EVALUATION OF SOME CHICKPEA GENOTYPES TO BACTERIAL INOCULATION

Field experiment was carried out during the two successive seasons (2015-2016 and 2016-2017) at Sids Research Station, Agricultural Research Center (ARC), Bani Swif Governorate, Egypt, to evaluate the response of some new chickpea genotypes cultivated under Egyptian soil conditions for rhizobial inoculation alone or in combination with foliar inoculation of Pink Pigmented Facultatively Methylotrophic bacteria (PPFMs) as Plant Growth Promoting Rhizobacteria (PGPR). Nodulation status, some vegetative growth and yield parameters were determined. The obtained results cleared that all chickpea genotypes tested were positively responded to the native soil rhizobia and formed root nodules. Rhizobial inoculation alone or in combination with foliar application of PPFMs bacteria (5 L fed) scored significant increases in nodule numbers, plant dry weight, yield per plant as well as seed yield at the both seasons as compared to untreated treatments. Using rhizobial inoculation and PPFMs bacteria emphasized the superiority and gave of the highest values at all tested parameters. Generally, the second season gave the highest values at all plant tested parameters as compared to uninoculated ones. Chickpea genotypes GT3, GT4 and GT7 emphasized higher response to cultivated under Egyptian soil conditions and gave higher values for nodules number and dry weight, growth parameters i.e. plant dry weight and plant N-content and yield parameters i.e. yield per plant, seed index and seed yield ton. fedas compared to chickpea variety G195.


INTRODUCTION
Legumes-Rhizobium symbiosis is undoubtedly the most important N2-fixing process and play a subtle role in providing nitrogen and maintaining/improving soil fertility. Symbiosis between legumes and rhizobia are of a considerable environmental and agricultural importance, since they are responsible for most of the atmospheric nitrogen fixed on land (Graham and Vance. 2003).
Chickpea (Cicer arietinum L.) is one of the earliest grain crops cultivated by man and has been found in Middle Eastern archeological sites dated at 7500-6800 B.C., (Williams and Singh. 1987). Chickpea is highly self-pollinating with an out crossing rate of less than 1%. Two main types of chickpea cultivars are grown globally, representing two diverse sub gene pools: Kabuli and Desi. The Kabuli types are generally grown in the Mediterranean region, southern Europe, western Asia, and northern Africa and the Desi types are grown mainly in Ethiopia and the Indian subcontinent. In spite of the above-mentioned constraints, extensive international breeding efforts have led to the development of over 300 improved varieties ( Gowda and Gaur .2004).
Chickpea is grown in about 50 countries, it can fix up to 140 kg nitrogen ha -1 and meet up to 80% of its nitrogen requirement from symbiotic nitrogen fixation (Abo Taleb (1998) and Al-hudaiji (2015)). Chickpea has the highest nutritional compositions and rich in fiber and minerals (phosphorus, calcium, magnesium, iron, and zinc). Its lipid fraction is high in unsaturated fatty acids in addition to having high protein content (20-22%). (Zohary and Hopf. 2000). Singh et al., (2008) illustrated the genetic relationships between the cultivated chickpea and its wild relatives is a prerequisite to track the evolution of cultivated species and also to determine the close relatives which can be exploited for introgression of Available online free at www.futurejournals.org

The Future Journal of Biology Print ISSN: 2572-3006 Online ISSN: 2572-3111
Future Science Association useful traits into the cultigen in plant breeding programs and many developing countries are substantial research programs to improve its yield, disease resistance and nutritional quality.
Vessey (2003) reported that numerous species of soil bacteria, which flourish in the rhizosphere of plants, but which may grow in, on, or around plant tissues, stimulate plant growth by various mechanisms. These bacteria are correctively known as Plant Growth Promoting Rhizobacteria (PGPR). The search for PGPR and investigation of their modes of action are increasing to exploit them commercially as biofertilizer. The mode of action of the biofertilizers includes fixing nitrogen, increasing the availability of nutrients in the rhizosphere, positively influencing both morphology and growth of roots, and promoting other beneficial plantmicrobe symbiosis. The combination of these modes of actions in PGPR is also addressed.

Madhiayan et al. (2005)
reported that The genus Methylobacterium-as PGPR -includes a variety of pink pigmented facultative methylotrophic bacteria (PPFMs) that promote plant growth by generating vitamins ,phytohormones (IAA, gibberellins and cytokinins) ,as well as supply nitrogen to plant through diazotrophy and indirectly reduce or prevent the deleterious effects of pathogenic microorganisms, through induced systemic resistance.
Etesami and Maheshwari (2018) stated that, combined use of PGPRs in agricultural environments may be a suitable approach to sustainably integrate with chemical fertilizers and lead to plant health improvements that play an important role in reducing the amount of chemicals to achieve sustainable agricultural productivity.
The present work aims to evaluate new chickpea genotypes response to rhizobial inoculation alone or in combination with application of PPFMs bacteria as PGPR bacterial inoculation and its role in enhancing the vegetative growth, seed yield and yield quality of chickpea plants under Egyptian soil conditions.

