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Tomato Yield, Quality, Mineral Composition and Antioxidants as Affected by Beneficial Microorganisms Under Soil Salinity Induced by Balanced Nutrient Solutions

Tomato Yield, Quality, Mineral Composition and Antioxidants as Affected by Beneficial... agriculture Article Tomato Yield, Quality, Mineral Composition and Antioxidants as A ected by Beneficial Microorganisms Under Soil Salinity Induced by Balanced Nutrient Solutions 1 2 1 Vincenzo Michele Sellitto , Nadezhda A. Golubkina , Laura Pietrantonio , 3 3 4 5 Eugenio Cozzolino , Antonio Cuciniello , Vincenzo Cenvinzo , Imbrea Florin and 4 , Gianluca Caruso * Msbiotech S.p.A., 86035 Larino, Campobasso, Italy; michele.sellitto@msbiotechspa.com (V.M.S.); laura.pietrantonio77@gmail.com (L.P.) Federal Scientific Center of Vegetable Production, Odintsovo District, 143072 Moscow, Russia; segolubkina45@gmail.com Council for Agricultural Research and Economics (CREA)—Research Center for Cereal and Industrial Crops, 81100 Caserta, Italy; eugenio.cozzolino@crea.gov.it (E.C.); antonio.cuciniello@crea.gov.it (A.C.) Department of Agricultural Sciences, University of Naples Federico II, 80055 Portici, Naples, Italy; vincenzo.cenvinzo2@unina.it Department of Agricultural Technologies, Banat’s University of Agricultural Sciences and Veterinary Medicine King Michael I of Romania, 300645 Timisoara, Romania; imbreaflorintm@gmail.com * Correspondence: gcaruso@unina.it; Tel.: +39-81-2539104 Received: 26 April 2019; Accepted: 21 May 2019; Published: 23 May 2019 Abstract: With the aim of assessing the e ects of beneficial microorganisms on greenhouse tomato “plum” grown under salinity conditions, research was carried out in southern Italy from summer to winter, by comparing two arbuscular mycorrhizal fungi (AMF) based formulates (Rizotech Plus, Myco Apply DR) and a non-inoculated control, in factorial combination with four soil electrical 1 1 conductivities (1.5, 3.0, 4.5, 6.0 mScm EC). The highest root colonization was 83% at 3.0 mScm under AMF-based treatments and 34% at 1.5 mScm in non-treated control; the latter attained lower values than AMF treatments at any soil EC. Harvest occurred 3.5 days earlier in control 1 1 plants, six days earlier under 6.0 mScm EC compared to 1.5 mScm . The inoculated plants always showed higher yield than the control ones and the highest production at 4.5 mScm EC; control plants attained the highest yield under 3.0–4.5 mScm EC. The highest values of most fruit quality indicators, mineral elements and antioxidant compounds and activity were recorded under AMF-based formulates inoculation and 6.0 mScm soil EC. Beneficial microorganisms proved to be an e ective environmentally friendly tool for improving tomato yield and quality performances in both normal and soil salinity conditions. Keywords: Solanum lycopersicum L. ‘plum’ type; root colonization; organic acids; antioxidant content and activity 1. Introduction Tomato (Solanum lycopersicum L.) is the most spread vegetable species worldwide [1], mainly cultivated in Asia (China, India, Turkey, Iran), Africa (Nigeria, Egypt), United States and Europe (Italy, Spain) and is rich in macronutrients, trace-elements and antioxidants [2]. Crop performances of tomato are a ected by farming management, which should promote ecient plant development as well as valuable fruit production, quality and antioxidant properties. The latter aspects are key goals of Agriculture 2019, 9, 110; doi:10.3390/agriculture9050110 www.mdpi.com/journal/agriculture Agriculture 2019, 9, 110 2 of 15 low environmental impact strategies, within which plant inoculation with beneficial microorganisms is a useful tool for preventing or reducing abiotic and biotic stresses [3]. Arbuscular mycorrhizal fungi (AMF) form symbiotic association with plants, thus changing their nutritional, biochemical and physiological status [4] and showing positive e ects on vegetable crops both in open field and greenhouse [5]. In this respect, AMF play an active role in plant nutrition due to their ability to improve mineral element uptake and plant growth, particularly phosphorus (P) which is an essential but low mobile nutrient [6]. Indeed, they interact with mineral and organic fertilizers, increasing phosphorus availability and improving plant physiological conditions, particularly in high salinity conditions causing osmotic stress and toxicity of some ions [7]. AMF inoculation results in the increase of P uptake, tomato plant growth and biomass under low P availability [8,9] and of P content in all tissues [10,11]. However, high fertilizer doses may reduce the mycorrhizal abundance [12] whose e ectiveness on yield depends on cultivar or ecotype [13,14], soil P concentration [15], mycorrhizal species [16]. According to Walder and van der Heijden [17], the eciency of the mycorrhizal symbiosis depends on the association specificity, reciprocity and multi-functionality, and the lifetime fitness should also be taken into account as the behaviour of symbionts may be antagonistic at an earlier life-stage but mutualistic at a later stage; the AMF-plant symbiosis is also influenced by other factors including the environment and the ability to exploit resources. In particular, most AMF have the ability to enhance plant growth and nutrition, and, in this respect, the beneficial e ects are strongly dependent on the availability of phosphorus and nitrogen in the soil; in phosphorus-limited soils, AM fungi have been shown to be beneficial to plants, whereas in nitrogen-limited soils, the same AM fungi can suppress growth. Indeed, in nature, plant roots are usually colonized simultaneously by AMF and other beneficial microorganisms belonging to di erent species, thus forming highly complex partnerships with intense resource exchange activity. In this respect, the co-inoculation of AMF and plant growth promoting bacteria targets to join the di erent benefits provided by the single microorganisms to plants, leading to yield increase and fruit quality modulation [18]. Though Juniper and Abbott [19] reported the negative e ect of soil salinity excess on the growth of AMF hyphae, contrastingly, in research on tomato, cucumber and lettuce [20,21] as well as on olive, apple and Citrus trees [22–24], the plant performances under salt stress conditions were enhanced by AMF inoculation. Indeed, the latter fungi encourage salt tolerance in the colonized plants by + + increasing water and nutrient uptake, K /Na ratio, osmoregulators synthesis, photosynthesis rate, water use eciency [20]. Under mild salt stress conditions, pot-grown Arundo donax plants showed a reduction of photosynthesis and growth, even in symbiosis with AMF such as Funneliformis mosseae and Rhizophagus irregularis, though the AM fungi elicited some plant metabolic changes consisting of proline and H O increase and higher isoprene emission [25]. Sánchez et al. [26] reported that the 2 2 e ects of salinity and water stress on the degree of leaf stomatal closure and photosynthesis reduction in A. donax is also dependent on the ecotype. Salt stress is a major cause of water stress and in this respect, Chitarra et al. [27] reported that AMF enhance the tomato leaf stomatal density, which is positively correlated with the plant CO absorption capacity, photosynthetic rate and relative water use eciency. In addition, the inoculation with Glomus deserticola, Claroideoglomus etunicatum and Funneliformis mosseae both in dicotyledonous and monocotyledonous plants showed a 24% higher e ect on stomatal conductance to water vapor than non-mycorrhizal (NM) control and a 100% higher e ect during moderate water deficit or over 400% under severe water stress compared to optimally watered conditions [28]. Indeed, the fungi hyphae can capillary explore wider soil volume than the root hair, thus allowing the plants to uptake water even under severe deficit conditions; in addition, the more the root colonization augments the more the stomatal conductance to water vapor increases up to 10 fold compared to lightly colonized plants [28]. Moreover, changes in cell wall composition of both roots and fruits may also be elicited by the inoculation of tomato plants with Funneliformis mosseae [29]. Chitarra et al. [30] showed that AMF symbiosis positively a ects the tolerance to water deficit in tomato, by improving water use eciency and, indeed, AMF-colonized plants better withstood Agriculture 2019, 9, 110 3 of 15 severe water stress conditions (1.3 MPa), as witnessed by the lower ABA content in roots and leaves compared to non-colonized plants. The inoculation with the Pseudomonas chlororaphis also encouraged tomato plant tolerance to mild water stress by enhancing antioxidant activity and proline content and by limiting the accumulation of reactive oxygen species [31]. Moreover, P. chlororaphis increased the ABA level in leaves of water-stressed plants, with a consequent improvement of stomatal closure modulation and water use eciency (WUE) and biomass accumulation. Volpe et al. [32] found that tomato plants inoculated with AMF show di erent patterns of adaptation to environmental stresses depending on the fungus species: Funneliformis mosseae elicited the production of volatile organic compound production and Rhizophagus intraradices resulted in a higher water use eciency under severe water stress. The present research aimed to assess the ability of beneficial microorganisms to valorise tomato crop nutrition by encouraging the plants to tolerate as high a soil electrical conductivity as possible, thus improving fruit production and quality. In this respect, the interaction between AMF-based formulates and di erent soil electrical conductivities induced by fertigation with balanced nutrient solutions was assessed on yield, quality, chemical composition and antioxidants content of tomato “plum” fruits grown in Mediterranean climate. 