bacterial inoculations during sowing.The field was plowed  twice prior to sowing the seed also weeds, unwanted materials were removed and cleaned manually. Plots were irrigated regularly with raw water without any mixture of chemical fertilizers.The crops were harvested during first week of April. The experiment had 6 treatments which are described below

R1- Seeds inoculated with PGPR Pseudomonas punonensis LMT03(Accession no.  MT677939)

R4- Seeds inoculated with PGPR Pseudomonas plecoglossicida (Accession  no.  MT883433)

R2- Seeds inoculated with PGPR Pseudomonas aeruginosa DSM 5007(Accession no.  MT845116)

DBHU5- Seeds  inoculated with PGPR Alcaligenes faecalis (Accession no. MT872514)

Consortium- Combined treatment of all the 4 PGPRs (R1, R4, R2  and  DBHU5)

Control- Without any PGPR treatment and any fertilizer (Non inoculated). Irrigation with  raw  water only

For evaluation of yield and yield attributing agronomic parameters ten randomly crop plants  were selected from each of three replicates, all the parameters were recorded from selected  plants.Yield was estimated through harvesting all the crop plants of each plot (three replication of each treatment). Mean value of all the three replications of each  treatments  were considerd for calculation of all agronomic traits. The agronomic parameters studied  was: Leaf surface area (cm²) – Leaf surface area were taken by measuring the  length  and  width of a leaf using scale.Plant height (cm)- At physiological maturity, height was  measured from the ground level to the top of the spike (excluding the awns) using a meter  rod. Number of productive/ fertile tillers- Number of fertile tillers per selected plants were  counted.

Spike length (cm)- Three spikes from each of the ten plants per plots  selected  and  length of spikes were recorded from the base to the apex of the spike through a meter rod.  Number of grains per spike- Three spikes of each selected ten plants from each replication  were threshed and the grains were separated from the spikes and were counted manually. 1000  grain weight (gm) – Thousand grains were counted after harvest and weighed  for  each  replication. After harvest, weight of thousand grains from each plot were taken using  weighing balance. The mean value of three replication was used in figure. Grain yield/ (kg/ha)- After harvesting, grains were threshed and seperated, grain  weight of all crop plant of each plot were taken in kg using electronic balance and  subsequently converted into kg/ha.

Straw yield (kg/ha)-After harvesting weight of sun-dried  above ground parts (excluding grains) of all crop plants from each plots were taken using  electronic balance in kg and subsequently converted into kg/ha. Total biomass/ Biological  yield (kg/ha) – All crops from each plot under that area were harvested, bundled, sun dried  and then weight the bundles in kg using electronic balance for estimation of total biomass ,  afterward converted into kg/ha. Harvest index%- Ratio between grain yield and total  biomass of all crops of each plot was determined by applying the following formula:  HI  (%)  =  (Grain yield  each  replication/ Total biomass (grain + straw) each replication ×100.

Histochemical deposition and distribution of lignin deposition in barley internodes: To estimate lignin deposition, a hand cut transverse section of fresh barley internode was mounted on a slide and stained with a solution of 0.5 percent saturated phloroglucinol (w/v) with addition of HCl and observed under an Olympus binocular microscope. The appearance of red-violet colour on the section defined the conformation of accurate lignin staining (Jensen 1962).

Statistical analysis: The IBM SPSS Statistics Ver.20 software was used for all statistical analysis and calculations. The statistical data were expressed as the mean of three independent replications, standard error of mean (SEM) of three replicates of each experiment, and thrice repetition data of each replicate, and were interpreted using one-way ANOVA followed by Duncan’s multiple range test at the P=0.001 significance level. The experiments in this study were carried out in triplicate, with each experiment being repeated three times using a completely randomised design.

