Nunzio Fiorentino, Youssef Rouphael, Armando De Rosa, Eugenio Cozzolino, Vincenzo Cenvinzo, Maria Giordano, Laura Gioia, Sheridan Woo, Massimo Fagnano
Dipartimento di Agraria, Università di Napoli, Via Università 100, 80055, Portici *[email protected]
Abstract
N fertilizer excess can cause the accumulation of high levels of nitrate in leafy vegetables, mainly when grown under reduced light levels, exposing consumers to important health risks. Appropriate agronomic practices can limit nitrate accumulation in vegetables and increase their quality, while producing optimal yields with low N inputs. In this work we report the results of two greenhouse experiments aimed at testing the effect of two Trichoderma strains (T. harzianum T22 and T. virens GV41) on lettuce and rocket quality under different N availability conditions. Both strains resulted in higher lettuce marketable yields (+19%) under optimal fertilization compared to the no inoculated control, while T.virens GV41 resulted in higher yields (+34% on the average) for both crops under low N availability conditions. Trichoderma inoculation with both strains enhanced total ascorbic acid in lettuce compared to the control and also in rocket at optimal and very high levels of nitrogen.
Keywords: Trichoderma; lettuce; rocket; nitrate; ascorbic acid Parole chiave: Trichoderma, lattuga; rucola; nitrati; acido ascorbico Introduction
Nitrate is the main nitrogen (N) source for most vegetables and a significant number of them, in particular leafy vegetables, require large quantities of nitrate fertilizer for ensuring maximum yield (Marschner, 1994). Balancing the amount of N required for optimum growth and development while minimizing the nitrate losses in surface and ground water represents a major sustainability challenge, since many vegetable growers are dealing with higher fertilizers costs, as well as restrictions and regulations imposed by several European countries. One of the most important and innovative approaches to tackle this important challenge is to use naturally derived biostimulants, which are gaining importance globally (Colla and Rouphael 2015). As defined by du Jardin (2015), plant biostimulants (PBs) correspond ‘to any substance or microorganism applied
to plants with the aim to enhance nutrition efficiency, abiotic stress tolerance and/or crop quality traits, regardless of its nutrients content’. Trichoderma is a genus of saprotrophic fungi that have also been reported to promote plant growth, in
addition to their biocontrol activities, acting by either the production of antimicrobial compounds or the parasitism of fungal plant pathogen (Lopez-Bucio et al., 2015). Some Trichoderma strains have a predominant biostimulant action, that make them interesting for their use in vegetables crop production (Lopez-Bucio et al., 2015). Two greenhouse experiments, one on lettuce (Lactuca sativa L.) and the other on rocket (Diplotaxis tenuifolia L) were carried out to assess the effectiveness of two Trichoderma strains under different N availability conditions. Inoculated and non inoculated plants were compared in terms of yield, yield components and nutritional quality of these two important and representative leafy vegetables.
Materials and Methods
Two consecutive experiments were conducted in the 2016 growing season from February 2nd to March 31st (experiment 1)
and from June 13th to July 11th (experiment 2) in a 240 m2 polyethylene greenhouse located in Portici (Campania region,
Southern-Italy). In experiment 1 Lactuca sativa var. iceberg cv. Silvinas (lettuce) was transplanted in double rows with a density of 11 pt m2, while in experiment 2 Diplotaxis tenuifolia L (rocket) was sowed with a density of 3000 seed m-2.
In both experiments, an optimal fertilization (100N) of 90 kg N ha-1 for lettuce and 60 kg N ha-1 for rocket was compared to
a non-fertilized control (0N) and an excess N-dose (200N) of 180 kg N ha-1 for lettuce and 120 kg N ha-1 for rocket. Two biostimulants containing a spore suspension (1 × 10sp ml-1) of different Trichoderma strains (T. harzianum T22 and T.
virens GV41, labelled as T1 and T2, respectively) were compared to a non-inoculated control (T0). Biostimulant
application to lettuce was done at transplanting (root dip) and during the crop cycle (24 days after transplanting, by watering with 50 ml plant-1), while a seed treatment was done for rocket. A split-plot design with 3 replicates (randomized
blocks) was adopted with fertilization (3 levels) as main factor and Trichoderma inoculation (3 levels) as sub-factor. The same experimental layout (blocks and plots) was adopted in both experiments.