Soil used
A field experiment was layout during the two successive seasons (2015-2016 and 2016-2017) at Sids Research Station, Bani Swif Governorate, Agricultural Research Center (ARC). Physicochemical properties of the used soil was carried out according to Jackson (1973) at soil analysis Lab., Soils, Water and Environment Research Institute (SWERI), ARC, Giza, and is shown in Table (1).

Bacterial strains used
3.1. Two strains of Mesorhizobium ciceri namely ICARDA 36 and NIFTAL 1148 specific to Chickpea grown on Yeast extract Mannitol agar (YEM) medium (Vincent, 1970) were used as mixture basal peat inoculant at rate 4g inoculant to 100 g seeds at the time of planting as seed coating method according to Abo Taleb (1998).

Fertilizers used
The recommended doses of P and K fertilizers: 100 Kg superphosphate (15.5 % P2O5 fed 1 ) and 50 Kg potassium sulphate (24 K2O fed -1 ) were added during field experiment preparation. N-fertilization as ammonium sulphate (20.5 % N) was applied at 15 and 50 Kg N fed -1 and were added at 15 ,21 and 35 days after planting.

Treatments
Three treatments with 3 replications were allocated in a completely randomized block design as follows: 1. Un-inoculated plants + 50 Kg N fed -1 .
The plot area was 3x 3.5 m 2 . 6. Determinations 6.1. Growth stage: Samples were taken after 75 days of planting to determine: nodulation status (number and dry weight of nodules) according to Vencent (1970) and some vegetative growth parameters (plant dry weight and plant nitrogen content).

Statistical analysis
Data were subjected to an analysis of variance (ANOVA) and the least significant difference test (LSD) at P <0.05, by using (MSTAT) Program according to Snedecor and Cochran (1980 (2) show that, the all chickpea genotypes responded to the native rhizobia and formed root nodules bacteria but scored the lowest values of nodules formation in both seasons which ranged from 11 to 22 (nod. no. plant -1 ) as compared to other tested treatments. Inoculation with specific rhizobia scored significant increases in both seasons and such increases ranged between 22.7 -254.5% as compared to un-inoculated ones. Inoculation with specific rhizobia in combination with PPFMs as foliar application scored highest value (53 nod. no. plant -1 ) and led to gave significant increases in number of nodules ranged from 8.6-34.4% in both seasons as compared to the treatments which received rhizobial inoculation. Table (3) reveal that, the un-inoculated treatment recorded the lowest value of nodules dry weight (30 mg plant -1 ) in both seasons as compared to other treatments. Inoculation with specific Mesorhizobium scored higher values (68-104 mg plant -1 ) as compared to un-inoculated treatment in both seasons. Application PPFMs as foliar spraying in the presence of rhizobial inoculation scored highest significant increases and recorded nodules dry weight up to (163 mg plant -1 ) as compared to inoculation with specific Mesorhizobium alone. Inoculated chickpea genotypes GT4 having the highest value in nodule dry weight (up to 104 mg plant -1 ) among all inoculated chickpea genotypes as compared to uninoculated treatments.    (4) show that, the high significant differences in plant dry weight were evident among the all tested treatments. Un-inoculated chickpea genotypes recorded the lowest plant dry weight ranged 1.5 to 2.9 g plant -1 . Inoculated plants scored higher values ranged from 2.5 to 3.1 g plant -1 , significant increases ranged from 11.5 to 80 % of plant dry weight as compared to uninoculated treatments. Rhizobial inoculation in-combination with foliar PPFMs bacteria gave the highest plant dry weight (2.9 -3.3 g plant -1 ) and recorded significant increases up to 11.1% were observed as compared to inoculated treatments as such. Chickpea genotypes GT3, GT4 and GT7 having the highest plant dry weight as compared to other chickpea genotypes tested among the two tested seasons.    (5) reveal that, un-inoculated treatment recorded the lowest values of plant Ncontent, such values ranged from 41.7 to 74.2 mg plant -1 as compared to other treatments among the two tested seasons. Inoculation with specific Mesorhizobium scored higher values ranged from 87.4 to 94.8 mg plant -1 . The highest values of plant N-content were found at treatments which received rhizobial inoculation in combination with PPFMs foliar application with chickpea genotypes GT3, GT4 and GT7 as well as chickpea variety G195. stated that inoculated peanut seedlings with rhizobia as such or incombination with PGPR scored significant increase in total nitrogen (N) content (up to 76%) over the non-inoculated control.

