Beauveria bassiana: An entomopathogenic fungus alleviates Fe chlorosis symptoms in plants grown on calcareous substrates

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Beauveria bassiana: An entomopathogenic fungus alleviates Fe chlorosis symptoms in plants grown on calcareous substrates Antonio R. Sánchez-Rodríguez a,∗ , María Carmen Del Campillo b , Enrique Quesada-Moraga a a b

Departamento de Ciencias y Recursos Agrícolas y Forestales, Universidad de Córdoba, Edificio C4, Campus de Rabanales, 14071 Córdoba, Spain Departamento de Agronomía, Universidad de Córdoba, Edificio C4, Campus de Rabanales, 14071 Córdoba, Spain

a r t i c l e

i n f o

Article history: Received 16 June 2015 Received in revised form 11 September 2015 Accepted 23 September 2015 Available online xxx Keywords: Iron nutrition Entomopathogenic fungus Endophyte Biofertilizer Calcareous soils

a b s t r a c t Entomopathogenic fungi are known due to their ability to kill insects and besides they can play other roles, such as promoting plant growth or improving nutrient uptake. The main aim of this work was to assess the potential of Beauveria bassiana strain EABb 04/01-Tip for improving iron (Fe) nutrition in plants grown on calcareous substrates. Plants of a dicot (tomato) and a monocot (wheat) were pot-grown on an artificial substrate consisting of 0–240 g kg−1 Fe oxide-coated sand (FOCS), 250 g kg−1 calcium carbonate sand and quartz sand. The plants were subjected to two different treatments, namely seed dressing, which involved inoculating seeds with a conidial suspension of B. bassiana before sowing, and a control without inoculation. Leaf chlorophyll concentrations (SPAD) and Fe uptake by plants were correlated with the FOCS content of the substrate, therefore the experimental design was appropriate to see different levels of Fe chlorosis symptoms. B. bassiana was able to colonise both tomato and wheat plants without a negative effect on plant height, plant dry weight, root development in tomato or grain production in wheat. In addition, B. bassiana alleviated Fe chlorosis symptoms (described for first time) in both crops during early growing stages (<50 days after sowing) but the intensity of the effect depended on the plant species and available Fe on substrate. Nutritional alterations in K due to fungal application were detected in tomato and wheat plants. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Endophytic fungi are microorganisms that can live in a host plant without causing any symptoms of disease. Plant–endophyte symbiotic relationships occur in the vast majority of ecosystems where plants need fungi to withstand adverse factors (Rodriguez et al., 2004) such as biotic (herbivory and pathogens) and abiotic (e.g. high temperatures, salt, drought, low content of nutrients in soil, heavy metals) stresses (Akello et al., 2009; Arnold et al., 2003; Ownley et al., 2010; Redman et al., 2002; Saikkonen et al., 2013; Vega et al., 2009; Waller et al., 2005). Ascomycetes, which constitute one of the most important group of endophytic fungi, have been found on a wide range of host plants (Ownley et al., 2010; Quesada-Moraga et al., 2014; Vega

et al., 2009). The entomopathogenic fungi in this division are used as biopesticides because of their ability to kill insects by producing secondary metabolites that are of industrial and agricultural interest (Aly et al., 2011; Schulz et al., 2002; Zhang et al., 2006). However, they can play other not fully understood roles such as promoting plant growth (Liao et al., 2014; Maag et al., 2013; Sasan and Bidochka, 2012; Vega et al., 2009) or even improving plant nutrition (García-López et al., 2013). Beauveria bassiana is an entomopathogenic fungus naturally present in soils that can infect insects, mites and ticks, and hence possesses a great potential for pest management (Meyling and Eilenberg, 2007). Some authors have shown B. bassiana to endophytically colonize crops such as opium, tomato, wheat, corn and sorghum (Gurulingappa et al., 2010; Quesada-Moraga et al., 2006; Tefera and Vidal, 2009), besides protecting crops against pest and disease (Lozano-Tovar et al., 2013). Also, this fungus has been found to contribute to rock and mineral bioweathering, and to alter the

∗ Corresponding author. E-mail address: [email protected] (A.R. Sánchez-Rodríguez). http://dx.doi.org/10.1016/j.scienta.2015.09.029 0304-4238/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Sánchez-Rodríguez, A.R., et al., Beauveria bassiana: An entomopathogenic fungus alleviates Fe chlorosis symptoms in plants grown on calcareous substrates. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2015.09.029

