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

Rootstock Influence on Growth and Mineral Content of Citrus limon and Citrus sinensis cv.... agronomy Article Rootstock Influence on Growth and Mineral Content of Citrus limon and Citrus sinensis cv. Valencia Inoculated with Candidatus Liberibacter Asiaticus 1 1 , 1 Criseida Alhelí Sáenz-Pérez , Eduardo Osorio-Hernández *, Benigno Estrada-Drouaillet , 1 1 2 Sergio Castro-Nava , Rafael Delgado-Martínez , Claudia Magdalena López-Badillo and Raúl Rodríguez-Herrera Division of Postgraduate Studies and Research, Faculty of Engineering and Sciences, Autonomous University of Tamaulipas, Adolfo López Mateos University Center, Victoria, Tamaulipas 87120, Mexico; alhe_saenz@outlook.com (C.A.S.-P.); benestrada@docentes.uat.edu.mx (B.E.-D.); scastro@docentes.uat.edu.mx (S.C.-N.); rdelgado@docentes.uat.edu.mx (R.D.-M.) School of Chemistry, Autonomous University of Coahuila, Boulevard Venustiano Carranza and Ing. José Cárdenas Valdés, Col. República, Saltillo, Coahuila 25280, Mexico; cllopezb@uadec.edu.mx (C.M.L.-B.); raul.rodriguez@uadec.edu.mx (R.R.-H.) * Correspondence: eosorio@docentes.uat.edu.mx Received: 4 September 2020; Accepted: 10 October 2020; Published: 14 October 2020 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 di erent 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 di erences were observed for Mn, P, and Zn concentration. Keywords: Huanglongbing (HLB); height; scion; Citrus volkameriana; X-ray fluorescence spectrometry 1. Introduction One of the factors that reduce citrus production is the presence of di erent 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 di erent citrus areas around the world, HLB is caused by di erent strains of Candidatus Liberibacter; however, Agronomy 2020, 10, 1564; doi:10.3390/agronomy10101564 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1564 2 of 14 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 a ected by secretions of the bacteria that consist of dependent e ectors 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 a ects plant growth and development [10]; more specifically, in leaves and fruits it induces changes in secondary metabolites content, a ecting 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 di erent 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 di erent 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 Agronomy 2020, 10, 1564 3 of 14 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, di erent 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 L 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]. Agronomy 2020, 10, 1564 4 of 14 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 di erences 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. 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 di erent patterns. C. limon C. sinensis C. aurantium C. macrophylla C. volkameriana C. aurantium C. volkameriana Plant Cq Value 1 26.28 32.62 35.34 34.42 30.26 2 35.43 35.11 36.58 33.12 30.23 3 33.12 34.67 34.85 32.56 29.71 4 34.32 34.39 36.36 34.34 28.50 5 32.18 34.08 35.29 34.27 30.64 6 33.57 37.25 32.92 32.03 30.15 7 33.75 33.30 34.02 24.05 29.75 8 35.26 34.81 32.56 32.96 29.25 9 35.36 36.20 34.74 33.28 31.57 10 34.02 35.48 33.75 31.9 30.12 11 32.32 37.23 38.11 29.05 29.44 12 34.04 35.29 35.02 34.64 31.14 13 31.59 36.37 34.53 31.23 31.09 14 36.34 37.27 36.28 32.93 21.11 15 34.72 37.64 37.47 35.88 27.65 16 34.66 36.68 36.15 26.30 31.02 17 34.18 36.04 36.11 26.02 30.18 18 32.95 36.82 36.41 25.50 30.12 19 32.55 36.39 34.54 27.07 30.76 20 35.12 36.28 35.06 24.06 31.43 21 32.19 36.95 34.22 24.60 29.74 22 33.96 31.04 34.65 27.11 32.84 23 32.08 37.12 34.63 28.12 29.52 24 35.07 36.07 34.32 28.17 32.02 25 35.47 35.61 34.46 28.91 29.45 26 34.42 37.06 36.46 19.44 18.46 27 34.16 36.06 36.21 23.79 31.26 28 36.4 36.93 36.22 25.46 31.44 29 34.75 36.86 34.42 24.39 31.60 30 34.81 36.91 34.24 26.53 28.39 31 34.90 37.06 36.37 26.97 29.38 32 36.08 36.97 35.87 28.78 30.82 33 35.25 36.95 35.02 