Results and Discussion

Yield and yield attributed agronomic parameters: Grain yield performance is heavily  influenced by agronomic traits. Seeds inoculated with PGPR strains P. punonensis, P. plecoglossicida, P.aeruginosa, A. faecalis alone and in combination (consortium) significantly increases plant height, number of fertile tillers, leaf surface area, spike length, number of grains per spike, 1000 grain weight, grain yield, straw yield, total biomass, and harvest  index. Performance of the plants on all the studied  parameters  was  superior  in PGPR inoculated  treatments  in comparison  to the  non-PGPR  inoculated  plants. The maximum increase in all the agronomic  and plant growth promoting parameters  was recorded  by consortium  treated  plants  followed  by P.  punonensis, P.  plecoglossicida, P.  aeruginosa,  A. faecalis, where as  the  least  value of data was obtained  from  control  plots. For  all the field parameters, mean value of all the three replications of each  treatments  were consider for calculation  and  yield  analysis  (Fig.  1).

Figure 1: Barley crop grown in experimental field plot of botanical  garden of Banaras Hindu University.

Leaf  surface  area  (cm²): Seeds  inoculated with PGPR significantly increases the leaf surface area in barley cultivars over untreated plants. The  mean result of all the treatments revealed that the combined effect of all the PGPRs (consortium) produced the highest leaf surface area (72.13 cm², followed  by  R1  (70.26  cm²),  R4  (68.60  cm²),  R2  (67.73  cm²)  and  DBHU5  (66.30  cm²),  while  the  least  value  (59.73  cm²)  was  recorded  by  control  treatment.  The  mean  data  with  respect  to  leaf  surface  area  have  been  summarized  in  Table  No.  1  and  Fig.  2a.  PGPR  treatments  in  barley  plants  results  in  increase  in  leaf  surface  area  which  may  enhances  the  gaseous  exchange  hence  rate  of  photosynthesis  increases  which  promotes  various  plant  metabolic  activities.  Purwanto  et  al.  have  reported  that  PGPR  inoculation  in  rice  plant  can  increase  leaf  surface  area  upto  91.10  percent  compared  to  control  plants  (Purwanto  et  al.   2019).

Plant height (cm): All the PGPR treatments had significantly increased the plant height  over untreated plants. The mean of plant height was observed to be in the ranges of  76.56-87.53 cm. The highest plant height (87.53 cm) was recorded by consortium followed by R1  (85.36 cm), R4 (84.60 cm), R2 (82.56 cm), and DBHU5 (80.80 cm), while the shortest plant  height was showed by control plants (76.56 cm). Result of mean plant height indicated in (Table No. 1, Fig. 2a). Plant height is an important factor and positively correlated with grain yield.Increase in plant height by the inoculation of PGPR indicates that PGPR  inoculation in barley plants can increase vegetative growth. Increase in plant height of  barley, wheat, corn, by PGPR treatment was already reported (Shaharoona et al.2007; Gholami et al. 2009; Shirinzadeh et al. 2013; Hussain et al. 2020).

Number of productive/fertile tillers: Seeds treated by consortium produced maximum number of productive tillers (10.23), followed by R1 (8.76), R4 (7.40), R2 (6.30) an DBHU5 (6.00), the reduced number of productive tillers was produced by control plants.The average number of total fertile tillers as indicated in (Table No. 1, Fig. 2a).The number of fertile tillers is an important agronomic factor that influences grain yield. Similar reports were foundby Shaharoona et al. in wheat crop as the number of fertile or productive tillers in plant increases the number of spikes along with grains, which play a vital role in grain yield (Shaharoona et al. 2007). Increase in number of tillers is one of the chief agronomic character as this may compensate the difference in number of plants, partially or totally after crop establishment and may allow crop recovery from early frost (Acevedo et al. 1998; Hussain et al. 2020).

Figure 2: (a-b) Graph representing effect of different PGPR strains and their consortium on (a.) leaf surface area, plant height, number of fertile/productive tillers, (b.) spike length, grain per spike, 1000 grain weight of barley plants in comparision to  control plants, illustrating increase over control in field condition. Data are means of  three replicates along with standard error of mean bars. Different letters above the standard error bars denotes significant differences over control (p<0.001).