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availability of metals such as Fe, Cu and Ag when it is directly applied to a metallic surface (Joseph et al., 2012). There are seemingly no literature references to the effect of B. bassiana on plant nutrition; however, experiments with other fungi used as biocontrol agents such as Trichoderma have provided evidence for increased nutrient (Fe) uptake by plants (de Santiago et al., 2009). Therefore, applying B. bassiana to crops grown on soils with low bio-availability of some nutrients might provide a twofold benefit: plant protection (well known) and improve nutrient uptake. Calcareous soils span 30% of all arable land in the world and typically have a high calcium carbonate (CaCO3 ) content and a pH of 7.5–8.5. Iron bioavailability at these pH levels is very low and results in deficiencies such as Fe chlorosis—a major problem in plants grown on calcareous soils (Tagliavini and Rombolà, 2001). The main symptoms of Fe chlorosis are interveinal yellowing of young leaves and diminished plant growth, because plants are unable to synthetize chlorophyll under these conditions. Necrotic spots can replace chlorosis and cause leaves to die or even decrease production yield and quality. Because Fe bioavailability depends on the specific surface area and crystallinity of Fe oxides (Schwertmann, 1991), it is increased by the presence in soils of non-crystalline Fe oxides such as ferrihydrite, wich has a high specific surface area (Diaz et al., 2009). Although some of the above-described experiments, in which B. bassiana was used to protect crops against pests [Bactrocera oleae (Gmelin.) (Diptera; Tephritidae)] and disease (Verticillium dahliae, Phytophthora megasperma and Phytophthora inundata), were conducted on calcareous soils, not much is known about the effect of this endophytic fungus on plant growth or nutrition. Gatarayiha et al. (2010) studied the interactions between B. bassiana and potassium (K) silicate fertilization, and observed a higher efficiency of the fungus used to control two spotted spider when K fertilization was applied to inoculated dicot and monocot plants (cucumber, eggplant, bean and maize). However, there is a lack of information on the effect that B. bassiana causes the plant in absence of insects. The objectives of this work were (i) to assess the colonization of leaf, stem and root of tomato (dicot) and wheat (monocot) plants after being inoculated with B. bassiana strain EABb 04/01-Tip (via seed) and (ii) to examine the effect of this entomopathogenic fungus on Fe nutrition and plant growth in tomato and wheat plants grown on artificial calcareous substrates.

2. Materials and methods 2.1. Substrate preparation In order to simulate the typical conditions of calcareous soils, quartz sand and calcium carbonate sand were sieved and particles 0.2–0.5 mm in size selected for optimum hydraulic performance. The resulting fractions were washed 6 times with a large volume of tap water containing Na2 CO3 at pH 9.5 (quartz sand) or plain water (calcium carbonate sand) to disperse clay and impurities. Both types of sand were also washed several times with de-ionized water to remove salts before drying in an oven at 40 ◦ C. The preparation procedure was repeated once. The specific surface area as measured by N2 adsorption (BET method, Brunauer et al., 1938) of the quartz sand was 0.14 m2 g−1 and that of the calcium carbonate sand 0.27 m2 g−1 . Citrate/bicarbonate/dithionite-extractable Fe, used as a proxy for the total content in Fe oxides (Fed , Mehra and Jackson, 1960), accounted for 40 mg kg−1 in quartz sand and 120 mg kg−1 for calcium carbonate sand. Available P (Olsen et al., 1954) was less than 1 mg kg−1 in both types of sand. Part of the quartz sand was coated with Fe oxides (ferrihydrite, called FOCS) as detailed in Rahmatullah (2000). FOCS comprised 380 mg kg−1 Fed

and 210 mg kg−1 acid oxalate-extractable Fe (Feox , Schwertmann, 1964). The latter is a measure of poorly crystalline Fe oxides (Reyes and Torrent, 1997), which constitute the main source of Fe for plants in calcareous media.

2.2. Plant material, culture, preparation of the conidial suspension and treatments Five substrates containing variable proportions of FOCS (viz., 20, 40, 80, 120 and 240 g kg−1 ), one part of calcium carbonate (250 g kg−1 ) and quartz sand to complete 100% (730, 710, 670, 630 and 510 g kg−1 , respectively) were prepared. A positive control in the form of a substrate inducing no Fe chlorosis and consisting of 100% quartz sand (0 g kg−1 FOCS) was also studied. Cylindrical PVC pots 12 cm high and 4.5 cm in diameter with a drainage hole in the bottom were filled with 250 g of the previous combinations following sterilization by heating twice at 121 ◦ C for 20 min before sowing the crops. Seeds of tomato (Lycopersicon esculentum Mill. cv. MarmandeCuarenteno, commercial seeds) and wheat (Triticum aestivum L. cv. Chinese Spring, kindly provided by Dr. Pilar Prieto from Institute for Sustainable Agriculture of Córdoba, Spain) were immersed in a 6% H2 O2 solution for 10 min and then gently washed with deionized water. After pre-germination at 25 ◦ C in the dark for 2 days, the seeds were subjected to two different treatments. One, called the “control”, involved immersing a part of the seeds in sterile, conidium-free de-ionized water containing Tween 80 (0.1% v/v) under shaking at 180 rpm for 4 h. The other one, called “seed dressing”, was performed under the same conditions but used a B. bassiana suspension containing 1 × 108 conidia mL−1 to inoculate the remaining seeds. This fungal strain was routinely grown on slants of malt agar (MA; Biocult, Madrid, Spain) at 25 ◦ C in the dark and stored at 4 ◦ C. Fungus cultures were grown at 25 ◦ C in the dark on malt agar containing 25 g malt agar and 7.5 g agar for 2 weeks, after which conidia were collected by scraping the surface of the culture with a sterile camel hairbrush into a 100 mL glass beaker containing 50 mL of sterile distilled water plus Tween 80 (0.1% v/v). The conidial suspension was stirred, filtered, adjusted to 1 × 108 conidia mL−1 and used to inoculate the seeds as described. A different fresh culture was prepared for each crop. The conidial suspension was prepared by using B. bassiana strain EABb 04/01-Tip isolated from a dead Iraella luteipes larva. This strain, which had previously exhibited an endophytic behaviour upon inoculation to opium plants is deposited in the CRAF Entomopathogenic Fungi Collection of the University of Cordoba and the Spanish Collection of Culture Types (CECT) of the University of Valencia (CECT 20,744, accession number). A fresh conidial suspension was prepared for each crop. Once treated, the seeds were dried under sterile conditions and 3 seeds sown in each pot. The pot experiment was performed in a growth chamber under the following conditions: 12 h day−1 , 250 ␮mol m−2 s−1 , 21 ◦ C and 70% relative humidity. Seven days after sowing (DAS), all plants except one in each pot were cut off and the pots weighed and watered daily to keep soil moisture near field capacity. Also, each crop and pot were irrigated with a modified Hoagland solution on a weekly basis (5–10 mL each week depending on the extent of plant growth). The Fe-free modified Hoagland nutrient solution applied to the plants grown on the FOCS-containing substrates consisted of Ca(NO3 )2 ·4H2 O (5 mM), KNO3 (5 mM), MgSO4 (2 mM), K Cl (0.1 ␮M), Ca(H2 PO4 )2 ·H2 O (0.3 ␮M), H3 BO3 (50 ␮M), MnSO4 ·H2 O (4 ␮M), ZnSO4 ·7H2 O (4 ␮M), CuSO4 ·5H2 O (0.1 ␮M) and Na2 MoO4 (6 ␮M). The solution was enriched with Fe (20 ␮M) for application to the pots containing no FOCS. This rate had previously proved effective to prevent Fe chlorosis.

