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New traits to identify physiological responses induced by different rootstocks after root-knot nematode inoculation (Meloidogyne incognita) in sweet pepper
Crop Protection 119 (2019) 126–133
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New traits to identify physiological responses induced by different rootstocks after root-knot nematode inoculation (Meloidogyne incognita) in sweet pepper pez-Marín a Amparo Galvez a, Francisco M. del Amor a, *, Caridad Ros b, Josefa Lo a b
Department of Crop Production and Agri-technology, Murcia Institute of Agri-Food Research and Development (IMIDA), C/Mayor s/n, 30150, Murcia, Spain Department of Crop Protection, Murcia Institute of Agri-Food Research and Development (IMIDA), C/Mayor s/n, 30150, Murcia, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Greenhouse Sustainability Amino acids Monocropping Meloidogyne incognita Capsicum annuum L
Root-knot nematodes (RKNs) are one of the major phytopathological problems that limit potential yields in intensive agriculture worldwide. Grafting with resistant rootstocks has been demonstrated to be a viable technique to manage Meloidogyne incognita in several crops (tomato, melon, watermelon, etc.). However, little research has been conducted on sweet pepper (Capsicum annuum L.) grown in greenhouses under Mediterraneanclimate conditions. Therefore, we studied a susceptible scion variety (Gacela) grafted onto resistant rootstocks (C19, C25, and RT17), in comparison with ungrafted and self-grafted (GAL) plants. The RKN produced significant root galling in ungrafted and GAL plants; however, RT17 was unaffected. This differing response to infection is discussed from physiological and agronomic perspectives. Thus, following the inoculation of susceptible rootstocks, leaf photosynthesis was impaired by altered stomatal regulation and photochemical efficiency (chlorophyll a fluorescence), which led to unbalanced fruit nutrient concentrations (especially Ca) and to enhanced allocation of carbon from photosynthesis to the production of biochemical defense compounds. Consequently, total phenolics and carotenoids were significantly affected, as was the amino acid profile. These results provide new insights into the traits that can identify RKN-resistant rootstocks, and therefore new tools to induce resistance, while avoiding the use of disinfectants in the soil.
1. Introduction
then migrate intercellularly to the vascular cylinder, where they establish feeding sites and disrupt the vascular tissue (Abad et al., 2003). These alterations have been found to interfere in many physiological and biochemical processes in plants related to photosynthesis and respiration, ion uptake and nutrient concentrations, pigments composition, growth promoters, and metabolism (Strajnar et al., 2012). Moreover, although the scion variety can affect the final yield and fruit quality, rootstock effects can alter these characteristics, depending on the crop management and production environment (Rouphael et al., 2010). Thus, in order to limit the effect of RKNs and to induce resistance, grafting is used as a common practice in many horticultural crops (Davis et al., 2008; King et al., 2008; Oda, 1998). Additionally, recent research has demonstrated the beneficial effects of using grafting to enhance nutrient uptake (Savvas et al., 2017) and fruit quality (Rouphael et al., 2010). Sweet pepper, an important horticultural crop species and a valuable
Root-knot nematodes (RKNs) are a major phytopathological soil problem, that affects sweet pepper (Capsicum annuum L.) (Guerrero-Díaz et al., 2013). In Spain alone, some 2000 ha have been continuously occupied for more than 20 years by a monocropping system for sweet ~ez et al., 2014). These pepper, grown over a 9–10-month cycle (Ros-Iban soils are mainly affected by the RKN, Meloidogyne incognita (Bello et al., 2004). In fact, in horticultural production under plastic the ideal soil conditions (relatively warm, moist, and with adequate texture) and continuous cropping can offer an ideal scenario for populations of RKNs to increase to damaging levels (Aguiar et al., 2014). The primary symptoms of RKN infestation in susceptible plants are anatomical changes in roots, such as giant-cell development and gall formation (Bleve-Zacheo et al., 1998). The RKNs invade the roots in the zone of elongation and
Abbreviations: RKNs, Root-knot nematodes; Pn, Leaf net photosynthesis; SPAD, Hand-held chlorophyll meter; gs, stomatal conductance; E, transpiration rate; iWUE, instantaneous water use efficiency; Fv/Fm, the maximum quantum use efficiency of PSII in the dark-adapted state. * Corresponding author. E-mail address: [email protected] (F.M. del Amor). https://doi.org/10.1016/j.cropro.2019.01.026 Received 6 June 2018; Received in revised form 23 January 2019; Accepted 24 January 2019 Available online 25 January 2019 0261-2194/© 2019 Elsevier Ltd. All rights reserved.
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humidity (%) was: 94.2/24.5/48.9; 95.0/19.9/44.4; 94.7/18.7/43.0; 94.1/18.0/43.5; 94.0/20.0/44.2; 94.1/19.7/45.0; 94.8/18.947.0; and the substrate temperature ( C) inside the pots was: 27.5/9.4/24.5; 28.0/9.8/24.0; 29.2/9.8/23.9; 30.5/10.4/24.4; 31.5/10.6/24.5; 31.0/11.0/24.8; 30.0/12.0/24.9.
source of nutritional and healthy components, is mainly grown in greenhouses (L opez-Marín et al., 2013). However, it is susceptible, on a worldwide basis, to RKNs. Empirically, the influence of roots on shoot behavior can be measured and described as changes in the grafted scion in relation to vegetative and generative growth. However, although the rootstock/scion composite has been used as an experimental system to understand root–shoot communication, the mechanisms governing rootstock x scion phenotypes still remain unclear (Albacete et al., 2015; Ghanem et al., 2008; Gregory et al., 2013). Plants often respond to pathogenic and parasitic pressure by modifying biochemical processes and increasing production of oxidant and antimicrobial metabolites (del Amor et al., 2008). The correct identification of such metabolites specifically involved in defense, which confers increased tolerance, is of paramount importance in developing new products, but also to establish new crop strategies for nematode control. On the other hand, as abiotic stress can trigger systemic defense systems in susceptible plants, there may be a shift in the allocation of carbon structures from photosynthesis to the production of chemical defense compounds, rather than growth (Atkinson et al., 2011); therefore, the yield and quality of the fruits can be impaired. We hypothesize that rootstocks can alter both agronomic and specific physiological responses of scions under the biotic stress provoked by RKNs. Therefore, the aim of this work was to obtain insights into the mechanisms by which grafted plants are able to overcome the deleterious effects of nematodes, and to identify the physiological response mechanisms in sweet pepper which confer this differential tolerance. We focused on three different rootstocks and self-grafted plants, comparing their abilities to mitigate this important biotic stress. Currently, information on how leaf gas exchange and the leaf chlorophylls content are influenced by the specific resistance of a rootstock to nematodes is still scarce, as are data for the nutritional profile and the physiological characterization based on phenolic compounds and carotenoids. Moreover, to our knowledge, this is the first report where the amino-acid profile is analyzed under the combined effects of grafting and nematodes in pepper, which could provide relevant information about its crucial role during pathogen infection. Thus, the overall goal of our research was to help maintain both crop profitability and sustainability in greenhouse systems, through the study of different physiological stress indicators and fruit production.
2.3. Inoculation procedure and gall index The rootstocks were infested by one population of M. incognita. This population is virulent to the Atlante (C25) rootstock and carries the gene Me3 (Sanchez-Solana et al., 2016). It was obtained from a single egg mass of naturally-infested roots of Atlante and belongs to pepper race 2 (Robertson et al., 2006) The isolate was identified in the reference laboratory of the Spanish National Research Council (CSIC, Madrid), where it was determined to be M. incognita isolates race 2, based on the North Carolina differential host test (Robertson et al., 2006, 2009). Each plant was inoculated with 10 mL of a suspension of 2400 eggs mL1, which were poured into two holes around the plant roots - following the methodology used by Djian-Caporalino et al. (1999). At 51, 85, 124, 150, 170, and 196 days after transplanting (DAT), three plants of each inoculation treatment were removed and taken to the laboratory. The roots were washed individually with tap water and examined under a magnifying glass to determine the percentage of plants infested by the nematode and the gall index (GI), which followed the 0–10 scale developed by Bridge and Page (2009); on this scale 0 is a well-developed and healthy root system, and 10 is plants and roots dead. 2.4. Leaf gas exchange Leaf water and gas exchange were monitored in fully-expanded leaves in the generative plant stage, in six leaves for each treatment. The leaf closest to the most-recently-set fruit was used to measure both parameters. Measurements were carried out 20 days after fruit set - 172, 182, and 188 DAT - and at the mature and dark-green fruit stage (196 DAT), from 9:00 a.m. to 11:00 a.m. The net CO2 assimilation rate (Pn), stomatal conductance (gs), transpiration rate (E), and water use efficiency (Pn/E) were measured in the steady state, at saturating light (800 μmol m2 s1 photon flux density) and 400 μmol mol1 CO2, with an LI-6400 portable photosynthesis system (LI-COR Inc., Lincoln, Nebraska, USA).