Number of pods (No. Plant -1 )
Data in Table (7) show that, un -inoculated treatment gave the lowest number of pods at all tested chickpea genotypes as compared to other treated treatments in the both two seasons, these values ranged from (21.2 to 39.9 No. plant -1 ).
Inoculation with specific Rhizobium as such or in combination with PPFM bacteria emphasized the superiority in number of pods and recorded values ranged from 33.3 to 69.3 and 35.4 to 72.4 for inoculated plants as such and rhizobial inoculation in-combination with PPFMs bacteria respectively as an average of the two tested seasons.  (Fatima et al., 2008).

Yield per plant (g plant -1 )
Untreated chickpea genotypes recorded lower values for yield per plants and these values ranged from 9.9 to 14.2 g plant -1 as shown in Table (8). Application of rhizobial inoculation as such did support plant yield and led to gave higher values and scored significant increases ranged from 27.5 to 175 %, as compared to un-inoculated treatments. On the other hand, rhizobial inoculation in-combination with PPFMs bacteria having the highest plant yield values among the all tested treatments in the both seasons and gave percentage increases ranged from 6.4 to 59.4 % as compared to inoculated chickpea genotypes as such.  of Methylobacterium isolates incombination with Bradyrhizobium japonicum strain SB120 had significant influence on different plant growth parameters, nutrient uptake and yield of soybean plants.

Seed index (g 100 seed -1 )
Data in Table (9) cleared that, both rhizobial inoculations as such or in-combination with PPFMs bacteria gave higher values for seed index at all tested chickpea genotypes as compared to un treated treatments. GT4 and GT7 chickpea genotypes as well as chickpea variety G195 recorded the highest values of seed index and these values were 36.9, 33.6 and 34.6 (g 100seed -1 ) for GT4, GT7 and G195 respectively among the two tested seasons. The above mentioned data are in harmony with those obtained by

Seed yield (kg plot -1 )
Application of various bacterial treatments led to enhance seed yield (kg plot -1 ) and recorded higher values as compared to untreated treatments which recorded the lowest values for seed yield (kg plot -1 ) as shown in Table (10). GT7, GT3, GT4 chickpea genotypes and variety G195 were responded to rhizobial inoculation in-combination with PPFMs bacteria and scored the highest seed yield (kg plot -1 ) and these values were 2.13,1.99,1. reported that , the production of the plant growth regulators like auxins, particularly indole-3-acetic acid (IAA) and indole-3-pyruvic acid, zeatin, zeatin riboside and reacted cytokinins by Methylotrophs and IAA production and nitrogen fixation by Rhizobium has been reported as the factors that enhances plant growth of legumes. The increase in the vegetative growth of the plant is attributed to the increase in the yield of a crop.  (11) show that un-inoculated treatment recorded the lowest value of seed yield (0.252 ton fed. -1 ) in both seasons as compared to other treatments. Inoculation with specific Mesorhizobium scored higher value (0.950 ton. fed. -1 ) as compared to un-inoculated treatment in both seasons. Application with PPFMs as foliar spraying in the presence of rhizobial inoculation scored highest value (0.960 ton fed. -1 ) as compared to rhizobial inoculation ones. Application of both rhizobial inoculations had a positive effect on seed yield (ton fed. -1 ) for all tested chickpea genotypes as shown in table (11). Untreated treatments recorded the lowest seed yield values as compared to inoculated treatments as such or in-combination with PPFMs bacteria. The values were 0.810 ,0.718, 0.765 and 0.950 seed yield (ton fed .-1 ) for inoculated chickpea genotypes GT3, GT4, GT7 and G195 respectively and corresponding values at rhizobial inoculation in combination with PPFMs bacteria were 0.796 ,0.753, 0.850 and 0.960 (ton fed. -1 ) in the same order. These data were in agreement with those obtained by (Kantar et al., 2003; Ozturk et al.,  2003) and Orf, Heba et al. (2006) who reported that significant increases in seed protein content due to bacterial inoculation supported the hypothesis that biological nitrogen fixation by the Rhizobium and PGPR-root associations could be responsible for the observed higher N uptake of inoculated plants. Shehata, Sawsan (2006) established The increase in the yield due to compatible nature of Methylobacterium  Rhizobium and they found that, combined influence on phyllosphere by methylotrophs, which are plant growth promoting phyllosphere (PGPP) bacteria, and on rhizosphere by Rhizobium, which is nitrogen fixing bacterium, might have resulted in increased plant growth and yield parameters.

CONCLUSION
In the present study it could be concluded that: under Egyptian soil conditions, necessity exists for inoculation with specific rhizobia alone or in combination with PGPR bacteria to maximizing the development and yield production of chickpea plants. All tested genotypes of chickpea emphasized the superiority of response to inoculation with specific rhizobia and foliar application with PPFM bacteria. PPFMs did support nodule form, plant growth and yield and reduce using chemical fertilizers specially nitrogen fertilizers. GT3, GT4 and GT7 chickpea genotypes gave a positive results and higher values for the all tested chickpea parameters in comparison to chickpea G195 variety under Egyptian soil conditions.