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Article

Rootstock Influence on Growth and Mineral Content of Citrus limon and Citrus sinensis cv. Valencia Inoculated with Candidatus Liberibacter Asiaticus

by
Criseida Alhelí Sáenz-Pérez
1,
Eduardo Osorio-Hernández
1,*,
Benigno Estrada-Drouaillet
1,
Sergio Castro-Nava
1,
Rafael Delgado-Martínez
1,
Claudia Magdalena López-Badillo
2 and
Raúl Rodríguez-Herrera
2
1
Division of Postgraduate Studies and Research, Faculty of Engineering and Sciences, Autonomous University of Tamaulipas, Adolfo López Mateos University Center, Victoria, Tamaulipas 87120, Mexico
2
School of Chemistry, Autonomous University of Coahuila, Boulevard Venustiano Carranza and Ing. José Cárdenas Valdés, Col. República, Saltillo, Coahuila 25280, Mexico
*
Author to whom correspondence should be addressed.
Agronomy 2020, 10(10), 1564; https://doi.org/10.3390/agronomy10101564
Submission received: 4 September 2020 / Revised: 2 October 2020 / Accepted: 10 October 2020 / Published: 14 October 2020
(This article belongs to the Special Issue Citrus Production and Protection from Pests and Diseases)
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Versions Notes

Abstract

:
Huanglongbing (HLB) reduces the growth and development of citrus and induces changes in secondary metabolites such as flavonoids, limonoids, and polyamines. Likewise, infected plants have a deficient absorption of nutrients such as zinc, potassium, manganese, and copper. Therefore, the objective of this study was to evaluate the influence of different rootstocks on morphology and mineral changes of Citrus limon and Citrus sinensis cv. Valencia plants inoculated with Candidatus Liberibacter asiaticus. In a greenhouse of the Experimental Station-Autonomous University of Tamaulipas, the Candidatus Liberibacter asiaticus bacteria were inoculated to Citrus limon plants (growing on Citrus volkameriana, Citrus macrophylla, and Citrus aurantium rootstocks) and Citrus sinensis cv. Valencia (growing on Citrus volkameriana and Citrus aurantium rootstocks). The experiment was established under a completely randomized design with 45 graft/rootstock repetitions. In each graft/rootstock combination, the plant height and stem diameter were determined using a tape measurer and a Vernier, respectively. In addition, the nutrient content of foliar samples was determined by an X-ray fluorescence spectrometer. In both citrus species, the C. aurantium rootstock promoted a higher concentration of the bacteria. On the other hand, the rootstock that showed the best agronomical results after inoculation with the bacteria was C. volkameriana, presenting the least variation in mineral content and conferring greater plant height (15%) and stem diameter (23%). In contrast, the presence of Ca. Liberibacter asiaticus decreased S content and increased Cu concentration in C. lemon plants. Similarly, plants infected with C. sinensis presented higher Fe content. Finally, in both species, no significant differences were observed for Mn, P, and Zn concentration.

