Nodulation , Nutrient Uptake and Yield of Common Bean Inoculated with Rhizobia and Trichoderma in an Acid Soil

Common bean is an important source of protein, fat, carbohydrate, vitamins and minerals for both human beings and livestock. In Zambia, common bean is produced mostly by smallholder farmers whose current yields fall far short of the potential 2 t/ha due to various challenges. Among the biophysical constraints, poor soil fertility and acidity pose the greatest challenges. This study investigated the individual and dual inoculation of Rhizobia and Trichoderma to common bean in a phosphorus deficient, acid soil. Soil in which the common bean was grown was characterized for selected chemical properties before planting and at harvest using standard laboratory procedures. Soils were amended with nitrogen and phosphorus fertilizer at the rates of 100 kg N and 80 kg P2O5 per ha; 1 g/kg of seed of Trichoderma harzianum; 100 g/kg of seed of Rhizobium tropici; or a combination of both at the recommended rates at planting. Nodulation and nodule effectiveness were determined at 51 days after planting. Nitrogen and phosphorus accumulation in the above ground biomass, biomass and grain yields were determined at maturity. To determine differences among soil amendments, data were analysed using Analysis of Variance and Least Significant Difference at 95% confidence limit. Relationships among parameters were determined using correlation analysis. The results showed that amending soils with inorganic N at high rates can depress nodulation even in the presence of high levels of inorganic phosphorus. Inoculating common bean with Rhizobia and Trichoderma either singly or in combination increases nodule number and effectiveness per plant but may not result in higher nitrogen and phosphorus accumulation, or in an increase in subsequent biomass or grain yields. The low phosphorus accumulation in Trichoderma inoculated plants observed in this study needs to be investigated further by studying the extent of colonization and the accompanying changes in root volume.