Spike  length  (cm): The impact of seed inoculation with PGPR treatment on spike length  was  significant. The maximum spike length (8.10) was obtained by consortium treated plant  followed by R1 (7.80 cm), R4  (6.53 cm), R2  (5.86 cm), DBHU5 (5.30 cm) and minimum  spike length (4.10 cm) was recorded by control plots indicated  in  (Table No. 1, Fig.  2b).  PGPR inoculated plants showed significant increase in the grain number per spike in  comparision to control plants so most highest number of grains per spike shown by  consortium (50.10) treated plants, while R1 (44.40) showed the second most highest  grains/spike followed by R4 (42.50), R2 (40.23) and DBHU5 (37.63).

The lowest  number  of  grain/spike was recorded by control plots (34.96) as indicated in (Table No. 1, Fig. 2b).  Increase in spike length, number of grains per spikeis directly proportional to the grain  yield, increase in spike length by PGPR treatments, results in more production of grains in  spike which consequently results in increase of grain yield of barley plant (Shahzad et al. 2007). In our study increase in spike length and number of grains per spike through the  inoculation of PGPR is according to the findings of  Shirinzadeh et al., on agronomic traits  of barley (Shirinzadeh et  al. 2013).  Inoculated barley plants had more grain number per  spike and hence more grain yield (Hussain et al. 2020).

1000  grain  weight  (gm)-  Based on the data of all treatments of each of the three  replicates, the highest 1000 grain weight was recorded by plants inoculated by consortium  (47.86 gm), further R1 showed the second highest (45.80 gm), later on R4, R2 and DBHU5  showed (44.50 gm), (44.10 gm) and (43.46 gm) 1000 grain weight respectively. The  lowest  value was recorded by the grains produced by control plants (38.53) indicated in Table No. 1 and Fig. 2b. 1000-grain weight is an essential yield determining factor of barley. Inoculation  of barley seeds with PGPR significantly increases the 1000-grain weight which results in  improved seed quality, has been reported by cakmakci et al (cakmakci et  al 2007). Increase in 1000-grain weight of barley plant, inoculated by Azotobacter,Azospirillium, Azotobacter+ Azospirillium is reported by Shirinzadeh et al. (Shirinzadeh et al. 2013). Wheat plant treated with consortia of Paenibacillus polymyxa, Bacillus subtilis and Bacillus aryabhattai as well as separate inoculation of these PGPRs significantly increased the 1000 grain weight  of wheat (Hussain et al. 2020).

Grain  yield/  Economic  yield  (kg/ha)-  Grain yield varied between 3200 kg/ha in without  treated till 7976 kg/ha in seed treated with PGPR. Maximum grain production was recorded  by consortium treated plants (7976 kg/ha) followed by R1 (6976 kg/ha), R4 (6263 kg/ha),  R2 (5400 kg/ha), and DBHU5 (4400 kg/ha), while control platts produced only 3200 kg/ha  indicated in (Table No.1, Fig. 3a). Grain yield is the main goal of agricultural practices by  farmers. Grain yield is one of the significant factor towards yield and yield attributing components. Increased grain yield is directly dependent on increase in number of productive tillers and grain per spike which is also supported by the study of Naeem et al. (Naeem et al. 2018). Enhancement in barley plant growth and grain yield through PGPR treatment is reported in a previous study (Cakmakci et al. 2007). Increase in yield of many cereals crops  through application of diazotrophs has been in a previous study (Dobbelaere et al. 2003). Bacteria inoculated plants such as corn, sugarcane, rice increases the yield upto 10 to 30 percent as reported in a previous study (Kloepper et al. 1992). Hussain et al. (2020) reported that application of novel Bacillus and Paenibacillus species bio-inoculants separately and in combination has a positive influence on yield of wheat crop. Application of Pseudmonas spp.

and Burkholderia caryophylli in wheat plant leads to increase in yield of wheat is reported in a previous study (Shaharoona et al. 2007). Significant increase in barley grain yield by the application of Azotobacter and Azospirilliu is reported by Shirinzadeh et al. (Shirinzadeh et al. 2013). Seed inoculation with Azospirillum brasilense significantly affects the yield of barley and wheat as reported in a previous study (Ozturk et al. 2003). Similar result was found by combined application of indigenous PGPR; B. megaterium, A. chlorophenolicus and Enterobacter on wheat grain yield as reported in a previous study (Kumar et al.  2014). Several studies were found in support of significant increase in grain yield by PGPR inoculated plants (Tiwari et al. 1989). Imran et al. (2015) have reported increase in grain yield in Ochrobactrum ciceri and Mesorhizobium ciceri inoculated chickpea (PUSA-372) plant (Imran et al. 2015). The maximum increase in grain yield of wheat was observed due to the consortium application of PGPR; Paenibacillus polymyxa, Bacillus subtilis and Bacillus aryabhattai  as investigated in a previous study (Hussain et al. 2020). Bacillus spp. significantly increased the grain yields of crops such as fingermillet, maize, amaranth, buckwheat and French bean (Pal 1988; Hussain et al. 2020).

