Growth and nutrient accumulation in mycorrhized papaya seedlings cultivated in a phosphorus-fertilized substrate

Submitted on 04/06/2015 and approved on 10/26/2015. 1 Universidade Federal da Paraíba, Departamento Solos e Engenharia Rural, Areia, Paraíba, Brasil. almeida_dacio@hotmail.com 2 Universidade Federal da Paraíba, Departamento Ciências Fundamentais e Sociais, Areia, Paraíba, Brasil. walterufpb@yahoo.com.br 3 Universidade Federal da Paraíba, Departamento de Solos e Engenharia Rural, Areia, Paraíba, Brasil. patriciareia@yahoo.com.br 4 Universidade Federal da Paraíba, Departamento de Solos e Engenharia Rural, Areia, Paraíba, Brasil. jarissonagro@hotmail.com 5 Universidade Federal do Tocantins, Núcleo de Estudos Ambientais, Porto Nacional, Tocantins, Brasil. wmelouft@yahoo.com * Corresponding author: walterufpb@yahoo.com.br Growth and nutrient accumulation in mycorrhized papaya seedlings cultivated in a phosphorus-fertilized substrate


INTRODUCTION
Brazil has favorable soil and climatic conditions for the cultivation of tropical fruit species and is among the major world producers of fruit and fruit juice.Papaya (Carica papaya L.) is one of the most harvested fruits in the country and its annual production is approximately two billion tons (Agrianual 2011).
One of the limitations in the papaya production chain is the production of high quality seedlings due to the elevated plant density per hectare and the need for orchard renewals every three years.Thus, seedling production is a fundamental step in the implementation of a productive orchard.
Mycorrhized plants exhibit developmental advantages when compared to non-mycorrhized ones under nutrientlimiting conditions.Scientific investigations have shown that inoculation of arbuscular mycorrhizal fungi (AMF) promotes the formation of good quality papaya seedlings (TRINDADE at al. 2001b;MOHANDAS 2012).However, the benefits maximized by that symbiosis in plant nurseries, such as increase in nitrogen (MIYAUCHI at al. 2008), potassium and phosphorus uptake (OLIVEIRA & OLIVEIRA 2005), tolerance to root diseases (BORGES at al. 2007), and improved seedling growth and vigor during its formation period (LIMA at al. 2011), can be jeopardized under field conditions.Most cultivated areas occur in soils featuring low-fertility, low pH, high aluminum and low phosphorus contents and, consequently, are highly fertilized, primarily with phosphorus, which is an essential nutrient for plant growth and influences root formation and growth, water use efficiency, and uptake and assimilation of other nutrients (Epstein & Blomm 2006).
Therefore, in phosphorus-rich soils, the advantages of that type of symbiosis are reduced since carbohydrate consumption by the fungi does not cease and, indeed acts as a strong sink.Consequently, the performance of mycorrhized plants can be poorer than that of nonmycorrhized ones (Janos, 2007) which risks seedling survival in the field and causes a decrease in vigor and productivity.
The present research aimed at studying the efficiency of arbuscular mycorrhizal fungi (AMF) on dry matter and nutrient accumulation in Sunrise Solo papaya seedlings, by applying doses of P 2 O 5 that are detrimental to the symbiosis.