Introduction
Common bean (Phaseolus vulgaris L.) is the most important food grain legume in the world (Beebe, 2009;Nakitto, Muyona, & Nakimbungwe, 2015).It is an important source of protein, fat, carbohydrates, vitamins and minerals for humans and livestock (Beebe, Gonzalez, & Rengifo, 2000).Protein content in most varieties is high and averages 25%.The crop is rich in the amino acids tryptophan and lysine, which are not typically found in the staple maize crop.Common bean therefore, provides a good alternative to animal protein to meet minimal daily requirements.In addition, common bean provides other health benefits that include weight control, reduced proneness to diabetes, colon cancer and heart problems (Nyau, 2014).
Current crop production in Zambia covers over 85,000 hectares of land mostly in the Northern, North western and Eastern provinces of Zambia (CSO, 2012).These provinces cover parts of agro-ecological regions II and III of the country, with average seasonal rainfall between 800 and 1000, and more than 1000 mm, respectively.In Zambia, the crop is grown mostly at subsistence and small-scale levels, with only a small portion being grown by commercial farmers.While the potential yield of most varieties is 2 t/ha, the average yield is only 0.58 t/ha (Hamazakaza et al., 2014).Constraints to common bean production have been cited as being the use of unimproved varieties, and attack by pests and diseases.Pests and diseases alone account for 25-50% of the total yield losses (Mweetwa, 2011).In addition, poor soil fertility prevalent in the common bean production areas limits its productivity.Specifically, the leaching of bases in high rainfall areas (agro-ecological region III) results in an accumulation of hydrogen and aluminium ions in the soils.The resulting acidity has both direct and indirect effects on plant growth; common bean grows well in soils with pH ranging from 6.5 to 7.5 (Izquierdo, 1990).On the other hand, common bean is particularly sensitive and highly susceptible to physical and chemical environmental stresses compared to other legumes (Kabahuma, 2013) making the crop very nutrient demanding (da Silva et al., 2014).
In Zambia, most small-scale producers of common bean do not typically use chemical fertilizers to enhance crop productivity.While chemical fertilizers are readily available and may provide immediate answers to current limiting nutrient levels in the soil, they tend to be expensive and in the long-term, cause soil acidification and pollution of soils and water.This has resulted in the promotion of more sustainable agricultural practices such as the use of organic fertilizers to address poor soil fertility.Organic fertilizers have the advantage of improving soil physical properties, acting as substrates for beneficial soil microorganisms, buffering soil reaction, controlling erosion and as well as controlling plant parasitic nematodes and fungi (Gupta, 2011;Panda, 2011) in addition to supplying plant nutrients to the soil.The challenge usually of using organic fertilizers, such as animal and green manures, is that they tend to be bulky and of variable quality, and nutrient release may also be slow.This, coupled with competition for alternative uses on most small-scale farms, limits their use as single solutions to limiting soil nutrients.
As a legume, common bean forms symbioses with a broad range of Rhizobia that biologically fix nitrogen in the nodules.Several factors influence nitrogen fixation in legumes; these include soil nutrient conditions, temperature and moisture (Kabahuma, 2013).In biological nitrogen fixation, phosphorus is critical for nodule formation and functioning as well as for specific nitrogenase activity (Kouas, Labidi, Debez, & Abdelly, 2005).In Zambia, regions of common bean production tend to be deficient in phosphorus and high in acidity, this limits biological nitrogen fixation and productivity of the crop.In addition, common bean crop productivity is often limited by nitrogen deficiency despite its ability to form symbiotic relationships with nitrogen fixing Rhizobia (Rodino, Santalla, De Ron, & Drevon, 2005); unlike other legumes, common bean is a poor nitrogen fixer; this is attributable to some genetic factors and its sensitivity to physical and environmental stresses (Yadegari & Rahmani, 2010).
The use of bio-fertilizers such as Rhizobia and other plant-growth promoting bacteria or fungi have been suggested to result in increased growth and subsequent yield in many crop species including legumes (Stajkovic et al., 2011).Rhizobia inoculation alone to seeds or application to the soil at planting has shown varied results in terms of stand count after emergence, stand count at harvest, biomass yield, number of nodules per plant, biological nitrogen fixation and grain yield of common bean (Musandu & Joshua, 2001;Mweetwa et al., 2014a).On the other hand, co-application of Rhizobia with other microorganisms such as Pseudomonas flourescens, Bacillus and Azospirillum lipoferum has been reported to result in improved dry weight, phosphorus uptake, nodulation, nitrogen fixation, enzyme activity, protein content, yield and yield components (El-Katany, 2010;Stajkovic et al., 2011;Yadegari & Rahmani, 2010;Samavat, Samavat, Mafakheri, & Shakour, 2012).The benefits of co-inoculating Rhizobia with other microorganisms are derived from the ability of these microorganisms to make available to the symbiosome essential nutrients such as phosphorus, to enhance water uptake by the host plant, to produce plant growth regulators and to inhibit some soil-borne plant pathogens through anti-fungal activity and siderophore production (Sharma, Jadega, Kataria, Anamika, & Kumar, 2014;Tatarani, Dichio, & Xiloyannis, 2012;Yadegari & Rahmani, 2010;Izquierdo, 1990).
Trichoderma species are filamentous fungi of the ascomycota known to exist in many soil types of the World (Tatarani et al., 2012).Many species of the Trichoderma genus are known to control various plant pathogens through a number of chemiotropic mycoparasitic interactions (Sharma et al., 2014).In addition, Trichoderma have been reported to enhance uptake of nutrients and water through stimulated production of lateral roots, and to promote plant growth and biomass yield through the stimulation of the production of several natural plant growth hormones (Tatarani et al., 2012).Dual inoculation of Trichoderma and Rhizobia to legumes has been reported with conflicting results in terms of the nature of the interactions between the two organisms, and their combined effect on nodulation, biomass production and grain yield.Inhibition of Rhizobia growth by Trichoderma in vitro has been attributed to competition for nutrients, antibiosis and lysis (Jayaraj & Ramabadran, 1999); on the other hand, compatibility, even synergism has been reported by others.
In view of the potential benefits to biological nitrogen fixation by enhanced phosphorus uptake through Trichoderma-plant interactions, a study was undertaken to investigate dual inoculation of Rhizobia and Trichoderma harzianum to common bean grown in a phosphorus deficient acid soil.This paper reports the effect of singly or co-inoculating Rhizobia and Trichoderma harzanium on nodulation, nutrient uptake, biomass production and grain yield of common bean under greenhouse conditions.