1. Introduction

One of the factors that reduce citrus production is the presence of different diseases, among them, Huanglongbing disease (HLB), which is one of the biggest problems in citrus worldwide, reduces production up to 50%, and in advanced stages of infection, may cause death of trees [1,2]. Globally, every year, the HLB causes losses of billions of dollars in the citrus industry [3]. In different citrus areas around the world, HLB is caused by different strains of Candidatus Liberibacter; however, in Mexico and the Caribbean, the asiaticus strain is responsible for this disease. In Mexico, the decrease in production has reached 25%, that is, 1.84 million tons of citrus fruits [4]. The Candidatus Liberibacter asiaticus bacterium is spread by the Asian citrus psyllid (Diaphorina citri) [5]. The dissemination of the bacteria begins in the phloem; this plant tissue is affected by secretions of the bacteria that consist of dependent effectors and virulence factors. Likewise, Candidatus Liberibacter asiaticus interferes with protein mobilization and causes cell death and phloem necrosis [1]. In addition, within the primary infection process, the bacterium reaches the roots through the phloem [6]. Later, a dark material appears in the roots between the plasma membrane and cell walls. As this material accumulates, the roots show a decline and a low starch content [7]. Therefore, the growth of vertical roots decreases by 20% compared to horizontal roots, and finally, the root system shows a decline and death [8]. However, root death could also be a secondary symptom of infection in the absence of carbohydrates [6].
The pathogen raises starch concentration in the vascular bundles, induces a nutritional deficiency that is expressed in the leaves through irregular spots, and causes thickening of midribs [9]. In addition, it affects plant growth and development [10]; more specifically, in leaves and fruits it induces changes in secondary metabolites content, affecting flavonoids, limonoids, hydroxycinnamic acids, and polyamines [11,12]. Besides, the pathogen uses the nutrients present in the vascular bundle for its growth and reproduction; therefore, the plant has a deficient absorption of nutrients such as zinc, potassium, manganese, and copper [13,14]. Thus, diseased plants show a decrease of 16% to 30% in canopy height and volume [15]. In addition, the concentrations of these minerals vary depending on the cultivar and rootstock used [16]. The scion-rootstock connection is essential for optimal growth, absorption, and transport of water and nutrients [17]. In addition, the transmissibility of scion impacts plant shape and physiology. This is due to long-distance communication pathways between cells and may involve hormones, metabolites, water, and nutrient availability, as well as other molecules (proteins, transcripts, and sRNA) that provide a direct link to the underlying genetic mechanisms [18].
Recently, the University of Florida and USDA/ARS in the USA have conducted joint research for the development of rootstocks resistant to HLB [19]. Evaluations have been made with different graft/rootstock combinations, for example: ‘Hamlin/Kinkoji’, ‘Hamlin/Cleopatra’, ‘Temple/Cleopatra’, ‘Fallglo/Kinkoji’, ‘Sugar Belle/Sour Orange’, ‘Tango/Kuharske’, and ’Ruby Red/Kinkoji’, to name a few [20].
The most used technology for this bacterium detection is based on polymerase chain reaction (PCR), while techniques to determine changes in chemical compounds and mineral contents include gas chromatography, spectroscopy, and chemical analysis of volatile organic compounds [21,22]. However, these detection techniques are expensive or require a long time [23]. Another methodology for assessing mineral concentration is X-ray fluorescence spectrometry [24], which has the advantage that can generate spectra of a wide range of samples (powders, liquids, among others) with minimum sample preparation, and its analysis is performed in a short time. In addition, it has quantitative applications based on the relationship between spectral, and reference data obtained by conventional methods to establish prediction models and qualitative applications, to illustrate the studied diversity and classifying the samples according to their spectral characteristics [25]. Therefore, the objective of this study was to evaluate the changes in minerals and morphology of Citrus limon and Citrus sinensis plants inoculated with Candidatus Liberibacter asiaticus grown on different rootstocks.

2. Materials and Methods

2.1. Plant Material and Experimental Design

The experiment was established in a greenhouse of the “Ingeniero Herminio García González”, Experimental Station-Autonomous University of Tamaulipas (23°56′26.5″ North Latitude and 99°05′59.9″ West Longitude, and 193 masl) at Güémez, Tamaulipas, México on January 2018. Italian lemon (Citrus limon) (which were grafted on Citrus aurantium, Citrus macrophylla, and Citrus volkameriana) and Valencia orange (Citrus sinensis) (grafted on Citrus aurantium and Citrus volkameriana) rootstocks were grown under a completely randomized design. In each scion/rootstock combination, 40 plants were inoculated, and 5 plants were used as a control. After 6 months of inoculation, the data were collected monthly for two years.

2.2. Bacterial Inoculation of Plant Materials

One-and-a-half-year-old Citrus limon and Citrus sinensis plants grown under greenhouse conditions were grafted on January 2018 with symptomatic HLB buds (the presence of Candidatus Liberibacter asiaticus was previously confirmed by PCR). In addition, different plants were grafted with pathogen-free buds, which were used as a control.

2.3. Bacteria Identification

DNA extraction was performed from the leaf tissue of infected and control plants. Briefly, 0.05 g of leaf tissues was homogenized and 1 mL of the genomic DNA isolation reagent (Bio Basic Canda Inc., Markham ON, Canada) was added; then samples were centrifuged at 12,000× g for 10 min at 4 °C. The precipitate (pastille) was washed with 75% ethanol and then centrifuged and re-suspended in 30 µL of molecular biology grade water. The confirmation of the bacteria was carried out using the methodology reported by Li [26].

2.4. Test Parameters and Data Analysis

2.4.1. Test Parameters

Response variables included plant height, which was measured from the base of the bag to the apex of the highest leaf using a measuring tape. Likewise, the stem diameter was measured at 15 cm below the graft union by employing an electronic vernier Steren HER-411 (Steren®, Mexico City, Mexico). Both variables were measured monthly over a period of two years.

