Effect of Co-Inoculation of Bradyrhizobium and Trichoderma on Growth, Development, and Yield of Arachis hypogaea L. (Peanut)

Cultivation of the peanut (Arachis hypogaea L.) on the same land contributes to the accumulation of root exudates, leading to increased soil pathogens and decreased yield. Trichoderma harzianum is a naturally occurring endophytic biocontrol fungus that can enhance plant growth, nutrient uptake, and tolerance to biotic and abiotic stresses. Separately, Bradyrhizobium spp. is a biological nitrogen-fixing (BNF) bacterium favoring nodule formation in peanut roots which promotes nitrogen fixation. The dynamics of the symbiotic association between these two organisms were evaluated in the laboratory and greenhouse conditions. Peanuts were cultivated in pots inoculated with either Bradyrhizobium or Trichoderma or both to evaluate growth, development, and yield. The in vitro study results showed that seeds treated with Trichoderma had better germination and seedling biomass (p = 0.0008) compared to the other treatments. On the other hand, the results of greenhouse studies showed that seeds inoculated with both microbes, and those inoculated with Bradyrhizobium alone had higher dry biomass (p < 0.0001) as well as higher chlorophyll content (p < 0.0001) compared to the other treatments. Understanding of the interactive effects of fungal endophytes and rhizobial bacteria on plant growth and development will help in both the nutrient and disease management of Arachis hypogaea L.


Introduction
Legumes have a prominent role in sustaining agricultural productivity [1,2]. Pulses, legumes, and grasslands are grown because of their ability to interact with nitrogen-fixing bacteria [3], and they can also be used in rotation systems to manage pests, diseases, and weeds [4]. Small quantities of arable land and intensive agro-industrialization have led to the cultivation of crops on the same land without crop rotation. Such exhaustive peanut production is particularly widespread in the subtropical regions of Asia. The consecutive monoculture may, however, have caused a steady decline in productivity and performance, and increased disease susceptibility [5].
Castro et al. [6] reported increased yield when peanut crops were inoculated with rhizobium, but Walker et al. [7] and Huang et al. [8] noted that continuous cropping of peanut on the same piece of land resulted in reduced yield even when there was evidence of rhizobium inoculation. Daimon et al. [9] showed that fertilization with nitrate prevents root hair infection and initiation, development, and formation of nodules. Molecular analysis of peanut nodule formation suggests

Viability of Inoculated Seeds In Vitro
The ability of the microorganisms to exhibit any kind of antagonistic properties during germination was evaluated in vitro by inoculating the sterilized peanut seeds with the above-specified concentrations of the inoculum. This included four different treatments in two trials with 10 seeds in each treatment and allowed to germinate in moist towel paper. The untreated seeds were considered the control (S), while the other treatments were seeds treated with Bradyrhizobium only (S + B), seeds with Trichoderma only (S + T), and seeds treated with both Bradyrhizobium and Trichoderma (S + B + T). To study the effects of these treatments on the germination of peanuts 10 days after inoculation (DAI), all the seeds with emerged radicle and open cotyledons were considered as germinated and their fresh biomass was recorded.

Greenhouse Studies to Assess the Microorganism Effect on Growth and Development
Peanut seeds were planted in 28 cm diameter by 23 cm height pots, filled with the soil mixture specified above. All the treatments were arranged in randomized complete block design (RCBD) to compensate and/or account for light gradient within the greenhouse as the pots were placed on one side of the greenhouse only. The greenhouse was maintained under natural light conditions and 60-70% RH. The temperatures varied from 28-35 • C during the day and 20-24 • C in the night. There were four treatments with 36 pots per treatment in three replications for a total of 432 pots, with three seeds sown in each pot. Each individual treatment was placed in nine rows of pots with four pots per row. The four treatments included: seed only (S); seeds inoculated with Bradyrhizobium and Trichoderma (S + B + T); seeds inoculated with Bradyrhizobium only (S + B); seeds inoculated with Trichoderma only (S + T).
Bradyrhizobium inoculum was sprayed on the seeds and then sowed immediately after drying, whereas Trichoderma inoculum was added directly to the potting mixture after sowing. Plants were irrigated with deionized water so that no extraneous nutrients were added to the growing medium. The crops were harvested after 125 days of sowing and the dry biomass of plant parts along with number of survived plants and mature pods were recorded.

Data Analysis
The following parameters were measured for statistical analyses. i.
Dry biomass of the harvested plant parts (g). Seed biomass per treatment and 100-seed weight per treatment.
In addition, data collected from these parameters were used to calculate the following indices: Harvest index (HI) = {Yield (seed or pod weight)/Biological yield (total dry weight)} × 100 (1) Shelling percentage = (weight of seeds per treatment/weight of pods in the same treatment) × 100 (2) Seedling vigor index = (germination percentage × mean shoot length) × 100 Relative growth rate (3) The data were analyzed by Analysis of Variance (ANOVA) in SAS version 9.4, and treatment means were compared using the Student-Newman-Keuls (SNK) method at 5% significance level.

