Evaluation of Growth and Morphological Pattern of Mycorrhization in Cowpea [Vigna unguiculata (L.)] Fertilized With Phosphorus

Microorganisms perform important functions in the soil and, among these organisms, the role of arbuscular mycorrhizal fungi (AMF) in plant growth should be highlighted. AMF colonize the roots of most plant species and their beneficial functions in plant development include increased absorption of nutrients from the soil, especially those of low mobility such as phosphorus (P). Evaluating agricultural practices conducted by farmers, such as phosphate fertilization, and observing how they will influence AMF activity in benefiting plant growth should be prioritized. Thus, an experiment was conducted in greenhouse to evaluate the effect of phosphate fertilization on the growth of cowpea plants colonized by AMF and to know which morphological pattern of colonization prevails in their roots. Five P doses and a control treatment, without fertilization, were added to the soil. Cowpea plants respond to phosphate fertilization up to the dose of 240.50 mg P kg soil, for shoot dry mass and in the dose of 150 mg P kg soil, for plant height. The morphological pattern observed in the roots was the intermediate type, characterized by the presence of intra and intercellular hyphae and vesicles, and there was no influence of phosphate fertilization on morphology. High P contents added to the soil led to a reduction in mycorrhizal colonization in cowpea roots.


Introduction
Cowpea [Vigna unguiculata (L.)] is one of the most important crops for the population of the North and Northeast regions of Brazil, where it is widely cultivated and consumed.In addition, its grains represent an important source of protein and minerals in the population's diet, besides having notorious socioeconomic relevance.It is a plant of the Fabaceae family which is able to fix nitrogen (N) through the association with bacteria of the genus Rhizobium, with low demand for soil fertility, adapted to regions of hot climate (humid or semi-arid), besides being considered as tolerant to drought and moderately tolerant to salinity (Dutra et al., 2015;Gomes do Ó et al., 2017).
Soils with low levels of fertility are one of the main factors limiting cowpea production, and phosphorus (P) stands out among the nutrients affecting its growth.Therefore, agricultural practices which favor the activity of soil microorganisms, especially those whose activity directly benefit plant growth through greater absorption of nutrients, such as arbuscular mycorrhizal fungi (AMF), should be encouraged.
AMF are considered as important components in the recovery and reestablishment of vegetation in degraded areas, as well as in maintaining plant biodiversity and functions of ecosystems.Additionally, they have a relevant nutritional effect on plant growth, for promoting greater absorption of water and nutrients, especially those with low mobility such as phosphorus (P) and copper (Cu), contributing to greater development of plant species (Santos & Carrenho 2011;Silva et al., 2017).
Cowpea plants, particularly those cultivated in low-fertility soils, use symbiotic interactions with AMF as a means of meeting their demand for nutrients.A more detailed study on the interaction of these fungi with these plants could represent a notable strategy in agricultural systems with limited resources, because it has been known that The morphological pattern of AMF colonization has been classified as Arum, Paris and intermediate, the latter of which has structures of the first two types.The Paris type has intracellular hyphae, some of which are coiled, and is common in forest plants, whereas the Arum type has intracellular arbuscule and intercellular hypha, present especially in cultivated herbaceous plants.Although the factors influencing the formation of the morphological pattern of mycorrhizal association have not been fully clarified, there is evidence of the importance of this plant species and AMF species in the formation of the predominant pattern in the plant (Dickson, 2004).
Nevertheless, it is important to know which morphological pattern of AMF colonization is developing in the host plant because the ability of the fungus to colonize and provide nutrients, such as P, may differ depending on the type of morphological pattern that will develop in the root.Considering the context presented, this study aimed to evaluate the growth and morphological pattern of AMF colonization in cowpea [Vigna unguiculata (L.)] plants cultivated in soil with increasing P doses.

Location and Climate Condition of the Experimental Area
The experiment was conducted in greenhouse at the Department of Soil Sciences of the Federal University of Ceará (UFC), Pici Campus, in Fortaleza-CE, Brazil, for a period of 38 days after germination.According to Köppen's classification (Köppen, 1931) the climate of the region is Aw', at an altitude of 20 m above sea level.The city has mean temperature of 26 °C, rains concentrated between February and May, and mean rainfall of 1600 mm, where April is the rainiest month and November is the driest one (Pessoa & Campos, 2015).The average minimum and maximum temperatures in the greenhouse along the experiments were 28 °C and 36 °C, respectively.

Soil
The soil used in the experiment was collected in an area of native forest, in the 0-20 cm layer, at the Raposa Experimental Farm of the Federal University of Ceará (UFC), located in the municipality of Maracanaú-CE (3º50′66″ S; 38º38′38″ W).
After collection, the soil was pounded to break up clods, homogenized and sieved through 2 mm mesh to obtain the ADFE (air-dried fine earth), and one sample was sent to the Laboratory of Analyses of Soil, Water and Plants of the Department of Soil Sciences of the Center of Agrarian Sciences of the UFC.Soil chemical and physical characteristics, determined according to the methodology proposed by EMBRAPA (1997), are presented in Table 1.Note.Organic matter (OM); Electrical conductivity (EC).