Straw yield (kg/ha), Biological yield/Total biomass (kg/ha)– Among all treatments maximum significant straw yield (14990 kg/ha) was recorded in consortium treated plants, followed by R1 (14133 kg/ha), R4 (13433 kg/ha), R2 (12033 kg/ha) and DBHU5 (11333 kg/ha), while lowest straw yield (10200 kg/ha) was obtained in control plants indicated in Table 1, Figure 3a. The maximum biological yield were recorded in consortium treated plants (22966 kg/ha) followed by R1 (21110 kg/ha), R4 (19696 kg/ha), R2 (17433 kg/ha) and DBHU5 (15733 kg/ha) while the lowest biological yield (13400 kg/ha) was obtained from control  plots (Table No. 1, Fig. 3a). Biological yield is also an important parameter because farmers were interested in straw in addition to grain (Tigabu and Asfaw 2016).

In our study maximum biological yield or total yield was produced from consortia treated barley plants and similar result was found in a previous study by the treatment of triple combination of PGPR B. megaterium A. chlorophenolicus and Enterobacter on wheat plant which significantly enhanced the straw yield in field conditions in comparision to uninoculated plant (Kumar et al. 2014). Increase in straw yield, total biomass and harvest index by application of phosphate solublizing bacteria on wheat in comparision to control plants (Turan et al. 2010). Combined effect of Azospirillum lipoferum, Arthrobacte  mysorens and Agrobacterium  radiobacter increases the grain and straw yield in 3 barley cultivars (Belimov et al. 1995). Seed inoculation with Bacillus polymyxa significantly enhanced total yield in rice and chickpea crops (Tiwari et al. 1989). Harvest index (%)- The maximum harvest index value was obtained from consortium treated plants (34.73%), followed by R1 (33.04 %), R4 (31.79 %), R2 (30.95 %) and DBHU5 (27.96%), while the lowest harvest index was recorded by control plants (23.85%), as explained in Table No. 1, Fig. 3b.

Figure 3(a-b). Graph representing effect of different PGPR strains and their  consortium on (a.) grain yield, straw yield and total biomass/ biological yield, (b.)  Harvest index % of barley plants in comparision to control plants, illustrating increase  over control in field condition. Data are means of three replicates along with standard  error of mean bars. Different letters above the standard error bars denotes  significant  differences over control (p<0.001).

Table 1. Effect of inoculation with PGPR strains and their consortium on yield and  yield attributing parameters of barley crop in comparision to control plants.

Treatments	PH	LSA	FT	SL	GPS	TGW	GY	SY	TB	HI
Consortia	87.53±0.20a	72.13±0.08a	10.23±0.14a	8.10±0.05a	50.1±0.05a	47.86±0.03a	7976±14.52a	1499±5.77a	22966±20.27a	34.73±0.03a
R1	85.36±0.27b	70.26±0.12b	8.76±  0.13b	7.8±  0.05a	44.40±0.30b	45.80±0.43b	6976±14.52b	1413±133.33b	21110±145.25b	33.04±0.17b
R4	84.60±0.20c	68.60±0.15c	7.40±  0.05c	6.53±  0.2b	42.50±0.28c	44.50±0.28c	6263±131.69c	1343±260c	19696±375.95c	31.79±0.26c
R2	82.56±0.17d	67.73±0.12d	6.30±  0.24d	5.86±0.44bc	40.23±0.14cd	44.10±0.05cd	5400±57.73d	1203±33.33d	17433±66.66d	30.95±0.23d
DBHU5	80.80±0.05e	66.30±0.30e	6.0±  0.06d	5.30±0.25c	37.63±0.91e	43.46±0.26d	4400±57.73e	1133±88.19e	15733±145.29e	27.96±0.11e
Control	76.56±0.28f	59.79±0.37f	4.10±0.16e	4.10±0.05d	34.96±0.54f	38.53±0.2e	3200±115.4f	10200±115.47f	13400±230.94f	23.85±0.44f