2. Materials and Methods 2.1. Experimental Protocol and Growing Conditions Research was carried out in 2016-17 and 2017-18 on tomato “plum” (Solanum lycopersicum L., cultivar Pixel F ) grown in greenhouse, at the Experimental Centre of the Department of Agricultural 0  0 Sciences, University Federico II of Naples, Italy (40 49 N, 14 20 E, 63 m above sea level) in the Mediterranean climate. Seedlings were transplanted on 22 August, in both 2016 and 2017, in plastic pots of 24 cm diameter filled with sandy-loam soil and perlite (10% in volume), placed on 10 cm-thick polystyrene sheets, with a density of 4 plants per m . The crops were grown under a three-span polytunnel, each span being 5 m wide, 2 and 3.5 m high at wall and roof respectively. The trend of temperature in the greenhouse is shown in Figure 1 as ten-day mean values from transplant to the end of harvests and as an average of 2016-17 and 2017-18 since the year of research did not significantly a ect the variables examined. Comparisons were made between two arbuscular mycorrhizal fungi (AMF) based formulates (Rizotech Plus, Myco Apply DR) and a non-inoculated control, in factorial combination with four soil electrical conductivities (1.5, 3.0, 4.5, 6.0 mScm EC). A split plot design was used with three replicates and each treatment covered a 4.5 m surface area. Both AMF-based formulates predominantly contain Claroideoglomus etunicatum, Funneliformis mosseae, Glomus aggregatum, Rhizophagus intraradices (10% in Rizotech Plus and 1% in Myco Apply DR) and, in addition, fungi and bacteria species belonging to genera Trichoderma, Streptomyces, Bacillus, 3 1 6 1 Pseudomonas (10 UFCg in Rizotech Plus and 210 UFCg in Myco Apply DR). Notably, Rizotech powder was applied in the soil holes made for seedling transplant, whereas Myco Apply was supplied to the soil as a water solution upon transplanting. The four soil ECs examined (1.5 to 6.0 mScm ) were carried out by supplying nutrient solutions with EC ranging from 1.2 to 4.8 mScm and pH 6.0, by 1 1 using drip irrigation method with 2 Lmin emitters. The ratios between the concentrations (mgL ) of N, P, K, Ca, Mg, S in the nutrient solutions were 1.0:0.4:1.4:1.1:0.4:0.4; the microelement concentration (molL ) was constant in the four nutrient solutions: 35.0 Fe; 1.8 Cu; 24.0 Mn; 11.0 Zn; 82.0 B; 1.0 Mo. R.H. (%) Agriculture 2019, 9, 110 4 of 15 Agriculture 2019, 9, x FOR PEER REVIEW 4 of 15 40 100 0 0 AugSept II III Oct II III Nov II III Dec II III Jan II III Feb III I I I I I I Minimum R.H. Maximum R.H. Maximum T Minimum T Figure 1. Ten-day means of air temperature (T, C) and relative humidity (R.H., %) in greenhouse in Portici Figure 1. (Naples, Ten-da southern y means of ai Italy) r temperature (T, from August to °C) and re February, as lative hu an average midity of ( 2016–2017 R.H., %) in g and re 2017–2018. enhouse in Portici (Naples, southern Italy) from August to February, as an average of 2016–2017 and 2017–2018. 2.2. Determinations of Root Mycorrhizal Colonization, Plant Growth Indices and Yield Components 2.2. Determinations of Root Mycorrhizal Colonization, Plant Growth Indices and Yield Components The mycorrhizal colonization was assessed twice, sixty days after transplant and at the crop end, according to Trouvelot et al. [33] by calculating the frequency of mycorrhization (%). In this respect, The mycorrhizal colonization was assessed twice, sixty days after transplant and at the crop end, 30 randomly chosen 1 cm-long pieces were cut from the root apparatus of 5 plants per treatment and according to Trouvelot et al. [33] by calculating the frequency of mycorrhization (%). In this respect, cleared in 10% KOH for 45 min at 60 C, stained with 1% methyl blue in lactic acid and mounted on 30 randomly chosen 1 cm-long pieces were cut from the root apparatus of 5 plants per treatment and a slide. cleared in 10% KOH for 45 min at 60 °C, stained with 1% methyl blue in lactic acid and mounted on At the end of the crop cycles, the following growth determinations were performed on plants a slide. taken from all plots: the maximum leaf area, using a bench top electronic leaf area meter (Li-Cor 3000, At the end of the crop cycles, the following growth determinations were performed on plants Li-Cor, Lincoln, NE, USA); the aboveground dry biomass in an oven at 70 C until constant weight. taken from all plots: the maximum leaf area, using a bench top electronic leaf area meter (Li-Cor 3000, Fruit harvest began on 25 or 27 October in 2016 and 2017 respectively and ended on 1 or 5 February Li-Cor, Lincoln, NE, USA); the aboveground dry biomass in an oven at 70 °C until constant weight. in 2017 and 2018 respectively; during this time interval, five ripe fruit trusses were picked up from each Fruit harvest began on 25 or 27 October in 2016 and 2017 respectively and ended on 1 or 5 plant and, in coincidence with each harvest, total weight, number and mean weight of marketable February in 2017 and 2018 respectively; during this time interval, five ripe fruit trusses were picked fruits (regular-shaped and undamaged) from 15 plants per treatment were assessed. up from each plant and, in coincidence with each harvest, total weight, number and mean weight of marketable fruits (regular-shaped and undamaged) from 15 plants per treatment were assessed. 2.3. Determinations of Fruit Quality, Mineral Composition and Antioxidant Compounds and Activity 2.3. Determinations Fruit samples of wer Fruit Quality, Mineral e collected at the second Compositio truss n and harvest Antio (6xand idant 9Compounds and November in 2017 Activity and 2018 respectively) and transferred to the laboratory, where determinations of dry residue, soluble solids, Fruit samples were collected at the second truss harvest (6 and 9 November in 2017 and 2018 organic acids (malic, oxalic, citric, isocitric), minerals (K, Ca, Mg, Na, P, S, NO , Cl), lycopene, total respectively) and transferred to the laboratory, where determinations of dry residue, soluble solids, phenols, total ascorbic acid, lipophilic and hydrophilic antioxidant activities were performed by using organic acids (malic, oxalic, citric, isocitric), minerals (K, Ca, Mg, Na, P, S, NO3, Cl), lycopene, total the following procedures: The dry residue was assessed in oven at 70 C until constant weight; phenols, total ascorbic acid, lipophilic and hydrophilic antioxidant activities were performed by the soluble solids ( Brix) at 20 C on the supernatant obtained by centrifuging the raw homogenate, using the following procedures: The dry residue was assessed in oven at 70 °C until constant weight; using a digital refractometer by Bellingham and Stanley, model RFM 81. The organic acids were the soluble solids (°Brix) at 20 °C on the supernatant obtained by centrifuging the raw homogenate, determined as previously described [34]. The mineral elements were assessed according to Rouphael using a digital refractometer by Bellingham and Stanley, model RFM 81. The organic acids were et al. [35]. Lycopene determination was performed referring to De Sio et al. [36]; total phenols and determined as previously described [34]. The mineral elements were assessed according to Rouphael ascorbic acid, as described by Golubkina et al. [37]. The antioxidant activity was assessed according to et al. [35]. Lycopene determination was performed referring to De Sio et al. [36]; total phenols and Brand-Williams et al. [38]. ascorbic acid, as described by Golubkina et al. [37]. The antioxidant activity was assessed according to Brand-Williams et al. [38]. 2.4. Statistical Processing 2.4. Statistical Processing The data statistical processing was performed by the two-way analysis of variance and mean separation through Tukey’s test with reference to 0.05 probability level, using SPSS software version 21. The data statistical processing was performed by the two-way analysis of variance and mean Data expressed as a percentage underwent angular transformation before processing. The variables separation through Tukey’s test with reference to 0.05 probability level, using SPSS software version 21. Data expressed as a percentage underwent angular transformation before processing. The T (°C) Agriculture 2019, 9, x FOR PEER REVIEW 5 of 15 Agriculture 2019, 9, 110 5 of 15 variables examined in our research were not significantly affected by the research year and, therefore, examined in our research were not significantly a ected by the research year and, therefore, only mean only mean data of the two years are reported. data of the two years are reported. 3. Results and Discussion 3. Results and Discussion 3.1. Root Mycorrhizal Colonization 3.1. Root Mycorrhizal Colonization The frequency of root fragments colonized by mycorrhizal hyphae in tomato plants did not The frequency of root fragments colonized by mycorrhizal hyphae in tomato plants did not significantly change between the two determinations performed sixty and eighty-four days after significantly change between the two determinations performed sixty and eighty-four days after transplant and therefore only their mean values are shown in Figure 1. This index was significantly transplant and therefore only their mean values are shown in Figure 1. This index was significantly affected by the interaction between AMF-based formulate and soil EC (Figure 2): the increase of a ected by the interaction between AMF-based formulate and soil EC (Figure 2): the increase of nutrient -1 nutrient availability in the soil from 1.5 to 3.0 mS cm EC enhanced the mycorrhizal root colonization availability in the soil from 1.5 to 3.0 mScm EC enhanced the mycorrhizal root colonization from from 60% to 83% in plants inoculated with AMF; interestingly, the further soil enrichment with 60% to 83% in plants inoculated with AMF; interestingly, the further soil enrichment with balanced -1 balanced nutrient solution up to 6.0 mS cm EC did not inhibit the root colonization by mycorrhizal nutrient solution up to 6.0 mScm EC did not inhibit the root colonization by mycorrhizal hyphae. -1 hyphae. In non-treated plants, soil salinity over 1.5 mS cm EC impaired the symbiotic relationship In non-treated plants, soil salinity over 1.5 mScm EC impaired the symbiotic relationship between -1 between AMF and tomato roots, which was lowest at 6.0 mS cm EC (26%). The percentages of AMF and tomato roots, which was lowest at 6.0 mScm EC (26%). The percentages of colonization colonization recorded in the present research in the roots of tomato plants inoculated with AMF- recorded in the present research in the roots of tomato plants inoculated with AMF-based consortia based consortia fell in the middle of the range including reports from other authors relevant to the fell in the middle of the range including reports from other authors relevant to the same species. same species. Indeed, a 55.7% and 63% root colonization were recorded upon the inoculation with Indeed, a 55.7% and 63% root colonization were recorded upon the inoculation with Funneliformis Funneliformis mosseae and Claroideoglomus etunicatum respectively, in a clay-silty soil in the mosseae and Claroideoglomus etunicatum respectively, in a clay-silty soil in the Mediterranean region of Mediterranean region of Adana, Turkey [39]. The 84.1% and 100% of roots were colonized by Adana, Turkey [39]. The 84.1% and 100% of roots were colonized by mycorrhizal hyphae upon AMF mycorrhizal hyphae upon AMF inoculation in a silty-loam soil along Basilicata coast (southern Italy), inoculation in a silty-loam soil along Basilicata coast (southern Italy), compared to 51% and 92.6% of compared to 51% and 92.6% of non-inoculated control, at mid and end growing season respectively non-inoculated control, at mid and end growing season respectively [40]. Contrary to the conditions of [40]. Contrary to the conditions of soil medium-high phosphorus content set up in our research, soil medium-high phosphorus content set up in our research, Kowalska et al. [9] recorded the 51% of Kowalska et al. [9] recorded the 51 % of root colonization induced by AMF application under low P root colonization induced by AMF application under low P availability in Poland. availability in Poland. a A a A a A b A 40 ab B ab B b B a B 1.5 3.0 4.5 6.0 -1 Soil EC (mS·cm ) Non-inoculated control Rizotech Plus Myco Apply DR Figure 2. Interaction between arbuscular mycorrhizal fungi (AMF)-based formulate and soil electrical Figure 2. Interaction between arbuscular mycorrhizal fungi (AMF)-based formulate and soil electrical conductivity (EC) on tomato root colonization (%). Di erent letters mean significant di erence in the conductivity (EC) on tomato root colonization (%). Different letters mean significant difference in the comparison between soil ECs (lowercase letters) or between AMF-based formulates (capital letters), com accor parison be ding to Tukey’s tween so test il EC at p s (lower  0.05. case letters) or between AMF-based formulates (capital letters), according to Tukey’s test at p ≤ 0.05. 