Please cite this article in press as: Sánchez-Rodríguez, A.R., et al., Beauveria bassiana: An entomopathogenic fungus alleviates Fe chlorosis symptoms in plants grown on calcareous substrates. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2015.09.029

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2.3. Determinations Tomato plants were sampled 15 (with four leaves), 30 (with six leaves) and 48 (with eight leaves) DAS, and wheat plants 10 (2 leaves), 22 (tillering), 50 (jointing) and 90 (flowering) DAS, corresponding with different phenological stages. In each sampling, four randomly selected plants per fungal application (4 for control and 4 for seed dressing) were removed from the pots and separated in different plant tissues (leaf, stem and root). Then, the different plant tissues were separately immersed in a 2% H2 O2 solution for 2 min and washed twice with sterile de-ionized water for 2 min before plating onto Sabouraud Dextrose Chloramphenicol Agar (SDCA; Biocult, Madrid, Spain) to assess re-isolation of B. bassiana: 8–10 portions of 0.5 cm2 from leaves (young leaves), 6 pieces of 0.5-cm long pieces from roots and 8 pieces 0.5-cm long of stems per plant. The Petri dishes were incubated at 25 ◦ C in the dark for approximately 2 weeks and examined for the presence of B. bassiana. The earliest Fe chlorosis symptoms appeared at the end of the second week in both tomato and wheat plants. Thereafter, leaf chlorophyll concentrations were weekly estimated in the last three youngest leaves by using a SPAD 502 Portable Chlorophyll Meter (Minolta Camera Co., Osaka, Japan). SPAD readings were validated by using 48 younger, completely expanded leaves with a variable degree of Fe chlorosis from each crop. To this end, the leaflet (tomato) or a 1-cm2 piece of the central part of younger leaves (wheat) was weighed, its surface area calculated, SPAD measured and chlorophyll extracted in 96% ethanol for measurement according to Wintermans and de Mots (1965). SPAD values and leaf chlorophyll concentrations per unit surface were highly correlated in both tomato (r = 0.71, P < 0.001) and wheat (r = 0.75, P < 0.001). Plant height was measured weekly since 20 DAS in both crops. At harvest [viz., 5 plants per treatment 48 DAS for tomato because the artificial substrate was unable to supply enough moisture and 4 plants per treatment 150 DAS for wheat], aerial biomass and root were weighed, root length and diameter estimated (only for tomato) with the software WinRhizo Pro 2004 (Regent Instruments, Inc., Quebec, Canada), and wheat spikes and grains counted and weighed. Mineral element in aerial biomass of tomato and in grain of wheat were determined after drying at 70 ◦ C for at least 72 h and digestion in a nitric–perchloric acid mixture (Zasoski and Burau 1977), using atomic absorption spectrophotometry for Ca, Mg, Fe, Mn, Zn and Cu; flame emission spectrometry for K; and the Molybdenum Blue Method for P (Murphy and Riley 1962). Nutrients in biomass were not analysed in the case of bread wheat because it was completely dried at the end of the crop. 2.4. Experimental design and statistical analysis A completely randomized design with two treatments (seed dressing and control), six FOCS rates (0 or positive control, 20, 40, 80, 120 and 240 mg kg−1 ) and seven replicates per treatment, was used for each crop. The experimental unit was a pot with a plant. The median test (2 , Siegel and Castellan, 1998) was used to compare SPAD (leaf chlorophyll content), plant height, dry weight, root development in tomato, grain production in wheat, and nutrient uptake by tomato plants and stored in wheat grains of control vs seed dressing plants. This non-parametric test was used as an alternative to the analysis of variance because the homogeneity of variance was not satisfied for the fungal application. The results were subjected to the two sample Student’s T-test (T) to compare the effect of fungal application at each FOCS rate and to identify differences between the effects of applying Fe in the Hoagland solution (positive control or plants grown on the substrate inducing no Fe chlorosis, 0 g kg−1 in calcium carbonate sand) and that of adding Fe