Site Description, Soil Collection and Characterization
Greenhouse experiments were conducted at the University of Zambia, 15 o 24′ South and 28 o 19′ East.Soils used in the study were collected from the Liempe University of Zambia Farm (15 o 24′ South and 28 o 28′ East).This site was specifically selected for soil collection because soils in that location are known to be acid and because the land had been left fallow for a number of seasons.
Soils were collected to a depth of 20 cm from ten (10) random spots and mixed thoroughly to make a composite sample.Soil intended for chemical characterization was air-dried and passed through a 2-mm sieve.Soil was then characterized for reaction (pH) and exchangeable bases.Soil reaction was determined in 0.01 M Calcium chloride using a soil: solution ratio of 1:2.5 using glass calomel combination electrode connected to a pH meter.The ammonium acetate method (Thomas, 1982) was used to determine exchangeable bases of the soil.These soil characteristics were also measured at harvest to determine the effects of the different soil amendments.

Fertilizer and Inoculation Treatments and Experimental Setup
The study was conducted as a greenhouse experiment during the 2013/14 cropping season.Common bean seeds of the common variety Lyambai were planted in pots with the following fertilizer or inoculation treatments: unamended controls; nitrogen and phosphorus fertilizer application to the soil at planting (100 kg N/ha and 80 kg P 2 O 5 /ha, respectively); 1 g/kg seed of Trichoderma harzanium (Eco-T); 100 g/kg seed of Rhizobium tropici (Biofix); a combination of Trichoderma harzanium and Rhizobium tropici as one application.Microbial inoculants were applied to the seeds just before planting.Each treatment was replicated four times and arranged in a completely randomized design (CRD).Four seeds were sown in each pot (5 kg soil) and thinned to one plant at 14 days after emergence.In order to determine both nodulation and yield of common bean due to these amendments, two sets of experiments were setup exactly as described above.

Determination of Nodulation and Nodule Effectiveness
At 51 days after planting, plant stems were cut just above the soil level.The remaining below ground biomass was rinsed under running water to remove soil particles from the roots.Sieves were placed at the bottom of the biomass while rinsing to capture any nodules that could have detached during the processing.Nodules from each plant were physically counted from the roots to determine number per plant.In addition, nodule effectiveness was determined by selecting four equal sized nodules; nodules were sliced open with a sharp surgical razor to expose their centre.Nodule colour was then scored as follows: pink and red nodules were scored as effective, while white, brown and/ or green nodules were scored as ineffective.Nodule effectiveness was then calculated as a percent.

Determination of Nitrogen and Phosphorus Uptake
At maturity, plants were cut just above the soil level.The harvested above ground biomass was dried and weighed and oven dried at 60 o C for 72 hours.Using the modified Kjedahl and Dry Ashing methods, tissue nitrogen and phosphorus were determined (Doll & Lucas, 1975;Kalra & Maynard, 1991).

Determination of above Ground Biomass and Grain Yield
Above ground biomass yield was determined by cutting the plant stems just above the soil, oven drying at 60 o C to constant weight and weighing;while the grain yield was determined by weighing the shelled pods and as well as the actual seed after shelling.

Data Management and Statistical Analyses
Effects of different amendments were compared using analysis of variance (ANOVA).Means were compared at 95% confidence using Least Significance Difference (LSD).Other relationships between parameters were made using correlation analysis in Excel Windows software.

Effect of N and P Fertilizer, Rhizobium tropici and Trichoderma harzianum on Number of Nodules per Plant and Nodule Effectiveness in Common Bean
The number of nodules across amendments ranged from 0 to 228 per plant and was significantly affected by the type of amendment.The application of inorganic nitrogen and phosphorus did not yield any nodules, while inoculating with Rhizobia and Trichoderma resulted in more nodules per plant than the unamended plants.The number of nodules per plant was not significantly different between the single Rhizobia and Trichoderma treatments with 197 and 189 nodules per plant, respectively (Figure 1).The most number of nodules was observed in co-inoculated plants (228 nodules per plant).
Nodule effectiveness ranged from 25 to 88% and was highest in plants co-inoculated with Rhizobia and Trichoderma (Figure 1).Single inoculations with Trichoderma or Rhizobia did not significantly increase nodule effectiveness compared to the unamended controls.In summary, co-inoculating common bean seeds at planting with Trichoderma and Rhizobia gave the greatest response of both nodule number per plant and percent effectiveness, while single inoculations only significantly increased nodule number per plant and not nodule effectiveness.