MATERIAL AND METHODS
The experiment was carried out from January through May 2012 under a shaded environment, at the Department of Plant and Environmental Sciences, Agricultural Sciences Center, Campus II of the Federal University of Paraíba, located in the Municipality of Areia, Paraíba.The climate of the area, classified as As according to the Köppen system, is tropical, hot and humid, exhibiting a rainy season along fall and winter, with an average relative humidity of 80% (Gondim 1999).During the experiment, the average temperature and relative humidity varied from 22.5 to 23.6 o C and 60 to 80%, respectively.
The evaluated factors consisted of four P 2 O 5 doses (0; 672; 1386 and 2100 mg dm -3 ), based on Melo at al. (2007), and three species of mycorrhizal fungi supplied by Embrapa Agrobiology: Gigaspora margarita, Entrophospora colombiana and Scutellospora heterogama.The control treatment was mycorrhiza-and P 2 O 5 -free.The source of P 2 O 5 was triple superposphate.The experiment was set up in a randomized block design in the factorial scheme, with four blocks and four seedlings per experimental unit.
Seeds of the cultivar Sunrise Solo, produced by Agristar, were sown in germination trays (60 cm x 40 cm) containing sand autoclaved at 121 o C. When seedlings reached 5.0 cm in height they were transferred to PVC tubes (25 cm length and 8 cm in diameter) containing 0.7 dm -3 of a substrate mix composed of 75% soil (Yellow Latosol, total sand = 506 g kg -1 , silt = 103 g kg -1 , clay = 391 g kg -1 ), 15% sand and 10% manure.The soil was sandy clay loam and was mixed with cow manure.The mix was passed through a 4 mm mesh sieve.The physical-chemical characteristics of the mix are shown in Table 1.After autoclaving, N, K, Ca, Mg, S and B were added to the mix, whose amounts were based on its chemical analysis.At transplanting, 3.0 g of the mix containing fungal spores were added to each tube.Seedlings were irrigated twice a day with chlorine-free water [electrical conductivity -EC = 0.22 dS.m -1 and sodium adsorption rate -SAR = 2.41 (mmol c L -1 ) 0.5 ], in order to keep the substrate under field capacity and avoid excessive water retention.Data collection was carried out 102 days after the beginning of the experiment and the following variables were analyzed: root and shoot dry matter, nitrogen, phosphorus and P 2 O 5 .A inoculação do fungo Entrophospora colombiana promoveu maiores ganhos de biomassa.A adubação com P 2 O 5 aumentou os teores de fósforo foliar e radicular.
For dry matter and plant tissue analysis, leaves and roots were washed in running water and rinsed with distilled water.Following that, they were placed in paper bags and dried in a drying oven with forced air circulation at 65 o C until constant weight was reached.After dry matter determination, the plant material was ground in a Willey grinder and then passed through a 0.5 mm mesh sieve.For nutrient content analysis, the material was digested by use of sulfuric acid (H 2 SO 4 ) and hydrogen peroxide (H 2 O 2 ) in a block digester at 350 o C. Nitrogen content was determined through distillation in 13N sodium hydroxide (NaOH) followed by titration with 0.07143N hydrochloric acid.Phosphorous was quantified by spectrophotometry upon measuring the color intensity of the phosphomolybdic complex, produced as result of the reduction of molybdate with ascorbic acid, and potassium was quantified by the flame photometry method (Embrapa 2011).
Mycorrhizal colonization was assessed by collecting secondary roots of seedlings and immersing them in a sodium hypochlorite solution for 30 seconds followed by another 30 second immersion in 70 o ethyl alcohol.Roots were then rinsed with distilled water, placed in plastic pots containing 50 mL of 70 o ethyl alcohol and 3.0 mL of formalin, and kept under refrigeration at 4 o C. For clarification and staining (Phillips & Hayman 1970) roots were immersed in a 10% KOH solution for 15 days at room temperature.After that period, they were washed, acidified in 1% HCl for 3 minutes and stained with 1 mL trypan blue.After 10 minutes, the stained roots were sliced in 1 cm pieces and uniformly spread in a 1.27 cm square Pedtri Dish with grid and analyzed in a microscope (each intersection of roots and grid) for the presence or absence of fungal structures (Giovannetti & Mosse 1980).
Analysis of variance (ANOVA) was used to test the significance of the model sources of variation.The effect of fungal species was compared by the Tukey test and that of triple superphosphate doses by a polynomial regression.The comparison with the control treatment was conducted by the Dunett test.SAS/STAT 9.3 software (SAS 2011) was used for all statistical analyses.