27.02 33.52 34 34.81 36.31 34.52 27.01 31.67 35 36.12 36.89 34.44 26.95 32.78 36 36.13 37.55 34.41 26.83 33.12 37 35.32 37.15 34.62 29.58 31.51 38 34.31 36.39 34.67 27.41 33.40 39 36.40 36.57 34.63 23.46 32.63 40 35.28 36.10 34.77 28.34 31.84 Agronomy 2020, 10, 1564 5 of 14 Table 2. Means comparison of quantification cycle (Cq) values of C. limon and C. sinensis developed on di erent rootstocks. Specie Rootstock Cq Values C. aurantium 34.24 b C. limon C. macrophylla 36.06 a C. volkameriana 35.13 ab C. aurantium 28.61 d C. sinensis C. volkameriana 30.23 c Values with the same letter within a column are not statistically di erent as determined by the Tukey test (p 0.05). MSD: Minimum significant di erence. 3.2. Plant Height and Stem Diameter No significant di erences (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 di erent rootstocks in each of the species, it was observed that uninfected Italian lemon and orange plants did not show significant di erences in plant height and stem diameter between each of the rootstocks (Table 4). Table 3. Plant height and stem diameter of uninfected and infected C. limon and C. sinensis plants. Species Presence Height (cm) Stem (mm) Not infected 167.66 a 16.44 a C. limon Infected 179.93 a 16.81 a MSD 13.14 1.25 Not infected 149.40 a 15.26 a Infected 154.43 a 15.74 a C. sinensis MSD 19.83 2.08 Values with the same letter within a column are not statistically di erent as determined by the Tukey test (p 0.05). MSD: Minimum significant di erence. Table 4. Plant height and rootstock stem diameter of uninfected and infected C. limon and C. sinensis plants. C. limon Not Infected Infected Rootstock Height (cm) Stem (mm) Height (cm) Stem (mm) C. volkameriana 170.60 a 17.82 a 196.88 a 19.35 a C. macrophylla 162.40 a 16.86 a 176.27 b 16.32 b C. aurantium 167.0 a 15.18 a 166.63 b 14.76 c MSD 51.95 4.31 10.70 0.67 C. sinensis Not Infected Infected Rootstock Height (cm) Stem (mm) Height (cm) Stem (mm) C. volkameriana 149.20 a 18.02 a 169.45 a 17.48 a C. aurantium 149.60 a 13.46 b 131.61 b 13.04 b MSD 22.05 2.73 12.17 0.99 Values with the same letter within a column are not statistically di erent as determined by the Tukey test (p 0.05). MSD: Minimum significant di erence. 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 a ected by HLB. With C. sinensis, none of the rootstocks showed Agronomy 2020, 10, 1564 6 of 14 significant di erences 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 di erences (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%). Table 5. Means comparison of mineral concentration (ppm) in Citrus limon and Citrus sinensis. C. limon (ppm) Mg Al Si P S Cl K Ca NI 7.40 a 1.02 a 0.32 a 1.38 a 3.97 a 6.15 a 27.89 a 50.33 a I 8.47 a 1.04 a 0.56 a 1.40 a 3.27 b 7.63 a 24.56 a 51.47 a MSD 1.23 0.34 0.96 0.49 0.32 2.01 4.98 6.7 Sc Ti V Cr Mn Fe Cu Zn NI 0.11 a 0.0009 a 0.00002 a 0.00001 a 0.08 a 0.23 a 0.08 b 0.03 a I 0.11 a 0.00009 a 0.00002 a 0.00001 a 0.08 a 0.24 a 0.01 a 0.03 a MSD 0.03 0.01 0.0000005 0.0000002 0.01 0.12 0.02 0.02 C. sinensis (ppm) Mg Al Si P S Cl K Ca NI 10.18 a 0.98 a 0.56 a 1.51 a 2.99 a 9.88 a 20.52 a 51.79 a I 9.8 a 0.99 a 0.46 a 1.48 a 3.63 a 8.38 a 17.42 a 56.22 a MSD 8.75 1.10 0.60 0.38 6.51 10.30 54.12 27.70 Sc Ti V Cr Mn Fe Cu Zn NI 0.11 a 0.0050 a 0.00003 a 0.00002 a 0.08 a 0.20 b 0.10 a 0.03 a I 0.07 a 0.00008 a 0.00003 a 0.000009 a 0.07 a 0.25 a 0.08 a 0.03 a MSD 0.98 0.06 0.0001 0.0000006 0.29 0.03 0.04 0.06 Values with the same letter within a column are not statistically di erent as determined by the Tukey test (p 0.05). MSD: Minimum significant di erence. Status: NI (not infected), I (Infected). 