Evaluation of interaction between Piriformospora indica, animal manure and NPK fertilizer on quantitative and qualitative yield and absorption of elements in sunflower

Abstract Microbial endophytes are considered as one of the most important soil microorganisms which increase their yield per unit area by generating genetic, physiological, and ecological changes in their host plants. We conducted an experiment as factorial in a completely randomized manner with three replications at the Ghasre Shirin greenhouse of Kermanshah province in order to evaluate the interaction of Piriformospora indica (inoculation–noninoculation) with manure (25% of the flowerpot volume—without using manure as a control) and NPK chemical fertilizer (10 g per flowerpot—without the use of fertilizer as a control), the quantitative and qualitative yield and absorption of elements in sunflower. The results showed that the interaction of P. indica, manure, and chemical fertilizer on the colonization percentage, seed oil percentage, nitrogen concentration, phosphorus concentration, and the 1000‐seed weight was significant at 1% probability level, and on yield index, growth, plant height, and concentration of potassium element it was at 5% probability level. Bilateral effects of P. indica with chemical fertilizer and the manure treated with chemical fertilizer on the budding time were significant at the probability level of 1% and 5%, respectively. It seems that the coexistence between sunflower root and P. indica increases the growth of the root system of the plant; thereby it increases the height of plant because of the absorption of essential elements such as nitrogen, phosphorus, and potassium by the root, and increases the quantitative and qualitative yield of sunflower.

showed that the grain yield was significantly influenced by the combined treatment of manure, poultry manure along with the nitrogen concentration chemical fertilizer. The reason is more access to the food needed during the critical stages of plant growth. The combined system of organic and chemical fertilizer has been reported in other researches on sunflower (Munir, Malik, & Saleem, 2007) and peanut (Basu, Bhadoria, & Mahapatra, 2008). Shehata and El-Khawas (2003), in the study about the effect of biological manures on growth parameters and the yield of sunflowers, found that fertilizers improve quality and yield compared to inoculum. Due to mass production of sunflower in the country and excessive use of chemical fertilizers, the present study was conducted to prevent overuse of chemical fertilizers and to replace P. indica with chemical fertilizers and also increasing yield, growth, increasing oil content, and nutrient uptake in sunflower plant was treated with P.
indica endophyte and organic fertilizer and chemical fertilizer.

| MATERIAL S AND ME THODS
The present study was conducted to evaluate the effect of P. indica cultivar which has been used. The minimum and maximum temperatures were 28°C and 14°C, respectively, and relative humidity was about 55% up to 60%. Sunflower seeds were also exposed to 14 hr of light (a combination of Fluorescent and Tungsten lumens). Strain of P. indica was obtained from the Department of Horticulture of Maragheh University and was used as seed priming. After 1 month (shahabivand, Parvaneh, & Aliloo, 2017), samples of thin roots of sunflower were isolated and the colonization percentage was measured using the Phillips and Hayman (1970) method and the following equation (Equation 1).
The final harvest (88 days after planting), the final height of the plants, the diameter of the head, and the grain yield were measured.
Percentage of seed oil was also measured using the Soxhlet ex- Data were analyzed using SAS 9.1 software. A comparison of the mean of data was done using Duncan's multi-domain test at the 5% probability level. Graphics were designed using software

| RE SULTS AND D ISCUSS I ON
The results of the variance of data analysis (Table 3) showed that the effect of P. indica, animal manure, and chemical fertilizer on root colonization, phosphorus concentration, oil percentage, and nitrogen percentage of the grain was meaningful at a probability level of 1% and on potassium concentration at 5% probability level.

| Percentage of colonization
According to the table of the comparison of averages (Table 4) (2) GDD = minimum temperature + maximum temperature 2 − base temperature TA B L E 3 Analysis of variance of the influence of P. indica, animal manure, and chemical fertilizer on concentration of nitrogen, phosphorus, potassium, and oil of sunflower may be due to increasing the growth of the poisonous properties of elements in high concentration, which reduces the potential of inoculum, and prevent the budding and natural growth of the spore.
Despite of reducing the colonization in high concentrations of the elements (Table 4), simultaneous use of fungi and chemicals is very useful to increase the yield and other growth parameters.

| Concentration of NPK elements
Nitrogen, potassium, and phosphorus concentration in seeds showed that the highest percentage of nitrogen, phosphorus, and potassium concentration was related to fungal treatments with manure and fertilizer in inoculated plants with P. indica (Table 4).
It seems that the absorption of elements is high around the root due to the effect of colonized fungus on increasing root growth (Padash et al., 2016). Alizadeh (2010)  and also increasing the capacity to maintain moisture (Ahmadian, Ghanbari, & Galavi, 2004). According to various scholars, the use of animal manure increases the organic substance, useable phosphorus (Antoun, Beauchamp, Goussard, Chabot, & Lalande, 1998), nitrate nitrogen (Graham & Vance, 2000), and other nutrients of the plant, and improves soil texture (Sharpley, McDowell, & Kleinman, 2004), which ultimately increases the quality of product slightly.
According to the results of the test and the results of previous reports, it seems that manure mixed with soil plays a significant role in the growth of fungus on roots. It also provides useful elements for the plant.