2.4.2. Mineral Content

To determine the mineral content from each specie/rootstock combination, a foliar sample was taken from the 40 inoculated plants to obtain a combined sample. In the same way, a combined sample was obtained from the 5 repetitions of healthy plants in each specie/rootstock combination. Then, 1 g of sampled graft leaves (previously dried and crushed) was placed on the energy dispersion X-ray fluorescence spectrometer (ED-XRF) (Epsilon 1, Malvern Panalytical, Almelo, The Netherlands). The underside of the tube was completely covered and, between each reading, the reader was cleaned with methanol. Mineral content analysis (Mg, Al, Si, P, S, Cl, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, and Zn) was performed using the software Omnian® (Malvern Panalytical, Almelo, The Netherlands) in a period of 20 min per sample.

2.4.3. Data Analysis

A discriminant canonical analysis was performed to identify if the nutrients, plant height, and stem diameter were associated with the phytosanitary state of Italian lemon and Valencia orange scions, which were inoculated with Ca. Liberibacter asiaticus. In addition, a cluster analysis was carried out with each citrus species in their respective rootstock and the concentration of minerals in order to determine whether the scion/rootstock combination and phytosanitary status influence nutrient absorption. Finally, the data were analyzed using ANOVA, and treatment means of plant height and rootstock stem diameter were compared using the Tukey multiple range tests (p < 0.05), and standard deviations were also determined. Data were analyzed using the SAS software Version 9.0 (SAS Institute Inc., Cary, NC, USA) [27].

3. Results

3.1. Bacteria Identification

Table 1 shows the Cq values for each graft/rootstock combination. Likewise, during means comparison of Cq values (Table 2), significant differences were observed among the rootstocks in the two cultivars. When C. limon was developed on C. aurantium, the concentration of the bacteria rose 5% compared to the rest of the slides. Similarly, in the combination C. sinensis/C. aurantium, the concentration of Ca. Liberibacter asiaticus increased by 5%. Likewise, both lemon and orange plants did not present symptoms of HLB.

3.2. Plant Height and Stem Diameter

No significant differences (p ≤ 0.05) for plant height and stem diameter were observed between healthy and infected Italian lemon and Valencia orange plants in a general way (Table 3). However, when the variables were evaluated taking into account different rootstocks in each of the species, it was observed that uninfected Italian lemon and orange plants did not show significant differences in plant height and stem diameter between each of the rootstocks (Table 4).
In contrast, infected plants that were developed on rootstocks of C. volkameriana were significantly larger in height (15%) and stem diameter (23%) compared to C. aurantium and C. macrophylla. Like Italian lemon, other lemon species were affected by HLB. With C. sinensis, none of the rootstocks showed significant differences in plant height. However, when plants were infected, C. volkameriana showed a higher height (17%) compared to C. aurantium. In addition, for uninfected and infected plants, the stem diameter in C. volkameriana was 25% higher than C. aurantium (Table 4).

3.3. Mineral Concentration of Citrus limon and Citrus sinensis in Uninfected and Infected Plants

When a comparison of means was performed (Table 5), it was observed that most of the mineral contents in both citrus plants did not show significant differences (p ≤ 0.05). However, in plants infected with C. limon, Cu significantly increased (77%) but sulfur decreased by 18%. In C. sinensis infected with HLB, the Fe content increased significantly (20%).

3.4. Standardized Canonical (Can) Nutrient Coefficients, Plant Height, and Rootstock Stem Diameter

The discriminant canonical analysis showed that the first canonical correlation was significant (0.0049 < 0.05) and explains 99.97% of the variability that was recorded on nutrient concentrations, plant height, and stem diameter (Table 6). Therefore, the first principal component is associated with the variables of nutrient concentration, plant height, and rootstock stem diameter and helped to discriminate the phytosanitary state of the Italian lemon and Valencia orange scions. The second canonical correlation was not significant (0.88 > 0.05). That is, the second principal component has no association with the variables and, therefore, does not help to determine the phytosanitary status of the evaluated citrus.
In order of importance, the variable that shapes the component and achieves a separation between the groups is Ca (90.20), then Cl (89.39), and in a smaller proportion K (57.59) and height (28.91). On the contrary, stem diameter (−15.01) does not contribute anything to the model (Table 1).