3.4. Standardized Canonical (Can) Nutrient Coecients, 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. Table 6. Standardized canonical (Can) nutrient coecients, plant height, and rootstock stem diameter. Variable Can1 Can2 Can3 Cl 89.39737621 2.96360649 0.78333747 K 57.79235462 3.53450965 1.92557449 Ca 90.20094892 2.60397644 2.05580445 Height 28.91402537 1.13598256 0.16235885 Stem 15.01864614 0.50627275 0.78861065 Agronomy 2020, 10, x FOR PEER REVIEW 7 of 14 Table 6. Standardized canonical (Can) nutrient coefficients, plant height, and rootstock stem diameter. Variable Can1 Can2 Can3 Cl 89.39737621 2.96360649 −0.78333747 K 57.79235462 3.53450965 −1.92557449 Agronomy 2020, 10, 1564 7 of 14 Ca 90.20094892 2.60397644 −2.05580445 Height 28.91402537 1.13598256 −0.16235885 In order of importance, the variable that shapes the component and achieves a separation between Stem −15.01864614 −0.50627275 0.78861065 the groups is Ca (90.20), then Cl (89.39), and in a smaller proportion K (57.59) and height (28.91). On 3.5. the Signif contrary icance of t , stem he Ma diameter halanobi ( s Distan 15.01) c does es bet not ween Eac contribute h of thanything e Groups and to the Canoni model cal Disper (Table sio 1).ns of Uninfected and Infected Species 3.5. Significance of the Mahalanobis Distances between Each of the Groups and Canonical Dispersions of Table 7 shows the significance of the Mahalanobis distances between each of the groups. That Uninfected and Infected Species is, it shows the separation between uninfected and infected plants of Italian lemon and Valencia Table 7 shows the significance of the Mahalanobis distances between each of the groups. That is, orange cv. Valencia, respectively. The distances of uninfected lemon with the remaining groups, it shows the separation between uninfected and infected plants of Italian lemon and Valencia orange cv. infected lemon (5695), uninfected orange (7398), and infected orange (15914), were significant (p < Valencia, respectively. The distances of uninfected lemon with the remaining groups, infected lemon 0.05). However, there was no significant distance between infected lemon (group 1) and uninfected (5695), uninfected orange (7398), and infected orange (15914), were significant (p < 0.05). However, orange (group 2); therefore, the distance between these two groups is equal to 0. On the other hand, there was no significant distance between infected lemon (group 1) and uninfected orange (group 2); there is a significant distance of 2573 between the uninfected lemon and infected oranges. Finally, the therefore, the distance between these two groups is equal to 0. On the other hand, there is a significant distance of 1614 between the groups of uninfected and infected orange (2 and 3, respectively) is distance of 2573 between the uninfected lemon and infected oranges. Finally, the distance of 1614 significant, that is, the distance is non-zero. Therefore, Figure 1 shows the separation between each between the groups of uninfected and infected orange (2 and 3, respectively) is significant, that is, of the evaluated groups (uninfected lemon, infected lemon, uninfected orange, and infected orange) the distance is non-zero. Therefore, Figure 1 shows the separation between each of the evaluated plants. groups (uninfected lemon, infected lemon, uninfected orange, and infected orange) plants. Table 7. The significance of Mahalanobis distances between uninfected and diseased Italian lemon Table 7. The significance of Mahalanobis distances between uninfected and diseased Italian lemon and and Valencia orange plants. Valencia orange plants. Presence 0 1 2 3 Presence 0 1 2 3 0 0 5695 7398 15914 0 0 5695 7398 15914 1 5695 * 0 114.04 2573 1 5695 * 0 114.04 2573 2 7398 * 114.04 § 0 1614 2 7398 * 114.04 § 0 1614 3 15914 * 2573 * 1614 * 0 3 15914 * 2573 * 1614 * 0 *: p ≤ 0.05, §: p > 0.05. 0 = not infected Italian lemon, 1 = infected Italian lemon, 2 = not infected Valencia *: 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. orange and 3 = infected Valencia orange. Figure 1. Canonical dispersions of uninfected Italian lemon (A), infected Italian lemon Figure 1. Canonical dispersions of uninfected Italian lemon (A), infected Italian lemon (B), uninfected (B), uninfected Valencia orange (B’), and infected Valencia orange (C). The ovals represent each Valencia orange (B’), and infected Valencia orange (C). The ovals represent each of the of the aforementioned groups. Likewise, figures represent the values of the canonical dispersions, aforementioned groups. Likewise, figures represent the values of the canonical dispersions, the white the white figures are the centroids and in the case of group C, the centroid is located in the larger X. figures are the centroids and in the case of group C, the centroid is located in the larger X. 