2. Materials and methods 2.5. Chlorophyll content and fluorescence 2.1. Plant material Chlorophyll fluorescence was determined in the same leaf used for gas exchange determinations, with an ADC Fim 1500 pulse modulated
Using the Japanese top-graft procedure (Oda, 1998), the sweet pepper cultivar ‘Gacela’ F1 (Syngenta Seeds, USA) was grafted onto two commercial rootstocks, Atlante (C25) (Ramiro Arnedo Seeds S.A, La Rioja, Spain) and Robusto (RT17) (Syngenta Seeds S.A, Almeria, Spain), and one non-commercial rootstock, (C19) (Ramiro Arnedo Seeds S.A, La Rioja, Spain), and was also self-grafted (GAL). Ungrafted ‘Gacela’ plants were used as control plants. 2.2. Greenhouse conditions Grafted and ungrafted plants were transplanted into 10-L pots (50% sand, 30% peat, 20% vermiculite) on December 18, 2015, in an unheated, arch-shaped multispan greenhouse covered with thermal polyethylene, located at the ‘Torreblanca’ Experimental Farm of the Murcia Institute of Agri-Food Research and Development (IMIDA) in Murcia, SE Spain (lat. 37 450 N, long. 0 590 W). Irrigation was applied based on estimations of the weekly crop evapotranspiration; the surplus water lost by drainage represented more than 20–25% of the irrigation solution, to maintain the nutrient balance in the rhizosphere and avoid excessive salinity (del Amor and Gomez-Lopez, 2009). The maximum, minimum, and average air temperatures ( C) from December to June were: 29.5/11.0/17.5; 31.1/11.0/17.0; 31.9/11.4/18.0; 35.0/11.8/20.0; 34.4/14.9/22.1; 34.5/16.4/24.1; 35.1/17.0/25.4; the air relative
Fig. 1. Progression of the gall index in the rootstocks of sweet pepper after inoculation with M. incognita. Values are means (n ¼ 3) and vertical bars represent the standard error of the mean. 127
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Fig. 2. Effects of inoculation with M. incognita on A,B) net photosynthesis; C,D) stomatal conductance; E,F) water use efficiency; G,H) chlorophyll fluorescence, and I,J) relative chlorophyll content. Values are means (n ¼ 6) SE. Different letters within a column and graph indicate significant (p 0.05) differences between treatments. Analysis of variance for each parameter: ns, not significant; *p 0.05; **p 0.005; ***p 0.001.
128
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129
* * ns
bc bc a bc b 62.4 59.9 38.9 56.7 53.7
* * ns
bc c a ab a 51.3 41.9 35.1 40.3 53.0
ns * *
a ab ab bc d 0.81 2.21 1.10 2.99 4.32
ns ns ns
ab a a a a 210.3 176.5 179.4 157.1 179.5
** ** *
d c ab bc ab 165.3 132.2 107.6 123.7 107.2
ns * ns
bc c a ab a 741.8 1035.3 324.9 494.5 354.4
ns * ns
bc bc ab ab c 17783.3 17963.0 15902.2 16529.6 19970.4
ns *** ns
ab ab ab ab c 2331.3 2746.3 2618.0 3007.0 3849.4
* ** ns
a a b ab b 28758.6 29122.3 33573.1 31337.6 34447.6
Main factors Rootstocks RKN Rootstocks x RKN
ns * **
a bc b b c Ungrafted GAL C19 C25 RT17 Inoculated
28262.7 37508.3 36542.8 37415.9 45380.8
cd d a bc bc 68.1 71.3 42.9 62.1 61.3 bc d ab bc c 48.4 63.6 36.4 46.6 55.5 ab cd cd cd bc 1.93 3.42 3.35 3.34 2.55 b a a a a 250.1 188.6 165.3 173.2 170.9 bc bc a abc ab 130.0 121.2 96.5 117.9 105.5 a a a a a 294.2 348.1 273.7 255.5 259.2 a ab ab ab bc 31524.5 31070.6 34916.3 35090.0 39840.8
ab ab b b c
2857.4 3672.0 3459.0 3868.7 3510.9
ab c bc c bc
15078.0 15770.1 16798.5 16269.4 17809.8
Mn (mg kg1) Cu (mg kg1) Fe (mg kg1) B (mg kg1)
ab bc bc bc bc 33997.1 40648.9 39232.7 39030.7 37861.3 Ungrafted GAL C19 C25 RT17
Fig. 3. Effects of inoculation with M. incognita on total fruit yield of the scion Gacela grafted onto different rootstocks. Values are means (n ¼ 20) SE. Different letters indicate significant (p 0.05) differences between treatments. Analysis of variance: ns, not significant; *p 0.05; **p 0.005; ***p 0.001.
Uninoculated
The total phenolic compounds were extracted from 0.4 g of frozen fruits (80 C) according to the procedure described in Serrano-Martínez et al. (2008) for sweet peppers, using chlorogenic acid for the quantification. The total phenolic content was expressed as gallic acid equivalents, in mg/100 g fresh weight. The analyses were performed in triplicate for each sample. Determination of total carotenoids was carried out on an aliquot of the hexane extract, by measuring the absorbance at 450 nm in a Varian Cary 50-Bio spectrophotometer (Melbourne, Victoria, Australia). Total carotenoids were calculated according to Almela et al. (1991), using a commercial standard of β-carotene from Sigma (Madrid, Spain). Fifteen replicates were analyzed for each treatment (each replicate contained three fruits).
Na (mg kg1)
2.8. Total phenolic compounds and carotenoids
Mg (mg kg1)
Pepper fruits were dried at 65 C in an oven for 72 h. Cations were extracted from ground material (0.4 g) with 20 mL of deionized water, and determined by inductively coupled plasma–optical emission spectrometry (ICP-OES) (Varian Vista-MPX, Varian Australia, Mulgrave, Victoria, Australia). Fifteen replicates were analyzed for each treatment (each replicate contained three fruits).
P (mg kg1)
2.7. Fruit mineral concentrations
Ca (mg kg1)
Sweet pepper fruits were harvested from 20 plants (5 plants per block, 4 blocks) per treatment. The quality of the marketable commercial fruits was evaluated according to commercial practices, as were discarded fruits with physiological disorders (sun scald, BER, etc.) that were unmarketable (del Amor, 2007).
K (mg kg1)
2.6. Fruit yield
Treatments
Table 1 Effects of inoculation with M. incognita on fruit macro and micro nutrient concentrations. Means in a column followed by the same letter are not significantly different at p 0.05, according to Duncanʼs multiple range test (n ¼ 15). Analysis of variance: ns, not significant; *p 0.05; **p 0.005; ***p 0.001.
fluorometer (Analytical Development Company Ltd., Hoddesdon, UK). Minimal fluorescence values in the dark-adapted state (Fo) were obtained by application of a low-intensity, red-measuring light source (630 nm), while maximal fluorescence values (Fm) were measured after applying a saturating light pulse of 800 μmol m2 s1. In this way, the maximum quantum use efficiency of PSII in the dark-adapted state (30 min) was calculated as Fv/Fm ¼ (Fm Fo)/Fm. The relative chlorophyll content of leaves was determined with a Minolta SPAD-502 m (Konica Minolta Optics, Inc., Japan). This device determines the greenness and the interaction of thylakoid chlorophyll with incident light, taking instant readings without destroying the plant tissue.
Zn (mg kg1)
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and RT17 plants showed higher photosynthetic rates and stomatal conductance than GAL and ungrafted plants, after inoculation, at the end of the crop season. The instantaneous water use efficiency (iWUE: Pn/E) (Fig. 2E and F) was, in general, less affected by inoculation than photosynthesis or stomatal conductance; however, at the end of the study, ungrafted plants showed higher iWUE values than grafted plants (Fig. 2F). The leaf chlorophyll fluorescence was not significantly altered when plants were not inoculated (Fig. 2 G) and the distinct treatments maintained values of between 0.75 and 0.8. Inoculation differentially affected this response, and GAL and RT17 plants maintained the same values as uninoculated plants. The rest of the treatments showed reduced values, but not below 0.75. (Fig. 2H). By contrast, the SPAD values (relative chlorophyll content) were more affected - especially at the last sampling date (Fig. 2J), when the ungrafted plants and C25 rootstock had SPAD values that were 31% and 26% lower, respectively, compared with the uninoculated rootstock. The SPAD values were unaltered for the RT17 rootstock after inoculation.