RESULTS AND DISCUSSION
The results revealed no difference between the mycorrhizal treatments and the control for root dry matter accumulation (Table 2), except for E. colombiana at the lowest phosphate dose, which was 25% lower when compared to the control.Phosphorous can alter plant growth by allocating large amounts of carbohydrates to increase root biomass.Phosphorus availability also regulates several processes such as phytomass distribution pattern.Fernandes at al. (2000) observed insignificant effects of phosphate fertilization on root dry matter accumulation since the substrate studied already had adequate amounts of phosphorus for seedling growth.
The fact that the control did not differ from the other treatments, in terms of root dry matter accumulation, can be related to the time the seedlings remained in the tubes containing 0.7 dm -3 of substrate (102 days), which was long enough for their roots to occupy the entire substrate volume.Trindade at al. (2000), working with the production of papaya seedlings colonized by mycorrhizal fungi, which were grown in plastic bags containing 1 dm - 3 of substrate, also observed that 50 days after transplanting, the roots had spread throughout the whole substrate volume, because it is a fruit species with a high growth index.
The phosphate doses had distinct effects on root and shoot dry matter, depending on the species of fungus inoculated (Figures 1 A and B, respectively).In the phosphorus-free treatment, S. heterogama and G. margarita promoted 34 and 24% increase in root dry matter accumulation, respectively, in comparison with E. colombiana (Table 2).Khade at al. (2010), studying the variation in the level of native mycorrhizal colonization in papaya varieties, detected the influence of root colonization on phosphatase activity, which increased as colonization increased.
Regarding E. colombiana, the maximum estimated value of root dry matter (9.03 g seedling -1 ) was obtained with 1386 mg dm -3 P 2 O 5 and the responses decreased at higher doses (Figure 1A).This behavior can be related to the inhibitory effect of phosphorus on mycorrhizal colonization, as verified with S. heterogama (Figure 2   Inoculation with E. colombiana, G. margarita and S. heterogama promoted a 32, 33 and 36% increase in shoot dry matter, respectively, in relation to the control treatment, but did not differ among the fungal species (Table 3).Lima at al. (2011) reported that papaya seedlings inoculated with mycorrhizal fungi exhibited higher growth rates than those not inoculated, regardless of the amount of phosphorus in the soil.
Shoot dry matter behavior was similar to that observed for root dry matter (Figure 1 B).It is worth highlighting the effects on E. colombiana, which exhibited quadratic growth as fertilization doses increased, reaching a maximum value of 22.98 g seedling -1 , which corresponds to an estimated dose of 1104 mg dm -3 of P 2 O 5 .This superiority in relation to the control (15.88 g seedling -1 ) expresses the importance of mycorrhizal fungi in promoting one of the main advantages of that symbiosis: nutrient uptake.The increments in root uptake capacity, provided by hyphae development and branching, which in turn increase root-soil contact area, favor a greater absorption of nutrients such as phosphorus.Trindade at al. (2001a) demonstrated that papaya cultivars can benefit from inoculation with Glomus clarum and G. margarita, reducing up to seven times the need for P fertilization so that maximum shoot growth can be achieved.Lima at al. (2011), evaluating growth of papaya cv Golden seedlings, showed that fungal inoculation treatments promoted an increase in seedling leaf area, with an increment of 19.3% in relation to the control treatment, which influenced dry matter accumulation but did not differ among the fungal species studied.
No differences for leaf N content of seedlings were detected among mycorrhizal fungi (Table 4).However, all fungi treatments exhibited increases in foliar N, whose values were 24, 23 and 21% higher than the control for G. margarita, E. colombiana and S. heterogama, respectively, at 672 mg dm -3 P 2 O 5 (Table 5).Chu at al. (2001) also reported no differences in terms of foliar N content of Annona muticata seedlings when inoculated with the same fungi species assessed in the present study and using fumigated and nonfumigated soil.Rev. Ceres, Viçosa, v. 63, n.1, p. 086-094, jan-fev, 2016 Leaf N content exhibited a quadratic behavior as P 2 O 5 doses increased (Figure 3A), reaching a maximum value of 45.38 g kg -1 at 336 mg dm -3 P 2 O 5 .Nitrogen uptake by mycorrhizal fungi becomes more efficient because of the small hyphae diameter that penetrate more easily in decomposing organic matter, competing more effectively for recently mineralized N, especially simple nitrogen organic compounds (Hodge, 2003).Contrasting results were reported by Melloni & Cardoso (1999) in Citrus seedlings.The increasing phosphorus doses favored linear increases in the total amount of macronutrients absorbed by the rootstocks, including nitrogen.
Phosphorus content in the seedlings increased linearly as P 2 O 5 doses applied to the substrate increased (Figure 3B).Leaf phosphorus content of seedlings inoculated with mycorrhizal fungi also increased in relation to the control treatment at the highest P 2 O 5 doses.That increase was detected at P 2 O 5 doses that promoted the highest total phosphorus concentration in the seedlings, with over 80% increases at P 2 O 5 doses equal and above 1386 mg dm -3 .The positive influence of mycorrhizal inoculation on plant growth is especially associated with the uptake of low mobility nutrients such as phosphorus, copper and zinc, which reach the roots by slow diffusion mechanisms.The fungi can access soluble forms of phosphorus that are also available for non-inoculated plants, but hyphae end up exploring the soil more thoroughly and guarantee better nutrient absorption (Kanno at al. 2006).The increase in leaf phosphorus content as P 2 O 5 doses increased may not be related to mycorrhizal benefits at higher doses since there is a phosphorus balance between the soil and the plant tissue that controls the symbiotic association (Costa at al. 2001).
The effect of P 2 O 5 doses on nitrogen and phosphorus content of roots are displayed in Figure 4. Root nitrogen content decreased linearly as P 2 O 5 doses increased (Figure 4A).However, at lower doses the mycorrhizal effect was more distinct.According to a few authors, mycorrhizal fungi can form associations with nitrogen-fixing bacteria.
The fungus plays a central role in providing phosphorus for the plant and for the bacteria in the nodules, a process that is ATP-dependent (Barea at al. 1992).Nitrogen   Root phosphorus content exhibited a quadratic tendency in response to an increase in the amount of that nutrient in the substrate (Figure 4B).Based on the studies conducted by Moreira & Siqueira (2002), it can be assumed that in conditions of high phosphorus supply, phospholipid biosynthesis is increased, and as a result, cell permeability is affected.Thus, root exudation (sugars and amino acids), infection and colonization decline, as proved by colonization of S. heterogama (Figure 4B).The data revealed a higher phosphorus content in the tissues of inoculated seedlings in relation to the control (Table 5), reaching the highest value (12.3 g kg -1 ) at 1978 mg dm -3 P 2 O 5 (Figure 4B); these results were more evident than those reported by Trindade at al. (2001b).This can be due to the high doses used in the present study.Differences were only detected at doses equal or higher than 672 mg dm -3 P 2 O 5 .Increments of 201, 302 and 361% were verified at 672, 1386 and 2100 mg dm -3 P 2 O 5 , respectively, when comparing fungi treatments with the control.No difference was detected among the fungi used (Table 4).
The increase in phosphorus content of roots and leaves is a result of a higher amount of P 2 O 5 in the substrate.As it is a highly mobile nutrient, its concentration was higher in the tissues of those two organs.Mesquita at al. (2010), studying leaf mineral composition of papaya fertilized with an enriched biofertilizer, detected phosphorus increments of 4.2 g kg -1 , which were inferior to the results obtained in the present study.
Production of quality papaya seedlings, aiming at the enhancement of their performance after transfer to the field, makes the potential use of AMF more attractive since it is responsible for increasing the efficiency of nutrient uptake, thus reducing the use of fertilizers without affecting productivity.** *: Significant at (P<0.01) and (P<0.05)respectively, according to the F test