Nitrogen and Phosphorus Accumulation in Common Beans
Applying inorganic nitrogen and phosphorus resulted in the most nitrogen and phosphorus accumulation in plants at harvest than in the control plants and those treated with Trichoderma, Rhizobia, and Rhizobia and Trichoderma.Among the bio-fertilized, Trichoderma inoculated plants accumulated the least phosphorus, but did not differ significantly from the unamended control plants, plants inoculated with Rhizobia, and Rhizobia and Trichoderma (Table 1).Compared to the controls, amending soils with inorganic fertilizers resulted in approximately 11 fold increase in P accumulation, while Rhizobia, and Rhizobia and Trichoderma inoculation resulted in approximately 3 fold increases in P accumulation.While there was an increase in the total nitrogen in plants singly or co-inoculated with Rhizobia and Trichoderma, this increase was not significantly different from the control (Table 1).
Table 1.Nitrogen and phosphorus accumulation in common bean plants supplied with inorganic N and P, and inoculated with Rhizobia and Trichoderma at planting

Biomass and Grain Yield of Common Bean Due to N and P Fertilizer, Rhizobia and Trichoderma Application
Applying inorganic nitrogen and phosphorus to the soil resulted in the highest biomass and grain yields compared to the unamended and bio-fertilized common bean crops (Figure 3).While grain yield increased by 314, 163 and 277%, respectively from the unamended controls with Rhizobia, Trichoderma, and Rhizobia and Trichoderma application, respectively, these increases were not statistically significant.On the other hand, biomass yield increased significantly from the control by 747, 187, 23 and 230% with fertilizer, Rhizobia, Trichoderma, and Rhizobia and Trichoderma application, respectively.Inoculation with Trichoderma alone did not result in any significant increase in biomass yield from the unamended control (Figure 3).Grain yield was positively correlated with the amounts of accumulated phosphorus and nitrogen by the plant with r 2 values of 1 and 0.97, respectively.On the other hand, there was a positive but weak relationship between grain yield and number of nodules per plant (r 2 = 0.21).The addition of Rhizobia appeared to depress pH and magnesium levels.
Calcium levels in soils amended with fertilizer, Trichoderma, and Rhizobia and Trichoderma were comparable to the levels at the start of the experiment (no differences).Unamended soils and soils to which Rhizobia was added alone had lower levels of calcium compared to the initial calcium levels.Amending soils with fertilizer resulted in the lowest levels of potassium at harvest.