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as FOCS in the sand mixture (plants grown on the substrate with 20, 40, 80, 120 or 240 g kg−1 FOCS). These statistical analyses were performed using STATISTIX 9.0 software (Analytical Software, Tallahassee, FL, USA). Besides that, regression analyses were done by using SigmaPlot 10.0 (Systat Software Inc., Chicago, IL, USA). Reisolation of B. bassiana from different vegetal tissues was calculated as 100 × number of plant pieces from which the fungus was isolated in relation to the total number of pieces (Landa et al., 2013; Tefera and Vidal, 2009). 3. Results 3.1. Colonization of plant tissues B. bassiana was found in no tissue from the control or noninoculated plants. Table 1 shows the proportion of re-isolated fungus from leaf, stem and root in tomato and wheat plants inoculated before sowing (i.e., seed-dressed plants) in different samplings. As a rule, re-isolation was higher in wheat than in tomato-stem tissue excepted. Re-isolation decreased considerably with time in tomato leaf (10.3–0.0%), wheat leaf (43.3–20.0%) and wheat stem (20.0–10.3%). However, re-isolation in tomato roots could only be determined in the first sampling owing to the emergence of other, faster-growing fungi of the genera Aspergillus and Penicillium. In addition, re-isolation in wheat roots changed erratically between samplings: from 75.0% in the first to 41.3, 33.3 and 58.3% in the next three. 3.2. Leaf chlorophyll content (SPAD) Fig. 1 shows the mean SPAD values of 4 measurements from 20 to 48 DAS (a) for tomato and from 21 to 45 DAS (b) for wheat grown on different substrates and treated with two fungus rates. After that, no effect of B. bassiana on SPAD values of wheat plants was observed. As a rule, SPAD for plants increased with increasing FOCS until 48 DAS for tomato and 45 DAS for wheat crops. Linear and logarithmic correlations between estimated leaf chlorophyll concentrations (SPAD in tomato and wheat plants) and FOCS were found in both crops: Fig. 1a, lineal (r = 0.95, P = 0.016) and logarithmic (r = 0.98, P = 0.004) correlations for control and seed dressing plants, respectively; Fig. 1b, lineal correlation for seed dressing plants (r = 0.86, P = 0.046). These results testifies to the antichlorotic effect of Fe oxides present in calcareous substrates on especially sensitive plants. The substrate not inducing Fe chlorosis (viz., that containing no FOCS or calcium carbonate sand) was not included in the curve fitting, because it served as a positive control being supplemented with Fe as was explained in Section 2. The mean SPAD values 20-48 DAS for the tomato grown on the positive control (0 g kg−1 in FOCS and calcium carbonate sand) were significantly higher than those for tomato grown on the substrates containing 20–240 g kg−1 FOCS (T = −2.19, P = 0.030), but it did not occur for wheat plants in the period 21–45 DAS (T = −1.68, P = 0.099). Therefore, the amount of Fe added sufficed to prevent Fe chlorosis in tomato plants grown on 0 g kg−1 FOCS substrates (Fig.1a) but it only increased the SPAD values of wheat plants in comparison with plants grown on the lowest containing FOCS substrates (Fig. 1b). A general positive effect of the fungal application on SPAD was detected in tomato and wheat plants in the 21–48/21–45 DAS period (2 = 4.0, P = 0.045; 2 = 8.2, P = 0.004, respectively). Furthermore, the seed dressing application significantly increased SPAD in tomato plants grown on on the 240 g kg−1 FOCS substrate in the period 20–48 DAS (T = 17.5, P = 0.014; Fig. 1a). A similar positive effect was observed in inoculated wheat plants grown on the 0, 20 and 80 g kg−1 FOCS substrate in the 21–45 DAS period

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Table 1 Re-isolation of Beauveria bassiana (mean percentage ± standard error) in plant tissues from inoculated (seed-dressed) plants as a function of crop and time (days after sowing, DAS). Four inoculated and four non-inoculated (control) plants per sampling were used. B. bassiana was absent from the control plants. Growth of other fungi precluded re-isolation of B. bassiana. Vegetal tissue

Leaf Stem Root

Tomato

Wheat

15 DAS

30 DAS

48 DAS

10 DAS

22 DAS

50 DAS

90 DAS

10.3 ± 4.2

8.3 ± 3.0 12.0 ± 3.0 ×

0.0 ± 0.0 25.0 ± 5.0 ×

43.3 ± 12.0

25.0 ± 5.0

75.0 ± 14.4

41.3 ± 13.6

20.0 ± 11.5 20.0 ± 11.5 33.3 ± 17.6

20.0 ± 12.5 10.3 ± 5.2 58.3 ± 30.0

8.3 ± 8.3

×: Growth of other fungi precluded re-isolation of B. bassiana.

Fig. 1. Mean SPAD of the period 20-48 for tomato and 21-45 DAS for wheat plants grown on different substrates and treated with different fungal rates. FOCS: ferrihydritecoated sand. Plants grown on the 0 g kg−1 FOCS substrate were watered with 5–10 mL of full Hoagland solution containing 20 ␮M Fe on a weekly basis. The other plants were weekly watered with the same amount of Hoagland solution containing no Fe.

(T = 4.1, P = 0.089; T = 31.4, P < 0.001; and T = 4.1, P = 0.090, respectively; Fig. 1b). These positive effects due to seed inoculation with B. bassiana vanished in new wheat leaves growing after 45 DAS.