Here ‘ha’, hectare; ‘gm’, grams, PH- plant heigh, LSA- leaf surface area, FT- number of  fertile tillers, SL- spike length, GPS-grain per spike, TGW-thousand grain weight, GY-grain  yield, SY- straw yield, TB- total  biomass, HI- harvest index. Different letters on  mean±standard error denotes significant differences over control (p<0.001).

Barley seeds inoculation with PGPR Azotobacter and Azospirillium enhanced the plant  growth promoting parameters such as plant height, spike length, number of spike per area,  grains per spike, 1000 grain weight significantly and could enhance grain yield to an  acceptable level. PGPR inoculation in crop plants can increase the crop productivity and  yield as high as upto approx 57% depending on the crop plant (AsgharMet al. 2004; Shirinzadeh et al. 2013). The PGPR strains; Bacillus megaterium, Bacillus subtilis, and  Azospirillum brasilense were reported to enhance the grain yield, straw yield, total yield,  and plant nutrient element content of barley and wheat crop (Baris et al.  2014; Hussain et  al. 2020).

Linear Correlations Between All Agronomic Parameters of all Treatments: Table No. 2 represents the types of correlations between the agronomic growth and yield  parameters of barley. All the 10 agronomic parameters were positively and significantly  correlated with each other of all the six treatments, this implies that increase in the value of  one parameter leads to increase in the other parameter to which it is significantly correlated  and their growth were dependent on each other. As grain yield is our main objective of  this  study and for the farmers it is the main factor so we recorded that plant height showed  highly significant and maximum correlation with grain yield  r  =0.988**,1Leaf surface area  showed r  = 0.953** with grain yield, Number of fertile tiller have r  =0.985** with grain  yield, while spike length r  =0.987**, grain per spike r =0.981**, thousand grain weight r  =0.947**  also showed significant correlation coefficient with grain yield. Here ‘r’ is  correlation coefficient.

From the present correlation data it was concluded that in this study  plant height and spike length is positively interlinked with grain yield, if plant height and  spike length increases there will be increase in the grain yield also. It was also observed  from the data that if plant height increases there will be increase in the harvest index %.  Correlation between different agronomic traits provides necessay information and guidance  to the farmers for the selection of yield enhancing traits. In the present study correlation  between all the 10 traits; plant height, leaf surface area, number of fertile tillers, spike  length, grains per spike, 1000 grain weight, grain yield, straw yield  total biomass, harvest  index% were analyzed for all the six treatments. All  the traits have positive and  highly  significant interrelationship with each other (Hussain et al. 2020).

Table  2. Correlation (Pearson coefficients) among agronomic parameters of all the six  treatments.

Parameters	PH	LSA	FT	SL	GPS	TGW	GY	SY	TB	HI
PH	1
LSA	.983**	1
FT	.969**	.943**	1
SL	.970**	.947**	.985**	1
GPS	.953**	.908*	.983**	.957**	1
TGW	.975**	.995**	.951**	.940**	.920**	1
GY	.988**	.953**	.985**	.987**	.981**	.947**	1
SY	.978**	.933**	.984**	.987**	.974**	.926**	.995**	1
TB	.984**	.944**	.986**	.988**	.978**	.938**	.999**	.999**	1
HI	.992**	.984**	.948**	.962**	.935**	.972**	.979**	.979**	.960**	1

*  Correlation  is  significant  at  the  p  ≤  0.05  level

Deposition and Distribution of Lignin In Internodes of Barley: Deposition of lignin in pink to violet color were observed in the region of sclerenchyma ring  of cortex, epidermis, parenchyma and vascular tissue of all the 4 rhizobacterial isolates (R1,  R4, R2 and DBHU5) and their consortium treated barley plants, while there is light or  moderate pink color were developed which showed thin or less lignin deposition on  sclerenchyma ring of cortex, epidermis, parenchyma and vascular tissue of control plants.  PGPR treated plants showed thickness in the cell wall due to lignin deposition in  comparision to the non treated plants as observed in Fig 4 (a-f).