Discussion
The ability of legumes to form nodules has been attributed to many soil and biological factors that include levels of mineralizable N, levels of available P, soil reaction in the form of pH, type and vigour of legume, Rhizobia populations and their effectiveness in infecting and nodulating the host (Mohammadi, Shohrabi, Gholamreza, Khalesro, & Majidi, 2012).Phosphorus plays a significant role in legume nodulation through its ability to enhance root development and proliferation thereby, affording the Rhizobia more sites for infection and initiation of nodule formation.For this reason, addition of phosphorus to the soil has been reported to result in enhanced nodulation of common bean in particular and other legumes, in general.Attar, Blavet, Selim, Abdelhamid, and Drevon (2012) reported higher nodule numbers in plants fertilized with phosphorus; in which case fertilization with 45 and 90 kg P 2 O 5 per ha resulted in 31 and 37 nodules per plant, respectively compared to 20 in unfertilized plants.In addition, Muthamia, Kimani, Chemining'wa, and Esilaba (2015) also reported that phosphorus nutrition has a strong influence on the nodulation of common bean, with unfertilized plants having much lower numbers of nodules even in the presence of microbial inoculants.Adding phosphorus to the soil in other forms such as manure, has also been shown to have a positive effect on nodulation (Otieno et al., 1997).Our study on the other hand, reports a complete absence of nodules with the application of 80 kg P 2 O 5 /ha.We attribute the lack of nodulation to the addition of high levels of inorganic nitrogen at planting (100 kg N/ha).Application of nitrogen in high amounts to common bean at sowing has previously been reported to reduce nodule number, nodule fresh weight and nitrogenase activity (Izquierdo, 1990;Muller & Pereira, 1995;Otieno et al., 2007).Inorganic sources of nitrogen in the form of nitrate and ammonium are preferred by both the Rhizobia and legume hosts to biologically fixed dinitrogen which is energetically costly to acquire.Therefore, when levels of soil nitrogen are sufficient, nodulation is inhibited and existing nodules can degrade (Otieno et al., 1997).On the other hand, small starter doses of inorganic nitrogen have been shown to stimulate nodule initiation (Musandu & Joshua, 2001).
Because of the role that phosphorus plays in nodulation, number of nodules is severely restricted when it is limiting (Kouas et al., 2005).While low soil P may limit the numbers of nodules initiated, it does not directly influence functional characteristics nor restrict nitrogenase enzyme activity (Kouas et al., 2005).It must, however, still be noted that phosphorus plays a major role in determining the energy budget of the symbiosome in the form of ATP.
The current study reports significantly increased numbers of nodules with Rhizobia alone, Trichoderma alone, and Rhizobia co-inoculated with Trichoderma compared to the control.This increase could be attributed to both direct and indirect interactions between the host plant with Rhizobia and Trichoderma.Indirectly, Rhizobia and Trichoderma play some roles in modifying the fertility of the soil mainly through their participation in the decomposition of organic matter, mineral nutrient transformations such as phosphorus solubilisation (Mweetwa et al., 2014b) and modification of soil reaction.Directly, Trichoderma increases uptake of nutrients and water due to its ability to increase contact of the plant with the rhizospheric soil through enhanced plant lateral root production and mycelial extensions from the roots (Samolski, Rincón, Pinzón, Viterbo, & Monte, 2012;Pereira et al., 2014).This increases the surface area from which the plant can absorb nutrients required for the formation of nodules and for their functioning.In this paper, we report a 4 fold increase in the number of nodules per plant due to inoculation with Trichoderma alone.Rhizobia on the other hand, infect the host root system and therefore, directly have an influence on the nodulation process.Inoculating legume seeds at planting with Rhizobia has been shown to increase number of nodules per plant (Mweetwa et al., 2014a).Our results show a 4 fold increase in the number of nodules per plant and corroborate with earlier findings that inoculating common bean with Rhizobia can cause a significant increase in the number of nodules per plant (Otieno et al., 1997).It must be noted, however, that inoculation with Rhizobia may not always result in enhanced nodulation due to many other factors; these include the presence of many but ineffective Rhizobia strains; the ensuing competition between the introduced and the indigenous strains; poor quality of the inoculum and unfavourable soil conditions (Mcloughlin & Dunican, 1985;Montealegre & Graham, 1996;Mohammadi et al., 2012).This study shows that there is added value with respect to number of nodules per plant as well as their effectiveness when Rhizobia and Trichoderma are co-inoculated compared to when they are applied singly.The co-inoculation, brings together both the direct and indirect influences of the individual microbial symbionts.
Total nitrogen and phosphorus accumulated in common bean plants differed significantly among amendments.Inorganic N and P fertilization provided high amounts of available forms of these nutrients explaining why significantly higher amounts were accumulated during plant growth.On the other hand, inoculating with Rhizobia or Trichoderma alone or together did not result in significantly higher amounts of total nitrogen at harvest.Our results agree with earlier reports that have shown that while inoculating groundnuts, cowpea and soybean with Rhizobia resulted in increased nodule number and fresh weigh per plant, there was no corresponding increase in total nitrogen accumulated at harvest (Mungai & Karubiu, 2010;Mweetwa et al., 2014a).The observations in this study suggest that apart from nodule number, other nodule factors such as nodule efficiency play a crucial role in influencing the amount of total accumulated N at harvest (Mungai & Karubiu, 2010).In addition, inoculation with Rhizobia potentially introduces moderately effective strains that occupy the nodules, resulting in unimproved nitrogen accumulation or biomass yield (Singleton & Tavares, 1986).