3.3. Plant growth All plants were found to grow vigorously. FOCS content had no effect on plant height, aerial biomass or root weight wheat except for a linear correlation with plant height in the control tomato plants (r = 0.98, P = 0.003; Fig. 2a) and an inverse first-order correlation with root weight in the control wheat plants (r = 0.97, P = 0.006; Fig. 2f). As can be seen from Fig. 2, B. bassiana had no systematic effect on these growth-related parameters. However, plant height and root weight were smaller in seed-dressed wheat plants grown on the substrate containing no FOCS (positive control) than in the control plants. Table 2 shows the root length and diameter of tomato plants at harvest (48 DAS) as a function of FOCS content and fungal application. The FOCS content of the substrate affected root length and diameter differently. Thus, fine roots in control tomato plants increased logarithmically with increasing FOCS content (r = 0.99, P < 0.001). A similar correlation was found in seed-dressed tomato plants (r = 0.98, P = 0.004), and so was a logarithmic increase in root length (r = 0.95, P = 0.012) with increasing FOCS content. On the other hand, FOCS content and root diameter in seed-dressed tomato plants exhibited a negative linear correlation (r = −0.96, P < 0.011). Plants grown on the substrate containing no FOCS, which were supplied with Fe via the Hoagland solution, had significantly longer roots than those grown on the Fe chlorosis-inducing substrates (Table 2). A non-consistent effect on fungal application was observed in root parameters.

Table 3 shows the production-related parameters and yield at harvest of wheat plants grown on the different substrates and treated with B. bassiana or subjected to no fungal application. As can be seen, FOCS content and fungal application had no significant effect on spike weight, number of spikes per plant, grain weight or number of grains in wheat plants. However, an increase in spikes per plant was observed in the comparison between the positive control (0 g kg−1 FOCS) and the rest of the substrates (Table 3). 3.4. Mineral nutrient uptake No nutritional deficiencies other than that of Fe in young leaves were observed. The concentrations of mineral nutrients (not shown) were above the critical values established by Jones and Wolf (1991) and Reuter et al. (1997). Calcium, Mg and Mn in tomato biomass and wheat grains were not correlated with FOCS content or fungal application. Fig. 3 shows the concentration of Fe, P, K and Zn in tomato biomass (Fig. 3a, c, e and g) and wheat grains (Fig. 3b, d, f and h) as function of FOCS content and fungal application. Iron and P uptake by tomato plants or stored in wheat grains depended on the FOCS content of the substrate. As can be seen from Fig. 3a, Fe uptake by tomato control plants and FOCS content were linearly correlated (r = 0.98, P = 0.004). In addition, Fe uptake by control tomato plants was linearly correlated with SPAD 48 DAS (r = 0.72, P < 0.001), and Fe in grains of seed-dressed wheat was logarithmically correlated with FOCS (r = 0.97, P = 0.006; Fig. 3b). Phosphorus uptake was logarithmically correlated with FOCS content in control (r = 0.98, P = 0.002) and seed-dressed tomato plants (r = 0.94, P = 0.006; Fig. 3c). Uptake of both nutrients by tomato plants grown on the substrate containing no FOCS was significantly greater than in the other plants although this was not the case with grains of wheat plants, however (Fig. 3a–d). The application of B.

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Fig. 2. Plant height, aerial biomass and root weight at the end of the experiment (48 DAS in tomato and 150 DAS in wheat) in tomato and wheat plants grown on different calcareous substrates and treated with different fungal rates. FOCS: ferrihydrite-coated sand. Plants grown on the 0 g kg−1 FOCS substrate were watered with 5–10 mL of full Hoagland solution containing 20 ␮M Fe on a weekly basis. The other plants were weekly watered with the same amount of Hoagland solution containing no Fe.

bassiana had no significant effect on Fe or P uptake by tomato or stored in wheat grains. Potassium and zinc uptake by tomato plants or stored in wheat grains were not correlated with FOCS content (Fig. 3e–h); by exception, K stored in grain of wheat decreased linearly with increasing FOCS content (r = 0.90, P = 0.038). Potassium uptake by tomato plants was increased by fungal application in relation to control plants (2 = 14.4, P < 0.001; Fig 3e). A similar trend was observed in Zn uptake by tomato plants but the differences were not significant (2 = 0.9, P = 0.342; Fig 3g). No significant differences were observed for K or Zn in wheat grain. Uptake of the two nutrients was linearly correlated in tomato plants (r = 0.32, P = 0.041). 4. Discussion The length of tomato (48 DAS) and wheat (150 DAS) crops was different because the first one has a high water demand and the

artificial substrate was not able to supply it. Wheat plants showed a great homogeneity and were harvested at the end of ripening phenological stage according to Zadoks et al. (1994). B. bassiana was re-isolated from all tomato and wheat plant tissues (leaf, root and stem; Table 1), which indicates that the fungus succeeded in colonizing the plants and establishing itself as an endophyte in different vegetal tissues. This ability of B. bassiana was previously identified in a number of crops (Akello et al., 2009; Gurulingappa et al., 2010; Ownley et al., 2008; Posada et al., 2007; Quesada-Moraga et al., 2014; Tefera and Vidal, 2009; Vega et al., 2009). The percentages of re-isolation values obtained in this work are lower than others previously reported by Gurulingappa et al. (2010) for tomato (Lycopersicum esculentum Mill. cv. Grosse Lisse; 40–60% in leaves) and wheat (Triticum aestivum L. cv. Morocco; 55–100% in leaves), whether foliarly inoculated or in contact with inoculated soil (ca. 73% in wheat leaves and 47% in stems). Only wheat roots led to greater re-isolation than the crops studied by

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Fig. 3. Total Fe, P, K and Zn at the end of the experiment (48 DAS in tomato and 150 DAS in wheat) in aerial biomass of tomato and wheat (grain) grown on different substrates and treated with different fungal rates. FOCS: ferrihydrite-coated sand. Plants grown on the 0 g kg−1 FOCS substrate were watered with 5–10 mL of full Hoagland solution containing 20 ␮M Fe on a weekly basis. The other plants were weekly watered with the same amount of Hoagland solution containing no Fe.