2.9. Free amino acids Free amino acids were extracted from fruits frozen at 80 C: sap was extracted after vortexing at 5000 g (10 min, 4 C) and analyzed with the AccQTag-ultra ultra-performance liquid chromatography (UPLC) method (Waters, UPLC Amino Acid Analysis Solution. Waters Corporation, Milford, MA, 2006). For derivatization, 70 μL of borate buffer were added to the hydrolyzed sample or to 10 μL of the fruit sap. Next, 20 μL of reagent solution were added. The reaction mixture was mixed immediately and heated at 55 C for 10 min. After cooling, an aliquot of the reaction mixture was used for UPLC injection. The UPLC was performed on an Acquity system (Waters, Milford, Massachusetts, USA), equipped with a fluorescence detection (FLR) system. The column used was a BEH C18 100 mm 2.1 mm, 1.7 μm (Waters). The flow rate was 0.7 mL min1 and the column temperature was kept at 55 C. The injection volume was 1 μL. Wavelength excitation (λex) and emission (λem) were set at 266 and 473 nm, respectively. The solvent system consisted of two eluents: (A) AccQTag-ultra eluent A concentrate (5%, v/v) and water (95%, v/v); (B) AccQTag ultra eluent B. The following elution gradient was used: 0–0.54 min, 99.9% A–0.1% B; 5.74 min, 90.9% A–9.1% B; 7.74 min, 78.8% A–21.2% B; 8.04 min, 40.4% A–59.6% B; 8.05–8.64 min, 10% A–90% B; 8.73–10 min, 99.9% A–0.1% B. Empower 2 (Waters) software was used for system control and data acquisition. External standards (Thermo Scientific) were used for quantification of (Ala) alanine; (Arg) arginine; (Asp) aspartic acid; (Glu) glutamic acid; (Gly) glycine; (His) histamine; (Ile) isoleucine; (Leu) leucine; (Lys) lysine; (Met) methionine; (Phe) phenylalanine; (Pro) proline; (Ser) serine; (Thr) threonine; (Tyr) tyrosine; and (Val) valine. Fifteen replicates were analyzed for each treatment (each replicate contained three fruits). 2.10. Experimental design and statistical analysis The experimental design was a randomized complete block. For yield monitoring, five plants were used in each of four blocks, whilst for destructive sampling (gall index) one plant was harvested in each of three blocks, and for each treatment, at every sampling date (51, 85, 124, 150, 170, and 196 days after transplanting (DAT)). The Statgraphics statistical package was used to calculate significant differences by ANOVA, and means were compared at a probability of p 0.05 according to the Duncan test. The data were checked for normality and homogeneity of variances and were transformed, where required, to their arcsin √x (x ¼ percentage of infested plants) or log10 (x þ 1) (x ¼ the gall index) values.
Fig. 4. Effects of inoculation with M. incognita on of total phenolic in fruits of the scion Gacela grafted onto different rootstocks. Values are means (n ¼ 15) SE. Different letters indicate significant (p 0.05) differences between treatments. Analysis of variance: ns, not significant; *p 0.05; **p 0.005; ***p 0.001.
3. Results 3.1. Root-knot infection Our study shows the effect of inoculation with M. incognita on different rootstocks of sweet pepper during the crop season. The ungrafted plants had the highest GI, whilst C25 and C19 were relatively less affected, with decreases in the GI of 26.3% and 38.9%, respectively, at the last evaluation, compared with the ungrafted plants (Fig. 1). The self-grafted scion (GAL) showed a moderate reduction in the GI (8.9%), but the RT17 rootstock exhibited no progression of galling - indicating its higher tolerance of M. incognita. 3.2. Leaf gas exchange and relative chlorophyll content The leaf CO2 assimilation rate (Fig. 2A and B) and stomatal conductance (Fig. 2C and D) were severely impaired after inoculation in ungrafted plants, and at the end of the study the values of these parameters were reduced by 62.8% and 88.1%, respectively, compared with the control (ungrafted and uninoculated) plants. Self-grafted plants showed a lower impact, but the values of both parameters were reduced with respect to control plants: by 34.5% and 57.4%, respectively. Clearly, C19
Fig. 5. Effects of inoculation with M. incognita on carotenoids in fruits of the scion Gacela grafted onto different rootstocks. Values are means (n ¼ 15) SE. Different letters indicate significant (p 0.05) differences between treatments. Analysis of variance: ns, not significant; *p 0.05; **p 0.005; ***p 0.001. 130
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Table 2 Effects of inoculation with M. incognita on fruit amino acid concentrations. Means in a column followed by the same letter are not significantly different at p 0.05, according to Duncanʼs multiple range test (n ¼ 15). Analysis of variance: ns, not significant; *p 0.05; **p 0.005; ***p 0.001. Treat. Uninoculated
Inoculated
Ser
Arg
Ungraf GAL C19 C25 RT17
604.1 672.1 581.3 546.2 603.4
ab
Ungraf GAL C19 C25 RT17
509.6 596.5 787.8 473.5 509.9
a
Main factors Rootstocks RKN Rootstocks x RKN
ns ns ns
ab ab ab ab
ab b a a
Gly
593.0 495.1 525.1 464.6 541.0
a
425.0 538.6 626.5 392.8 410.6
a
ns ns ns
a a a a
a a a a
Asp
81.3 61.1 56.0 52.3 91.5
cd
42.0 51.5 71.9 38.1 46.1
ab
abc abc abc d
abc bcd a ab
ns ** ns
Glu
94.6 130.4 111.7 61.4 68.8
abc
88.1 96.0 155.7 57.2 64.6
abc
ns ns ns
bc abc ab ab
abc c a ab
Thr
292.9 136.0 86.8 126.9 183.4
bc
384.5 378.4 134.5 123.7 129.8
c
*** * ns
a a a ab
c a a a
Ala
264.3 224.8 155.8 164.2 215.4
c
129.3 161.7 149.9 136.4 148.2
a
* *** **
b b b b
b a a a
296.8 279.5 195.1 171.3 326.6
c
135.3 115.0 275.4 173.8 164.7
a
c b b c
a c b b
ns * ns
in ungrafted plants were significantly lessened, specifically for Gly, Thr, Ala, Cys, Val, Ile, Leu, and Phe; in contrast, Pro was the only amino acid whose concentration increased due to inoculation in these plants, and the same response of Pro was also found in self-grafted (GAL) plants.
3.3. Fruit yield The severity of the RKN symptoms on the sweet pepper plants was clearly reflected in the fruit yield (Fig. 3), as was expected since it integrates many of the physiological processes affected by the action of nematodes in the roots. Firstly, in the absence of biotic stress the rootstock significantly altered the response of the scion; thus, plants on C19 or RT17 gave lower yields than ungrafted, self-grafted, and C25 plants. However, the rootstock/scion combinations that gave lower yields under control conditions were less affected by RKNs and - in contrast ungrafted, GAL, and C25 plants showed higher impacts.
4. Discussion Grafting is used in many countries, in protected cultivation - in which land is intensively used and crops are not rotated, allowing improvements in both productivity and fruit quality (Kyriacou et al., 2017). Moreover, the correct choice of rootstock is of paramount importance for the performance of a vulnerable crop (L opez-Perez et al., 2006). Our study shows that RKNs induced severe changes in the roots, as indicated by the progression of the GI in susceptible rootstocks. A promising way to improve pepper resistance to RKNs is the grafting of commercial cultivars onto nematode-resistant rootstocks, but the complexity of the response and differences among genotypes and growth environments could minimize its effectiveness. Our study shows the disturbance of root anatomy and of several physiological and biochemical processes caused by M. incognita in sweet pepper under greenhouse cultivation. Dorhout et al. (1991) reported that infestation increased axial resistance to water flow and reduced total water uptake in tomato. Our data show the differential impact of the rootstock on water use efficiency and, especially, stomatal conductance - which were severely impaired in ungrafted plants after inoculation. Leaf photosynthesis was also reduced in plants on susceptible rootstocks. Melakeberhan et al. (1987) reported, for soybean, that M. incognita can contribute to premature leaf abscission and chlorosis, which can affect photosynthesis. We observed that the reduction in the photosynthesis rate was linked to decreases in stomatal conductance and also in chlorophyll fluorescence (maximum quantum yield of the primary photochemistry of PSII). Thus, the RT17 and GAL treatments maintained the Fv/Fm values, but for ungrafted, C19, and C25 plants the values of this parameter were reduced at the end of the crop season, after inoculation. Therefore, we suggest that the main effect of M. incognita on the process of photosynthesis in sweet pepper is on stomatal closure and diffusive resistance, but there is also metabolic uncoupling. This agreed with the reduced relative chlorophyll content in the leaves. This important effect was not observed when plants were only submitted to nutritional depletion (del Amor, 2006). Thus, Campos et al. (2014) indicated that further processes beyond nutritional impairment are implicated in the photosynthetic response, and emphasized the importance of maintaining a proper water balance to avoid slowdown of the electron transfer in the reaction centers of the photosystem, which could become more relevant as a result of the effect of M. incognita on stomatal closure. This response of susceptible rootstocks with regard to stomatal regulation has important consequences for an appropriate and balanced nutrient uptake