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 di erent 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 di erent combinations of scion/rootstock, it was observed that inoculation with Ca. Liberibacter asiaticus a ected the mineral content since two di erent combinations of scion/rootstock did not have Agronomy 2020, 10, x FOR PEER REVIEW 8 of 14 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 Agronomy 2020, 10, 1564 8 of 14 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 the same content. Branches of di erent lengths were observed, the smallest branches corresponded grouped with treatment 7 (uninfected C. sinensis/C. aurantium). On the other hand, the combination to C. limon/C. volkameriana (infected and uninfected), which suggests that there were fewer changes C. limon/C. aurantium (in its two versions) had a great disparity in concentration and different in mineral content due to inoculation with the bacteria. minerals. Figure 2. Dendrogram of treatments in Citrus limon and Citrus sinensis (graft/rootstock) plants. 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 Uninfected (−) and infected (+) graft/rootstock, 1: −C. limon/C. aurantium), 2: +C. limon/C. aurantium 3: 3: C. limon/C. macrophylla, 4: +C. limon/C. macrophylla, 5: C. limon/C. volkameriana, 6: +C. limon/C. −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 volkameriana, 7: −C. sinensis/C. aurantium, 8: +C. sinensis/C. aurantium, 9: −C. sinensis/C. volkameriana and and 10: +C. sinensis/C. volkameriana. 10: +C. sinensis/C. volkameriana. The next least a ected treatments were infected and uninfected C. sinensis/C. volkameriana due to 4. Discussion being grouped, although they had larger branches than those of C. limon/C. volkameriana. The mineral concentration of the other combinations was greatly a ected since the infected and uninfected versions 4.1. Detection of Ca. Liberibacter Asiaticus were not grouped. For example, according to its mineral content, the uninfected C. limon/C. macrophylla The number of PCR cycles ranged from 18 to 38, indicating abundant to moderate target nucleic forms a group with treatment 1 (uninfected C. limon/C. aurantium) and then is grouped with treatment acid in the sample, respectively, and samples are accurately identified as infected or uninfected, as 7 (uninfected C. sinensis/C. aurantium). On the other hand, the combination C. limon/C. aurantium (in its they are considered positive for Ca. Liberibacter asiaticus when the cycle quantification values (Cq) two versions) had a great disparity in concentration and di erent minerals. <40 [28]. Likewise, there is a high correlation between bacterial concentration and the severity of HLB. 4. ThDiscussion at 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 4.1. Detection of Ca. Liberibacter Asiaticus bacteria inoculum. On the contrary, C. macropylla and C. volkameriana present lower Cq values. 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% Agronomy 2020, 10, 1564 9 of 14 to 28% significantly lower in infected plants in comparison to uninfected plants [31]. In the same way, another study [32] evaluated the e ects 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 di erences 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 di erent rootstocks was evaluated to determine if rootstocks have an e ect 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 e ects 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 e ect 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 di erences 