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Table 2 Root length and diameter (mean ± standard error) at the end of the experiment (48 DAS) in tomato plants grown on artificial substrate as a function of the content in ferrihydrite-coated sand (FOCS) and treatment in control (untreated) plants and plants previously seed-dressed with Beauveria bassiana. Five plants per FOCS rate and treatment were used. FOCSa (g kg−1 ) 0×

20

40

80

120

240

Comparisons: 0 vs FOCS Control vs Seed dress.

Root length

Diameter

Root length (cm) per diameter (mm)

Control Seed dressing T, p Control Seed dressing T, p Control Seed dressing T, p Control Seed dressing T, p Control Seed dressing T, p Control Seed dressing T, p

(cm) 1697 ± 141 1689 ± 156 0.03, 0.979 525 ± 60 522 ± 68 0.03, 0.980 607 ± 26 734 ± 39 −2.03, 0.089 1162 ± 18 961 ± 58 2.39, 0.075 986 ± 114 1104 ± 68 −0.83, 0.426 1115 ± 45 1062 ± 85 0.49, 0.642

(mm) 0.41 ± 0.02 0.42 ± 0.01 −0.54, 0.625 0.58 ± 0.02 0.51 ± 0.02 1.94, 0.110 0.44 ± 0.05 0.51 ± 0.01 −1.23, 0.286 0.49 ± 0.01 0.46 ± 0.02 0.99, 0.380 0.41 ± 0.02 0.41 ± 0.02 −0.01, 0.989 0.42 ± 0.03 0.37 ± 0.02 1.09, 0.325

<0.2 743 ± 35 714 ± 39 0.41, 0.710 107 ± 8 147 ± 29 −1.00, 0.363 200 ± 31 189 ± 7 0.27, 0.803 351 ± 28 322 ± 34 0.53, 0.626 431 ± 42 432 ± 17 −0.03, 0.974 507 ± 78 574 ± 82 −0.49, 0.645

0.2–0.8 875 ± 92 887 ± 107 −0.06, 0.770 361 ± 45 333 ± 44 0.36, 0.731 372 ± 36 497 ± 29 −2.01, 0.092 737 ± 41 587 ± 43 2.08, 0.106 511 ± 79 622 ± 72 −0.94, 0.370 557 ± 46 449 ± 27 1.57, 0.176

>0.8 79 ± 14 88 ± 14 −0.35, 0.752 58 ± 7 41 ± 5 1.66, 0.158 36 ± 10 48 ± 5 −0.92, 0.410 74 ± 5 52 ± 6 2.39, 0.076 45 ± 6 50 ± 6 −0.47, 0.653 52 ± 5 40 ± 3 1.55, 0.181

T’, p’ X2 , p”

5.94, <0.001 1.53, 0.217

−1.07, 0.293 0.00, 0.999

4.92, <0.001 0.76, 0.537

4.64, <0.001 0.38, 0.536

4.51, <0.001 3.44, 0.064

T, Student’s statistic and p, p values of the Student’s T-test in the comparison of control vs seed dressing treatment at each FOCS rate. T’, Student’s statistic and p’, p values of the Student’s T-test in the comparison of 0 FOCS (positive control) vs 20, 40, 80, 120 and 240 FOCS rate (g kg−1 ). X2 , chi-square statistic for median comparison, Control vs Seed dressing, and p , p values for the median test control vs seed dressing treatment at each FOCS rate. ×: Fe (20 ␮M) was added in 5-10 mL of Hoagland solution per pot on a weekly basis. The other plants were watered weekly with the same amount of Hoagland solution containing no Fe. a FOCS: ferrihydrite-coated sand. Table 3 Yield parameters (mean ± standard error) at the end of the experiment (150 DAS) in wheat plants grown on artificial substrate as a function of the content in ferrihydrite-coated sand (FOCS) and Beauveria bassiana treatment in control (untreated) and seed-dressed plants. Four plants per FOCS rate and treatment were used. FOCSa −1

(g kg 0×

Treatment

Spike weight

Spikes plant−1

Grain weight

Grain number

Weight grain−1

Control Seed dressing T, p Control Seed dressing T, p Control Seed dressing T, p Control Seed dressing T, p Control Seed dressing T, p Control Seed dressing T, p

(g) 2.1 ± 0.1 2.2 ± 0.3 −0.48, 0.651 1.9 ± 0.2 2.1 ± 0.3 −0.46, 0.663 2.3 ± 0.3 2.2 ± 0.1 0.07, 0.949 1.5 ± 0.3 2.3 ± 0.3 −1.83, 0.118 2.0 ± 0.4 1.5 ± 0.2 1.13, 0.301 2.2 ± 0.3 1.6 ± 0.2 1.97, 0.107