3.4. Mineral concentrations For inoculated plants, the less susceptible rootstocks, RT17 and C19, gave Ca concentrations that were higher than in ungrafted or self-grafted (GAL) plants (Table 1) (e.g. the Ca concentration was 16.5% higher in RT17 than in ungrafted plants). However, this pattern was reversed for Na, B, and Mn: RT17 and C19 showed lower concentrations compared with the ungrafted or self-grafted (GAL) plants. The concentrations of P and, especially, Cu were notably higher in the RT17 plants; in the fruits they were increased by 39.4% and 81.3%, respectively, relative to the uninoculated plants. 3.5. Total phenolics and carotenoids Regarding the concentration of total phenolics in the fruits, there were significant differences among the studied rootstocks (Fig. 4). Thus, the plant response to RKN inoculation boosted total phenolics by 34% for the ungrafted plants, and by 37% and 33% for GAL and C25, respectively, when compared with the uninoculated plants. However, this increase in total phenolics was attenuated for the C19 rootstock, and the level in RT17 was unaffected by inoculation. The total carotenoids (Fig. 5) showed a similar response to inoculation, the C19 and RT17 rootstocks being unaffected. 3.6. Total amino acid content The effect of grafting on the total amino acid concentration was relevant in the absence of inoculation (Table 2). Thus, the most noteworthy effect was on RT17, which had higher concentrations of Gly and Met than the rest of the treatments. Moreover, the ungrafted plants had the highest levels of Thr, Cys, Lys, Val, Leu, and Phe. In general, the selfgrafted (GAL) plants showed a pattern similar to that of the C19 and C25 plants in the absence of stress (RKNs). However, after inoculation, the previously observed effect was altered. Thus, the higher concentrations 131
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Pro
Cys
bc 55.8 a 49.3 ab 51.3 bc 56.6 a 48.2 e 68.8 e 69.9 cd 60.3 b 66.3 b 51.7 Main factors * *** ns
388.1 262.9 198.3 225.6 245.5 210.0 246.4 331.2 179.3 198.4 * ns **
Lys c ab a a ab a ab bc a a
459.8 308.1 306.6 319.2 318.9 469.4 428.7 373.3 361.2 367.6 ** ns ns
Met c a a ab ab c bc abc abc abc
10.4 12.3 10.8 8.5 17.9 7.2 5.4 5.4 5.5 14.4 ns ** ns
Val bc cd cde abc d ab a a a cd
Ile d
356.0 242.7 243.8 228.6 245.8 183.1 212.9 215.2 168.9 193.8
c c bc c ab abc abc a abc
** *** ***
154.2 128.3 146.1 96.8 108.1 108.6 91.8 111.0 79.0 86.9
Leu d cd d ab bc bc ab bc a ab
* * ns
134.2 84.4 76.0 95.9 101.4 93.6 112.0 107.4 68.3 79.3 * ns *
Phe e bc ab bc cd bc de cd a ab
55.8 39.4 48.9 36.8 52.1 37.8 33.6 30.0 32.1 33.6 ns ** ns
Total AA e bc c ab d ab ab a ab ab
3841.2 3138.4 3137.5 2697.0 3168.1 2892.4 2859.7 2858.4 2480.7 2766.3
d c c b c b b b a b
** *** *
Our study highlights the necessity of a more physiological approach to the effects of RKNs on sweet pepper - to improve techniques, select resistant rootstocks, and develop efficient monitoring traits to detect potential yield impairments at reduced environmental cost. Future studies should analyze the impairment by M. incognita of stomatal regulation and the quantum efficiency of PSII, as both reduce the rate of photosynthesis and, consequently, plant growth and yield. Moreover, regulation of water uptake and transport linked with the appropriate nutritional control - to counterbalance reduced Ca, P, and Cu uptake together with exogenous addition of the target amino acids whose levels were significantly impaired after infection should be part of the solution to improve tolerance of M. incognita in sweet pepper.
by these plants. Thus, as the Ca concentration in horticultural products has been linked to mass flow (water uptake) (del Amor and Marcelis, 2006), our results indicate that the significant effect of inoculation on stomatal regulation clearly affected Ca uptake and lowered the Ca concentration in fruits on susceptible rootstocks and, especially, in ungrafted plants. We also observed higher fruit concentrations of P and Cu for the resistant RT17 rootstock, compared with the sensitive ones, which indicates that the general advantages of grafting with respect to RKNs could not be generalized to the rest of the nutrients under the studied stress. The rootstock/graft interaction can potentially affect yield and fruit quality, while it confers tolerance of this biotic stress (King et al., 2008). This effect is shown in our study, where the beneficial (and differential) influence of grafting on total fruit yield was clearly affected after RKN infestation. Thus, the combined effects of the RKNs on leaf gas exchange and specific nutrient concentrations were less severe for the RT17 and C19 rootstocks; however, such rootstocks did not show higher yields in the absence of infestation (compared to susceptible rootstocks or ungrafted plants). This important result indicates that these RKN-resistant rootstocks can deliver significant advantages only for infested soils. In general, phenolics have been shown to be related to plant resistance to different pests and pathogens, and previous research found a correlation of elevated levels of phenolics with resistance or a greater response of plants to nematode infection (Chitwood, 2002). However, our results show a bigger response for ungrafted, GAL, and C25 plants, and consequently a greater correlation with pathogen infection (GI) than with resistance, which agrees with recent research for tomato (Lobna et al., 2017). Carotenoids had a similar behavior after inoculation for the RT17 and C19 treatments, in which the inoculation effect was very limited. Other studies of nematodes and carotenoids showed similar results (Atkinson et al., 2011) and no response was found. However, Arimboor et al. (2015) recently indicated that changes in the content and structure of carotenoids in plants can also be markers of environmental stress, which agrees with our results. Amino acids can act as substrates for the pathogen or have fungistatic effects, the response being genotype-dependent, and changes in their individual abundances can be attributed to the susceptibility of the genotype (Ndoumou et al., 1996). Our results indicate that inoculation significantly reduced the sum of the amino acids for all treatments; therefore, this parameter cannot be considered a tolerance trait, but, individually, differences were found. Thus, pathogen infection has been shown to lead to specific changes in the expression of many genes involved in amino acid metabolism and transport (Pratelli and Pilot, 2014). In sweet pepper a fraction of the amino acids in susceptible rootstocks could be used for the synthesis of compounds, like phenolic derivatives, involved in plant defense mechanisms.
Acknowledgements The authors thank Carmen M. Rodriguez Sanchez, for technical assistance, and Dr. David J. Walker, for assistance with the correction of the English. Funding for this research was provided by the 80% Murcia FEDER 1420-08 project –CCI number: 2014ES16RFOP019. References Abad, P., Favery, B., Rosso, M.N., Castagnone-Sereno, P., 2003. Root-knot nematode parasitism and host response: molecular basis of a sophisticated interaction. Mol. Plant Pathol. 4, 217–224. Aguiar, J.L., Bachie, O., Ploeg, A., 2014. Response of resistant and susceptible bell pepper (Capsicum annuum) to a southern California Meloidogyne incognita population from a commercial bell pepper field. J. Nematol. 46, 346–351. Albacete, A., Martínez-Andújar, C., Martínez-Perez, A., Thompson, A.J., Dodd, I.C., PerezAlfocea, F., 2015. Unravelling rootstockscion interactions to improve food security. J. Exp. Bot. 66, 2211–2226. https://doi.org/10.1093/jxb/erv027. Almela, L., L opez-Roca, J.M., Candela, M.E., Alcazar, M.D., 1991. Carotenoid composition of new cultivars of red pepper for paprika. J. Agric. Food Chem. 39, 1606–1609. https://doi.org/10.1021/jf00009a013. Arimboor, R., Natarajan, R.B., Menon, K.R., Chandrasekhar, L.P., Moorkoth, V., 2015. Red pepper (Capsicum annuum) carotenoids as a source of natural food colors: analysis and stability—a review. J. Food Sci. Technol. 52, 1258–1271. https://doi.org/10. 1007/s13197-014-1260-7. Atkinson, N.J., Dew, T.P., Orfila, C., Urwin, P.E., 2011. Influence of combined biotic and abiotic stress on nutritional quality parameters in tomato (Solanum lycopersicum). J. Agric. Food Chem. 59, 9673–9682. https://doi.org/10.1021/jf202081t. Bello, A., L opez-Perez, J.A., García Alvarez, A., Arcos, S.C., Ros, C., Guerrero, M.M., Guirao, P., 2004. Biofumigaci on con solarizaci on para el control de nematodos en cultivo de pimiento. In: Lacasa, A., Guerrero, M.M., Oncina, M., Mora, J.A. (Eds.), Desinfecci on de suelos en invernaderos de pimiento. Regi on de Murcia, Espa~ na, pp. 129–208. Consejería de Agricultura, Agua y Medio Ambiente. Bleve-Zacheo, T., Bongiovanni, M., Melillo, M.T., Castagnone-Sereno, P., 1998. The pepper resistance genes Me1 and Me3 induce differential penetration rates and temporal sequences of root cell ultrastructural changes upon nematode infection. Plant Sci. 133, 79–90. https://doi.org/10.1016/S0168-9452(98)00021-1. Bridge, J., Page, S.L.J., 2009. Estimation of root-knot nematode infestation levels on roots using a rating chart. Trop. Pest Manag. 26, 296–298. https://doi.org/10.1080/ 09670878009414416. Campos, H., Trejo, C., Pe, C.B., García-Nava, R., Conde-Martínez, F.V., 2014. Stomatal and non-stomatal limitations of bell pepper ( Capsicum annuum L .) plants under 132