3.2. Plant Growth and Yield 3.2. Plant Growth and Yield The harvest of the first fruit truss began 64 days after transplant in the non-inoculated plants and 3.5 days later in those treated with mycorrhizal-based formulates (Table 1). The harvest of the first fruit truss began 64 days after transplant in the non-inoculated plants Regarding soil EC (Table 1), the earliest fruit ripeness was elicited by the highest EC (6 mScm ), and 3.5 days later in those treated with mycorrhizal-based formulates (Table 1). whereas the most diluted soil solution (1.5 mScm ) caused a six-day delay. −1 Regarding soil EC (Table 1), the earliest fruit ripeness was elicited by the highest EC (6 mS·cm ), −1 whereas the most diluted soil solution (1.5 mS cm ) caused a six-day delay. Root colonization (%) Agriculture 2019, 9, 110 6 of 15 The described physiological behaviour is explainable by the fact that the inoculated plants showed a more enhanced vigour compared to control ones, with higher leaf surface area and total dry matter (0.38 m and 89.7 g per plant vs 0.34 and 58.7) and this resulted in a longer vegetative phase with consequent delay in fruit ripeness. The plants grown in the most diluted soil solution had the smallest leaf expansion and dry matter accumulation (0.33 m and 47.7 g respectively), whereas those subjected 1 2 to 4.5 mScm the highest (0.40 m and 107.2 g respectively). Balestrini et al. [41] reported that, after 3 months of greenhouse rearing, no di erence in grapevine plant growth was recorded between the inoculation with sole Funneliformis mosseae and with an AMF-PGPB consortium containing Trichoderma spp., Pochonia chlamidosporia, Streptomyces spp., Bacillus subtilis, Pseudomonas spp., Funneliformis mosseae, Glomus spp. After 3 months, the roots of the plants inoculated with the mixed inoculums showed a low frequency of AMF colonization (2.6%) compared to the high colonization frequency in the roots inoculated solely with Funneliformis mosseae (80.7%). Moreover, several AM marker genes were upregulated upon the F. mosseae treatment, with the mixed inoculum however eliciting an important transcriptional regulation. The expression of the genes associated to nutrient transport, transcription factors and cell wall was significantly but di erently a ected by both the two treatments. The crop system plays a major role in determining the e ect of AMF on the growth of plants which, in particular, may benefit from mycorrhizal contribution to valorise the nutrient supply [22]. Indeed, the increase of soil EC by enhancing the salt concentration of the balanced nutrient solutions applied through fertigation may improve yield and produce quality up to a salinity threshold depending on both the genotype and the growing management [23]. Among the latter, soil P availability is a key factor [42] and, in this respect, in our research the e ect of AMF application increased with the increase of nutrient uptake including P up to 4.5 mScm , whereas other authors [43] reported that the e ect of AMF is independent on soil P content. The fruit yield was significantly a ected by the interaction between AMF-based formulate and soil EC (Figure 3). Indeed, both the 3.0 and 4.5 mScm soil ECs resulted in the highest production of non-inoculated plants, whereas the 4.5 mScm EC led to the best performances of those treated with beneficial microorganisms, independently on mycorrhizal formulate. However, both in mycorrhized plants and in non-inoculated ones a yield reduction was recorded when the 6.0 mScm EC treatment was applied. Moreover, AMF inoculation was more e ective than non-treated control on yield at any soil EC but there were no significant di erences between the two formulates (Figure 3). Table 1. Tomato precocity, growth indices and yield components as a ected by mycorrhizal-based formulate and soil electrical conductivity. Leaf Dry Precocity Marketable Fruits Area Matter Days from g Yield Mean Experimental Treatment Transplant to Number per Plant per Plant (g per Plant) Weight (g) First Harvest Mycorrhizal-based formulate Rizotech 69.5 a 0.37 a 58.7 b 520.8 a 21.6 a 24.0 a Myco Apply 69.5 a 0.38 a 88.8 a 536.2 a 22.0 a 24.3 a Non-inoculated control 66.0 b 0.34 b 90.5 a 395.8 b 18.2 b 21.7 b Soil electrical conductivity 1.5 mScm 71.3 a 0.33 c 47.7 c 373.6 c 17.5 c 21.3 c 3.0 mScm 69.7 ab 0.37 ab 79.1 b 509.4 b 21.2 b 24.0 ab 4.5 mScm 67.0 bc 0.40 a 107.2 a 568.0 a 22.8 a 24.7 a 6.0 mScm 65.3 c 0.36 bc 83.3 b 485.9 b 20.8 b 23.2 b Within each column, means followed by di erent letters are significantly di erent according to Tukey’s test at p 0.05. Agriculture 2019, 9, 110 7 of 15 Agriculture 2019, 9, x FOR PEER REVIEW 7 of 15 The fruit yield was significantly enhanced by AMF-based formulate application that indeed The fruit yield was significantly enhanced by AMF-based formulate application that indeed promoted a higher establishment of fruits as well as higher mean weight, compared to non-inoculated promoted a higher establishment of fruits as well as higher mean weight, compared to non- control; the two production components were also significantly a ected by the interaction between inoculated control; the two production components were also significantly affected by the interaction AMF-based formulate and soil EC, showing similar trends as yield (Figure 3). between AMF-based formulate and soil EC, showing similar trends as yield (Figure 3). Yield a A b A a A b A b A b A c A a B a B c A b B c B No. of fruits a A b A a A b A b A b A c A a B a B c A b B c B Mean fruit weight a A b A b A a A b A c A b A a B c A a B b B c B 1.5 3.0 4.5 6.0 -1 Soil EC (mS·cm ) Non-inoculated control Rizotech Plus Myco Apply DR Figure 3. Interaction between AMF-based formulate and soil EC on yield performances of tomato ‘plum’ Pixel F . Di erent letters mean significant di erence in the comparison between soil ECs (lowercase Figure 3. Interaction between AMF-based formulate and soil EC on yield performances of tomato letters) or between AMF-based formulates (capital letters), according to Tukey’s test at p 0.05. ‘plum’ Pixel F1. Different letters mean significant difference in the comparison between soil ECs (lowercase letters) or between AMF-based formulates (capital letters), according to Tukey’s test at p ≤ In the present research, the soil EC values best enhancing fruit number and mean weight resulted 0.05. accordingly in the highest yield. In this respect, the AMF-based formulate application allowed the plants to better tolerate the salinity increase, thus valorising the fertigation up to 4.5 mScm soil EC, g no. per plant g per plant Agriculture 2019, 9, 110 8 of 15 which corresponded to the highest production; whereas the non-inoculated plants su ered from mild salt stress over 3.0 mScm , showing a tendency to yield decrease. However, the production of both the mycorrhized and control plants was depressed at 6 mScm EC, as a consequence of plant adaptation to water stress through vegetative growth reduction [44]. As a confirmation of the importance of genotype and crop system on plant tolerance to salinity, di erent findings compared to our results arose from previous research: a production drop with higher than 2.5 mScm nutrient solution EC, made of either balanced element composition or sodium chloride addition [45]; no negative e ects of nutrient solution EC increase from 4 to 7 dSm [46]. The benefits from the application of mixed microorganisms inocula can be targeted to improve crop performances by enhancing the fertigation as carried out in the present study or to valorise the AMF ability to enhance the plant nutrient use eciency by reducing the fertilization rate. The latter goal was achieved by Bona et al. [18], who recorded higher tomato mean fruit weight upon the co-inoculation of AMF and Pseudomonas fluorescens C7 or Pseudomonas sp. 19 Fv1T along with 30% reduction of the traditional fertilization, compared to the controls with full or 30% reduced fertilization; the latter e ect stemmed from the significant interaction between the arbuscular mycorrhizal fungi and the bacteria applied, as the co-inoculation was more e ective than the inoculation with the sole AMF or with the sole Pseudomonas. However, in another investigation on tomato [40] a mixed AMF-based inoculum did not result in better yield than the sole AMF inoculation, which witnessed the prevailing e ect of arbuscular mycorrhizal fungi on plant response; a 10.8% production increase upon the beneficial microorganism application was recorded in comparison with the non-inoculated control, as a consequence of the higher fruit number. Consistently with our results, in research carried out on lettuce and zucchini [8], the co-inoculation with Rhizophagus intraradices and Trichoderma atroviride led to a yield increase compared to non-inoculated control; indeed, the beneficial microorganisms showed a synergic e ect in enhancing the uptake of both macronutrients (N, P, K, Mg) and micronutrients (Fe, Mn, Zn and B) and better promoted plant growth compared to the inoculation with the sole R. intraradices and T. atroviride. The intensification of activity and biomass of soil microbial community [47] resulted in improving the nutrient absorption eciency, thus increasing the fruit number, weight and yield [48]. In particular, the yield increase promoted by AMF inoculation is connected to the biostimulant action of these fungi on plant uptake and growth [16,49], which is the consequence of eliciting the root auxin production in mycorrhized plants [50]. In this respect, plant growth and yield are dependent on the nutrient availability during the phenological development and, indeed, in previous research [51,52] the e ect of AMF inoculation was emphasized by soil P deficiency. Similarly, in Mediterranean field conditions, Rafique and Ortas [39] recorded the increase of tomato yield by as much as 37.8% and 76.1% upon the inoculation of Funneliformis mosseae and Claroideoglomus etunicatum respectively with no P supply, whereas with the soil application of 100 kgha P the production was only 29.1% and 6.8% higher compared to the non-inoculated control; moreover, a 31.8% increase of plant P uptake was recorded only with no P supply. However, consistently with the production increase which in the present investigation has been connected to the enhanced nutrient supply up to a certain threshold, in previous study [53] the number of flowers and fruits in tomato was encouraged by phosphorus availability, thus leading to yield increase; in this respect, Mahanta et al. [54] found a positive correlation between P and production. 