Experiment Installation, Conduction and Design
The soil was distributed in 5 L plastic pots, 4 kg of soil in each pot.Cowpea seeds were donated by the Company of Technical Assistance and Rural Extension of Ceará (EMATERCE).Based on chemical analysis results, soil fertility was corrected with respect to the elements nitrogen and potassium, according to the adaptation of the recommendation of the Manual of Fertilization and Liming of Ceará (Aquino et al., 1993).
Nitrogen (N) fertilization was split into two portions, with the first application at sowing, 0.5 g of N per pot.The second application, with the same dose, was carried out 20 days after the first one.Potassium (K) fertilization was also split in the same way as N.At planting, the first application with 0.5 g of K per pot and the second application with the same amount of K after 20 days, along with N. The sources of N and K were Urea (NH 2 CONH 2 ) and Potassium Chloride (KCl).
To increase the pH of the soil solution, which was initially acid, 4.6 (Table 1), each pot received 4 g of dolomitic limestone, whose chemical composition is: CaO (32%) and MgO (13%), with RNV of 45%.After application, the soil was incubated for a 25-day period of reaction, and soil moisture was kept close to field capacity with periodic irrigations until obtaining pH 6.05.Then, five seeds of cowpea were planted at 3 cm depth and thinning was carried out 10 days after germination, leaving two plants per plot, which were chosen based on size and uniformity.
Plants were irrigated every day with 100 mL of water from the Water and Sewage Company of Ceará (CAGECE), in order to maintain soil moisture near field capacity, along the entire experimental period, which corresponded to 38 days after thinning.The experimental design adopted was completely randomized (0; 50; 100; 150; 200; 250 mg of P kg -1 of soil), with six treatments and five replicates, totaling 30 plots.The source used for phosphate fertilization was Triple Superphosphate (Ca(H 2 PO 4 )2H 2 O), with 46% of phosphorus in the form of P 2 O 5 , applied in a single dose at sowing.

Plant Height, Shoot Dry Mass and Phosphorus Content (P)
Plant height (cm) was measured with a graduated ruler and was considered as the mean of the heights of both plants in each pot.The collected plants were divided into shoots (leaves and stems or stalks) and root system.
Shoot samples were separately placed in paper bags and dried in a forced air circulation oven at temperature of 65-70 ºC for 72 hours until reaching constant weight.Then, these samples were separately weighed on a scale with 0.001 g sensitivity.The root system was washed in running water to remove any soil debris and kept under refrigeration in a 70% alcohol solution until evaluation of root colonization.
After drying in the oven, the material was ground in Wiley-type mill and sieved through a 2.0 mm mesh for chemical analysis.The extracts for analysis of P contents were obtained by nitric-perchloric digestion and determined by colorimetry, following the procedures described by Malavolta et al. (1997).

Root Mycorrhizal Colonization and Morphological Pattern of Mycorrhization
To determine root mycorrhizal colonization, roots were kept in alcoholic solution until the procedure was carried out.Roots were cleared and stained for colonization analysis following the methodology adapted by Koske and Gemma (1989), and colonization percentage was obtained according to McGonigle et al. (1990).
The identification of AMF structures observed in the roots after the staining procedure was used to classify the morphological pattern of mycorrhization as Arum, Paris or intermediate, according to Dickson (2004).

AMF Spore Density
AMF spores were extracted from 100 g of soil by wet sieving according to the procedures described by Gedermann and Nicolson (1963), followed by centrifugation in 50% sucrose (Jenkins, 1964).

Statistical Analysis
The data were initially subjected to Shapiro-Wilk test at 0.05 probability level to check if they had normal distribution.If normality was observed, the parameters were subjected to the test of homogeneity of variances (Levene's test).For variables with normal distribution and homogeneous variances, analysis of variance (ANOVA) was carried out and, if significant (p < 0.05), their means were compared by Tukey test (p < 0.05).Variables with normal distribution, but heterogeneous variances, were subjected to Brown-Forsythe test and, when significant value (p < 0.05) was found, their means were compared by Games-Howell test.Variables with non-parametric distribution were subjected to Kruskal-Wallis test and, when significant value (p < 0.05) was observed, their means were compared by the stepwise method (Field, 2009).
Regression analysis was carried to verify which was the best model to represent the variables analyzed as a function of P doses.Statistical analysis was performed in the software package SPSS (Statistical Package for the Social Sciences), Version 20 (IBM, 2011).