3.5. Significance of the Mahalanobis Distances between Each of the Groups and Canonical Dispersions of Uninfected and Infected Species

Table 7 shows the significance of the Mahalanobis distances between each of the groups. That is, it shows the separation between uninfected and infected plants of Italian lemon and Valencia orange cv. Valencia, respectively. The distances of uninfected lemon with the remaining groups, infected lemon (5695), uninfected orange (7398), and infected orange (15914), were significant (p < 0.05). However, there was no significant distance between infected lemon (group 1) and uninfected orange (group 2); therefore, the distance between these two groups is equal to 0. On the other hand, there is a significant distance of 2573 between the uninfected lemon and infected oranges. Finally, the distance of 1614 between the groups of uninfected and infected orange (2 and 3, respectively) is significant, that is, the distance is non-zero. Therefore, Figure 1 shows the separation between each of the evaluated groups (uninfected lemon, infected lemon, uninfected orange, and infected orange) plants.

3.6. Dendrogram of Mineral Content in Citrus limon and Citrus sinensis (Graft/Rootstock) Treatments

When a division of the dendrogram (Figure 2) was performed at 70% of the total distance, four different groups were observed: group 1 formed by treatment 8, group 2 composed of treatments 2, 9, and 10, group 3 with treatments 4 and 7, and group 4 formed by treatments 1, 3, 5, and 6. In the different combinations of scion/rootstock, it was observed that inoculation with Ca. Liberibacter asiaticus affected the mineral content since two different combinations of scion/rootstock did not have the same content. Branches of different lengths were observed, the smallest branches corresponded to C. limon/C. volkameriana (infected and uninfected), which suggests that there were fewer changes in mineral content due to inoculation with the bacteria.
The next least affected treatments were infected and uninfected C. sinensis/C. volkameriana due to being grouped, although they had larger branches than those of C. limon/C. volkameriana. The mineral concentration of the other combinations was greatly affected since the infected and uninfected versions were not grouped. For example, according to its mineral content, the uninfected C. limon/C. macrophylla forms a group with treatment 1 (uninfected C. limon/C. aurantium) and then is grouped with treatment 7 (uninfected C. sinensis/C. aurantium). On the other hand, the combination C. limon/C. aurantium (in its two versions) had a great disparity in concentration and different minerals.

4. Discussion

4.1. Detection of Ca. Liberibacter Asiaticus

The number of PCR cycles ranged from 18 to 38, indicating abundant to moderate target nucleic acid in the sample, respectively, and samples are accurately identified as infected or uninfected, as they are considered positive for Ca. Liberibacter asiaticus when the cycle quantification values (Cq) <40 [28]. Likewise, there is a high correlation between bacterial concentration and the severity of HLB. That is, as the inoculum of the bacteria increases, is greater the expression of severity [29]. Therefore, C. aurantium rootstock is not suitable for C. limon and C. sinensis since it had the highest level of bacteria inoculum. On the contrary, C. macropylla and C. volkameriana present lower Cq values. Furthermore, the symptoms of HLB did not occur in either species; this is because at times the expression of symptoms can be sporadic, or the plant can be asymptomatic [30].

4.2. Plant Height and Stem Diameter

In previous research, the susceptibility of 18 citrus species to HLB was evaluated 6 months after inoculating the plants with bacteria. They mentioned that plant height and stem diameter were 2% to 28% significantly lower in infected plants in comparison to uninfected plants [31]. In the same way, another study [32] evaluated the effects of HLB on seven-year-old Valencia orange trees and observed a significant reduction (33% and 21%) in fruit yield and height of plants that tested positive for the pathogen. Probably, there were no significant differences due to a scarce or zero overproduction of starch that would obstruct the vascular bundles and prevent the absorption of water and nutrients [13]. Furthermore, other authors mentioned that other lemon varieties, such as rough lemon, have less sensitivity to HLB attack [33]. Moreover, C. aurantifolia showed lower height (17.7%) and stem diameter (20%) when they were positive to Ca. Liberibacter asiaticus [31].
In previous works, the development of Valencia orange plants on 15 different rootstocks was evaluated to determine if rootstocks have an effect on HLB development. They observed an increase in plant height (16.3%) and stem diameter (19.1%) when orange was grown on C. volkameriana in comparison when it was grown on C. aurantium. It should be noted that these authors mentioned that rootstocks conferred vigor to the trees on C. volkameriana and showed higher tolerance to HLB and growth and less canopy damage. Therefore, they suggested that this rootstock can allow trees in younger stages to overcome the adverse effects of the disease [34]. The tolerance mechanism of this rootstock against HLB occurs through the expression of proteins not susceptible to diseases (lectin-related proteins, hitinase, and miraculin-like protein), the photosynthesis protein, and redox homeostasis [35].
Citrus sinensis is less sensitive to HLB when it grows on C. volkameriana; species such as Siem Pontianak (C. nobilis Lour) and Keprok Tejakula (C. reticulata Blanco) show lower bacterial concentration, less intensity of canopy damage, and greater plant height and stem diameter when grafted on C. volkameriana [36]. In addition, C. aurantium, C. macrophylla, and C. volkameriana are varieties moderately tolerant to HLB [37].
Previous studies indicate that the susceptibility of citrus fruits is closely related to the content of secondary metabolites; the most susceptible citrus have high levels of proline, serine, aspartic acid, galactose, among others. In contrast, less susceptible citrus species have high levels of glycine and mannose [38]. In addition to metabolites, rootstocks also have a direct effect on the stem in the presence of hormones, availability of water and nutrients, as well as other molecules (proteins, transcripts, and RNAs) that can act as a defense mechanism in the presence of pathogens [18].