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]. Agronomy 2020, 10, 1564 10 of 14 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, a ecting chloroplasts and functions related to sugar accumulation and metabolic activities [47]. Since there were no significant di erences 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 e ects 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 e ects of HLB for its antibacterial activity [56,57]. Some of the results of this study di er with those reported in the literature, this is because di erent 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 di erences 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 a ect 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 di erences 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. Agronomy 2020, 10, 1564 11 of 14 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. References 1. Wang, N.; Pierson, E.A.; Setubal, J.A.; Xu, J.; Levy, J.G.; Zhang, Y.; Li, J.; Rangel, L.T.; Martins, J. The Candidatus Liberibacter–host interface: Insights into pathogenesis mechanisms and disease control. Ann. Rev. Phytopathol. 2017, 55, 451–482. [CrossRef] 2. Robles-González, M.M.; Orozco-Santos, M.; Manzanilla-Ramírez, M.A.; Velázquez-Monreal, J.J.; Medina-Urrutia, V.M.; Sanches-Stuchi, E. Experiencias con Huanglongbing en limón mexicano, en el estado de Colima, México. Citrus Res. Technol. 2018, 39, 2–12. [CrossRef] 3. Rao, M.J.; Ding, F.; Wang, N.; Deng, X. Metabolic mechanisms of host species against citrus Huanglongbing (Greening Disease). Crit. Rev. Plant Sci. 2019, 37, 1–16. [CrossRef] 4. Salcedo-Baca, D.; Hinojosa, R.A.; Mora-Aguilera, G.; Covarrubias-Gutierrez, I.; DePaolis, F.J.; Mora-Flores, J.S.; Cíntora, C. Evaluación del impacto económico de la enfermedad de los cítricos Huanglongbing (hlb) en la cadena citrícola mexicana. IICA, SAGARPA, SENASICA. México. 2011, pp. 40–47. Available online: http://repiica.iica.int/docs/b2146e/b2146e.pdf (accessed on 20 June 2020). 5. Hall, D.G. Incidence of “Candidatus Liberibacter asiaticus” in a Florida population of Asian citrus psyllid. J. Appl. Entomol. 2018, 142, 97–103. [CrossRef] 6. Johnson, E.G.; Wu, J.; Bright, D.B.; Graham, J.H. Association of ‘Candidatus Liberibacter asiaticus’ root infection, but not phloem plugging with root loss on huanglongbing-a ected trees prior to appearance of foliar symptoms. Plant Pathol. 2014, 63, 290–298. [CrossRef] 7. Achor, D.; Welker, S.; Mahmoud, S.B.; Wang, C.; Folimonova, S.Y.; Dutt, M.; Gowda, S.; Levy, A. Dynamics of Candidatus Liberibacter asiaticus Movement and Sieve-Pore Plugging in Citrus Sink Cells. Plant Physiol. 2020, 182, 882–891. [CrossRef] [PubMed] 8. Louzada, E.S.; Vasquez, O.E.; Braswell, W.E.; Yanev, G.; Devanaboina, M.; Kunta, M. Distribution of ‘Candidatus Liberibacter asiaticus’ Above and Below Ground in Texas Citrus. Phytopathology 2016, 106, 702–709. [CrossRef] [PubMed] 9. Deng, H.; Achor, D.; Extberria, E.; Yu, Q.; Du, D.; Stanton, D.; Liang, G.; Gmitter, F.G., Jr. Phloem Regeneration Is a Mechanism for Huanglongbing-Tolerance of “Bearss” Lemon and “LB8-9” Sugar Belle Mandarin. Plant Sci. 2019, 10, 1–13. [CrossRef] [PubMed] 10. Da-Graca, J.V.; Douhan, G.W.; Halbert, S.E.; Keremane, M.L.; Lee, R.F.; Vidalakis, G. Huanglongbing: An overview of a complex athosystem ravaging the world’s citrus. J. Integr. Plant Biol. 2016, 58, 373–387. [CrossRef] [PubMed] 11. Suh, J.H.; Niu, Y.S.; Wang, Z.; Gmitter, F.G.; Wang, Y. Metabolic analysis reveals altered long-chain fatty acid metabolism in the host by Huanglongbing disease. J. Agric. Food. Chem. 2018, 66, 1296–1304. [CrossRef] [PubMed] 12. Dala-Paula, B.M.; Plotto, A.; Bai, J.; Manthey, J.A.; Baldwin, E.A.; Ferrarezi, R.S. E ect of Huanglongbing or greening disease on orange juice quality, a Review. Plant Sci. 2019, 9, 1–19. [CrossRef] [PubMed] 13. Slisz, A.M.; Breksa, A.P.; Mishchuk, D.O.; McCollum, G.; Slupsky, C.M. Metabolomic analysis of citrus infection by ‘Candidatus Liberibacter ’ reveals insight into pathogenicity. J. Proteome Res. 2012, 11, 4223–4230. [CrossRef] 14. Gómez-Flores, W.; Garza-Saldaña, J.J.; Varela-Fuentes, S.E. Detection of Huanglongbing disease based on intensity-invariant texture analysis of images in the visible spectrum. Comput. Electron. Agric. 