In Vitro Study of Germination of Seeds Inoculated with Bradyrhizobium and Trichoderma Assay 10 Days after Inoculation (DAI)
Statistical analysis indicated that the seeds inoculated with Trichoderma had a significantly higher germination rate (Table 1) and the seedling fresh biomass ( Figure 1) (p = 0.0008) at 10 DAI. Table 1. Effect of inoculants on germination percentage and seedling biomass 10 days after inoculation (DAI) at in-vitro conditions. In addition, data collected from these parameters were used to calculate the following indices: Harvest index (HI) = {Yield (seed or pod weight)/Biological yield (total dry weight)} ×100 (1) Shelling percentage = (weight of seeds per treatment/weight of pods in the same treatment) ×100 (2) Seedling vigor index = (germination percentage × mean shoot length) ×100 Relative growth rate (3) The data were analyzed by Analysis of Variance (ANOVA) in SAS version 9.4, and treatment means were compared using the Student-Newman-Keuls (SNK) method at 5% significance level.

In Vitro Study of Germination of Seeds Inoculated with Bradyrhizobium and Trichoderma Assay 10 Days after Inoculation (DAI).
Statistical analysis indicated that the seeds inoculated with Trichoderma had a significantly higher germination rate (Table 1) and the seedling fresh biomass ( Figure 1) (p = 0.0008) at 10 DAI.

Germination and Seedling Vigor
Unlike in vitro results, inoculated treatments did not have any effect on both germination and seedling vigor (Table 2) on the 10th day after sowing (DAS). However, 20 DAS, treatments inoculated with Trichoderma showed an increase in germination (p = 0.0506). The study also concluded that seedling vigor index, 20 DAS, was not affected by the inoculation of Bradyrhizobium, Trichoderma or their co-inoculation. In fact, Table 6 shows that co-inoculation of Bradyrhizobium and Trichoderma could be antagonistic to the enhancement effect of Trichoderma on peanut seed germination.

Relative Growth Rate (RGR)
Plant height, calculated as a mean of five plants, was recorded every two weeks. The co-inoculated treatment and treatment inoculated with Bradyrhizobium had higher relative plant heights compared to the control and treatment inoculated with Trichoderma alone (p = 0.0008, Table 3). Table 3. Effect of individual and co-inoculation of Bradyrhizobium and Trichoderma on relative height growth and chlorophyll content.

Chlorophyll Content
Minolta developed the Soil Plant Analysis Development (SPAD) values as the proportional amount of chlorophyll present in the leaves [43]. Chlorophyll content analysis indicates that inoculation of peanut seedlings with Bradyrhizobium increased chlorophyll content (p < 0.0001, Table 3) and the difference was visible 10 weeks after sowing, as the leaves in the control and Trichoderma inoculated treatments started to turn yellow while the co-inoculated and Bradyrhizobium treatments were green. The SPAD readings were at peak 30 DAS; thereafter, they declined gradually in Bradyrhizobium inoculated treatments, but the decline was abrupt in the control and Trichoderma inoculated treatments ( Figure 2). Table 4 shows the treatment effects on the biomass of peanut plant parts (p < 0.0001) along with the number of plants that survived in each pot (p = 0.0488) at the time of harvest. Co-inoculation with Bradyrhizobium and Trichoderma has the highest survival rate, shoot and root biomass, followed by the treatment inoculated with Bradyrhizobium alone. These findings agree with Badawi et al. (2011) conclusions that, co-inoculations resulted to significant difference in the biomass of plants compared to the control. The number of pods, dry weight of pods, seeds and shells were higher in the treatment inoculated with Bradyrhizobium and treatment co-inoculated with Bradyrhizobium and Trichoderma (Table 4). Crops co-inoculated with Bradyrhizobium and Trichoderma, and those inoculated with Bradyrhizobium alone significantly increased biomass and yield of the species. Tables 5 and 6 show the interactive effects of these microbes on peanut growth, development, and yield. Agronomy 2020, 10, x FOR PEER REVIEW 6 of 12  Table 4 shows the treatment effects on the biomass of peanut plant parts (p < 0.0001) along with the number of plants that survived in each pot (p = 0.0488) at the time of harvest. Co-inoculation with Bradyrhizobium and Trichoderma has the highest survival rate, shoot and root biomass, followed by the treatment inoculated with Bradyrhizobium alone. These findings agree with Badawi et al. (2011) conclusions that, co-inoculations resulted to significant difference in the biomass of plants compared to the control. The number of pods, dry weight of pods, seeds and shells were higher in the treatment inoculated with Bradyrhizobium and treatment co-inoculated with Bradyrhizobium and Trichoderma (Table 4). Crops co-inoculated with Bradyrhizobium and Trichoderma, and those inoculated with Bradyrhizobium alone significantly increased biomass and yield of the species. Tables 5 and 6 show the interactive effects of these microbes on peanut growth, development, and yield.     In peanuts, kernel size analysis, computed with 100 seed weight [44], and the number of seeds per pod are used to determine the qualitative commercial value of the crop [45]. The weight of 100 peanut seeds was recorded after drying and the inoculations did not enhance the kernel mass (p = 0.0898). Moreover, shelling percentage was not enhanced either (p = 0.0770), whereas analysis of pod and seed HI data showed significant difference (p < 0.0001). Seed HI and pod HI, measures of yield efficiency in peanut, were assessed as a fraction of economic biomass to total crop biomass. Results of seed and pod harvest indices (Table 5) indicate that inoculation with the microorganisms separately and co-inoculation had no statistically significant change in the computed commercial attributes of peanut except the treatment with Trichoderma alone for pod and seed HI, and treatment with Bradyrhizobium alone for seed HI. However, the computed interaction effects of these microbes show the synergistic effect of Bradyrhizobium and Trichoderma in improving the commercial attributes of peanut (Table 6).