4.3. Mineral Content of Citrus limon and Citrus sinensis

Mineral deficiencies such as Zn, P, Mn, and Cu usually occur in HLB-infected plants [13,14,39]. However, in the specific case of Zn, this nutrient did not decrease in any of the infected combinations compared to healthy plants. However, high levels of Zn were reported in citrus with HLB located in Punjab, Pakistan; the plants presented up to 11% more concentration of this nutrient than healthy plants. Similar results were also observed in Florida, United States, in lemon plants (C. limon cv. ‘Todo del Ano’ grafted on C. paradisi cv. ‘Duncan’ rootstock) infected with HLB, where the Zn content tended to increase in lemon varieties and decrease in others citrus [40]. These results are contradictory with those presented in other investigations, and they may have their origin in the physiological changes caused by HLB or the coexistence of Phytophthora [41].
In previous studies, it has been observed that HLB-infected C. sinensis plants show significant decreases in Cu and Mn because C. sinensis is one of the most sensitive species to HLB compared to other citrus species [31]. Therefore, one of the measures to mitigate the HLB attack is the applications of Cu [42]. However, in the present investigation, no significant differences were observed in the concentrations of Cu and Mn. On the contrary, Cu increased in plants infected with C. lemon.
In the case of P, deficiencies of up to 26% are more likely in older infected trees [43]. Potassium has great mobility and is a promoter of flowering, increasing the number of fruits, and enhancing the transport of water, sugars, and other nutrients [44]. Consequently, when the plant lacks potassium, symptomatic fruits have a lower sugar concentration and high acidity [45].
Therefore, as in the present investigation there were no significant variations of P in either of the two species, this behavior may be correlated with the age of the plants due to the fact that C. limon and C. sinensis were too young [43].
Another mineral found in lower concentrations in plants infected with HLB is Mg [46]. This mineral participates in photosynthesis, affecting chloroplasts and functions related to sugar accumulation and metabolic activities [47]. Since there were no significant differences in the concentration of Mg in either of the two species, the absence of green and irregular spots on the leaf, characteristic symptoms of the disease, would be explained [46].
Ca concentration changed in none of the species. On the contrary, in other investigations, it has been observed that Citrus sinensis cv. Navelina has a higher Ca content when infected with HLB [48]. The literature mentions that citrus fruits infected with HLB can present Ca deficiencies because bacteria use this mineral for their growth [49]. Therefore, the plant presents an active abiotic or biotic stress through the signaling pathways of Ca kinase and calmodulin-like proteins as defense mechanisms; and Ca is another nutrient that improves the health status of the plant [50,51]. This mechanism occurs across the plasma membrane via permeable ion channels [39]. In addition, calcium strengthens cell walls and provides greater protection against pathogens, which is one of the reasons why symptomatic fruits with HLB are harder and have a thicker shell [48,52].
K also did not vary between healthy and infected plants of C. lemon and C. sinensis. In previous research, a 34% reduction in K was observed in seven citrus species infected with HLB [53]. This mineral can mitigate the adverse effects of Ca. Liberibacter asiaticus, as it performs an antibacterial function by being present in protein synthesis, carbohydrate metabolism, and enzyme activation [54,55].
Like the aforementioned nutrients, HLB also causes Fe deficiencies [42,56]. On the contrary, the concentration of this mineral was higher (20%) in infected plants of C. sinensis and there were no variations in the content of C. limon. In this same species, a sulfur deficiency was observed; it should be clarified that other works do not report deficiencies of this mineral but it is an essential nutrient in foliar nutrition programs to counteract the adverse effects of HLB for its antibacterial activity [56,57].
Some of the results of this study differ with those reported in the literature, this is because different species were evaluated. In addition, factors such as soil, climate, and stage of development of HLB-infected plants have a direct influence on the data analyzed [58]. Finally, in some cases, there are no differences in nutrient content between healthy and HLB-infected plants [59]. The way each nutrient contributes to a plant’s response varies, and in general, the interactions between nutrients and pathogens are not well understood [46]. Nutrient deficiencies or excesses in citrus fruits negatively affect susceptibility to HLB through metabolic changes in plants, thus creating a more favorable environment for this disease development [60].