2019, 162, 825–835. [CrossRef] 15. Li, J.; Li, L.; Kolbasov, V.; Ehsani, R.; Carter, E.; Wang, N. Developing citrus Huanglongbing management strategies based on the severity of symptoms in HLB-endemic citrus-producing regions. Phytopathology 2018, 109, 582–592. [CrossRef] [PubMed] 16. Albrecht, U.; Tripathi, I.; Kim, H.; Bowman, K.D. Rootstock e ects on metabolite composition in leaves and roots of young navel orange (Citrus sinensis L. Osbeck) and pummelo (C. grandis L. Osbeck) trees. Trees 2018, 33, 243–265. [CrossRef] Agronomy 2020, 10, 1564 12 of 14 17. Martínez-Ballesta, M.C.; Alcaraz-López, C.; Muries, B.; Mota-Cadenas, C.; Carvajal, M. Physiological aspects of rootstock–scion interactions. Sci. Hortic. 2010, 127, 112–118. [CrossRef] 18. Warschefsky, E.J.; Klein, L.L.; Frank, M.H.; Chitwood, D.H.; Londo, J.P.; Wettberg, V.E.J.B.; Miller, A.J. Rootstocks: Diversity, domestication, and impacts on shoot phenotypes. Plant. Sci. 2016, 21, 418–437. [CrossRef] 19. Graham, J.; Gottwald, T.; Setamou, M. Status of Huanglongbing (HLB) outbreaks in Florida, California and Texas. Trop. Plant Pathol. 2020, 45, 265–278. [CrossRef] 20. Stover, E.; Inch, S.; Richardson, M.L.; Hall, D.G. Conventional Citrus of Some Scion/Rootstock Combinations Show Field Tolerance under High Huanglongbing Disease Pressure. Hort. Sci. 2016, 51, 127–132. [CrossRef] 21. Aksenov, A.A.; Pasamontes, A.; Peirano, D.J.; Zhao, W.; Dandekar, A.M.; Fiehn, O.; Eshani, N.; Davis, C.E. Detection of Huanglongbing Disease Using Di erential Mobility Spectrometry. Anal. Chem. 2014, 86, 2481–2488. [CrossRef] 22. Garza-Saldaña, J.J.; Varela-Fuentes, S.; Gómez-Flores, W. Métodos para la detección presuntiva de Huanglongbing (HLB) en cítricos. Ciencia UAT 2017, 11, 93–104. [CrossRef] 23. Arredondo-Valdés, R.; Delgado-Ortíz, J.C.; Beltrán-Beache, M.; Anguiano-Cabello, J.; Cerna-Chávez, E.; Rodríguez-Pagasa, Y.; Ochoa-Fuentes, Y.M. A review of techniques for detecting Huanglongbing (greening) in citrus. Can. J. Microbiol. 2016, 62, 803–811. [CrossRef] 24. Hitpold, I.; Demarta, L.; Johnson, S.N.; Moore, B.D.; Power, S.A.; Mitchel, C. Silicon and other essential element composition in roots sing X-ray fluorescence spectroscopy: A high throughput approach. In Proceedings of the Ninth ACGIE, 1st ed.; Invertebrate Ecology of Australasian Grasslands: Hawkesbury, Australia, 2017; pp. 191–196. 25. Canteri, M.H.G.; Renard, C.M.G.; Le Bourvellec, C.; Bureau, S. ATR-FTIR spectroscopy to determine cell wall composition: Application on a large diversity of fruits and vegetables. Carbohydr. Polym. 2019, 212, 186–196. [CrossRef] [PubMed] 26. Li, W.; Hartung, J.S.; Lew, L. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus Huanglongbing. J. Microbiol. Meth. 2006, 66, 104–115. [CrossRef] [PubMed] ® ® 27. SAS Institute Inc. The SAS System for Windows (Ver. 9.0); SAS Institute Inc: Cary, NC, USA, 2004. 28. Ammar, E.D.; Hall, D.G.; Alvarez, J.M. E ect of Cyantraniliprole, a Novel Insecticide, on the Inoculation of Candidatus Liberibacter Asiaticus Associated with Citrus Huanglongbing by the Asian Citrus Psyllid (Hemiptera: Liviidae). J. Econ. Entomol. 2015, 108, 399–404. [CrossRef] [PubMed] 29. Flores-Sánchez, J.-L.; Mora-Aguilera, G.; Loaeza-Kuk, E.; López-Arroyo, J.I.; Domínguez-Monge, S.; Acevedo-Sánchez, G.; Robles-García, P. Yield loss caused by Candidatus Liberibacter asiaticus in Persian lime, in Yucatan Mexico. Rev. Mex. Fitopatol. 2015, 33, 195–210. 