3.8 ± 0.5 2.8 ± 0.5 1.48, 0.190 2.3 ± 0.3 2.0 ± 0.0 0.14, 0.850 2.0 ± 0.4 2.2 ± 0.1 −1.00, 0.356 1.8 ± 0.5 2.5 ± 0.3 −0.93, 0.387 2.3 ± 0.5 1.5 ± 0.3 1.34, 0.228 2.0 ± 0.0 2.0 ± 0.4 0.07, 0.950

(g) 1.3 ± 0.2 1.2 ± 0.1 0.29, 0.785 1.3 ± 0.2 1.2 ± 0.1 0.14, 0.893 1.5 ± 0.1 1.4 ± 0.2 0.42, 0.659 1.0 ± 0.2 1.3 ± 0.1 −1.13, 0.302 1.3 ± 0.2 1.1 ± 0.1 1.23, 0.263 1.4 ± 0.2 1.1 ± 0.1 1.42, 0.206

51.2 ± 3.8 44.8 ± 4.9 1.06, 0.332 46.3 ± 8.4 47.8 ± 6.0 −0.14, 0.890 50.0 ± 2.8 49.5 ± 6.2 0.07, 0.944 38.5 ± 6.4 46.8 ± 4.2 −1.08, 0.322 43.3 ± 6.0 32.3 ± 3.5 1.16, 0.292 49.8 ± 5.8 38.0 ± 3.1 1.78, 0.125

(mg) 25.3 ± 2.7 28.3 ± 2.9 −0.77, 0.469 27.6 ± 1.2 25.8 ± 1.5 0.96, 0.374 28.9 ± 1.5 27.6 ± 1.1 0.69, 0.516 27.0 ± 2.1 27.5 ± 1.8 −0.28, 0.790 30.6 ± 1.1 30.8 ± 2.3 −0.09, 0.934 28.6 ± 1.5 29.7 ± 0.4 −0.74, 0.488

T’, p’ X2 , p”

1.02, 0.315 1.33, 0.248

3.87, <0.001 0.08, 0.999

0.14, 0.887 3.00, 0.083

0.85, 0.402 1.39, 0.238

−1.14, 0.260 0.33, 0.564

)

20

40

80

120

240

Comparisons: 0 vs FOCS Control vs Seed dress.

T, Student’s statistic and p, p values of the Student’s T-test in the comparison of control vs seed dressing treatment at each FOCS rate. T’, Student’s statistic and p’, p values of the Student’s T-test in the comparison of 0 FOCS (positive control) vs 20, 40, 80, 120 and 240 FOCS rate (g kg−1 ). X2 , chi-square statistic for median comparison, Control vs Seed dressing, and p , p values for the median test control vs seed dressing treatment at each FOCS rate. ×: Fe (20 ␮M) was added in 5–10 mL of Hoagland solution per pot on a weekly basis. The other plants were watered weekly with the same amount of Hoagland solution containing no Fe. a FOCS: ferrihydrite-coated sand.

Gurulingappa et al. (2010) (ca. 31% 21 days after soil inoculation). This was the likely result of the longer duration of our experiment (48 DAS for tomato and 150 DAS for wheat) in comparison with Gurulingappa et al. (2010) (only 21 days). Tefera and Vidal (2009) obtained B. bassiana re-isolation values similar to ours for wheat in inoculated sorghum seeds grown on sterile soil for 20 days. Besides length of the experiment, the use of different cultivars of tomato

and wheat in our experiment and other inoculation methods could affect the percentages of fungal re-isolation. Some authors found that the B. bassiana effect on plant is influenced by nutrient bio-availability and fertilization (Diniz et al., 2009 Gatarayiha et al., 2010), but very little seems to have been published on the effects of B. bassiana on leaf chlorophyll concentrations or mineral nutrient uptake by inoculated plants. SPAD and Fe uptake