3.3. Fruit Quality, Mineral Composition and Antioxidant Compounds and Activity The fruit quality indicators examined, that is, dry residue, soluble solids and organic acids were significantly a ected by AMF application to rhizosphere. In fact, these parameters attained higher values in the fruits obtained from inoculated plants, as compared to control ones but the two AMF-based formulates did not di er from each other (Table 2). Consistently with our findings, in previous research [18] a higher dry residue was recorded in tomato fruits harvested from plants co-inoculated with AMF and Pseudomonas sp. The increase of Agriculture 2019, 9, 110 9 of 15 soluble solids but not of dry residue was recorded in shallot bulbs upon the application of a mixed beneficial microorganism formulate [55]. Contrastingly to the results of the present investigation, Candido et al. [40] did not record significant di erences between AMF-inoculated and control plants in terms of tomato fruit dry weight and soluble solids. The increase of soil EC from 1.5 to 6.0 mScm enhanced the fruit quality attributes (Table 2). Presumably, the highest availability of nutrients corresponding to the highest salt concentrations led to more enhanced accumulation in tomato fruits [52] and, accordingly, to higher dry residue and soluble solids, the latter being notoriously correlated to sugar content. Similar e ects of salinity on fruit quality properties also arose in previous research [52] and, in particular, Adams and Ho [56] recorded enhancement in sugars as an e ect of applied salinity increase. However, in other investigations fruit quality worsening was detected with over 5 mScm EC [57] or sugar content lowering caused by the fruit respiration enhancement under salt stress [58]. Table 2. Quality indicators of tomato fruits as a ected by mycorrhizal-based formulate and soil electrical conductivity. Organic Acids (gkg d.w.) Dry Soluble Solids Experimental Treatment Residue (%) ( Brix) Malic Oxalic Citric Isocitric Mycorrhizal-based Formulate Rizotech 8.9 a 7.6 a 5.7 a 2.2 a 40.1 a 0.71 a Myco Apply 8.9 a 7.6 a 6.0 a 2.4 a 41.6 a 0.70 a Non-inoculated control 8.5 b 7.3 b 4.6 b 1.5 b 31.5 b 0.49 b Soil electrical Conductivity 1.5 mScm 8.2 c 7.0 d 4.2 d 1.2 d 31.7 d 0.49 c 3.0 mScm 8.5 c 7.3 c 4.8 c 1.7 c 35.3 c 0.55 c 4.5 mScm 9.0 b 7.7 b 5.7 b 2.2 b 39.1 b 0.66 b 6.0 mScm 9.4 a 8.0 a 7.0 a 2.9 a 44.8 a 0.83 a d.w.: dry weight. Within each column, means followed by di erent letters are significantly di erent according to Tukey’s test at p 0.05. As for fruit mineral composition (Table 3), the application of AMF-based formulates resulted in higher content of K, Ca, Mg, P, S and NO compared to the control, whereas Cl was not significantly a ected by the beneficial microorganisms. Interestingly, the fruits produced by the mycorrhized plants showed a lower Na concentration than those obtained from the non-inoculated ones, presumably due to the concurrent higher accumulation of K, Ca and Mg. Zouari et al. [59] found that the nutrient content of tomato fruits produced by mycorrhized plants under low fertilization is similar to that of fruits from non-inoculated plants grown in optimal nutrient conditions, which means that the use of AMF can reduce the negative environmental impact of mineral fertilizers. Moreover, 712 genes were found to be di erentially expressed in fruits from mycorrhized or control plants. In particular, the fruits of mycorrhized plants showed genes characteristic of a climacteric fleshy fruit and genes related to mycorrhizal status, such as phosphate and sulphate transporters. In other research [60], plants inoculated with Glomus intraradices produced fruits with higher content of potassium, calcium, phosphorus and zinc compared to control plants. Consistently with our findings, Ndung’u Magiroi et al. [61] reported that the beneficial microorganism inoculation led to increased fruit content of calcium, potassium and magnesium, the latter soil exchangeable form being positively correlated with bacteria community solubilizing phosphorus. Indeed, the presence of AMF results in the siderophore release in the rhizosphere, which promotes P solubilization and availability for plants, thus leading to higher P concentration in plant tissues [62]. The high level of trehalose in mycorrhized plants could be the reason for high intracellular P concentration, which mobilizes polyphosphates [63]. Thompson et al. [64] also recorded an improved P status in tomato upon the inoculation with Funneliformis mosseae under field conditions and low soil phosphorus. Agriculture 2019, 9, 110 10 of 15 Other authors [65] reported that the composition of soil microbial community can contribute to nutrient accumulation in plants more than the microbial biomass. Table 3. Mineral composition of tomato fruits as a ected by mycorrhizal-based formulate and soil electrical conductivity. Experimental Treatment K Ca Mg Na P S NO Cl gkg d.w. Mycorrhizal-Based Formulate Rizotech 32.4 a 0.66 a 1.31 a 0.60 b 1.06 a 0.60 a 0.17 a 5.46 Myco Apply 32.5 a 0.70 a 1.32 a 0.63 b 1.00 a 0.64 a 0.18 a 5.43 Non-inoculated control 28.8 b 0.49 b 1.06 b 0.69 a 0.64 b 0.36 b 0.09 b 5.42 n.s. Soil Electrical Conductivity 1.5 mScm 29.0 c 0.52 d 1.10 b 0.75 a 0.77 c 0.42 c 0.10 c 5.40 30.4 bc 0.59 c 1.15 b 0.69 b 0.86 b 0.53 bc 0.11 c 5.47 3.0 mScm 4.5 mScm 31.8 ab 0.65 b 1.32 a 0.60 c 0.93 b 0.57 b 0.17 b 5.32 33.7 a 0.72 a 1.35 a 0.52 d 1.04 a 0.62 a 0.21 a 5.57 6.0 mScm n.s. d.w., dry weight. Within each column, n.s. no statistically significant di erence; means followed by di erent letters are significantly di erent according to Tukey’s test at p 0.05. Similar to the quality indicators and mineral elements described above, both the antioxidant compounds and activities examined in tomato fruits were better a ected by the beneficial microorganism inoculation compared to non-treated control, but no significant di erence was recorded between the two AMF-based formulates (Table 4). Consistently with our findings, in previous research the content of lycopene in tomato fruits was enhanced by the inoculation of Glomus intraradices [60], Funneliformis mosseae or Rhizophagus irregularis [10]. A positive e ect of beneficial microorganisms was recorded on ascorbic acid and polyphenols with increasing nitrogen [66], whereas conversely Le Bot et al. [67] reported the polyphenols synthesis limitation caused by nitrogen increase in soil solution. Other authors [68] reported the e ect of Glomus intraradices inoculation in enhancing the phenolic profile of rosemary leaves. Amanifar et al. [69] recorded the increased antioxidant synthesis promoted by Funneliforms mosseae in liquorice under salt conditions, compared to non-inoculated control. Unlike the results of the present research, Nzanza et al. [70] did not detect overall benefits for antioxidant compounds or the activity of tomato fruits upon inoculation with Trichoderma harzianum and Glomus mosseae. Soil salinity also had a significant e ect on the fruit antioxidant status, as the values of all the variables (lycopene, phenols and ascorbic acid as well as hydrophilic and lipophilic antioxidant activities) increased from 1.5 to 6.0 mScm EC. Consistently with our results, in previous investigation [71] similar trends were recorded for lycopene as a response to salinity. Navarro et al. [72] reported that the use of a moderately saline water was beneficial to pepper red fruits by increasing both the hydrophilic and lipophilic antioxidant activities but it did not a ect the concentration of lycopene, ascorbic acid and total phenolics; the two latter antioxidant compounds along with carotenoids and -tocopherol were positively a ected up to 4.1-4.4 mScm EC in green pepper type [73]. Agriculture 2019, 9, 110 11 of 15 Table 4. Antioxidant content and activity in tomato fruits as a ected by mycorrhizal-based formulate and soil electrical conductivity. Lipophilic Hydrophilic Total Phenols Antioxidant Antioxidant Lycopene Ascorbic Acid mg Gallic Activity mmol Activity mmol 1 1 mg100 g f.w. mg100 g f.w. Acid100 g d.w. Trolox Ascorbic Acid 1 1 eq100 g d.w. eq100 g d.w. Mycorrhizal-based formulate Rizotech 338.2 a 1.93 a 18.5 a 10.2 a 8.6 a Myco Apply 350.0 a 2.04 a 20.4 a 11.3 a 9.0 a Non-inoculated control 285.6 b 1.63 b 14.4 b 7.5 b 7.8 b Soil electrical conductivity 1.5 mScm 207.7 d 1.75 b 11.0 d 7.5 d 7.4 c 3.0 mScm 297.3 c 1.80 b 15.3 c 9.1 c 8.2 b 4.5 mScm 360.6 b 1.95 a 20.6 b 10.0 b 8.8 ab 6.0 mScm 432.9 a 1.97 a 24.2 a 11.9 a 9.4 a f.w., fresh weight; d.w., dry weight. Within each column, means followed by di erent letters are significantly di erent according to Tukey’s test at p 0.05. 4. Conclusions From research carried out in southern Italy, enhancement of tomato fruit yield, quality, mineral composition and antioxidant status arose upon the application of arbuscular mycorrhizal fungi (AMF) 1 1 based formulates to plants grown in saline soils (1.5 mScm to 6.0 mScm ). Taking into account both the higher consumer expectations for healthy products and the current policies oriented to environmentally friendly crop systems, the use of beneficial microorganisms represents an e ective and eco-compatible farming technique aiming to reduce chemical inputs, even more under salt stress conditions. Author Contributions: G.C. conceived the research idea and experimental protocol, coordinated the research and wrote the manuscript; V.M.S., N.A.G., L.P. and I.F. critically commented on the manuscript; E.C., A.C. and V.C. were involved in crop management and performed the greenhouse determinations; N.A.G., L.P. and E.C. performed the laboratory analyses. 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E ects of cultural cycle and nutrient solution electrical conductivity on plant growth, yield and fruit quality of “Friariello” pepper grown in hydroponics. Hortic. Sci. 2017, 44, 91–98. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Agriculture Multidisciplinary Digital Publishing Institute

Tomato Yield, Quality, Mineral Composition and Antioxidants as Affected by Beneficial Microorganisms Under Soil Salinity Induced by Balanced Nutrient Solutions