Figure 2 :
Figure 2: Mycorrhizal colonization in papaya cv.Sunrise Solo seedlings inoculated with mycorrhizal fungi and fertilized with P 2 O 5.
or negatively interfere with the development of plant mycorrhizal dependency.Santos at al. (2001) observed a decrease in the number of spores as nitrogen fertilization increased.

Figure 3 :
Figure 3: Nitrogen (NL) "A" and phosphorus (PL) "B" contents in leaves of papaya cv.Sunrise Solo seedlings inoculated with mycorrhizal fungi and fertilized with P 2 O 5 .
(Nogueira & Cardoso 2007)8)y E. colombiana and G. margarita it was not significantly affected by P 2 O 5 doses.AccordingBalzergue at al. (2011), elevated phosphorus concentration in the medium is accompanied by a decreased strigolactone content, decreasing the mycorrhizal colonization.Similar results were verified in studies involving mycorrhizal colonization in fruit species, such as Hancornia speciosa(Cardoso Filho at al. 2008)and Citrus species(Nogueira & Cardoso 2007).

Table 2 :
Root dry matter (g seedling -1 ) of papaya cv.Sunrise Solo seedlings inoculated with mycorrhizal fungi and fertilized with P 2 O 5 Mean values followed by the same block letter within lines are not significantly different according to the Tukey test (P < 0.05).ns and *: not significant and significantly different, respectively, from the control treatment, according to the Dunnett's test at (P < 0.05); (P < 0.05).Control = without fungal inoculation.

Table 3 :
Shoot dry matter (g seedling -1 ) of papaya cv.Sunrise Solo seedlings inoculated with mycorrhizal fungi and fertilized with P 2 O 5 Mean values followed by the same block letter within lines are not significantly different according to the Tukey test (P<0.05).and*: significantly different, respectively, from the control treatment, according to the Dunnett's test at (P<0.05); (P<0.05).Control = without fungal inoculation.**Significant at (P<0.01) according to the F test.

Table 4 :
Nutrient content (g kg -1 ) in roots and leaves of papaya cv.Sunrise Solo seedlings inoculated with mycorrhizal fungi

Table 5 :
Nutrient content (g kg -1 ) in roots and leaves of papaya cv.Sunrise Solo seedlings inoculated with mycorrhizal fungi and fertilized with P 2 O 5 ns and *: not significant and significantly different at (P < 0.05), respectively, in relation to the control treatment according to the Dunnett's test (P < 0.05).Control = without fungal inoculation and fertilization.