Abstract: To study the effect of co-inoculation with plant growth-promoting rhizobacteria (PGPR) and Rhizobium, on yield and yield components of common bean (Phaseolus vulgaris L.) cultivars was investigated in 2 consecutive years under field condition of plant growing evidence indicates that soil beneficial bacteria can positively affect symbiotic performance of rhizobia. PGPR strains Pseudomonas fluorescens P-93 and Azospirillum lipoferum S-21 as well as two highly effective Rhizobium strains were used in this study. Common bean seeds of three cultivars were inoculated with Rhizobium singly or in a combination with PGPR to evaluate their effect on growth characters. A significant variation of plant growth in response to inoculation with Rhizobium strains was observed. Treatment with PGPR significantly increased pod per plant, number of seeds per pod, weight of 100 seed, weight of seeds per plant, weight of pods per plant, total dry matter in R6 as well as seed yield and protein content. Co-inoculation with Rhizobium and PGPR demonstrated a significant increase in the yield and yield components. The results showed that all treatments of bacteria increased yield; however, strains Rb-133 with Pseudomonas fluorescens P-93 gave the highest seed yield, number of pods per plant, weight of 100 seed, seed protein yield, number seed per pod, seed protein yield.
INTRODUCTION
The common bean (Phaseolus vulgaris L.) is an important legume for human nutrition and a major protein and calorie source in the world (Sharon, 2003). Phaseolus vulgaris can grow by assimilation on mineral nitrogen or molecular N fixation. A board range of Rhizobium species are able nodule and fix N2 with beans including R. leguminosarum biovar phaseoli, R. tropic and R. etli. The most Iran soils N deficiency, N2 fixation Rhizobium bacteria could increase yield at a low cost and preserve water resources from pollution by nitrates. It occupies more than 125,000 ha in Iran but yield remains low to moderate due to the scarce nodulation, high inputs of chemical fertilizers and low technologies applied. Common bean is usually considered a poor nitrogen-fixing legume however; its promising potential to fix nitrogen has been shown in several studies (Peix et al., 2001; Garcia et al., 2004; Remans et al., 2008). Poor nodulation and variable response to inoculation is mainly attributed to intrinsic characteristics of the host plant, particularly the nodulation promiscuity as well as the great sensitivity to other nodulation-limiting factors, such as high rates of N fertilizer used in intensive agriculture, high temperatures and soil dryness (Bais et al., 2006; Egamberdiyeva et al., 2007). Genotypic variation in beans as well as compatibility of Rhizobium-plant cultivars can also greatly affect the efficiency of symbiosis established. This variability often limits the nitrogen-fixing performance of soil native rhizobia or use of commercially available inocula. As a result, application of high amounts of inorganic nitrogen fertilizers is becoming a common practice which has detrimental environmental consequences.
MATERIALS AND METHODS
Plant material and bacterial strains: Three kidney bean cultivars
Sayad, Akhtar and Goli were obtained from Seeds and Plant Improvement
Institute (SPII) housed at Karaj, Iran and used throughout this research.
Two Rhizobium strains Rb-133 and Rb-136 with high nitrogen-fixing
effectiveness were used in this study. These strains were selected during
our previous screening program and demonstrated a good potential to nodulate
beans and increase plant growth and yield in greenhouse and field experiments
(Asadi Rahmani et al., 2005). PGPR strains Pseudomonas fluorescens
P-93 and Azospirillum lipoferum S-21 positive effects in this
study (Table 1). For preparation of inoculants, bacteria
were grown in appropriate growth media for three days and 150 mL of each
strain suspension was added to a polypropylene plastic bag containing
50 g of sterile powdered perlite and mixed thoroughly.
| Table 1: | Some characteristics of PGPR strains used in this study |
Experimental conditions: Field trails established in 2006 and 2007 at Shahrekord (50° 51`N 32° 17`E) south western in Iran. Experiments were arranged in a randomized complete block design using a split plot layout with three replications. Seeds of common bean cultivars Sayad, Akhtar and Goli were planted after they were moistened with a 20% solution of sucrose and then inoculated (7 g inoculant per kg seed) with Rhizobium alone or in combination with P. fluorescens P-93 or A. lipoferum S-21. The experiment also included a non-inoculated control as well as one N-fertilized (100 kg N ha-1) treatments. The main-plots units consisted of 8 treatments (six bacterial combinations, one uninoculated control and one N-fertilized) applied.
RESULTS AND DISCUSSION
We found significant different effects induced by rhizobia on growth parameters of common bean. Plant cultivars also showed different responses due to inoculation with rhizobial isolates.
Inoculation with Rhizobium significantly (p<0.01) increased number seed per pod, number pod per plant, weight of 100 seed, seed yield, weight of seed per plant, weight of pod per plant, protein seed yield and total dry matter in R6 in both years. Co-inoculation with PGPR promoted nodulation over Rhizobium alone, where the highest values for seed yield and yield components were observed by combination of Rhizobium and Pseudomonas fluorescens P-93. Similar result was obtained for shoot dry matter of plants in R6 growth stage. Rhizobium inoculation by isolates Rb-133 and Rb-136 increased dry matter production of the shoots. Co-inoculation with A. lipoferum S-21 improved dry matter production over Rhizobium treatment. This beneficial effect was greater for PGPR strain P. fluorescens P-93 and demonstrated the highest dry matter production. Application of PGPR enhanced seed yield compared to either control or Rhizobium alone. The highest seed yield was obtained from plants inoculated with Rb-133+ P. fluorescens P-93 which showed a 240% increase over control plants.