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agriculture Article Tomato Yield, Quality, Mineral Composition and Antioxidants as A ected by Beneficial Microorganisms Under Soil Salinity Induced by Balanced Nutrient Solutions 1 2 1 Vincenzo Michele Sellitto , Nadezhda A. Golubkina , Laura Pietrantonio , 3 3 4 5 Eugenio Cozzolino , Antonio Cuciniello , Vincenzo Cenvinzo , Imbrea Florin and 4 , Gianluca Caruso * Msbiotech S.p.A., 86035 Larino, Campobasso, Italy; michele.sellitto@msbiotechspa.com (V.M.S.); laura.pietrantonio77@gmail.com (L.P.) Federal Scientific Center of Vegetable Production, Odintsovo District, 143072 Moscow, Russia; segolubkina45@gmail.com Council for Agricultural Research and Economics (CREA)—Research Center for Cereal and Industrial Crops, 81100 Caserta, Italy; eugenio.cozzolino@crea.gov.it (E.C.); antonio.cuciniello@crea.gov.it (A.C.) Department of Agricultural Sciences, University of Naples Federico II, 80055 Portici, Naples, Italy; vincenzo.cenvinzo2@unina.it Department of Agricultural Technologies, Banat’s University of Agricultural Sciences and Veterinary Medicine King Michael I of Romania, 300645 Timisoara, Romania; imbreaflorintm@gmail.com * Correspondence: gcaruso@unina.it; Tel.: +39-81-2539104 Received: 26 April 2019; Accepted: 21 May 2019; Published: 23 May 2019 Abstract: With the aim of assessing the e ects of beneficial microorganisms on greenhouse tomato “plum” grown under salinity conditions, research was carried out in southern Italy from summer to winter, by comparing two arbuscular mycorrhizal fungi (AMF) based formulates (Rizotech Plus, Myco Apply DR) and a non-inoculated control, in factorial combination with four soil electrical 1 1 conductivities (1.5, 3.0, 4.5, 6.0 mScm EC). The highest root colonization was 83% at 3.0 mScm under AMF-based treatments and 34% at 1.5 mScm in non-treated control; the latter attained lower values than AMF treatments at any soil EC. Harvest occurred 3.5 days earlier in control 1 1 plants, six days earlier under 6.0 mScm EC compared to 1.5 mScm . The inoculated plants always showed higher yield than the control ones and the highest production at 4.5 mScm EC; control plants attained the highest yield under 3.0–4.5 mScm EC. The highest values of most fruit quality indicators, mineral elements and antioxidant compounds and activity were recorded under AMF-based formulates inoculation and 6.0 mScm soil EC. Beneficial microorganisms proved to be an e ective environmentally friendly tool for improving tomato yield and quality performances in both normal and soil salinity conditions. Keywords: Solanum lycopersicum L. ‘plum’ type; root colonization; organic acids; antioxidant content and activity 1. Introduction Tomato (Solanum lycopersicum L.) is the most spread vegetable species worldwide [1], mainly cultivated in Asia (China, India, Turkey, Iran), Africa (Nigeria, Egypt), United States and Europe (Italy, Spain) and is rich in macronutrients, trace-elements and antioxidants [2]. Crop performances of tomato are a ected by farming management, which should promote ecient plant development as well as valuable fruit production, quality and antioxidant properties. The latter aspects are key goals of Agriculture 2019, 9, 110; doi:10.3390/agriculture9050110 www.mdpi.com/journal/agriculture Agriculture 2019, 9, 110 2 of 15 low environmental impact strategies, within which plant inoculation with beneficial microorganisms is a useful tool for preventing or reducing abiotic and biotic stresses [3]. Arbuscular mycorrhizal fungi (AMF) form symbiotic association with plants, thus changing their nutritional, biochemical and physiological status [4] and showing positive e ects on vegetable crops both in open field and greenhouse [5]. In this respect, AMF play an active role in plant nutrition due to their ability to improve mineral element uptake and plant growth, particularly phosphorus (P) which is an essential but low mobile nutrient [6]. Indeed, they interact with mineral and organic fertilizers, increasing phosphorus availability and improving plant physiological conditions, particularly in high salinity conditions causing osmotic stress and toxicity of some ions [7]. AMF inoculation results in the increase of P uptake, tomato plant growth and biomass under low P availability [8,9] and of P content in all tissues [10,11]. However, high fertilizer doses may reduce the mycorrhizal abundance [12] whose e ectiveness on yield depends on cultivar or ecotype [13,14], soil P concentration [15], mycorrhizal species [16]. According to Walder and van der Heijden [17], the eciency of the mycorrhizal symbiosis depends on the association specificity, reciprocity and multi-functionality, and the lifetime fitness should also be taken into account as the behaviour of symbionts may be antagonistic at an earlier life-stage but mutualistic at a later stage; the AMF-plant symbiosis is also influenced by other factors including the environment and the ability to exploit resources. In particular, most AMF have the ability to enhance plant growth and nutrition, and, in this respect, the beneficial e ects are strongly dependent on the availability of phosphorus and nitrogen in the soil; in phosphorus-limited soils, AM fungi have been shown to be beneficial to plants, whereas in nitrogen-limited soils, the same AM fungi can suppress growth. Indeed, in nature, plant roots are usually colonized simultaneously by AMF and other beneficial microorganisms belonging to di erent species, thus forming highly complex partnerships with intense resource exchange activity. In this respect, the co-inoculation of AMF and plant growth promoting bacteria targets to join the di erent benefits provided by the single microorganisms to plants, leading to yield increase and fruit quality modulation [18]. Though Juniper and Abbott [19] reported the negative e ect of soil salinity excess on the growth of AMF hyphae, contrastingly, in research on tomato, cucumber and lettuce [20,21] as well as on olive, apple and Citrus trees [22–24], the plant performances under salt stress conditions were enhanced by AMF inoculation. Indeed, the latter fungi encourage salt tolerance in the colonized plants by + + increasing water and nutrient uptake, K /Na ratio, osmoregulators synthesis, photosynthesis rate, water use eciency [20]. Under mild salt stress conditions, pot-grown Arundo donax plants showed a reduction of photosynthesis and growth, even in symbiosis with AMF such as Funneliformis mosseae and Rhizophagus irregularis, though the AM fungi elicited some plant metabolic changes consisting of proline and H O increase and higher isoprene emission [25]. Sánchez et al. [26] reported that the 2 2 e ects of salinity and water stress on the degree of leaf stomatal closure and photosynthesis reduction in A. donax is also dependent on the ecotype. Salt stress is a major cause of water stress and in this respect, Chitarra et al. [27] reported that AMF enhance the tomato leaf stomatal density, which is positively correlated with the plant CO absorption capacity, photosynthetic rate and relative water use eciency. In addition, the inoculation with Glomus deserticola, Claroideoglomus etunicatum and Funneliformis mosseae both in dicotyledonous and monocotyledonous plants showed a 24% higher e ect on stomatal conductance to water vapor than non-mycorrhizal (NM) control and a 100% higher e ect during moderate water deficit or over 400% under severe water stress compared to optimally watered conditions [28]. Indeed, the fungi hyphae can capillary explore wider soil volume than the root hair, thus allowing the plants to uptake water even under severe deficit conditions; in addition, the more the root colonization augments the more the stomatal conductance to water vapor increases up to 10 fold compared to lightly colonized plants [28]. Moreover, changes in cell wall composition of both roots and fruits may also be elicited by the inoculation of tomato plants with Funneliformis mosseae [29]. Chitarra et al. [30] showed that AMF symbiosis positively a ects the tolerance to water deficit in tomato, by improving water use eciency and, indeed, AMF-colonized plants better withstood Agriculture 2019, 9, 110 3 of 15 severe water stress conditions (1.3 MPa), as witnessed by the lower ABA content in roots and leaves compared to non-colonized plants. The inoculation with the Pseudomonas chlororaphis also encouraged tomato plant tolerance to mild water stress by enhancing antioxidant activity and proline content and by limiting the accumulation of reactive oxygen species [31]. Moreover, P. chlororaphis increased the ABA level in leaves of water-stressed plants, with a consequent improvement of stomatal closure modulation and water use eciency (WUE) and biomass accumulation. Volpe et al. [32] found that tomato plants inoculated with AMF show di erent patterns of adaptation to environmental stresses depending on the fungus species: Funneliformis mosseae elicited the production of volatile organic compound production and Rhizophagus intraradices resulted in a higher water use eciency under severe water stress. The present research aimed to assess the ability of beneficial microorganisms to valorise tomato crop nutrition by encouraging the plants to tolerate as high a soil electrical conductivity as possible, thus improving fruit production and quality. In this respect, the interaction between AMF-based formulates and di erent soil electrical conductivities induced by fertigation with balanced nutrient solutions was assessed on yield, quality, chemical composition and antioxidants content of tomato “plum” fruits grown in Mediterranean climate. 2. Materials and Methods 2.1. Experimental Protocol and Growing Conditions Research was carried out in 2016-17 and 2017-18 on tomato “plum” (Solanum lycopersicum L., cultivar Pixel F ) grown in greenhouse, at the Experimental Centre of the Department of Agricultural 0  0 Sciences, University Federico II of Naples, Italy (40 49 N, 14 20 E, 63 m above sea level) in the Mediterranean climate. Seedlings were transplanted on 22 August, in both 2016 and 2017, in plastic pots of 24 cm diameter filled with sandy-loam soil and perlite (10% in volume), placed on 10 cm-thick polystyrene sheets, with a density of 4 plants per m . The crops were grown under a three-span polytunnel, each span being 5 m wide, 2 and 3.5 m high at wall and roof respectively. The trend of temperature in the greenhouse is shown in Figure 1 as ten-day mean values from transplant to the end of harvests and as an average of 2016-17 and 2017-18 since the year of research did not significantly a ect the variables examined. Comparisons were made between two arbuscular mycorrhizal fungi (AMF) based formulates (Rizotech Plus, Myco Apply DR) and a non-inoculated control, in factorial combination with four soil electrical conductivities (1.5, 3.0, 4.5, 6.0 mScm EC). A split plot design was used with three replicates and each treatment covered a 4.5 m surface area. Both AMF-based formulates predominantly contain Claroideoglomus etunicatum, Funneliformis mosseae, Glomus aggregatum, Rhizophagus intraradices (10% in Rizotech Plus and 1% in Myco Apply DR) and, in addition, fungi and bacteria species belonging to genera Trichoderma, Streptomyces, Bacillus, 3 1 6 1 Pseudomonas (10 UFCg in Rizotech Plus and 210 UFCg in Myco Apply DR). Notably, Rizotech powder was applied in the soil holes made for seedling transplant, whereas Myco Apply was supplied to the soil as a water solution upon transplanting. The four soil ECs examined (1.5 to 6.0 mScm ) were carried out by supplying nutrient solutions with EC ranging from 1.2 to 4.8 mScm and pH 6.0, by 1 1 using drip irrigation method with 2 Lmin emitters. The ratios between the concentrations (mgL ) of N, P, K, Ca, Mg, S in the nutrient solutions were 1.0:0.4:1.4:1.1:0.4:0.4; the microelement concentration (molL ) was constant in the four nutrient solutions: 35.0 Fe; 1.8 Cu; 24.0 Mn; 11.0 Zn; 82.0 B; 1.0 Mo. R.H. (%) Agriculture 2019, 9, 110 4 of 15 Agriculture 2019, 9, x FOR PEER REVIEW 4 of 15 40 100 0 0 AugSept II III Oct II III Nov II III Dec II III Jan II III Feb III I I I I I I Minimum R.H. Maximum R.H. Maximum T Minimum T Figure 1. Ten-day means of air temperature (T, C) and relative humidity (R.H., %) in greenhouse in Portici Figure 1. (Naples, Ten-da southern y means of ai Italy) r temperature (T, from August to °C) and re February, as lative hu an average midity of ( 2016–2017 R.H., %) in g and re 2017–2018. enhouse in Portici (Naples, southern Italy) from August to February, as an average of 2016–2017 and 2017–2018. 