Marketable and unmarketable yield were measured on 1 m2 reference area within each plot 60 days after transplanting for
In both experiments, the dried leaf tissues were finely ground in a mill (IKA, MF10.1, Germany) to pass through a 0.5-mm sieve. Nitrate were extracted from 250-mg samples with deionized water at 80 °C in a shaking water bath for 10 min. The resulting solution was filtered, diluted, and analyzed by ion chromatography (ICS-3000, Dionex, USA). An IonPac AG11- HC guard column and IonPac AS11-HC analytical column were used (Dionex) for nitrate determination. The total ascorbic acid defined as ascorbic acid (ASA) and dehydroascorbate (DHA) acid was assessed by spectrophotometric detection on fresh plant tissues as reported by Kampfenkel et al. (1995). The absorbance of the solution was measured at 525 nm, and data were expressed as mg of ascorbic acid on 100 g fresh weight. All data were subjected to ANOVA and means separated according to LSD test (p<0.05).
Results and Discussion
Figure 1. Effect of fertilization doses and biostimulants on lettuce (a) and rocket (b) marketable (black bars) and non-
marketable (grey bars) yield. 0N: non-fertilized control; 100N: 90 and 60 kg N ha-1 for lettuce and rocket, respectively;
200N : 180 and 120 kg N ha-1for lettuce and rocket, respectively; T0: non-inoculated control; T1: biostimulant containing
T. harzianum; T2: biostimulant containing T.virens. Different letters within each column indicate means different at p<0.05. Letters at the top of the bars are referred to total yield. n.s: not significant.
Figura 1. Effetto della fertilizzazione e dei biostimolanti sulla produzione commerciale (barre in nero) e sullo scarto (barre
in grigio) di lattuga (a) e rucola (b). 0N: non concimato; 100N: 90 and 60 kg Nha-1 per lattuga e rucola, rispettivamente;
200N: 180 and 120 kg N ha-1 per lattuga e rucola, rispettivamente; T0 non inoculato; T1 biostimolante contenente T.
harzianum; T2: biostimolante contenente T.virens. Lettere differenti indicano medie differenti per ciascuna variabile (p<0.05). Le lettere posizionate all’estremità alle barre sono riferite alla produzione totale. n.s: non significativo
Biostimulants containing Trichoderma positively affected lettuce (Fig.1a) and rocket (Fig.1b) yield compared to T0 in non fertilized plots, while yield response to biostimulants varied between the two crops when fertilizers were applied. In absence of external N inputs (0N), T2 increased total yield by 34% for both lettuce and rocket, respectively; marketable yield followed the same pattern for rocket while no effect was recorded for lettuce with an average commercial weight of 292 g FW plant-1. These results are probably due to the positive effects of Trichoderma in enhancing root N uptake under
sub optimal conditions (Marschner, 1994). No increase in yields was recorded for both crops when an excess N dose was applied.
No biostimulant effect was recorded for rocket under optimal N supply (100N), while an average increase was recorded in lettuce total (+14%) and marketable (+19%) yield compared to 100N-T0. The excessive N dose (200N) was not different from 100N-T0 for both crops. In addition 200N significantly suppressed biostimulant effect on rocket marketable yield with values lower than T0 (4707 vs 3786 g FW m-2 for 200N-T0 vs 200N-T2, respectively), while values recorded for
lettuce with 200N-T2 (409 g FW plant-1) were found significantly lower than 100N-T2 (509 g FW plant-1). The different
response of the two crops to biostimulants in fertilized plots was probably due to the length of the growth cycle who allowed a more marked effect in lettuce (60 days cycle) than in rocket (30 days cycle). A different root-Trichoderma interaction in the two tested crops cannot be excluded.