5. Conclusions

In both citrus species, the C. aurantium rootstock showed a higher concentration of the bacteria. On the other hand, the rootstock that showed the best results after inoculation with the bacteria was C. volkameriana, presenting the least variation in mineral content and conferring greater plant height (15%) and stem diameter (23%). In contrast, the presence of Ca. Liberibacter asiaticus decreased S content and increased Cu concentration in C. lemon plants. Similarly, infected plants with C. sinensis presented higher Fe content. Finally, in both species, no significant differences were observed in the concentration of Mn, P, and Zn.

Author Contributions

Conceptualization, E.O.-H.; methodology, C.A.S.-P., E.O.-H., and R.D.-M.; software, C.A.S.-P.; validation, E.O.-H., C.M.L.-B., and R.R.-H.; formal analysis, C.A.S.-P., C.M.L.-P., and R.R.-H.; investigation, C.A.S.-P.; resources, E.O.-H., C.M.L.-B., and R.R.-H.; data curation, C.A.S.-P., B.E.-D., and R.R.-H.; writing—original draft preparation, C.A.S.-P.; writing—review and editing, C.A.S.-P., E.O.-H., R.D.-M., S.C.-N., and R.R.-H.; visualization, E.O.-H., S.C.-N., and R.R.-H.; supervision, E.O.-H.; project administration, E.O.-H.; funding acquisition, C.A.S.-P. and E.O.-H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Autonomous University of Tamaulipas, grant number UATINVES20-24.