2.2. Determinations of Root Mycorrhizal Colonization, Plant Growth Indices and Yield Components 2.2. Determinations of Root Mycorrhizal Colonization, Plant Growth Indices and Yield Components The mycorrhizal colonization was assessed twice, sixty days after transplant and at the crop end, according to Trouvelot et al. [33] by calculating the frequency of mycorrhization (%). In this respect, The mycorrhizal colonization was assessed twice, sixty days after transplant and at the crop end, 30 randomly chosen 1 cm-long pieces were cut from the root apparatus of 5 plants per treatment and according to Trouvelot et al. [33] by calculating the frequency of mycorrhization (%). In this respect, cleared in 10% KOH for 45 min at 60 C, stained with 1% methyl blue in lactic acid and mounted on 30 randomly chosen 1 cm-long pieces were cut from the root apparatus of 5 plants per treatment and a slide. cleared in 10% KOH for 45 min at 60 °C, stained with 1% methyl blue in lactic acid and mounted on At the end of the crop cycles, the following growth determinations were performed on plants a slide. taken from all plots: the maximum leaf area, using a bench top electronic leaf area meter (Li-Cor 3000, At the end of the crop cycles, the following growth determinations were performed on plants Li-Cor, Lincoln, NE, USA); the aboveground dry biomass in an oven at 70 C until constant weight. taken from all plots: the maximum leaf area, using a bench top electronic leaf area meter (Li-Cor 3000, Fruit harvest began on 25 or 27 October in 2016 and 2017 respectively and ended on 1 or 5 February Li-Cor, Lincoln, NE, USA); the aboveground dry biomass in an oven at 70 °C until constant weight. in 2017 and 2018 respectively; during this time interval, five ripe fruit trusses were picked up from each Fruit harvest began on 25 or 27 October in 2016 and 2017 respectively and ended on 1 or 5 plant and, in coincidence with each harvest, total weight, number and mean weight of marketable February in 2017 and 2018 respectively; during this time interval, five ripe fruit trusses were picked fruits (regular-shaped and undamaged) from 15 plants per treatment were assessed. up from each plant and, in coincidence with each harvest, total weight, number and mean weight of marketable fruits (regular-shaped and undamaged) from 15 plants per treatment were assessed. 2.3. Determinations of Fruit Quality, Mineral Composition and Antioxidant Compounds and Activity 2.3. Determinations Fruit samples of wer Fruit Quality, Mineral e collected at the second Compositio truss n and harvest Antio (6xand idant 9Compounds and November in 2017 Activity and 2018 respectively) and transferred to the laboratory, where determinations of dry residue, soluble solids, Fruit samples were collected at the second truss harvest (6 and 9 November in 2017 and 2018 organic acids (malic, oxalic, citric, isocitric), minerals (K, Ca, Mg, Na, P, S, NO , Cl), lycopene, total respectively) and transferred to the laboratory, where determinations of dry residue, soluble solids, phenols, total ascorbic acid, lipophilic and hydrophilic antioxidant activities were performed by using organic acids (malic, oxalic, citric, isocitric), minerals (K, Ca, Mg, Na, P, S, NO3, Cl), lycopene, total the following procedures: The dry residue was assessed in oven at 70 C until constant weight; phenols, total ascorbic acid, lipophilic and hydrophilic antioxidant activities were performed by the soluble solids ( Brix) at 20 C on the supernatant obtained by centrifuging the raw homogenate, using the following procedures: The dry residue was assessed in oven at 70 °C until constant weight; using a digital refractometer by Bellingham and Stanley, model RFM 81. The organic acids were the soluble solids (°Brix) at 20 °C on the supernatant obtained by centrifuging the raw homogenate, determined as previously described [34]. The mineral elements were assessed according to Rouphael using a digital refractometer by Bellingham and Stanley, model RFM 81. The organic acids were et al. [35]. Lycopene determination was performed referring to De Sio et al. [36]; total phenols and determined as previously described [34]. The mineral elements were assessed according to Rouphael ascorbic acid, as described by Golubkina et al. [37]. The antioxidant activity was assessed according to et al. [35]. Lycopene determination was performed referring to De Sio et al. [36]; total phenols and Brand-Williams et al. [38]. ascorbic acid, as described by Golubkina et al. [37]. The antioxidant activity was assessed according to Brand-Williams et al. [38]. 2.4. Statistical Processing 2.4. Statistical Processing The data statistical processing was performed by the two-way analysis of variance and mean separation through Tukey’s test with reference to 0.05 probability level, using SPSS software version 21. The data statistical processing was performed by the two-way analysis of variance and mean Data expressed as a percentage underwent angular transformation before processing. The variables separation through Tukey’s test with reference to 0.05 probability level, using SPSS software version 21. Data expressed as a percentage underwent angular transformation before processing. The T (°C) Agriculture 2019, 9, x FOR PEER REVIEW 5 of 15 Agriculture 2019, 9, 110 5 of 15 variables examined in our research were not significantly affected by the research year and, therefore, examined in our research were not significantly a ected by the research year and, therefore, only mean only mean data of the two years are reported. data of the two years are reported. 3. Results and Discussion 3. Results and Discussion 3.1. Root Mycorrhizal Colonization 3.1. Root Mycorrhizal Colonization The frequency of root fragments colonized by mycorrhizal hyphae in tomato plants did not The frequency of root fragments colonized by mycorrhizal hyphae in tomato plants did not significantly change between the two determinations performed sixty and eighty-four days after significantly change between the two determinations performed sixty and eighty-four days after transplant and therefore only their mean values are shown in Figure 1. This index was significantly transplant and therefore only their mean values are shown in Figure 1. This index was significantly affected by the interaction between AMF-based formulate and soil EC (Figure 2): the increase of a ected by the interaction between AMF-based formulate and soil EC (Figure 2): the increase of nutrient -1 nutrient availability in the soil from 1.5 to 3.0 mS cm EC enhanced the mycorrhizal root colonization availability in the soil from 1.5 to 3.0 mScm EC enhanced the mycorrhizal root colonization from from 60% to 83% in plants inoculated with AMF; interestingly, the further soil enrichment with 60% to 83% in plants inoculated with AMF; interestingly, the further soil enrichment with balanced -1 balanced nutrient solution up to 6.0 mS cm EC did not inhibit the root colonization by mycorrhizal nutrient solution up to 6.0 mScm EC did not inhibit the root colonization by mycorrhizal hyphae. -1 hyphae. In non-treated plants, soil salinity over 1.5 mS cm EC impaired the symbiotic relationship In non-treated plants, soil salinity over 1.5 mScm EC impaired the symbiotic relationship between -1 between AMF and tomato roots, which was lowest at 6.0 mS cm EC (26%). The percentages of AMF and tomato roots, which was lowest at 6.0 mScm EC (26%). The percentages of colonization colonization recorded in the present research in the roots of tomato plants inoculated with AMF- recorded in the present research in the roots of tomato plants inoculated with AMF-based consortia based consortia fell in the middle of the range including reports from other authors relevant to the fell in the middle of the range including reports from other authors relevant to the same species. same species. Indeed, a 55.7% and 63% root colonization were recorded upon the inoculation with Indeed, a 55.7% and 63% root colonization were recorded upon the inoculation with Funneliformis Funneliformis mosseae and Claroideoglomus etunicatum respectively, in a clay-silty soil in the mosseae and Claroideoglomus etunicatum respectively, in a clay-silty soil in the Mediterranean region of Mediterranean region of Adana, Turkey [39]. The 84.1% and 100% of roots were colonized by Adana, Turkey [39]. The 84.1% and 100% of roots were colonized by mycorrhizal hyphae upon AMF mycorrhizal hyphae upon AMF inoculation in a silty-loam soil along Basilicata coast (southern Italy), inoculation in a silty-loam soil along Basilicata coast (southern Italy), compared to 51% and 92.6% of compared to 51% and 92.6% of non-inoculated control, at mid and end growing season respectively non-inoculated control, at mid and end growing season respectively [40]. Contrary to the conditions of [40]. Contrary to the conditions of soil medium-high phosphorus content set up in our research, soil medium-high phosphorus content set up in our research, Kowalska et al. [9] recorded the 51% of Kowalska et al. [9] recorded the 51 % of root colonization induced by AMF application under low P root colonization induced by AMF application under low P availability in Poland. availability in Poland. a A a A a A b A 40 ab B ab B b B a B 1.5 3.0 4.5 6.0 -1 Soil EC (mS·cm ) Non-inoculated control Rizotech Plus Myco Apply DR Figure 2. Interaction between arbuscular mycorrhizal fungi (AMF)-based formulate and soil electrical Figure 2. Interaction between arbuscular mycorrhizal fungi (AMF)-based formulate and soil electrical conductivity (EC) on tomato root colonization (%). Di erent letters mean significant di erence in the conductivity (EC) on tomato root colonization (%). Different letters mean significant difference in the comparison between soil ECs (lowercase letters) or between AMF-based formulates (capital letters), com accor parison be ding to Tukey’s tween so test il EC at p s (lower  0.05. case letters) or between AMF-based formulates (capital letters), according to Tukey’s test at p ≤ 0.05. 