Plant Height, Shoot Dry Mass and Phosphorus Content (P)
According to the Shapiro-Wilk test, the variables plant height (PH), shoot dry mass (SDM) and phosphorus content (P) did not follow normal distribution (p < 0.05).Thus, the Kruskal-Wallis test was applied and, when significance was observed (p < 0.05), treatment means were compared by the stepwise method.Phosphate fertilization had significant influence on the variables analyzed (Table 2).The quadr dose of 15 (Figure 1A doses of or doses high fertilizatio ha -1 . Figure 1 org Plant height (PH and cultivated atments 0 mg P kg -1 soil 50 mg P kg -1 soi 100 mg P kg -1 so 150 mg P kg -1 so 200 mg P kg -1 so 250 mg P kg -1 so ans followed by es added to the kg -1 soil, sign hosphate fertiliz miting factor f lants grown u on to non-fertil ratic regression 50 mg P kg -1 so A).2012) observed greater growth due to the presence of AMF when plants were grown in soil with low availability of nutrients, because soils with high levels of fertility, especially of P, as in treatments T5 (200 mg P kg -1 soil) and T6 (250 mg P kg -1 soil), are not favorable to mycorrhizal colonization in plant roots.Consequently, the beneficial effect of AMF, which promotes plant growth, will be smaller.
For shoot dry mass (SDM), a quadratic model had the best fit (Figure 1B).SDM increased with the addition of P to the soil and its maximum value was obtained at dose of 240.50 mg P kg -1 soil.Higher doses limited the growth of cowpea plants.As can be seen in Figure 1B, the treatment with highest P dose (T6: 250 mg P kg -1 soil) reduced plant growth by up to 14.41% compared to the treatment with highest value of SDM.As already observed by Schiavo et al. (2010), high contents of nutrients, and more specifically of P, can reduce the growth of mycorrhized plants because, in general, these plants reach maximum development at low doses of P.
The attributes which can control P demand and supply to the plant, and thus its dependence on the nutrient, are related to the morphological and physiological characteristics of the host.Because of that, the most adequate P content in the soil for the response to mycorrhizae is highly variable between plant species and cultivars, and differences occur even between plants that are genetically very close (Clement & Habte, 1995).Fernandes et al. (2013) reported increase in the SDM of cowpea plants grown in soil fertilized with P, and the estimated doses of 45 and 48 kg ha -1 of P 2 O 5 led to highest SDM production.Higher doses of P caused reduction in SDM, as also observed in the present study.
Phosphate fertilization through increasing doses of P in the soil led to significant increase in P content in cowpea shoots (Figure 1C).Similar results have been found in several previous studies, and this response is related to the increase in P availability in the soil solution due to the addition of soluble fertilizer (triple superphosphate).P contents in the tissues increased with increasing doses of P (Figure 1C).2014) also observed increment in P content in the shoots of common beans with the increase of P doses in the soil in a field experiment.According to these authors, P is an essential element to the metabolism of common bean, which has a well-defined response to the addition of P in the soil, thus contributing significantly to the increase in grain yield and root development, favoring the increase in the number of pods and grain mass (Zucareli et al., 2010).Consequently, the importance of phosphate fertilization in cowpea cultivation, especially in soils with reduced fertilized, is unequivocal.

Root Mycorrhizal Colonization and Morphological Pattern of Mycorrhization
Mycorrhization in cowpea plants occurred naturally, due to the abundance of native spores in the soil.The data of arbuscular mycorrhizal colonization had a non-parametric distribution according to the Shapiro-Wilk test (p < 0.05).Since this variable had a non-parametric distribution, Kruskal-Wallis test was applied and, when significance was observed at 0.05 level, treatment means were compared by the stepwise method (Table 3).The treatment T3: 100 mg P kg -1 soil had the highest percentage of mycorrhizal colonization, significantly differing from the others.There was no statistically significant difference between T1: 0 mg P kg -1 soil and T4: 150 mg P kg -1 soil treatments and from the treatment T4, there was a reduction in mycorrhizal colonization, a behavior related to the high contents of P added to the soil, which was also observed by Vitorazi Filho et al. (2012).ratic regression Figure 4).

Figure
Figure 4. AM

Table 1 .
Physical and chemical characteristics of the soil collected at the Raposa Farm in Maracanaú-CE

Table 3 .
Mycorrhizal colonization in the roots of cowpea grown in soil fertilized with increasing doses of P Note.Means followed by the same letter in the column do not differ by the stepwise method (p < 0.05).
Alguacil et al. (2010)found similar result evaluating mycorrhizal colonization in Centrosema macrocarpum Benth.fertilized with different doses of P (rock phosphate or diammonium phosphate).Regardless of the source used in fertilization, at the highest dose applied to the soil the mycorrhizal colonization was significantly reduced in comparison to the lowest dose of P used in the experiment.According to these authors, although the mechanism determining the influence of phosphate fertilization on AMF community is mostly unknown, the increase in soil jas.ccsenet.