30. Folimonova, S.Y.; Robertson, C.J.; Garnsey, S.M.; Gowda, S.; Dawson, W.O. Examination of the Responses of Di erent Genotypes of Citrus to Huanglongbing (Citrus Greening) Under Di erent Conditions. Phytopathology 2009, 99, 1346–1354. [CrossRef] 31. Shokrollah, H.; Abdullah, T.L.; Sijam, K.; Abdullah, S.N.A. Potential use of selected citrus rootstocks and interstocks against HLB disease in Malaysia. Crop Prot. 2011, 30, 521–525. [CrossRef] 32. Bowman, K.D.; McCollum, G.; Albrecht, U. Performance of ‘Valencia’ orange (Citrus sinensis [L.] Osbeck) on 17 rootstocks in a trial severely a ected by huanglongbing. Sci. Hortic. 2016, 201, 355–361. [CrossRef] 33. Fan, J.; Chen, C.; Yu, Q.; Khalaf, A.; Achor, D.S.; Brlansky, R.H.; Moore, G.A.; Li, Z.G.; Gmitter, F.G. Comparative Transcriptional and Anatomical Analyses of Tolerant Rough Lemon and Susceptible Sweet Orange in Response to “Candidatus Liberibacter asiaticus” Infection. Mol. Plant-Microbe Interact. 2012, 25, 1396–1407. [CrossRef] 34. Albrecht, U.; McCollum, G.; Bowman, K.D. Influence of rootstock variety on Huanglongbing disease development in field-grown sweet orange (Citrus sinensis [L.] Osbeck) trees. Sci. Hortic. 2012, 138, 210–220. [CrossRef] 35. Song, X.; Bhattarai, K.; Lv, D.; Gao, F.; Ying, X. Can CRISPR Win the Battle against Huanglongbing? J. Plant Pathol. Microbiol. 2017, 8, 1–5. [CrossRef] 36. Widyaningsih, S.; Utami, S.N.H.; Joko, T.; Subandiyah, S. Development of disease and growth on six scion/rootstock combinations of citrus seedlings under Huanglongbing pressure. J. Agric. Sci. 2017, 9, 229–238. [CrossRef] Agronomy 2020, 10, 1564 13 of 14 37. Munir, S.; He, P.; Wu, Y.; He, P.; Khan, S.; Huang, M.; Cui, W.; He, Y. Huanglongbing Control: Perhaps the End of the Beginning. Microb. Ecol. 2017, 76, 192–204. [CrossRef] [PubMed] 38. Albrecht, U.; Fiehn, O.; Bowman, K.D. Metabolic variations in di erent citrus rootstock cultivars associated with di erent responses to Huanglongbing. Int. J. Plant Physiol. Biochem. 2016, 107, 33–44. [CrossRef] 39. Eticha, D.; Kwast, A.; Chiachia, T.R.S.; Horowitz, N.; Stützel, H. Calcium nutrition of orange and its impact on growth, nutrient uptake and leaf cell wall. Citrus Res. Technol. 2017, 38, 62–70. [CrossRef] 40. Killiny, N.; Etxeberria, E.; Ponce Flores, A.; González Blanco, P.; Flores Reyes, T.; Ponce Cabrera, L. Laser-induced breakdown spectroscopy (LIBS) as a novel technique for detecting bacterial infection in insects. Sci. Rep. 2019, 9, 1–7. [CrossRef] 41. Fageria, N.K. The Use of Nutrients in Crop Plants; CRC Press Taylor & Francis Group: Boca Raton, FL, USA, 2009; 448p. 42. Ebel, R.C.; Hamido, S.; Morgan, K.T. Interaction of Huanglongbing and Foliar Applications of Copper on Growth and Nutrient Acquisition of Citrus sinensis cv. Valencia. HortScience 2019, 54, 297–302. [CrossRef] 43. Ranty, B.; Aldon, D.; Cotelle, V.; Galaud, J.P.; Thuleau, P.; Mazars, C. Calcium sensors as key hubs in plant responses to biotic and abiotic stresses. Plant Sci. 2016, 7, 327. [CrossRef] 44. Demidchik, V.; Shabala, S.; Isayenkov, S.; Cuin, T.A.; Pottosin, I. Calcium transport across plant membranes: Mechanisms and functions. New. Phytol. 2018, 220, 49–69. [CrossRef] 45. Chen, H.; McCollum, G.; Baldwin, E.; Bai, J. Impacts of Huanglongbing symptom severity on fruit detachment force and mechanical properties of sweet oranges (Citrus sinensis). Hort. Sci. 2016, 51, 356–361. [CrossRef] 46. Gilani, K.; Naz, S.; Aslam, F.; Gurley, W. A Comparison of Zinc, Phosphorous and Potassium levels in leaves and fruit pulp of healthy and Huanglongbing a ected citrus cultivars. J. Plant Physiol. Pathol. 2018, 7, 1–8. [CrossRef] 47. Hasanuzzaman, M.; Bhuyan, M.; Nahar, K.; Hossain, M.; Mahmud, J.; Hossen, M.; Fujita, M. Potassium: A vital regulator of plant responses and tolerance to abiotic stresses. Agronomy 2018, 8, 31. [CrossRef] 48. Souza-Ferraz, R.L.; De Andrade-Barbosa, M.; Araújo-Wanderley, M.J.; Da Silva-Costa, P.; Dourado-Magalhães, I.; De Souza-Medeiros, A.; Miranda-Faria, H.; Marchioro, V.; Soares-de Melo, A.; Alves-dos Anjos, F. Potassium phosphite reduction of Candidatus Liberibacter spp. population on leaves of Ponkan tangerines tree with Huanglongbing. Afr. J. Microbiol. Res. 2018, 12, 248–253. [CrossRef] 49. Magbalot-Fernández, A.; De Guzman, C.C. Potassium fertilization for higher flowering and fruit yield in ‘Magallanes’ Pummelo (Citrus maxima). AJAHR 2019, 3, 1–8. [CrossRef] 50. Minzanova, S.T.; Mironov, V.F.; Mironova, L.G.; Nizameev, I.R.; Kholin, K.V.; Voloshina, A.D.; Milyukov, V.A. Synthesis, properties, and antimicrobial activity of pectin complexes with cobalt and nickel. Chem. Nat. Compd. 