3.2. Plant Growth and Yield 3.2. Plant Growth and Yield The harvest of the first fruit truss began 64 days after transplant in the non-inoculated plants and 3.5 days later in those treated with mycorrhizal-based formulates (Table 1). The harvest of the first fruit truss began 64 days after transplant in the non-inoculated plants Regarding soil EC (Table 1), the earliest fruit ripeness was elicited by the highest EC (6 mScm ), and 3.5 days later in those treated with mycorrhizal-based formulates (Table 1). whereas the most diluted soil solution (1.5 mScm ) caused a six-day delay. −1 Regarding soil EC (Table 1), the earliest fruit ripeness was elicited by the highest EC (6 mS·cm ), −1 whereas the most diluted soil solution (1.5 mS cm ) caused a six-day delay. Root colonization (%) Agriculture 2019, 9, 110 6 of 15 The described physiological behaviour is explainable by the fact that the inoculated plants showed a more enhanced vigour compared to control ones, with higher leaf surface area and total dry matter (0.38 m and 89.7 g per plant vs 0.34 and 58.7) and this resulted in a longer vegetative phase with consequent delay in fruit ripeness. The plants grown in the most diluted soil solution had the smallest leaf expansion and dry matter accumulation (0.33 m and 47.7 g respectively), whereas those subjected 1 2 to 4.5 mScm the highest (0.40 m and 107.2 g respectively). Balestrini et al. [41] reported that, after 3 months of greenhouse rearing, no di erence in grapevine plant growth was recorded between the inoculation with sole Funneliformis mosseae and with an AMF-PGPB consortium containing Trichoderma spp., Pochonia chlamidosporia, Streptomyces spp., Bacillus subtilis, Pseudomonas spp., Funneliformis mosseae, Glomus spp. After 3 months, the roots of the plants inoculated with the mixed inoculums showed a low frequency of AMF colonization (2.6%) compared to the high colonization frequency in the roots inoculated solely with Funneliformis mosseae (80.7%). Moreover, several AM marker genes were upregulated upon the F. mosseae treatment, with the mixed inoculum however eliciting an important transcriptional regulation. The expression of the genes associated to nutrient transport, transcription factors and cell wall was significantly but di erently a ected by both the two treatments. The crop system plays a major role in determining the e ect of AMF on the growth of plants which, in particular, may benefit from mycorrhizal contribution to valorise the nutrient supply [22]. Indeed, the increase of soil EC by enhancing the salt concentration of the balanced nutrient solutions applied through fertigation may improve yield and produce quality up to a salinity threshold depending on both the genotype and the growing management [23]. Among the latter, soil P availability is a key factor [42] and, in this respect, in our research the e ect of AMF application increased with the increase of nutrient uptake including P up to 4.5 mScm , whereas other authors [43] reported that the e ect of AMF is independent on soil P content. The fruit yield was significantly a ected by the interaction between AMF-based formulate and soil EC (Figure 3). Indeed, both the 3.0 and 4.5 mScm soil ECs resulted in the highest production of non-inoculated plants, whereas the 4.5 mScm EC led to the best performances of those treated with beneficial microorganisms, independently on mycorrhizal formulate. However, both in mycorrhized plants and in non-inoculated ones a yield reduction was recorded when the 6.0 mScm EC treatment was applied. Moreover, AMF inoculation was more e ective than non-treated control on yield at any soil EC but there were no significant di erences between the two formulates (Figure 3). Table 1. Tomato precocity, growth indices and yield components as a ected by mycorrhizal-based formulate and soil electrical conductivity. Leaf Dry Precocity Marketable Fruits Area Matter Days from g Yield Mean Experimental Treatment Transplant to Number per Plant per Plant (g per Plant) Weight (g) First Harvest Mycorrhizal-based formulate Rizotech 69.5 a 0.37 a 58.7 b 520.8 a 21.6 a 24.0 a Myco Apply 69.5 a 0.38 a 88.8 a 536.2 a 22.0 a 24.3 a Non-inoculated control 66.0 b 0.34 b 90.5 a 395.8 b 18.2 b 21.7 b Soil electrical conductivity 1.5 mScm 71.3 a 0.33 c 47.7 c 373.6 c 17.5 c 21.3 c 3.0 mScm 69.7 ab 0.37 ab 79.1 b 509.4 b 21.2 b 24.0 ab 4.5 mScm 67.0 bc 0.40 a 107.2 a 568.0 a 22.8 a 24.7 a 6.0 mScm 65.3 c 0.36 bc 83.3 b 485.9 b 20.8 b 23.2 b Within each column, means followed by di erent letters are significantly di erent according to Tukey’s test at p 0.05. Agriculture 2019, 9, 110 7 of 15 Agriculture 2019, 9, x FOR PEER REVIEW 7 of 15 The fruit yield was significantly enhanced by AMF-based formulate application that indeed The fruit yield was significantly enhanced by AMF-based formulate application that indeed promoted a higher establishment of fruits as well as higher mean weight, compared to non-inoculated promoted a higher establishment of fruits as well as higher mean weight, compared to non- control; the two production components were also significantly a ected by the interaction between inoculated control; the two production components were also significantly affected by the interaction AMF-based formulate and soil EC, showing similar trends as yield (Figure 3). between AMF-based formulate and soil EC, showing similar trends as yield (Figure 3). Yield a A b A a A b A b A b A c A a B a B c A b B c B No. of fruits a A b A a A b A b A b A c A a B a B c A b B c B Mean fruit weight a A b A b A a A b A c A b A a B c A a B b B c B 1.5 3.0 4.5 6.0 -1 Soil EC (mS·cm ) Non-inoculated control Rizotech Plus Myco Apply DR Figure 3. Interaction between AMF-based formulate and soil EC on yield performances of tomato ‘plum’ Pixel F . Di erent letters mean significant di erence in the comparison between soil ECs (lowercase Figure 3. Interaction between AMF-based formulate and soil EC on yield performances of tomato letters) or between AMF-based formulates (capital letters), according to Tukey’s test at p 0.05. ‘plum’ Pixel F1. Different letters mean significant difference in the comparison between soil ECs (lowercase letters) or between AMF-based formulates (capital letters), according to Tukey’s test at p ≤ In the present research, the soil EC values best enhancing fruit number and mean weight resulted 0.05. accordingly in the highest yield. In this respect, the AMF-based formulate application allowed the plants to better tolerate the salinity increase, thus valorising the fertigation up to 4.5 mScm soil EC, g no. per plant g per plant Agriculture 2019, 9, 110 8 of 15 which corresponded to the highest production; whereas the non-inoculated plants su ered from mild salt stress over 3.0 mScm , showing a tendency to yield decrease. However, the production of both the mycorrhized and control plants was depressed at 6 mScm EC, as a consequence of plant adaptation to water stress through vegetative growth reduction [44]. As a confirmation of the importance of genotype and crop system on plant tolerance to salinity, di erent findings compared to our results arose from previous research: a production drop with higher than 2.5 mScm nutrient solution EC, made of either balanced element composition or sodium chloride addition [45]; no negative e ects of nutrient solution EC increase from 4 to 7 dSm [46]. The benefits from the application of mixed microorganisms inocula can be targeted to improve crop performances by enhancing the fertigation as carried out in the present study or to valorise the AMF ability to enhance the plant nutrient use eciency by reducing the fertilization rate. The latter goal was achieved by Bona et al. [18], who recorded higher tomato mean fruit weight upon the co-inoculation of AMF and Pseudomonas fluorescens C7 or Pseudomonas sp. 19 Fv1T along with 30% reduction of the traditional fertilization, compared to the controls with full or 30% reduced fertilization; the latter e ect stemmed from the significant interaction between the arbuscular mycorrhizal fungi and the bacteria applied, as the co-inoculation was more e ective than the inoculation with the sole AMF or with the sole Pseudomonas. However, in another investigation on tomato [40] a mixed AMF-based inoculum did not result in better yield than the sole AMF inoculation, which witnessed the prevailing e ect of arbuscular mycorrhizal fungi on plant response; a 10.8% production increase upon the beneficial microorganism application was recorded in comparison with the non-inoculated control, as a consequence of the higher fruit number. Consistently with our results, in research carried out on lettuce and zucchini [8], the co-inoculation with Rhizophagus intraradices and Trichoderma atroviride led to a yield increase compared to non-inoculated control; indeed, the beneficial microorganisms showed a synergic e ect in enhancing the uptake of both macronutrients (N, P, K, Mg) and micronutrients (Fe, Mn, Zn and B) and better promoted plant growth compared to the inoculation with the sole R. intraradices and T. atroviride. The intensification of activity and biomass of soil microbial community [47] resulted in improving the nutrient absorption eciency, thus increasing the fruit number, weight and yield [48]. In particular, the yield increase promoted by AMF inoculation is connected to the biostimulant action of these fungi on plant uptake and growth [16,49], which is the consequence of eliciting the root auxin production in mycorrhized plants [50]. In this respect, plant growth and yield are dependent on the nutrient availability during the phenological development and, indeed, in previous research [51,52] the e ect of AMF inoculation was emphasized by soil P deficiency. Similarly, in Mediterranean field conditions, Rafique and Ortas [39] recorded the increase of tomato yield by as much as 37.8% and 76.1% upon the inoculation of Funneliformis mosseae and Claroideoglomus etunicatum respectively with no P supply, whereas with the soil application of 100 kgha P the production was only 29.1% and 6.8% higher compared to the non-inoculated control; moreover, a 31.8% increase of plant P uptake was recorded only with no P supply. However, consistently with the production increase which in the present investigation has been connected to the enhanced nutrient supply up to a certain threshold, in previous study [53] the number of flowers and fruits in tomato was encouraged by phosphorus availability, thus leading to yield increase; in this respect, Mahanta et al. [54] found a positive correlation between P and production. 