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water stress and re-watering : Delayed restoration of photosynthesis during recovery, 98, pp. 56–64. https://doi.org/10.1016/j.envexpbot.2013.10.015. Chitwood, D.J., 2002. Phytochemical strategies for nematode control. Annu. Rev. Phytopathol. 40, 221–249. https://doi.org/10.1146/annurev.phyto.40.032602. 130045. Davis, A.R., Perkins-Veazie, P., Sakata, Y., L opez-Galarza, S., Maroto, J.V., Lee, S.G., Huh, Y.C., Sun, Z., Miguel, A., King, S.R., Cohen, R., Lee, J.M., 2008. Cucurbit grafting. CRC Crit. Rev. Plant Sci. 27, 50–74. https://doi.org/10.1080/ 07352680802053940. del Amor, F.M., 2007. Yield and fruit quality response of sweet pepper to organic and mineral fertilization. Renew. Agric. Food Syst. 22, 233–238. https://doi.org/10. 1017/S1742170507001792. del Amor, F.M., 2006. Growth, photosynthesis and chlorophyll fluorescence of sweet pepper plants as affected by the cultivation method. Ann. Appl. Biol. 148, 133–139. https://doi.org/10.1111/j.1744-7348.2006.00048.x. del Amor, F.M., Gomez-Lopez, M.D., 2009. Agronomical response and water use efficiency of sweet pepper plants grown in different greenhouse substrates. Hortscience 44, 810–814. del Amor, F.M., Marcelis, L.F.M., 2006. Differential effect of transpiration and Ca supply on growth and Ca concentration of tomato plants. Sci. Hortic. (Amsterdam). 111, 17–23. https://doi.org/10.1016/j.scienta.2006.07.032. del Amor, F.M., Serrano-Martínez, A., Fortea, I., Nu~ nez-Delicado, E., 2008. Differential effect of organic cultivation on the levels of phenolics, peroxidase and capsidiol in sweet peppers. J. Sci. Food Agric. 88, 770–777. https://doi.org/10.1002/jsfa.3140. Djian-Caporalino, C., Pijarowski, L., Januel, A., Lefebvre, V., Daubze, A., Palloix, A., Dalmasso, A., Abad, P., 1999. Spectrum of resistance to root-knot nematodes and inheritance of heat-stable resistance in in pepper (Capsicum annuum L.). Theor. Appl. Genet. 99, 496–502. https://doi.org/10.1007/s001220051262. Dorhout, R., Gommers, F.J., Koll€ offel, C., 1991. Water trasport through tomato roots infected with Meloidogyne incognita. Phytopathology 81, 379–385. https://doi.org/ 10.1094/Phyto-81-379. Ghanem, M.E., Albacete, A., Martínez-Andújar, C., Acosta, M., Romero-Aranda, R., Dodd, I.C., Lutts, S., Perez-Alfocea, F., 2008. Hormonal changes during salinityinduced leaf senescence in tomato (Solanum lycopersicum L.). J. Exp. Bot. 59, 3039–3050. https://doi.org/10.1093/jxb/ern153. Gregory, P.J., Atkinson, C.J., Bengough, A.G., Else, M.A., Fernandez-Fernandez, F., Harrison, R.J., Schmidt, S., 2013. Contributions of roots and rootstocks to sustainable, intensified crop production. J. Exp. Bot. 64, 1209–1222. https://doi.org/ 10.1093/jxb/ers385. Guerrero-Díaz, M.M., Lacasa-Martínez, C.M., Hernandez-Pi~ nera, A., Martínez-Alarc on, V., Lacasa-Plasencia, A., 2013. Evaluation of repeated biodisinfestation using Brassica carinata pellets to control Meloidogyne incognita in protected pepper crops. Span. J. Agric. Res. 11, 485–493. King, S.R., Davis, A.R., Liu, W., Levi, A., 2008. Grafting for disease resistance. Hortscience 43, 1673–1676. https://doi.org/AG-675. Kyriacou, M.C., Rouphael, Y., Colla, G., Zrenner, R., Schwarz, D., 2017. Vegetable grafting: the implications of a growing agronomic imperative for vegetable fruit quality and nutritive value. Front. Plant Sci. 8, 1–23. https://doi.org/10.3389/fpls. 2017.00741. Lobna, H., Aymen, E.M., Hajer, R., Naima, M.B., Najet, H.R., 2017. Biochemical and plant nutrient alterations induced by Meloidogyne javanica and Fusarium oxysporum f.Sp.radicis lycopersici co-infection on tomato cultivars with differing level of
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New traits to identify physiological responses induced by different rootstocks after root-knot nematode inoculation (Meloidogyne incognita) in sweet pepper pez-Marín a Amparo Galvez a, Francisco M. del Amor a, *, Caridad Ros b, Josefa Lo a b
Department of Crop Production and Agri-technology, Murcia Institute of Agri-Food Research and Development (IMIDA), C/Mayor s/n, 30150, Murcia, Spain Department of Crop Protection, Murcia Institute of Agri-Food Research and Development (IMIDA), C/Mayor s/n, 30150, Murcia, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords: Greenhouse Sustainability Amino acids Monocropping Meloidogyne incognita Capsicum annuum L
Root-knot nematodes (RKNs) are one of the major phytopathological problems that limit potential yields in intensive agriculture worldwide. Grafting with resistant rootstocks has been demonstrated to be a viable technique to manage Meloidogyne incognita in several crops (tomato, melon, watermelon, etc.). However, little research has been conducted on sweet pepper (Capsicum annuum L.) grown in greenhouses under Mediterraneanclimate conditions. Therefore, we studied a susceptible scion variety (Gacela) grafted onto resistant rootstocks (C19, C25, and RT17), in comparison with ungrafted and self-grafted (GAL) plants. The RKN produced significant root galling in ungrafted and GAL plants; however, RT17 was unaffected. This differing response to infection is discussed from physiological and agronomic perspectives. Thus, following the inoculation of susceptible rootstocks, leaf photosynthesis was impaired by altered stomatal regulation and photochemical efficiency (chlorophyll a fluorescence), which led to unbalanced fruit nutrient concentrations (especially Ca) and to enhanced allocation of carbon from photosynthesis to the production of biochemical defense compounds. Consequently, total phenolics and carotenoids were significantly affected, as was the amino acid profile. These results provide new insights into the traits that can identify RKN-resistant rootstocks, and therefore new tools to induce resistance, while avoiding the use of disinfectants in the soil.
1. Introduction
then migrate intercellularly to the vascular cylinder, where they establish feeding sites and disrupt the vascular tissue (Abad et al., 2003). These alterations have been found to interfere in many physiological and biochemical processes in plants related to photosynthesis and respiration, ion uptake and nutrient concentrations, pigments composition, growth promoters, and metabolism (Strajnar et al., 2012). Moreover, although the scion variety can affect the final yield and fruit quality, rootstock effects can alter these characteristics, depending on the crop management and production environment (Rouphael et al., 2010). Thus, in order to limit the effect of RKNs and to induce resistance, grafting is used as a common practice in many horticultural crops (Davis et al., 2008; King et al., 2008; Oda, 1998). Additionally, recent research has demonstrated the beneficial effects of using grafting to enhance nutrient uptake (Savvas et al., 2017) and fruit quality (Rouphael et al., 2010). Sweet pepper, an important horticultural crop species and a valuable
Root-knot nematodes (RKNs) are a major phytopathological soil problem, that affects sweet pepper (Capsicum annuum L.) (Guerrero-Díaz et al., 2013). In Spain alone, some 2000 ha have been continuously occupied for more than 20 years by a monocropping system for sweet ~ez et al., 2014). These pepper, grown over a 9–10-month cycle (Ros-Iban soils are mainly affected by the RKN, Meloidogyne incognita (Bello et al., 2004). In fact, in horticultural production under plastic the ideal soil conditions (relatively warm, moist, and with adequate texture) and continuous cropping can offer an ideal scenario for populations of RKNs to increase to damaging levels (Aguiar et al., 2014). The primary symptoms of RKN infestation in susceptible plants are anatomical changes in roots, such as giant-cell development and gall formation (Bleve-Zacheo et al., 1998). The RKNs invade the roots in the zone of elongation and
Abbreviations: RKNs, Root-knot nematodes; Pn, Leaf net photosynthesis; SPAD, Hand-held chlorophyll meter; gs, stomatal conductance; E, transpiration rate; iWUE, instantaneous water use efficiency; Fv/Fm, the maximum quantum use efficiency of PSII in the dark-adapted state. * Corresponding author. E-mail address: [email protected] (F.M. del Amor). https://doi.org/10.1016/j.cropro.2019.01.026 Received 6 June 2018; Received in revised form 23 January 2019; Accepted 24 January 2019 Available online 25 January 2019 0261-2194/© 2019 Elsevier Ltd. All rights reserved.