Interaction of Bradyrhizobium and Trichoderma in Peanut Growth, Development, and Yield
Colby [46] published an easily adoptable mathematical method to decipher synergism or antagonism due to the interaction of two or three combined chemicals in a cultural practice. He worked with a combination of herbicides, but the mathematical model is applicable to a variety of conditions. We adopted his equation to understand the interactive effects of these microorganisms on growth, development, and yield of peanut in greenhouse-controlled environment. Table 6 summarizes the effects of the microbes on the growth, development, and yield of peanut.

Discussion
Legume plants are closely linked to microbes that affect the plant features related to the provision of nutrients, plant growth, defenses, and abiotic stress [47]. It has been suggested that the thorough use of soil microbial potential is essential for better nutrient management [4]. The current research evaluated the effect of co-inoculations of nitrogen-fixing rhizobia and plant growth-promoting (PGP) endophyte in the enhancement of peanut germination, growth, development and yield under greenhouse conditions. The data show that the co-inoculation of Bradyrhizobium and Trichoderma had enhanced effect on growth, development, and yield of peanut.
The results from the in vitro germination tests were consistent with the observations of Yedidia et al. [48] on cucumber. They observed that Trichoderma enhanced seedling emergence and dry weight of cucumber five days after inoculation. The effect of Trichoderma on peanut germination was statistically significant from the control and, in fact, the co-inoculation of Trichoderma and Bradyrhizobium may be antagonistic to peanut germination (Table 6). This result makes sense since seed germination requires primarily abiotic conditions of conducive temperature for the species, moisture and oxygen. These conditions also favor the growth of some microbes which could explain the germination enhancement observed in this study.
From the greenhouse studies the trends in relative plant height (RPH) synchronized with the physiological maturity of the crop and the highest RPH was recorded 76 DAS and thereafter, height growth declined gradually indicating crop reproductive maturity (Figure 3). The efficiency of nitrogen fixation in peanuts resulted in the accumulation of nitrogen in plant tissues which in turn reflected the synthesis of chlorophyll [49]. SPAD readings for the Bradyrhizobium inoculated treatments are higher compared to others and this might be because of the Bradyrhizobium's fundamental function of biological nitrogen fixation [50] (Table 3). Treatments co-inoculated with Bradyrhizobium enhanced the synthesis of chlorophyll and height growth, resulting in significant increases in plant dry weight and yield [51]. Although the co-inoculated treatment had similar statistical effects as the treatment with Bradyrhizobium alone in all measured growth and development parameters of peanut except seed HI, but the interactive test showed that co-inoculation had an advantageous effect on plant biomass which can be correlated to the PGP-characteristics of Trichoderma such as stimulating substantial and vigorous plant roots and synthesis of plant hormones (31,32) along with biological nitrogen fixation by Bradyrhizobium (see Table 6). This postulation supports the highest mean biomass of the shoot, root, pod, seed, and shell in the co-inoculated treatment compared to the other treatments. Although the results of the 100 seed weight, shelling percentage, seed HI and pod HI showed no statistical difference between the treatments, the interactive effects of co-inoculation with Bradyrhizobium and Trichoderma had qualitative value in peanut. This is an indication of added market value to yield when peanut cropping adopts co-inoculation with these microbes in cultural practices. Furthermore, seed HI was significantly higher in the co-inoculation treatment than the treatment with Bradyrhizobium alone. It is important to note the sensitivity of Colby's [46] mathematical interactive method on the qualitative value of the crop. Statistical analysis showed no significant difference on the qualitatively computed information about the species but the slight differences between the treatments were discernable by Colby [46]. Although Badawi et al. [51] concluded that co-inoculation resulted to increased peanut growth and development in field conditions, this study, performed in controlled pathogen free conditions, confirms their results. Additionally, this study revealed that plant growth promotion is not a common function of Trichoderma [52].
Nieto-Jacobo et al. [53] demonstrated that the production of IAA was dependent on Trichoderma strains as influenced by different external stimuli. In addition, various Trichoderma species have been shown to affect plant growth and the growth of other pathogens in conducive soil environments [53][54][55][56][57]. However, a range of effects by Trichoderma secondary metabolites have been documented [54]. These effects range from no response to sensitive response by plants.
The positive interaction of Bradyrhizobium and Trichoderma increased the various yield attributes when compared to the control, as indicated by the interactive effects of these microorganisms (Table 6). Zhang et al. [58] noted that Trichoderma inoculation increased the secretion of flavonoids by peanut roots and this induced rhizobial nod genes leading to improved Bradyrhizobial colonization. Crops co-inoculated with Bradyrhizobium and Trichoderma, and those inoculated with Bradyrhizobium alone increased the biomass and yield of the species. Increase in biomass and yield demonstrate the compatibility between microorganisms and the synergism among them to promote and enhance crop growth, development, and yield.