3.3. Fruit Quality, Mineral Composition and Antioxidant Compounds and Activity The fruit quality indicators examined, that is, dry residue, soluble solids and organic acids were significantly a ected by AMF application to rhizosphere. In fact, these parameters attained higher values in the fruits obtained from inoculated plants, as compared to control ones but the two AMF-based formulates did not di er from each other (Table 2). Consistently with our findings, in previous research [18] a higher dry residue was recorded in tomato fruits harvested from plants co-inoculated with AMF and Pseudomonas sp. The increase of Agriculture 2019, 9, 110 9 of 15 soluble solids but not of dry residue was recorded in shallot bulbs upon the application of a mixed beneficial microorganism formulate [55]. Contrastingly to the results of the present investigation, Candido et al. [40] did not record significant di erences between AMF-inoculated and control plants in terms of tomato fruit dry weight and soluble solids. The increase of soil EC from 1.5 to 6.0 mScm enhanced the fruit quality attributes (Table 2). Presumably, the highest availability of nutrients corresponding to the highest salt concentrations led to more enhanced accumulation in tomato fruits [52] and, accordingly, to higher dry residue and soluble solids, the latter being notoriously correlated to sugar content. Similar e ects of salinity on fruit quality properties also arose in previous research [52] and, in particular, Adams and Ho [56] recorded enhancement in sugars as an e ect of applied salinity increase. However, in other investigations fruit quality worsening was detected with over 5 mScm EC [57] or sugar content lowering caused by the fruit respiration enhancement under salt stress [58]. Table 2. Quality indicators of tomato fruits as a ected by mycorrhizal-based formulate and soil electrical conductivity. Organic Acids (gkg d.w.) Dry Soluble Solids Experimental Treatment Residue (%) ( Brix) Malic Oxalic Citric Isocitric Mycorrhizal-based Formulate Rizotech 8.9 a 7.6 a 5.7 a 2.2 a 40.1 a 0.71 a Myco Apply 8.9 a 7.6 a 6.0 a 2.4 a 41.6 a 0.70 a Non-inoculated control 8.5 b 7.3 b 4.6 b 1.5 b 31.5 b 0.49 b Soil electrical Conductivity 1.5 mScm 8.2 c 7.0 d 4.2 d 1.2 d 31.7 d 0.49 c 3.0 mScm 8.5 c 7.3 c 4.8 c 1.7 c 35.3 c 0.55 c 4.5 mScm 9.0 b 7.7 b 5.7 b 2.2 b 39.1 b 0.66 b 6.0 mScm 9.4 a 8.0 a 7.0 a 2.9 a 44.8 a 0.83 a d.w.: dry weight. Within each column, means followed by di erent letters are significantly di erent according to Tukey’s test at p 0.05. As for fruit mineral composition (Table 3), the application of AMF-based formulates resulted in higher content of K, Ca, Mg, P, S and NO compared to the control, whereas Cl was not significantly a ected by the beneficial microorganisms. Interestingly, the fruits produced by the mycorrhized plants showed a lower Na concentration than those obtained from the non-inoculated ones, presumably due to the concurrent higher accumulation of K, Ca and Mg. Zouari et al. [59] found that the nutrient content of tomato fruits produced by mycorrhized plants under low fertilization is similar to that of fruits from non-inoculated plants grown in optimal nutrient conditions, which means that the use of AMF can reduce the negative environmental impact of mineral fertilizers. Moreover, 712 genes were found to be di erentially expressed in fruits from mycorrhized or control plants. In particular, the fruits of mycorrhized plants showed genes characteristic of a climacteric fleshy fruit and genes related to mycorrhizal status, such as phosphate and sulphate transporters. In other research [60], plants inoculated with Glomus intraradices produced fruits with higher content of potassium, calcium, phosphorus and zinc compared to control plants. Consistently with our findings, Ndung’u Magiroi et al. [61] reported that the beneficial microorganism inoculation led to increased fruit content of calcium, potassium and magnesium, the latter soil exchangeable form being positively correlated with bacteria community solubilizing phosphorus. Indeed, the presence of AMF results in the siderophore release in the rhizosphere, which promotes P solubilization and availability for plants, thus leading to higher P concentration in plant tissues [62]. The high level of trehalose in mycorrhized plants could be the reason for high intracellular P concentration, which mobilizes polyphosphates [63]. Thompson et al. [64] also recorded an improved P status in tomato upon the inoculation with Funneliformis mosseae under field conditions and low soil phosphorus. Agriculture 2019, 9, 110 10 of 15 Other authors [65] reported that the composition of soil microbial community can contribute to nutrient accumulation in plants more than the microbial biomass. Table 3. Mineral composition of tomato fruits as a ected by mycorrhizal-based formulate and soil electrical conductivity. Experimental Treatment K Ca Mg Na P S NO Cl gkg d.w. Mycorrhizal-Based Formulate Rizotech 32.4 a 0.66 a 1.31 a 0.60 b 1.06 a 0.60 a 0.17 a 5.46 Myco Apply 32.5 a 0.70 a 1.32 a 0.63 b 1.00 a 0.64 a 0.18 a 5.43 Non-inoculated control 28.8 b 0.49 b 1.06 b 0.69 a 0.64 b 0.36 b 0.09 b 5.42 n.s. Soil Electrical Conductivity 1.5 mScm 29.0 c 0.52 d 1.10 b 0.75 a 0.77 c 0.42 c 0.10 c 5.40 30.4 bc 0.59 c 1.15 b 0.69 b 0.86 b 0.53 bc 0.11 c 5.47 3.0 mScm 4.5 mScm 31.8 ab 0.65 b 1.32 a 0.60 c 0.93 b 0.57 b 0.17 b 5.32 33.7 a 0.72 a 1.35 a 0.52 d 1.04 a 0.62 a 0.21 a 5.57 6.0 mScm n.s. d.w., dry weight. Within each column, n.s. no statistically significant di erence; means followed by di erent letters are significantly di erent according to Tukey’s test at p 0.05. Similar to the quality indicators and mineral elements described above, both the antioxidant compounds and activities examined in tomato fruits were better a ected by the beneficial microorganism inoculation compared to non-treated control, but no significant di erence was recorded between the two AMF-based formulates (Table 4). Consistently with our findings, in previous research the content of lycopene in tomato fruits was enhanced by the inoculation of Glomus intraradices [60], Funneliformis mosseae or Rhizophagus irregularis [10]. A positive e ect of beneficial microorganisms was recorded on ascorbic acid and polyphenols with increasing nitrogen [66], whereas conversely Le Bot et al. [67] reported the polyphenols synthesis limitation caused by nitrogen increase in soil solution. Other authors [68] reported the e ect of Glomus intraradices inoculation in enhancing the phenolic profile of rosemary leaves. Amanifar et al. [69] recorded the increased antioxidant synthesis promoted by Funneliforms mosseae in liquorice under salt conditions, compared to non-inoculated control. Unlike the results of the present research, Nzanza et al. [70] did not detect overall benefits for antioxidant compounds or the activity of tomato fruits upon inoculation with Trichoderma harzianum and Glomus mosseae. Soil salinity also had a significant e ect on the fruit antioxidant status, as the values of all the variables (lycopene, phenols and ascorbic acid as well as hydrophilic and lipophilic antioxidant activities) increased from 1.5 to 6.0 mScm EC. Consistently with our results, in previous investigation [71] similar trends were recorded for lycopene as a response to salinity. Navarro et al. [72] reported that the use of a moderately saline water was beneficial to pepper red fruits by increasing both the hydrophilic and lipophilic antioxidant activities but it did not a ect the concentration of lycopene, ascorbic acid and total phenolics; the two latter antioxidant compounds along with carotenoids and -tocopherol were positively a ected up to 4.1-4.4 mScm EC in green pepper type [73]. Agriculture 2019, 9, 110 11 of 15 Table 4. Antioxidant content and activity in tomato fruits as a ected by mycorrhizal-based formulate and soil electrical conductivity. Lipophilic Hydrophilic Total Phenols Antioxidant Antioxidant Lycopene Ascorbic Acid mg Gallic Activity mmol Activity mmol 1 1 mg100 g f.w. mg100 g f.w. Acid100 g d.w. Trolox Ascorbic Acid 1 1 eq100 g d.w. eq100 g d.w. Mycorrhizal-based formulate Rizotech 338.2 a 1.93 a 18.5 a 10.2 a 8.6 a Myco Apply 350.0 a 2.04 a 20.4 a 11.3 a 9.0 a Non-inoculated control 285.6 b 1.63 b 14.4 b 7.5 b 7.8 b Soil electrical conductivity 1.5 mScm 207.7 d 1.75 b 11.0 d 7.5 d 7.4 c 3.0 mScm 297.3 c 1.80 b 15.3 c 9.1 c 8.2 b 4.5 mScm 360.6 b 1.95 a 20.6 b 10.0 b 8.8 ab 6.0 mScm 432.9 a 1.97 a 24.2 a 11.9 a 9.4 a f.w., fresh weight; d.w., dry weight. Within each column, means followed by di erent letters are significantly di erent according to Tukey’s test at p 0.05. 4. Conclusions From research carried out in southern Italy, enhancement of tomato fruit yield, quality, mineral composition and antioxidant status arose upon the application of arbuscular mycorrhizal fungi (AMF) 1 1 based formulates to plants grown in saline soils (1.5 mScm to 6.0 mScm ). Taking into account both the higher consumer expectations for healthy products and the current policies oriented to environmentally friendly crop systems, the use of beneficial microorganisms represents an e ective and eco-compatible farming technique aiming to reduce chemical inputs, even more under salt stress conditions. Author Contributions: G.C. conceived the research idea and experimental protocol, coordinated the research and wrote the manuscript; V.M.S., N.A.G., L.P. and I.F. critically commented on the manuscript; E.C., A.C. and V.C. were involved in crop management and performed the greenhouse determinations; N.A.G., L.P. and E.C. performed the laboratory analyses. Funding: This research received no external funding. Acknowledgments: The authors wish to thank: Roberto Maiello for his helpful assistance with laboratory analyses; the companies Msbiotech S.p.A. and Sumitomo S-p-A. for providing the two AMF-based formulates tested in the present research. Conflicts of Interest: The authors declare no conflict of interest. References 1. FAOSTAT. 2014. Available online: http://faostat3.fao.org/browse/Q/QC/E (accessed on 26 April 2019). 2. Dorais, M.; Ehret, D.L.; Papadopoulos, A.P. Tomato (Solanum lycopersicum) health components: From the seed to the consumer. Phytochem. Rev. 2008, 7, 231–250. [CrossRef] 3. Stefan, M.; Munteanu, N.; Stoleru, V.; Mihasan, M.; Hritcu, L. Seed inoculation with plant growth promoting rhizobacteria enhances photosynthesis and yield of runner bean (Phaseolus coccineus L.). Sci. Hortic. 2013, 151, 22–29. [CrossRef] 4. Pereira, J.A.P.; Vieira, I.J.C.; Freitas, M.S.M.; Prins, C.L.; Martins, M.A.; Rodrigues, R. 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E ects of cultural cycle and nutrient solution electrical conductivity on plant growth, yield and fruit quality of “Friariello” pepper grown in hydroponics. Hortic. Sci. 2017, 44, 91–98. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Journal

Agriculture – Multidisciplinary Digital Publishing Institute

Published: May 23, 2019

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