The amount of seed yield was affected by co-inoculation of Rhizobium with PGPR strains in cultivars used in this study (Table 2-4). P. fluorescens P-93 resulted in highest amount of seed yield and other characters followed by A. lipoferum S-21, when co-inoculated with Rb-133, indicating that P. fluorescens P-93 had the most promising effect on enhancement of symbiotic performance of rhizobial strains. Plants inoculated with Rhizobium alone showed different values for yield components. Plants inoculated with Rb-133+ P. fluorescens P-93 made the most seed yield, which showed over 50% increase compared to Rhizobium alone.
This research has shown the effects of co-inoculation with two PGPR strains
on the symbiotic performance of common bean nodulating rhizobia in three
P. vulgaris cultivars. Common bean is believed to be a poor nitrogen
fixer due to the genetic characteristics of symbiotic partners as well
as soil and environmental conditions. However, selecting rhizobia for
increased survival in specific soil types, greater compatibility with
crop species or cultivars, superior functioning under diverse climates,
improved compatibility and competitiveness with other soil micro organisms
and higher nitrogen-fixing efficiency have been shown that can improve
growth and yield components of inoculated legumes (Vessey, 2003). Beneficial
effects of rhizobia on common bean have been described in several studies
with different climatic and soil conditions (Peix et al., 2001;
Tilak et al., 2006; Remans et al., 2008). All plant factors
we measured in the study were positively affected by inoculation with
rhizobia strains Rb-133 and Rb-136. Rhizobia strains were able to increase
seed yield, number of pods per plant, number of seeds per pod, weight
of 100 seed, weight of seeds per plant, seed protein yield, total dry
matter in R6 and protein content over uninoculated control
plants. Amount of seed yield by inoculated plants was ranging from 1221
to 4693 kg ha-1 depending on the strain and cultivars used
during two years of the study. However, Rb-133 showed a greater symbiotic
efficiency than Rb-136. Plant cultivars also had different responses to
rhizobial inoculation. Cultivar Akhtar demonstrated highest potential
for seed yield, number of pods per plant, number of seeds per pod, weight
of 100 seed compared to cultivars Sayad and Goli. Differences among strains
of common bean rhizobia and plant cultivars in their nitrogen-fixing performance
were previously observed by Vladimir (2001), Bais et al. (2006),
Ahmad et al. (2006) and Remans et al. (2008). Co-inoculation
of the common bean with Rhizobium and PGPR resulted in better nodulation
which was translated into higher shoot dry matter and seed yield production.
This is in agreement with previous reports demonstrating the beneficial
effects of PGPR belonging to Pseudomonas spp. and Azospirillum
spp. on symbiotic efficiency of rhizobia nodulating different legume crops
(Preston, 2004; Bashan and Holguin, 2004; Valverde et al., 2006;
Figueiredo et al., 2007). The results revealed that application
of PGPR together with Rhizobium improved the growth and seed production
by inoculated beans. As a result, gross average of seed yield increased
from 1536-3000 kg ha-1 for Rhizobium alone to 1221-4693
kg ha-1 for those co-inoculated with A. lipoferum S-21
and P. fluorescens P-93, respectively. Present data showed that
P. fluorescens P-93 had better promoting effect on yield components
of rhizobia than A. lipoferum S-21. This difference is probably
attributable to siderophore production as well as higher ability for auxin
production and P-solubilizing activity of P. fluorescens P-93 (Table
1).
| Table 2: | Number pod per plant (PP), seed per pod (SP), weight of 100 seed (WS), weight of seed per plant (WSP), weight of pod per plant (WPP), total dry matter in R6 (TDM) and seed yield, seed protein yield by bean plants in the field experiment in the first year |
| Table 3: | Number pod per plant (PP), seed per pod (SP), weight of 100 seed (WS), weight of seed per plant (WSP), weight of pod per plant (WPP), total dry matter in R6 (TDM) and seed yield, seed protein yield by bean plants in the field experiment in the second year |
| Table 4: | Complex analysis of variance of number pod per plant, number seed per pod, weight of 100 seed, seed yield, weight of seed per plant, weight of pod per plant, protein seed yield, total dry matter in R6 in bean plants that affected by several bacteria treatments |
| ns, * and **: Non significant, significant at the 5 and 1% levels of probability, respectively | |
CONCLUSION
This study showed that plant growth and seed yield potential of Rhizobium-P. vulgaris varies with Rhizobium strains and plant cultivars. Co-inoculation of the common bean with Rb-133 and Pseudomonas fluorescens P-93 resulted in higher number seed per pod, weight of 100 seed, weight of seed per plant, weight of pod per plant, protein seed yield, total dry matter in R6 and thereby produced greater seed yield. The results indicate that in spite of the fact that Rb-133+ P. fluorescens P-93 can increase the proportion of seed per pod and productivity in plants, application of complementary inorganic nitrogen fertilizer in soils with low nitrogen content is needed.