| Oil percentage
Comparison of averages showed that application of manure in inoculated plants with P. indica increased the percentage of seed oil (25%) ( Table 4) and the lowest amount of oil was related to the application of chemical fertilizers ( which showed that the combined treatment had the lowest percentage of oil, and the control treatment had the highest oil concentration. The reduction of oil concentration with the high application of nitrogen fertilizers has been reported by other researchers (Khaliq, 2004). Kasem and EL-Mesilby (1992) reported that the percentage of seed oil decreases by increasing access to nitrogen. (Steer & Seiler, 1990) also found that there is a negative relationship between the amount of access to nitrogen and the amount of oil. Application of biologic fertilizers with organic fertilizers increased the amount of oil. Therefore, the P. indica fungi which has been combined with manure increases the amount of seed oil in the sunflower.
According to analyzing the table of variance (Table 5), it was determined that application of P. indica fungus with manure and chemical fertilizer on 1000-seed weight was significant at the probability level of 1% and for plant height, growth time, and yield at 5% probability level.
In addition, the treatment effects of P. indica with chemical fertilizer and animal manure on the appearance of sunflower head were significant at 1% probability level. Treatment of P. indica and animal manure TA B L E 4 Comparison of the effect of P. indica, animal manure, and chemical fertilizer on percentage of colonization and the amount of nitrogen, phosphorus, and potassium in sunflower plant

| Growth
Comparison of the meanings the effect of symbiotic fungus treatment with manure and chemical fertilizer on head maturity index in sunflower (Table 5)

| Height
Comparison of averages showed that the height of plants colonized with P. indica in combination with manure and chemical fertilizer had a significant difference compared to noninoculated plants (Table 6). (Skinner, Boddey, & Fendrik, 1987) reported that inoculation of seed with biological fertilizers increases root development and absorbs water and nutrients better. This can improve plant growth and increase plant height. The P. indica fungus makes most of the elements, especially nitrogen, to be absorbed with the effect on the root system. Animal manure provides a good basis for the pres-

| Grain yield
Grain yield in plants which was treated with P. indica was not significantly different from the control plants (noninoculation) ( Table 6).
However, the highest yield belongs to the use of fungi, along with animal manure and chemical fertilizer (Table 6). Probably, improving the physical properties of soil, better access to water and nutrients, also the ease of absorption of elements are the reason for increasing sunflower seed yield in manure (Mallanagouda, 1995). The introduction of organic matter into soil increases soil nutrient uptake and absorption capacity by the plant (Beare, 1997). The results of (Liang et al., 2005) showed that the manure provides a suitable ground for plant growth and ultimately boosts yield because of the presence of elements and water around the root.

TA B L E 5
Shows the analysis of variance of treatment with P. indica, animal manure, and chemical fertilizer on grain yield, heading, growth, height, and head diameter of sunflower Abbreviation: ns, nonsignificant. *Significant at a probability level of 5%.
**Significant at a probability level of 1%.

| 1000-seed weight
Comparison of mean values showed that the highest 1000-seed weight belonged to P. indica-inoculated plants with cadmium and chemical composition (Table 6). Studying the levels of nitrogen fertilizer on the yield of sorghum, Powell and Hons (1992) stated that there is a direct relationship between nitrogen fertilizer application and grain yield, the production of biomass and the weight TA B L E 6 Comparison of the effects of endophytic fungus Piriformospora indica, animal manure, and chemical fertilizer on growth indices and yield in sunflower plant indica, which was used in combination with chemical fertilizers and animal manure. Considering that fertilizer has significant amount of nitrogen, it is estimated that the plants, which are treated with this fertilizer, should have 1000-seed weight more than other plants.
On the other hand, the colonization of the plant with the combination of manure and chemical fertilizers increases the absorption of these elements.

| Head diameter
Comparison of averages showed that the combination of P. indica fungi with animal manure (Figure 1) and manure with chemical fertilizer ( Figure 2) has the greatest effect on head diameter. The results of various experiments showed that the use of some elements in sunflower production has a significant effect on plant height, head diameter, seed number, seed weight, seed oil percentage, number of leaves, and grain yield (Sepehr & Malakouti, 2004). In the comparison chart (Figures 1 and 2), the highest diameter was observed for treatments in which animal manure was used. The use of manure with chemical fertilizer (Figure 1) and fungi ( Figure 2) results in the absorption of most of the elements, and eventually created a thicker head with an effect on growth.

| Heading stage
The heading index, as the growth index, depends on the amount of nutrition during the plant growth period. The graph for comparison of the average application of chemical fertilizer with fungal F I G U R E 3 Shows the effect of P. indica and chemical fertilizer on head diameter index in sunflower plant F I G U R E 4 Shows the effect of chemical fertilizer and manure on heading index in sunflowers