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humidity (%) was: 94.2/24.5/48.9; 95.0/19.9/44.4; 94.7/18.7/43.0; 94.1/18.0/43.5; 94.0/20.0/44.2; 94.1/19.7/45.0; 94.8/18.947.0; and the substrate temperature ( C) inside the pots was: 27.5/9.4/24.5; 28.0/9.8/24.0; 29.2/9.8/23.9; 30.5/10.4/24.4; 31.5/10.6/24.5; 31.0/11.0/24.8; 30.0/12.0/24.9.
source of nutritional and healthy components, is mainly grown in greenhouses (L opez-Marín et al., 2013). However, it is susceptible, on a worldwide basis, to RKNs. Empirically, the influence of roots on shoot behavior can be measured and described as changes in the grafted scion in relation to vegetative and generative growth. However, although the rootstock/scion composite has been used as an experimental system to understand root–shoot communication, the mechanisms governing rootstock x scion phenotypes still remain unclear (Albacete et al., 2015; Ghanem et al., 2008; Gregory et al., 2013). Plants often respond to pathogenic and parasitic pressure by modifying biochemical processes and increasing production of oxidant and antimicrobial metabolites (del Amor et al., 2008). The correct identification of such metabolites specifically involved in defense, which confers increased tolerance, is of paramount importance in developing new products, but also to establish new crop strategies for nematode control. On the other hand, as abiotic stress can trigger systemic defense systems in susceptible plants, there may be a shift in the allocation of carbon structures from photosynthesis to the production of chemical defense compounds, rather than growth (Atkinson et al., 2011); therefore, the yield and quality of the fruits can be impaired. We hypothesize that rootstocks can alter both agronomic and specific physiological responses of scions under the biotic stress provoked by RKNs. Therefore, the aim of this work was to obtain insights into the mechanisms by which grafted plants are able to overcome the deleterious effects of nematodes, and to identify the physiological response mechanisms in sweet pepper which confer this differential tolerance. We focused on three different rootstocks and self-grafted plants, comparing their abilities to mitigate this important biotic stress. Currently, information on how leaf gas exchange and the leaf chlorophylls content are influenced by the specific resistance of a rootstock to nematodes is still scarce, as are data for the nutritional profile and the physiological characterization based on phenolic compounds and carotenoids. Moreover, to our knowledge, this is the first report where the amino-acid profile is analyzed under the combined effects of grafting and nematodes in pepper, which could provide relevant information about its crucial role during pathogen infection. Thus, the overall goal of our research was to help maintain both crop profitability and sustainability in greenhouse systems, through the study of different physiological stress indicators and fruit production.
2.3. Inoculation procedure and gall index The rootstocks were infested by one population of M. incognita. This population is virulent to the Atlante (C25) rootstock and carries the gene Me3 (Sanchez-Solana et al., 2016). It was obtained from a single egg mass of naturally-infested roots of Atlante and belongs to pepper race 2 (Robertson et al., 2006) The isolate was identified in the reference laboratory of the Spanish National Research Council (CSIC, Madrid), where it was determined to be M. incognita isolates race 2, based on the North Carolina differential host test (Robertson et al., 2006, 2009). Each plant was inoculated with 10 mL of a suspension of 2400 eggs mL1, which were poured into two holes around the plant roots - following the methodology used by Djian-Caporalino et al. (1999). At 51, 85, 124, 150, 170, and 196 days after transplanting (DAT), three plants of each inoculation treatment were removed and taken to the laboratory. The roots were washed individually with tap water and examined under a magnifying glass to determine the percentage of plants infested by the nematode and the gall index (GI), which followed the 0–10 scale developed by Bridge and Page (2009); on this scale 0 is a well-developed and healthy root system, and 10 is plants and roots dead. 2.4. Leaf gas exchange Leaf water and gas exchange were monitored in fully-expanded leaves in the generative plant stage, in six leaves for each treatment. The leaf closest to the most-recently-set fruit was used to measure both parameters. Measurements were carried out 20 days after fruit set - 172, 182, and 188 DAT - and at the mature and dark-green fruit stage (196 DAT), from 9:00 a.m. to 11:00 a.m. The net CO2 assimilation rate (Pn), stomatal conductance (gs), transpiration rate (E), and water use efficiency (Pn/E) were measured in the steady state, at saturating light (800 μmol m2 s1 photon flux density) and 400 μmol mol1 CO2, with an LI-6400 portable photosynthesis system (LI-COR Inc., Lincoln, Nebraska, USA).
2. Materials and methods 2.5. Chlorophyll content and fluorescence 2.1. Plant material Chlorophyll fluorescence was determined in the same leaf used for gas exchange determinations, with an ADC Fim 1500 pulse modulated
Using the Japanese top-graft procedure (Oda, 1998), the sweet pepper cultivar ‘Gacela’ F1 (Syngenta Seeds, USA) was grafted onto two commercial rootstocks, Atlante (C25) (Ramiro Arnedo Seeds S.A, La Rioja, Spain) and Robusto (RT17) (Syngenta Seeds S.A, Almeria, Spain), and one non-commercial rootstock, (C19) (Ramiro Arnedo Seeds S.A, La Rioja, Spain), and was also self-grafted (GAL). Ungrafted ‘Gacela’ plants were used as control plants. 2.2. Greenhouse conditions Grafted and ungrafted plants were transplanted into 10-L pots (50% sand, 30% peat, 20% vermiculite) on December 18, 2015, in an unheated, arch-shaped multispan greenhouse covered with thermal polyethylene, located at the ‘Torreblanca’ Experimental Farm of the Murcia Institute of Agri-Food Research and Development (IMIDA) in Murcia, SE Spain (lat. 37 450 N, long. 0 590 W). Irrigation was applied based on estimations of the weekly crop evapotranspiration; the surplus water lost by drainage represented more than 20–25% of the irrigation solution, to maintain the nutrient balance in the rhizosphere and avoid excessive salinity (del Amor and Gomez-Lopez, 2009). The maximum, minimum, and average air temperatures ( C) from December to June were: 29.5/11.0/17.5; 31.1/11.0/17.0; 31.9/11.4/18.0; 35.0/11.8/20.0; 34.4/14.9/22.1; 34.5/16.4/24.1; 35.1/17.0/25.4; the air relative
Fig. 1. Progression of the gall index in the rootstocks of sweet pepper after inoculation with M. incognita. Values are means (n ¼ 3) and vertical bars represent the standard error of the mean. 127
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Fig. 2. Effects of inoculation with M. incognita on A,B) net photosynthesis; C,D) stomatal conductance; E,F) water use efficiency; G,H) chlorophyll fluorescence, and I,J) relative chlorophyll content. Values are means (n ¼ 6) SE. Different letters within a column and graph indicate significant (p 0.05) differences between treatments. Analysis of variance for each parameter: ns, not significant; *p 0.05; **p 0.005; ***p 0.001.
128
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129
* * ns
bc bc a bc b 62.4 59.9 38.9 56.7 53.7
* * ns
bc c a ab a 51.3 41.9 35.1 40.3 53.0
ns * *
a ab ab bc d 0.81 2.21 1.10 2.99 4.32
ns ns ns
ab a a a a 210.3 176.5 179.4 157.1 179.5
** ** *
d c ab bc ab 165.3 132.2 107.6 123.7 107.2
ns * ns
bc c a ab a 741.8 1035.3 324.9 494.5 354.4
ns * ns
bc bc ab ab c 17783.3 17963.0 15902.2 16529.6 19970.4
ns *** ns
ab ab ab ab c 2331.3 2746.3 2618.0 3007.0 3849.4
* ** ns
a a b ab b 28758.6 29122.3 33573.1 31337.6 34447.6
Main factors Rootstocks RKN Rootstocks x RKN
ns * **
a bc b b c Ungrafted GAL C19 C25 RT17 Inoculated
28262.7 37508.3 36542.8 37415.9 45380.8
cd d a bc bc 68.1 71.3 42.9 62.1 61.3 bc d ab bc c 48.4 63.6 36.4 46.6 55.5 ab cd cd cd bc 1.93 3.42 3.35 3.34 2.55 b a a a a 250.1 188.6 165.3 173.2 170.9 bc bc a abc ab 130.0 121.2 96.5 117.9 105.5 a a a a a 294.2 348.1 273.7 255.5 259.2 a ab ab ab bc 31524.5 31070.6 34916.3 35090.0 39840.8
ab ab b b c
2857.4 3672.0 3459.0 3868.7 3510.9
ab c bc c bc
15078.0 15770.1 16798.5 16269.4 17809.8
Mn (mg kg1) Cu (mg kg1) Fe (mg kg1) B (mg kg1)
ab bc bc bc bc 33997.1 40648.9 39232.7 39030.7 37861.3 Ungrafted GAL C19 C25 RT17
Fig. 3. Effects of inoculation with M. incognita on total fruit yield of the scion Gacela grafted onto different rootstocks. Values are means (n ¼ 20) SE. Different letters indicate significant (p 0.05) differences between treatments. Analysis of variance: ns, not significant; *p 0.05; **p 0.005; ***p 0.001.
Uninoculated
The total phenolic compounds were extracted from 0.4 g of frozen fruits (80 C) according to the procedure described in Serrano-Martínez et al. (2008) for sweet peppers, using chlorogenic acid for the quantification. The total phenolic content was expressed as gallic acid equivalents, in mg/100 g fresh weight. The analyses were performed in triplicate for each sample. Determination of total carotenoids was carried out on an aliquot of the hexane extract, by measuring the absorbance at 450 nm in a Varian Cary 50-Bio spectrophotometer (Melbourne, Victoria, Australia). Total carotenoids were calculated according to Almela et al. (1991), using a commercial standard of β-carotene from Sigma (Madrid, Spain). Fifteen replicates were analyzed for each treatment (each replicate contained three fruits).