Acknowledgments

The authors would like to thank the Consejo Nacional de Ciencia y Tecnología (CONACYT) for the scholarship for the first author and the Comité Estatal de Sanidad Vegetal de Tamaulipas (CESAVETAM) for their support in carrying out this project.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 10 01564 g001 550
Figure 1. Canonical dispersions of uninfected Italian lemon (A), infected Italian lemon (B), uninfected Valencia orange (B’), and infected Valencia orange (C). The ovals represent each of the aforementioned groups. Likewise, figures represent the values of the canonical dispersions, the white figures are the centroids and in the case of group C, the centroid is located in the larger X.
Figure 1. Canonical dispersions of uninfected Italian lemon (A), infected Italian lemon (B), uninfected Valencia orange (B’), and infected Valencia orange (C). The ovals represent each of the aforementioned groups. Likewise, figures represent the values of the canonical dispersions, the white figures are the centroids and in the case of group C, the centroid is located in the larger X.
Agronomy 10 01564 g001
Agronomy 10 01564 g002 550
Figure 2. Dendrogram of treatments in Citrus limon and Citrus sinensis (graft/rootstock) plants. Uninfected (−) and infected (+) graft/rootstock, 1: −C. limon/C. aurantium), 2: +C. limon/C. aurantium 3: −C. limon/C. macrophylla, 4: +C. limon/C. macrophylla, 5: −C. limon/C. volkameriana, 6: +C. limon/C. volkameriana, 7: −C. sinensis/C. aurantium, 8: +C. sinensis/C. aurantium, 9: −C. sinensis/C. volkameriana and 10: +C. sinensis/C. volkameriana.
Figure 2. Dendrogram of treatments in Citrus limon and Citrus sinensis (graft/rootstock) plants. Uninfected (−) and infected (+) graft/rootstock, 1: −C. limon/C. aurantium), 2: +C. limon/C. aurantium 3: −C. limon/C. macrophylla, 4: +C. limon/C. macrophylla, 5: −C. limon/C. volkameriana, 6: +C. limon/C. volkameriana, 7: −C. sinensis/C. aurantium, 8: +C. sinensis/C. aurantium, 9: −C. sinensis/C. volkameriana and 10: +C. sinensis/C. volkameriana.
Agronomy 10 01564 g002
Table
Table 1. Values of the quantification cycle (Cq) of the bacterium Candidatus Liberibacter asiaticus in foliar samples of Citrus limon and Citrus sinensis developed in different patterns.
Table 1. Values of the quantification cycle (Cq) of the bacterium Candidatus Liberibacter asiaticus in foliar samples of Citrus limon and Citrus sinensis developed in different patterns.
C. limonC. sinensis
PlantC. aurantiumC. macrophyllaC. volkamerianaC. aurantiumC. volkameriana
Cq Value
126.2832.6235.3434.4230.26
235.4335.1136.5833.1230.23
333.1234.6734.8532.5629.71
434.3234.3936.3634.3428.50
532.1834.0835.2934.2730.64
633.5737.2532.9232.0330.15
733.7533.3034.0224.0529.75
835.2634.8132.5632.9629.25
935.3636.2034.7433.2831.57
1034.0235.4833.7531.930.12
1132.3237.2338.1129.0529.44
1234.0435.2935.0234.6431.14
1331.5936.3734.5331.2331.09
1436.3437.2736.2832.9321.11
1534.7237.6437.4735.8827.65
1634.6636.6836.1526.3031.02
1734.1836.0436.1126.0230.18
1832.9536.8236.4125.5030.12
1932.5536.3934.5427.0730.76
2035.1236.2835.0624.0631.43
2132.1936.9534.2224.6029.74
2233.9631.0434.6527.1132.84
2332.0837.1234.6328.1229.52
2435.0736.0734.3228.1732.02
2535.4735.6134.4628.9129.45
2634.4237.0636.4619.4418.46
2734.1636.0636.2123.7931.26
2836.436.9336.2225.4631.44
2934.7536.8634.4224.3931.60
3034.8136.9134.2426.5328.39
3134.9037.0636.3726.9729.38
3236.0836.9735.8728.7830.82
3335.2536.9535.0227.0233.52
3434.8136.3134.5227.0131.67
3536.1236.8934.4426.9532.78
3636.1337.5534.4126.8333.12
3735.3237.1534.6229.5831.51
3834.3136.3934.6727.4133.40
3936.4036.5734.6323.4632.63
4035.2836.1034.7728.3431.84
Table
Table 2. Means comparison of quantification cycle (Cq) values of C. limon and C. sinensis developed on different rootstocks.
Table 2. Means comparison of quantification cycle (Cq) values of C. limon and C. sinensis developed on different rootstocks.
SpecieRootstockCq Values
C. limonC. aurantium34.24 b
C. macrophylla36.06 a
C. volkameriana35.13 ab
C. sinensisC. aurantium28.61 d
C. volkameriana30.23 c
Values with the same letter within a column are not statistically different as determined by the Tukey test (p ≤ 0.05). MSD: Minimum significant difference.
Table
Table 3. Plant height and stem diameter of uninfected and infected C. limon and C. sinensis plants.
Table 3. Plant height and stem diameter of uninfected and infected C. limon and C. sinensis plants.
Species PresenceHeight (cm)Stem (mm)
C. limonNot infected167.66 a16.44 a
Infected179.93 a16.81 a
MSD13.141.25
C. sinensisNot infected149.40 a15.26 a
Infected154.43 a15.74 a
MSD19.832.08
Values with the same letter within a column are not statistically different as determined by the Tukey test (p ≤ 0.05). MSD: Minimum significant difference.
Table
Table 4. Plant height and rootstock stem diameter of uninfected and infected C. limon and C. sinensis plants.
Table 4. Plant height and rootstock stem diameter of uninfected and infected C. limon and C. sinensis plants.
C. limon
RootstockNot InfectedInfected
Height (cm)Stem (mm)Height (cm)Stem (mm)
C. volkameriana170.60 a17.82 a196.88 a19.35 a
C. macrophylla162.40 a16.86 a176.27 b16.32 b
C. aurantium167.0 a15.18 a166.63 b14.76 c
MSD51.954.3110.700.67
C. sinensis
RootstockNot InfectedInfected
Height (cm)Stem (mm)Height (cm)Stem (mm)
C. volkameriana149.20 a18.02 a169.45 a17.48 a
C. aurantium149.60 a13.46 b131.61 b13.04 b
MSD22.052.7312.170.99
Values with the same letter within a column are not statistically different as determined by the Tukey test (p ≤ 0.05). MSD: Minimum significant difference.
Table
Table 5. Means comparison of mineral concentration (ppm) in Citrus limon and Citrus sinensis.
Table 5. Means comparison of mineral concentration (ppm) in Citrus limon and Citrus sinensis.
C. limon
(ppm)
MgAlSiPSClKCa
NI7.40 a1.02 a0.32 a1.38 a3.97 a6.15 a27.89 a50.33 a
I8.47 a1.04 a 0.56 a1.40 a3.27 b7.63 a24.56 a51.47 a
MSD1.230.340.960.490.322.014.986.7
ScTiVCrMnFeCuZn
NI0.11 a0.0009 a0.00002 a0.00001 a0.08 a0.23 a0.08 b0.03 a
I0.11 a0.00009 a0.00002 a0.00001 a0.08 a0.24 a0.01 a0.03 a
MSD0.030.010.00000050.00000020.010.120.020.02
C. sinensis
(ppm)
MgAlSiPSClKCa
NI10.18 a0.98 a0.56 a1.51 a2.99 a9.88 a20.52 a51.79 a
I9.8 a0.99 a0.46 a1.48 a3.63 a8.38 a17.42 a56.22 a
MSD8.751.100.600.386.5110.3054.1227.70
ScTiVCrMnFeCuZn
NI0.11 a0.0050 a0.00003 a0.00002 a0.08 a0.20 b0.10 a0.03 a
I0.07 a0.00008 a0.00003 a0.000009 a0.07 a0.25 a0.08 a0.03 a
MSD0.980.060.00010.00000060.290.030.040.06
Values with the same letter within a column are not statistically different as determined by the Tukey test (p ≤ 0.05). MSD: Minimum significant difference. Status: NI (not infected), I (Infected).
Table
Table 6. Standardized canonical (Can) nutrient coefficients, plant height, and rootstock stem diameter.
Table 6. Standardized canonical (Can) nutrient coefficients, plant height, and rootstock stem diameter.
VariableCan1Can2Can3
Cl89.397376212.96360649−0.78333747
K57.792354623.53450965−1.92557449
Ca90.200948922.60397644−2.05580445
Height28.914025371.13598256−0.16235885
Stem−15.01864614−0.506272750.78861065
Table
Table 7. The significance of Mahalanobis distances between uninfected and diseased Italian lemon and Valencia orange plants.
Table 7. The significance of Mahalanobis distances between uninfected and diseased Italian lemon and Valencia orange plants.
Presence0123
005695739815914
15695 *0114.042573
27398 *114.04 §01614
315914 *2573 *1614 *0
*: p ≤ 0.05, §: p > 0.05. 0 = not infected Italian lemon, 1 = infected Italian lemon, 2 = not infected Valencia orange and 3 = infected Valencia orange.