In this study, total nitrogen accumulated was strongly and positively correlated with total phosphorous accumulated (r 2 = 0.97).This relationship was deemed important because of the role of phosphorus in nodule function and subsequent nitrogen fixation and accumulation.Unlike nitrogen accumulation, total phosphorus accumulation was significantly higher in microbial amendments than in the unamended.However, results did not show any added value in phosphorus accumulation by inoculating with Trichoderma alone or in the presence of Rhizobia when compared to Rhizobia inoculation alone.This result was rather unexpected; being relatively immobile in the soil, phosphorus acquisition can be enhanced by increasing the contact of root surface area with the soil.While the extent to which Trichoderma colonises and responds to the plant varies among species, the hyphae of the fungi when in symbiosis with the host plant have the potential to greatly increase the absorbing surface area of the roots and to increase the amount of phosphorus taken up by the plant (Rubio et al., 2014).In addition, Trichoderma has been shown to provide an alternative phosphorus uptake pathway which contributes to the total phosphorus plant uptake.This unexpected observation in this study requires further investigation by looking at the extent of Trichoderma colonization and the accompanying changes in root volume and span.
Biomass yield varied across amendments with the highest being observed in plants treated with inorganic P and N fertilizers.This observation has been previously reported by Otieno et al. (1997) and Mungai and Karubiu (2010).Biomass yield was positively and strongly correlated with both total N and P accumulation in the plants (r 2 = 0.90 and r 2 = 0.98, respectively; data not shown).Phosphorus and nitrogen as plant nutrients are important in plant metabolism as components of chlorophyll and amino acids; they are therefore key in photosynthesis, respiration, energy storage and transfer, cell division, cell enlargement and several other processes in plants.This explains why plants that had accumulated the most P and N also had the most biomass yield.In this study, inoculation with Rhizobium either alone or in combination with Trichoderma resulted in significant increases in biomass yield; this observation is incongruent with earlier reports of non-responsiveness of biomass yield to Rhizobia inoculation in other legumes (Mweetwa et al., 2014a).
In the current study, inoculating plants with Trichoderma alone, Rhizobia alone and Rhizobia with Trichoderma resulted in non-significantly higher grain yields than the unamended plants.Application of Rhizobia alone has previously been shown not to significantly influence common bean grain yields (Musandu & Joshua, 2001;Otieno et al., 1997); our results corroborate these earlier reports.In this study, grain yield was positively and strongly correlated with nitrogen accumulation (r 2 = 0.97); therefore, the low grain yield was attributed to the poor responsiveness of total nitrogen accumulation to the various microbial amendments.
Growth of legumes is typically known to depress soil pH due to their high uptake of bases such as calcium, magnesium and potassium in order to meet plant development demands (Liu, Lund, & Page, 1989).The results of this study show a decline in soil pH at harvest across all the amendments.Potassium levels in the soils at harvest had declined from the initial 0.35 cmol+/kg soil in all cases including the unamended soils, while calcium levels declined from their original levels in soils amended with Rhizobia alone and the unamended soils.Magnesium levels were generally unchanged by the time of harvest except for soils amended with Rhizobium alone.The decline in pH observed across all treatments can therefore be attributed to uptake of bases as indicated by lower soil solution concentrations.While Trichoderma has been suggested to buffer pH fluctuations in the soil, this was not true for soils amended with Trichoderma alone or in combination with Rhizobia.
In conclusion, amending soils with inorganic N at high rates can depress nodulation even in the presence of high levels of inorganic phosphorus which is typically beneficial for nodule initiation and development.Inoculating common bean with Rhizobia and Trichoderma either singly or in combination can enhance nodule number per plant but may not result in higher nitrogen and phosphorus accumulation, or in an increase in subsequent biomass or grain yields.The poor phosphorus accumulation in Trichoderma inoculated plants observed in this study needs to be investigated further by studying the extent of colonization and changes in root volume.

Figure 1 .
Figure 1.Number of nodules and nodule effectiveness of common bean plants in soils amended inorganic fertilizer, Rhizobia and Trichoderma

Figure 2 .
Figure 2. Relationship between Total P at harvest, number of nodules per plant at 51days after sowing and nitrogen accumulation in plants at harvest

Figure 3 .
Figure 3. Biomass and grain yield (t/ha) of common bean supplied with inorganic N and P, and inoculated with Rhizobia and Trichoderma

Figure 5 .
Figure 5. Changes in selected soil chemical properties due to to N and P fertilizer, Rhizobia and Trichoderma application