Na (mg kg1)
2.8. Total phenolic compounds and carotenoids
Mg (mg kg1)
Pepper fruits were dried at 65 C in an oven for 72 h. Cations were extracted from ground material (0.4 g) with 20 mL of deionized water, and determined by inductively coupled plasma–optical emission spectrometry (ICP-OES) (Varian Vista-MPX, Varian Australia, Mulgrave, Victoria, Australia). Fifteen replicates were analyzed for each treatment (each replicate contained three fruits).
P (mg kg1)
2.7. Fruit mineral concentrations
Ca (mg kg1)
Sweet pepper fruits were harvested from 20 plants (5 plants per block, 4 blocks) per treatment. The quality of the marketable commercial fruits was evaluated according to commercial practices, as were discarded fruits with physiological disorders (sun scald, BER, etc.) that were unmarketable (del Amor, 2007).
K (mg kg1)
2.6. Fruit yield
Treatments
Table 1 Effects of inoculation with M. incognita on fruit macro and micro nutrient concentrations. Means in a column followed by the same letter are not significantly different at p 0.05, according to Duncanʼs multiple range test (n ¼ 15). Analysis of variance: ns, not significant; *p 0.05; **p 0.005; ***p 0.001.
fluorometer (Analytical Development Company Ltd., Hoddesdon, UK). Minimal fluorescence values in the dark-adapted state (Fo) were obtained by application of a low-intensity, red-measuring light source (630 nm), while maximal fluorescence values (Fm) were measured after applying a saturating light pulse of 800 μmol m2 s1. In this way, the maximum quantum use efficiency of PSII in the dark-adapted state (30 min) was calculated as Fv/Fm ¼ (Fm Fo)/Fm. The relative chlorophyll content of leaves was determined with a Minolta SPAD-502 m (Konica Minolta Optics, Inc., Japan). This device determines the greenness and the interaction of thylakoid chlorophyll with incident light, taking instant readings without destroying the plant tissue.
Zn (mg kg1)
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and RT17 plants showed higher photosynthetic rates and stomatal conductance than GAL and ungrafted plants, after inoculation, at the end of the crop season. The instantaneous water use efficiency (iWUE: Pn/E) (Fig. 2E and F) was, in general, less affected by inoculation than photosynthesis or stomatal conductance; however, at the end of the study, ungrafted plants showed higher iWUE values than grafted plants (Fig. 2F). The leaf chlorophyll fluorescence was not significantly altered when plants were not inoculated (Fig. 2 G) and the distinct treatments maintained values of between 0.75 and 0.8. Inoculation differentially affected this response, and GAL and RT17 plants maintained the same values as uninoculated plants. The rest of the treatments showed reduced values, but not below 0.75. (Fig. 2H). By contrast, the SPAD values (relative chlorophyll content) were more affected - especially at the last sampling date (Fig. 2J), when the ungrafted plants and C25 rootstock had SPAD values that were 31% and 26% lower, respectively, compared with the uninoculated rootstock. The SPAD values were unaltered for the RT17 rootstock after inoculation.
2.9. Free amino acids Free amino acids were extracted from fruits frozen at 80 C: sap was extracted after vortexing at 5000 g (10 min, 4 C) and analyzed with the AccQTag-ultra ultra-performance liquid chromatography (UPLC) method (Waters, UPLC Amino Acid Analysis Solution. Waters Corporation, Milford, MA, 2006). For derivatization, 70 μL of borate buffer were added to the hydrolyzed sample or to 10 μL of the fruit sap. Next, 20 μL of reagent solution were added. The reaction mixture was mixed immediately and heated at 55 C for 10 min. After cooling, an aliquot of the reaction mixture was used for UPLC injection. The UPLC was performed on an Acquity system (Waters, Milford, Massachusetts, USA), equipped with a fluorescence detection (FLR) system. The column used was a BEH C18 100 mm 2.1 mm, 1.7 μm (Waters). The flow rate was 0.7 mL min1 and the column temperature was kept at 55 C. The injection volume was 1 μL. Wavelength excitation (λex) and emission (λem) were set at 266 and 473 nm, respectively. The solvent system consisted of two eluents: (A) AccQTag-ultra eluent A concentrate (5%, v/v) and water (95%, v/v); (B) AccQTag ultra eluent B. The following elution gradient was used: 0–0.54 min, 99.9% A–0.1% B; 5.74 min, 90.9% A–9.1% B; 7.74 min, 78.8% A–21.2% B; 8.04 min, 40.4% A–59.6% B; 8.05–8.64 min, 10% A–90% B; 8.73–10 min, 99.9% A–0.1% B. Empower 2 (Waters) software was used for system control and data acquisition. External standards (Thermo Scientific) were used for quantification of (Ala) alanine; (Arg) arginine; (Asp) aspartic acid; (Glu) glutamic acid; (Gly) glycine; (His) histamine; (Ile) isoleucine; (Leu) leucine; (Lys) lysine; (Met) methionine; (Phe) phenylalanine; (Pro) proline; (Ser) serine; (Thr) threonine; (Tyr) tyrosine; and (Val) valine. Fifteen replicates were analyzed for each treatment (each replicate contained three fruits). 2.10. Experimental design and statistical analysis The experimental design was a randomized complete block. For yield monitoring, five plants were used in each of four blocks, whilst for destructive sampling (gall index) one plant was harvested in each of three blocks, and for each treatment, at every sampling date (51, 85, 124, 150, 170, and 196 days after transplanting (DAT)). The Statgraphics statistical package was used to calculate significant differences by ANOVA, and means were compared at a probability of p 0.05 according to the Duncan test. The data were checked for normality and homogeneity of variances and were transformed, where required, to their arcsin √x (x ¼ percentage of infested plants) or log10 (x þ 1) (x ¼ the gall index) values.
Fig. 4. Effects of inoculation with M. incognita on of total phenolic in fruits of the scion Gacela grafted onto different rootstocks. Values are means (n ¼ 15) SE. Different letters indicate significant (p 0.05) differences between treatments. Analysis of variance: ns, not significant; *p 0.05; **p 0.005; ***p 0.001.
3. Results 3.1. Root-knot infection Our study shows the effect of inoculation with M. incognita on different rootstocks of sweet pepper during the crop season. The ungrafted plants had the highest GI, whilst C25 and C19 were relatively less affected, with decreases in the GI of 26.3% and 38.9%, respectively, at the last evaluation, compared with the ungrafted plants (Fig. 1). The self-grafted scion (GAL) showed a moderate reduction in the GI (8.9%), but the RT17 rootstock exhibited no progression of galling - indicating its higher tolerance of M. incognita. 3.2. Leaf gas exchange and relative chlorophyll content The leaf CO2 assimilation rate (Fig. 2A and B) and stomatal conductance (Fig. 2C and D) were severely impaired after inoculation in ungrafted plants, and at the end of the study the values of these parameters were reduced by 62.8% and 88.1%, respectively, compared with the control (ungrafted and uninoculated) plants. Self-grafted plants showed a lower impact, but the values of both parameters were reduced with respect to control plants: by 34.5% and 57.4%, respectively. Clearly, C19
Fig. 5. Effects of inoculation with M. incognita on carotenoids in fruits of the scion Gacela grafted onto different rootstocks. Values are means (n ¼ 15) SE. Different letters indicate significant (p 0.05) differences between treatments. Analysis of variance: ns, not significant; *p 0.05; **p 0.005; ***p 0.001. 130
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Table 2 Effects of inoculation with M. incognita on fruit amino acid concentrations. Means in a column followed by the same letter are not significantly different at p 0.05, according to Duncanʼs multiple range test (n ¼ 15). Analysis of variance: ns, not significant; *p 0.05; **p 0.005; ***p 0.001. Treat. Uninoculated
Inoculated
Ser
Arg
Ungraf GAL C19 C25 RT17
604.1 672.1 581.3 546.2 603.4
ab
Ungraf GAL C19 C25 RT17
509.6 596.5 787.8 473.5 509.9
a
Main factors Rootstocks RKN Rootstocks x RKN
ns ns ns
ab ab ab ab
ab b a a
Gly
593.0 495.1 525.1 464.6 541.0
a
425.0 538.6 626.5 392.8 410.6
a
ns ns ns
a a a a
a a a a
Asp
81.3 61.1 56.0 52.3 91.5
cd
42.0 51.5 71.9 38.1 46.1
ab
abc abc abc d
abc bcd a ab
ns ** ns
Glu
94.6 130.4 111.7 61.4 68.8
abc
88.1 96.0 155.7 57.2 64.6
abc
ns ns ns
bc abc ab ab
abc c a ab
Thr
292.9 136.0 86.8 126.9 183.4
bc
384.5 378.4 134.5 123.7 129.8
c
*** * ns
a a a ab
c a a a
Ala
264.3 224.8 155.8 164.2 215.4
c
129.3 161.7 149.9 136.4 148.2
a
* *** **
b b b b
b a a a
296.8 279.5 195.1 171.3 326.6
c
135.3 115.0 275.4 173.8 164.7
a
c b b c
a c b b
ns * ns
in ungrafted plants were significantly lessened, specifically for Gly, Thr, Ala, Cys, Val, Ile, Leu, and Phe; in contrast, Pro was the only amino acid whose concentration increased due to inoculation in these plants, and the same response of Pro was also found in self-grafted (GAL) plants.