As barley is a monocot grass plant which was known for less lignin deposition but in the  present result we found that stem vascular bundle section of all the four PGPR  treated plant  showed intense lignin deposition in comparision to control. Although it was studied and  found that lignin synthesis in barley plants occurs with very low intensity and lower  quantity, but in the present study it was found that all the four PGPR treated barley plants  showed significant and uniform lignin deposition in their vascular bundle region and cell  wall of  internodes section of stem in comparision to control plants. PGPR treated plants  develop maximum cell wall lignification which induces and activates higher concentrations  of defense related enzymes, it was found that rhizospheric bacteria Bacillus megaterium  enhances the lignin deposition in the cell wall of maize plants and protects against   Aspergillus niger (Jha 2019).

Similar result was observed by the treatment of PGPR inoculants Pseudomonas and   Rhizobium on lignin deposition in the vascular bundle of chickpea plant (Singh et al.  2013).  Study of the pattern of lignin deposition in the cell wall of internodes of barley (Begovic et  al. 2015). Maximum and dense lignin deposition was found in the secondary walls of xylem  vessels of strawberry plant treated by Azospirillum brasilense (Viejobueno et al. 2021).  Lignin deposition in plant stem perform vital role in conductance and movement of water  which develop resistance ability in plants under abiotic stress and also provide  rigidity  to  the cell wall (Ajao et al. 2018). Stem and root section of B. megaterium and P. fluorescens  treated mungbean plants showed significant increase in lignin deposition which also protect the mungbean plant from the infection of M. phaseolina (Javed et al. 2021).

Figure  4. (a-f)  Influence of PGPR strains (a.) Consortium (b.) R1, (c.) R4, (d) R2, (e.)  DBHU5 on lignification in barley stem by histochemical staining in comparision to  stem of (f.) non-inoculated control plant, illustrating increase lignin deposition  over  control. 

CONCLUSION

The findings of the present finding showed significant increase in the  yield and yield associated agronomic parameters of barley plants treated by separate  inoculation of PGPRs P. punonensi, P. plecoglossicida, P. aeruginosa  and  A. faecalis  along with combined inoculations of these PGPR strains in comparision to the control  plants. PGPR treated barley stem vascular bundle have more intense lignin deposition layer  as compared to the non treated plants. All the four PGPR treated isolates showed maximum  lignin deposition in the cell wall of barley internodes and consequentialy enhances cell wall  thickness in comparision to control. This  is the first study on the effect of PGPR; P.  punonensis, and  P. plecoglossicida strains treated barley plants on their yield and yield  attributing parameters under field condition. Also this the first study done on characterization  of lignin deposition on barley plants treated by P.punonensis and P. plecoglossicida PGPR  strains.

ACKNOWLEDGEMENTS

The study was supported by the department  of  botany, banaras hindu university, varanasi, uttar pradesh, india and human resource development group, council of scientificand  industrial research, new delhi, india under the research fellowship  as CSIR-JRF and CSIR-SRF.

Declarations

Funding:  KD wish to thanks Council of scientific and Industrial Research (award no.  09/013(0689)/2017-EMR-I), New Delhi, India for financial support as CSIR JRF and CSIR  SRF.

Conflicts of  Interests: Authors declares no conflicts of interests to disclose.

Availability of data and Material: All data/ results/ information is available with the  authors, were mentioned in the manuscript and can be shared on a reasonable request made  to the corresponding author when required.

Code  Availability: Not  applicable

Authors’ Contributions: KD: Conceived the research, wrote the manuscript, analyzed the data, acquire the funding.  KD: Performed the research. KD: Analyze the data. RSU: Wrote the manuscript, Supervised  the  research.

Ethics  approval:  Not applicable.

Consent to participate: All authors participated in the experiment.

Consent  for Publication: All authors read and aware of publishing the manuscript in  Bioscience Biotechnology Research Communications.

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