The nitrate content was not significantly influenced by biostimulant and N applications. Generally, nitrate accumulating leafy vegetables belong to the families of Brassicaceae, Chenopodiaceae, and also Asteraceae (Santamaria, 2006). This was also the case in the present study, where the highest nitrate accumulating leafy crop was rocket (3251.2 g kg-1 DW on the average), a known hyper-accumulator species (Santamaria et al. 2002) belonging to the family of Brassicaceae, whereas the values were lower in lettuce (1614.2 g kg-1 DW on the average). On the whole, values were below the
maximum threshold of nitrates (2500 mg NO3- kg-1) imposed by Commission Regulation (EC) No 1881/2006 for lettuce as
well as for rocket (6000 mg NO3- kg−1 FW in summer-grown rocket or 7000 mg NO3- kg−1 FW in winter-grown rocket).
a a a b ab b b b b 0 10 20 30 40 50 60 70 80 T0 T1 T2 T0 T1 T2 T0 T1 T2 m g ( 100 g FW) -1 Ascorbic acid in lettuce 0N 100N 200N b bc d c bc a bc cd b 0 10 20 30 40 50 60 70 80 T0 T1 T2 T0 T1 T2 T0 T1 T2 m g ( 100 g FW) -1 Ascorbic acid in rocket 0N 100N 200N
Figure 2. Effect of fertilization doses and biostimulants on Ascorbic acid content in lettuce (a) and rocket (b) leaves. 0N:
non-fertilized control; 100N: 90 and 60 kg N ha-1 for lettuce and rocket, respectively; 200N : 180 and 120 kg N ha-1for
lettuce and rocket, respectively; T0: non-inoculated control; T1: biostimulant containing T. harzianum; T2: biostimulant containing T.virens. Different letters indicate means different at p<0.05.
Figura 2. Effetto della fertilizzazione e dei biostimolanti sul contenuto di Acido ascorbico nelle foglie di lattuga (a) e
rucola (b). 0N: non concimato; 100N: 90 and 60 kg N-1ha-1 per lattuga e rucola, rispettivamente; 200N : 180 and 120 kg N
ha-1 per lattuga e rucola, rispettivamente; T0 non inoculato; T1 biostimolante contenente T. harzianum; T2: biostimolante
contenente T.virens. Lettere differenti indicano medie differenti (p<0.05).
Many leafy vegetables are regarded as a good source of vitamin C. The total ascorbic acid (AA) content, including ascorbic and dehydroascorbic acid, of the two leafy vegetables tested varied widely (figure 2). In experiment 1 the total ascorbic acid of lettuce ranged from 7.2 to 22.6 mg 100 g-1 FW whereas in experiment 2 the total ascorbic acid ranged from 2.4 to 72.7 mg 100 g-1 FW. In both experiments, the application of biostimulant and N doses affected significantly the total
ascorbic acid content. In experiment 1, the absence of N application exhibited the highest total ascorbic acid values irrespective of Trichoderma inoculation. Under optimal N supply (100N) the inoculation of T1 improved significantly the quality of lettuce leaves in comparison to T2 and non-inoculated plants. Finally, in rocket the application of 100N combined with inoculation with T2 could be considered an effective and sustainable way to biofortify the leaf quality of rocket since the total ascorbic acid was two-fold compared to the other treatments.
Conclusions
Recommended N doses for leafy vegetables allow the highest marketable yield, while an excessive N fertilization does not provide any yield benefit. Biostimulants containing Trichoderma can significantly increase yields in crops with a medium length cycle as lettuce when recommended N dose is applied. Our results also demonstrated that specific Trichoderma strains can enhance the nutritional quality of both leafy vegetables (i.e. higher total ascorbic acid content). Nitrate accumulation in lettuce and rocket leaves may not be a problem in Mediterranean cropping systems even when an excessive N dose is applied, meaning that in some soils with high N native availability the risk of contamination of groundwater could be the main concern instead of food security.
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