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Sáenz-Pérez, C.A.; Osorio-Hernández, E.; Estrada-Drouaillet, B.; Castro-Nava, S.; Delgado-Martínez, R.; López-Badillo, C.M.; Rodríguez-Herrera, R. Rootstock Influence on Growth and Mineral Content of Citrus limon and Citrus sinensis cv. Valencia Inoculated with Candidatus Liberibacter Asiaticus. Agronomy 2020, 10, 1564. https://doi.org/10.3390/agronomy10101564

AMA Style

Sáenz-Pérez CA, Osorio-Hernández E, Estrada-Drouaillet B, Castro-Nava S, Delgado-Martínez R, López-Badillo CM, Rodríguez-Herrera R. Rootstock Influence on Growth and Mineral Content of Citrus limon and Citrus sinensis cv. Valencia Inoculated with Candidatus Liberibacter Asiaticus. Agronomy. 2020; 10(10):1564. https://doi.org/10.3390/agronomy10101564

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Sáenz-Pérez, Criseida Alhelí, Eduardo Osorio-Hernández, Benigno Estrada-Drouaillet, Sergio Castro-Nava, Rafael Delgado-Martínez, Claudia Magdalena López-Badillo, and Raúl Rodríguez-Herrera. 2020. "Rootstock Influence on Growth and Mineral Content of Citrus limon and Citrus sinensis cv. Valencia Inoculated with Candidatus Liberibacter Asiaticus" Agronomy 10, no. 10: 1564. https://doi.org/10.3390/agronomy10101564

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