3.3. Fruit yield The severity of the RKN symptoms on the sweet pepper plants was clearly reflected in the fruit yield (Fig. 3), as was expected since it integrates many of the physiological processes affected by the action of nematodes in the roots. Firstly, in the absence of biotic stress the rootstock significantly altered the response of the scion; thus, plants on C19 or RT17 gave lower yields than ungrafted, self-grafted, and C25 plants. However, the rootstock/scion combinations that gave lower yields under control conditions were less affected by RKNs and - in contrast ungrafted, GAL, and C25 plants showed higher impacts.
4. Discussion Grafting is used in many countries, in protected cultivation - in which land is intensively used and crops are not rotated, allowing improvements in both productivity and fruit quality (Kyriacou et al., 2017). Moreover, the correct choice of rootstock is of paramount importance for the performance of a vulnerable crop (L opez-Perez et al., 2006). Our study shows that RKNs induced severe changes in the roots, as indicated by the progression of the GI in susceptible rootstocks. A promising way to improve pepper resistance to RKNs is the grafting of commercial cultivars onto nematode-resistant rootstocks, but the complexity of the response and differences among genotypes and growth environments could minimize its effectiveness. Our study shows the disturbance of root anatomy and of several physiological and biochemical processes caused by M. incognita in sweet pepper under greenhouse cultivation. Dorhout et al. (1991) reported that infestation increased axial resistance to water flow and reduced total water uptake in tomato. Our data show the differential impact of the rootstock on water use efficiency and, especially, stomatal conductance - which were severely impaired in ungrafted plants after inoculation. Leaf photosynthesis was also reduced in plants on susceptible rootstocks. Melakeberhan et al. (1987) reported, for soybean, that M. incognita can contribute to premature leaf abscission and chlorosis, which can affect photosynthesis. We observed that the reduction in the photosynthesis rate was linked to decreases in stomatal conductance and also in chlorophyll fluorescence (maximum quantum yield of the primary photochemistry of PSII). Thus, the RT17 and GAL treatments maintained the Fv/Fm values, but for ungrafted, C19, and C25 plants the values of this parameter were reduced at the end of the crop season, after inoculation. Therefore, we suggest that the main effect of M. incognita on the process of photosynthesis in sweet pepper is on stomatal closure and diffusive resistance, but there is also metabolic uncoupling. This agreed with the reduced relative chlorophyll content in the leaves. This important effect was not observed when plants were only submitted to nutritional depletion (del Amor, 2006). Thus, Campos et al. (2014) indicated that further processes beyond nutritional impairment are implicated in the photosynthetic response, and emphasized the importance of maintaining a proper water balance to avoid slowdown of the electron transfer in the reaction centers of the photosystem, which could become more relevant as a result of the effect of M. incognita on stomatal closure. This response of susceptible rootstocks with regard to stomatal regulation has important consequences for an appropriate and balanced nutrient uptake
3.4. Mineral concentrations For inoculated plants, the less susceptible rootstocks, RT17 and C19, gave Ca concentrations that were higher than in ungrafted or self-grafted (GAL) plants (Table 1) (e.g. the Ca concentration was 16.5% higher in RT17 than in ungrafted plants). However, this pattern was reversed for Na, B, and Mn: RT17 and C19 showed lower concentrations compared with the ungrafted or self-grafted (GAL) plants. The concentrations of P and, especially, Cu were notably higher in the RT17 plants; in the fruits they were increased by 39.4% and 81.3%, respectively, relative to the uninoculated plants. 3.5. Total phenolics and carotenoids Regarding the concentration of total phenolics in the fruits, there were significant differences among the studied rootstocks (Fig. 4). Thus, the plant response to RKN inoculation boosted total phenolics by 34% for the ungrafted plants, and by 37% and 33% for GAL and C25, respectively, when compared with the uninoculated plants. However, this increase in total phenolics was attenuated for the C19 rootstock, and the level in RT17 was unaffected by inoculation. The total carotenoids (Fig. 5) showed a similar response to inoculation, the C19 and RT17 rootstocks being unaffected. 3.6. Total amino acid content The effect of grafting on the total amino acid concentration was relevant in the absence of inoculation (Table 2). Thus, the most noteworthy effect was on RT17, which had higher concentrations of Gly and Met than the rest of the treatments. Moreover, the ungrafted plants had the highest levels of Thr, Cys, Lys, Val, Leu, and Phe. In general, the selfgrafted (GAL) plants showed a pattern similar to that of the C19 and C25 plants in the absence of stress (RKNs). However, after inoculation, the previously observed effect was altered. Thus, the higher concentrations 131
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Pro
Cys
bc 55.8 a 49.3 ab 51.3 bc 56.6 a 48.2 e 68.8 e 69.9 cd 60.3 b 66.3 b 51.7 Main factors * *** ns
388.1 262.9 198.3 225.6 245.5 210.0 246.4 331.2 179.3 198.4 * ns **
Lys c ab a a ab a ab bc a a
459.8 308.1 306.6 319.2 318.9 469.4 428.7 373.3 361.2 367.6 ** ns ns
Met c a a ab ab c bc abc abc abc
10.4 12.3 10.8 8.5 17.9 7.2 5.4 5.4 5.5 14.4 ns ** ns
Val bc cd cde abc d ab a a a cd
Ile d
356.0 242.7 243.8 228.6 245.8 183.1 212.9 215.2 168.9 193.8
c c bc c ab abc abc a abc
** *** ***
154.2 128.3 146.1 96.8 108.1 108.6 91.8 111.0 79.0 86.9
Leu d cd d ab bc bc ab bc a ab
* * ns
134.2 84.4 76.0 95.9 101.4 93.6 112.0 107.4 68.3 79.3 * ns *
Phe e bc ab bc cd bc de cd a ab
55.8 39.4 48.9 36.8 52.1 37.8 33.6 30.0 32.1 33.6 ns ** ns
Total AA e bc c ab d ab ab a ab ab
3841.2 3138.4 3137.5 2697.0 3168.1 2892.4 2859.7 2858.4 2480.7 2766.3
d c c b c b b b a b
** *** *
Our study highlights the necessity of a more physiological approach to the effects of RKNs on sweet pepper - to improve techniques, select resistant rootstocks, and develop efficient monitoring traits to detect potential yield impairments at reduced environmental cost. Future studies should analyze the impairment by M. incognita of stomatal regulation and the quantum efficiency of PSII, as both reduce the rate of photosynthesis and, consequently, plant growth and yield. Moreover, regulation of water uptake and transport linked with the appropriate nutritional control - to counterbalance reduced Ca, P, and Cu uptake together with exogenous addition of the target amino acids whose levels were significantly impaired after infection should be part of the solution to improve tolerance of M. incognita in sweet pepper.
by these plants. Thus, as the Ca concentration in horticultural products has been linked to mass flow (water uptake) (del Amor and Marcelis, 2006), our results indicate that the significant effect of inoculation on stomatal regulation clearly affected Ca uptake and lowered the Ca concentration in fruits on susceptible rootstocks and, especially, in ungrafted plants. We also observed higher fruit concentrations of P and Cu for the resistant RT17 rootstock, compared with the sensitive ones, which indicates that the general advantages of grafting with respect to RKNs could not be generalized to the rest of the nutrients under the studied stress. The rootstock/graft interaction can potentially affect yield and fruit quality, while it confers tolerance of this biotic stress (King et al., 2008). This effect is shown in our study, where the beneficial (and differential) influence of grafting on total fruit yield was clearly affected after RKN infestation. Thus, the combined effects of the RKNs on leaf gas exchange and specific nutrient concentrations were less severe for the RT17 and C19 rootstocks; however, such rootstocks did not show higher yields in the absence of infestation (compared to susceptible rootstocks or ungrafted plants). This important result indicates that these RKN-resistant rootstocks can deliver significant advantages only for infested soils. In general, phenolics have been shown to be related to plant resistance to different pests and pathogens, and previous research found a correlation of elevated levels of phenolics with resistance or a greater response of plants to nematode infection (Chitwood, 2002). However, our results show a bigger response for ungrafted, GAL, and C25 plants, and consequently a greater correlation with pathogen infection (GI) than with resistance, which agrees with recent research for tomato (Lobna et al., 2017). Carotenoids had a similar behavior after inoculation for the RT17 and C19 treatments, in which the inoculation effect was very limited. Other studies of nematodes and carotenoids showed similar results (Atkinson et al., 2011) and no response was found. However, Arimboor et al. (2015) recently indicated that changes in the content and structure of carotenoids in plants can also be markers of environmental stress, which agrees with our results. Amino acids can act as substrates for the pathogen or have fungistatic effects, the response being genotype-dependent, and changes in their individual abundances can be attributed to the susceptibility of the genotype (Ndoumou et al., 1996). Our results indicate that inoculation significantly reduced the sum of the amino acids for all treatments; therefore, this parameter cannot be considered a tolerance trait, but, individually, differences were found. Thus, pathogen infection has been shown to lead to specific changes in the expression of many genes involved in amino acid metabolism and transport (Pratelli and Pilot, 2014). In sweet pepper a fraction of the amino acids in susceptible rootstocks could be used for the synthesis of compounds, like phenolic derivatives, involved in plant defense mechanisms.
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