Conclusions
This study assessed the growth promoting properties of co-inoculation of microorganisms in peanut crop production. While co-inoculations are not a usual practice, this work confirms that coinoculation with Bradyrhizobium and Trichoderma enhanced growth, development, and yield in Arachis hypogaea. The inoculations improved growth attributes of the species, such as chlorophyll content, relative height growth, survival rate, biomass, and yield, as well as parameters such as 100 seed weight, shelling percentage, seed HI and pod HI. Moreover, the results of the study showed that the beneficial effects of co-inoculation occurred at seedling growth stage, leading us to conclude that the timing for co-inoculation of peanut with Bradyrhizobium and Trichoderma should be performed at seedling emergence. The findings of this study gave a better understanding of the effects of trilateral symbiotic association between plant-rhizobia-endophyte in leguminous crops. These associations will help in developing agro-ecosystems with minimal dependence on agrochemical inputs, and they simulate natural ecological interactions between crop plants and soil organisms. Although Badawi et al. [51] concluded that co-inoculation resulted to increased peanut growth and development in field conditions, this study, performed in controlled pathogen free conditions, confirms their results. Additionally, this study revealed that plant growth promotion is not a common function of Trichoderma [52].
Nieto-Jacobo et al. [53] demonstrated that the production of IAA was dependent on Trichoderma strains as influenced by different external stimuli. In addition, various Trichoderma species have been shown to affect plant growth and the growth of other pathogens in conducive soil environments [53][54][55][56][57]. However, a range of effects by Trichoderma secondary metabolites have been documented [54]. These effects range from no response to sensitive response by plants.
The positive interaction of Bradyrhizobium and Trichoderma increased the various yield attributes when compared to the control, as indicated by the interactive effects of these microorganisms (Table 6). Zhang et al. [58] noted that Trichoderma inoculation increased the secretion of flavonoids by peanut roots and this induced rhizobial nod genes leading to improved Bradyrhizobial colonization. Crops co-inoculated with Bradyrhizobium and Trichoderma, and those inoculated with Bradyrhizobium alone increased the biomass and yield of the species. Increase in biomass and yield demonstrate the compatibility between microorganisms and the synergism among them to promote and enhance crop growth, development, and yield.

Conclusions
This study assessed the growth promoting properties of co-inoculation of microorganisms in peanut crop production. While co-inoculations are not a usual practice, this work confirms that co-inoculation with Bradyrhizobium and Trichoderma enhanced growth, development, and yield in Arachis hypogaea. The inoculations improved growth attributes of the species, such as chlorophyll content, relative height growth, survival rate, biomass, and yield, as well as parameters such as 100 seed weight, shelling percentage, seed HI and pod HI. Moreover, the results of the study showed that the beneficial effects of co-inoculation occurred at seedling growth stage, leading us to conclude that the timing for co-inoculation of peanut with Bradyrhizobium and Trichoderma should be performed at seedling emergence. The findings of this study gave a better understanding of the effects of trilateral symbiotic association between plant-rhizobia-endophyte in leguminous crops. These associations will help in developing agro-ecosystems with minimal dependence on agrochemical inputs, and they simulate natural ecological interactions between crop plants and soil organisms.