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by plants were correlated with FOCS content (Figs. 1 and 3). The positive correlation between SPAD and Fe uptake by tomato contradicts previous results of Romheld (2000) in grapevine. This author described the “Fe chlorosis paradox” as a phenomenon whereby Fe accumulates in plants affected by Fe chlorosis. Our correlation, however, is consistent with the results of Sánchez-Rodríguez et al. (2013, 2014) in peanut, chickpea, lupin, and sorghum grown on artificial calcareous substrates and on calcareous soils. It should be noted that the Fe source in our Fe chlorosis-inducing substrates (20–240 g kg−1 FOCS) was ferrihydrite, which is a non-crystalline Fe oxide and the main source of Fe for plants growing on calcareous soils (Loeppert and Hallmark, 1985). Although an increase in Fe uptake was not detected for tomato plants or wheat grains (Fig. 3), B. bassiana improved Fe nutrition (SPAD values) from the third week to approximately 50 DAS in both crops (Fig. 1). The effect, however, was uneven after 45 days of growth in wheat plants. de Santiago et al. (2009) found Trichoderma asperellum strain T34, a widely used biocontrol agent, to increase Fe uptake by lupin grown on an artificial calcareous substrate containing 50 g kg−1 FOCS, but also to reduce chlorophyll synthesis and SPAD through increased activity in Fe-containing enzymes. However, in our experiment, the positive effect of B. bassiana on leaf chlorophyll content was evident for tomato grown on the highest FOCS content in the substrate and more apparent at low Fe contents (FOCS) in substrate for wheat plants (Figure 1). The fungus could increase the availability of Fe for our plants in these conditions. Monocot plants (wheat) have a strategy to uptake Fe based on the excretion of phytosiderophores, more efficient than the strategy of dicot plants (tomato), which consist on the release of protons that are neutralized by carbonate in calcareous conditions (Marschner and Römheld, 1994). It could explain the different fungal effect on both crops and the lower increase in SPAD for wheat plants grown on 0 g kg − − −1− − −1− − −1 FOCS substrate in comparison with tomate plants. In addition, de Santiago et al. (2013) found an increase of Fe in aerial biomass of cucumber plants after soil application of T. asperellum that was more evident when the availability of nutrients was not so restricted, that agrees with our results for SPAD in tomato plants. The positive effect of FOCS on plant growth (specifically, on the height and root of control tomato plants, and root length of seeddressed tomato plants, Figure 2 and Table 2) can be ascribed to an increase in Fe bioavailability when increasing FOCS content and alleviating limiting factors in calcareous substrates. In fact, increasing the FOCS content of the substrate decreased root length in tomato control plants and root diameter in seed-dressed plants, probably because these plants can readily absorb Fe without the need to explore the soil. No negative effect of the fungal application was observed on plant growth (Figure 2, Table 2) and wheat production (Table 3). This is consistent with the results of previous experiments in which B. bassiana was found to colonize the host plant without damage (Gurulingappa et al., 2010; Tefera and Vidal, 2009). García-López et al. (2013) found a decrease in dry matter-and Fe uptake-of cucumber plants inoculated with T. asperellum strain T34 and grown on artificial calcareous substrates containing less than 75 mg kg−1 Fe as ferrihydrite. This was a result of competition between plants and the fungus to absorb Fe. Endophytic fungi are known to require inputs from their host plants for both colonization and development (Meyling and Eilenberg, 2007); as a result, fungal inoculation can affect plant growth, but in our experiments it was insignificant. Phosphate has a high affinity for Fe oxide surfaces, where it is readily adsorbed (Torrent, 1987). Increasing the FOCS content of a substrate has the same effect as increasing the surface available for retaining P from the Hoagland solution. Therefore, tomato and

wheat plants absorb increased amounts of P and Fe after releasing organic acids (both) and phytosiderophores (wheat, monocot plant, Marschner and Römheld, 1994) at higher FOCS levels. In our experiment, B. bassiana altered K and Zn (slightly) uptake. This may have resulted from the fungus strengthening the role of the proton-releasing pump in the plasma membrane, acidifying the rhizosphere, and ultimately increasing the solubility of Zn and facilitating its absorption (Venkatraju and Marschner, 1981). The plasmatic pump increases proton release in response to an increased input of a cation such as K+ in order to regulate the membrane potential (Jolley and Brown, 1985). The difference in this respect between the two crops can be ascribed to their different response to Fe deficiency. Thus, dicots (tomato) typically exhibit acidification of the rhizosphere, whereas monocots (wheat) tend to produce phytosiderophores in order to chelate Fe in the rhizosphere and increase its availability to roots (Marschner and Römheld, 1994). Even if our experimental protocol does not allow quantitatively ascertaining the concentration of the fungal DNA in the plant, but the relative percentage of presence in different fragments, it has been reported that the potential benefits of the fungal endophyte to the plant could be “induce” in the early stages of colonization according to Soliman et al. (2013). One current drawback to the use of entomopathogenic fungi endophytes as biocontrol agents comes from the variability observed in the endophytic persistence of the fungi after the inoculation. The inoculation method, the fungal strain used, as well as the host plant genotype are key factors determining the persistence of entomopathogenic fungi endophytes and the compatibility of plant-endophyte associations (Quesada-Moraga et al., 2014). As a result of that, most known fungal endophytes seem to colonize their host plants in a non-systemic pattern (Rodriguez et al., 2009; Sánchez et al., 2012), probably due to a “balanced antagonism”, where the host plant can restrain the growth of the fungus, and the fungus can modulate the effectiveness of plant defence mechanisms (Schulz and Boyle, 2005). In such scenario, the improvement of Fe nutrition observed in our work may be related to the specific signalling pathways induce by the strain on each particular plant species, more than in the persistence and intensity of the endophytic colonisation.

5. Conclusions Using simple substrates is useful with a view to understanding plant–endophyte associations, which are usually complex and involve multiple interactions. B. bassiana strain EABb 04/01-Tip was able to colonize leaf, root and stem of tomato and wheat plants after seed inoculation during the whole experiment. Furthermore, B. bassiana was found to improve Fe nutrition (leaf chlorophyll concentration, for first time) in tomato and wheat plants grown on artificial substrates at early stages (<50 DAS), without affecting plant growth, root development (tomato) or yield (wheat). This positive effect that depends on plant vegetal species and Fe availability in soil increases the value of B. bassiana as a biological control agent in calcareous soils. Alterations in mineral nutrition (K) should be considered in designing these strategies. Innovative research on different plant species, soil types and growing conditions (growth chamber, field) is needed to better clarify how B. bassiana improves plant Fe nutrition and can alter availability of other nutrients. Our group is currently conducting in vitro and in vivo experiments on different entomopathogenic fungi (B. bassiana, Metarhizium brunneum and Isaria farinosa) to study the effect of the application method (foliar application, soil treatment, seed dressing) used to inoculate plants grown on soils with different properties.

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Please cite this article in press as: Sánchez-Rodríguez, A.R., et al., Beauveria bassiana: An entomopathogenic fungus alleviates Fe chlorosis symptoms in plants grown on calcareous substrates. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2015.09.029