2016, 52, 26–31. [CrossRef] 51. Xiao, J.X.; Hu, C.Y.; Chen, Y.Y.; Yang, B.; Hua, J. E ects of low magnesium and an arbuscular mycorrhizal fungus on the growth, magnesium distribution and photosynthesis of two citrus cultivars. Sci. Hortic. 2014, 177, 14–20. [CrossRef] 52. Li, Q.; Chen, H.H.; Qi, Y.P.; Ye, X.; Yang, L.T.; Huang, Z.R.; Chen, L.S. Excess copper e ects on growth, uptake of water and nutrients, carbohydrates, and PSII photochemistry revealed by OJIP transients in Citrus seedlings. Environ. Sci. Pollut. Res. 2019, 26, 30188–30205. [CrossRef] 53. McGollum, G.; Baldwin, E. Huanglongbing: Devastating Disease of Citrus. In Horticultural Reviews, 1st ed.; Janick, J., Ed.; John Wiley & Sons, Inc: Hoboken, NJ, USA, 2017; Volume 44, pp. 315–362. 54. Nwugo, C.C.; Duan, Y.; Lin, H. Study on Citrus Response to Huanglongbing Highlights a Down-Regulation of Defense-Related Proteins in Lemon Plants Upon ‘Ca. Liberibacter asiaticus’ Infection. PLoS ONE 2013, 8, e67442. [CrossRef] 55. Razi, M.F.D.; Khan, I.A.; Jaskani, M.J. Citrus plant nutritional profile in relation to Huanglongbing prevalence in Pakistan. Pak. J. Agric. Sci. 2011, 48, 299–304. 56. Shahzad, F.; Chun, C.; Schumann, A.; Vashisth, T. Nutrient Uptake in Huanglongbing-a ected Sweet Orange: Transcriptomic and Physiological Analysis. J. Amer. Soc. Hort. Sci. 2020, 1–14. [CrossRef] 57. Duan, Y.; Zhang, M.; Yang, C.; Powell, C.A.; Wang, J.; Huang, Y.; Avery, P.B. Field Evaluation of Integrated Management for Mitigating Citrus Huanglongbing in Florida. Plant Sci. 2019. [CrossRef] 58. Tian, S.; Lu, L.; Labavitch, J.M.; Webb, S.M.; Xiaoe Yang, X.; Brown, P.H.; He, Z. Spatial imaging of Zn and other elements in Huanglongbinga ected grapefruit by synchrotron-based micro X-ray fluorescence investigation. J. Exp. Bot. 2014, 65, 953–964. [CrossRef] Agronomy 2020, 10, 1564 14 of 14 59. Shen, W.; Cevallos-Cevallos, J.M.; Rocha, U.N.; Arevalo, H.A.; Stansly, P.A.; Roberts, P.D.; Bruggen, A.H.C. Relation between plant nutrition, hormones, insecticide applications, bacterial endophytes, and Candidatus Liberibacter Ct values in citrus trees infected with Huanglongbing. Eur. J. Plant Pathol. 2013, 137, 727–742. [CrossRef] 60. Ali, A. Best Practices of Integrated Plant Nutrition. In Best Practices of Integrated Plant Nutrition, System in SAARC Countries, 1st ed.; Jahan, F.N., Gurung, T.R., Eds.; SAARC Agriculture Centre: Dhaka, Bangladesh, 2017; Volume 8, pp. 104–134. 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Rootstock Influence on Growth and Mineral Content of Citrus limon and Citrus sinensis cv. Valencia Inoculated with Candidatus Liberibacter Asiaticus

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Abstract

agronomy Article Rootstock Influence on Growth and Mineral Content of Citrus limon and Citrus sinensis cv. Valencia Inoculated with Candidatus Liberibacter Asiaticus 1 1 , 1 Criseida Alhelí Sáenz-Pérez , Eduardo Osorio-Hernández *, Benigno Estrada-Drouaillet , 1 1 2 Sergio Castro-Nava , Rafael Delgado-Martínez , Claudia Magdalena López-Badillo and Raúl Rodríguez-Herrera Division of Postgraduate Studies and Research, Faculty of Engineering and Sciences, Autonomous University of Tamaulipas, Adolfo López Mateos University Center, Victoria, Tamaulipas 87120, Mexico; alhe_saenz@outlook.com (C.A.S.-P.); benestrada@docentes.uat.edu.mx (B.E.-D.); scastro@docentes.uat.edu.mx (S.C.-N.); rdelgado@docentes.uat.edu.mx (R.D.-M.) School of Chemistry, Autonomous University of Coahuila, Boulevard Venustiano Carranza and Ing. José Cárdenas Valdés, Col. República, Saltillo, Coahuila 25280, Mexico; cllopezb@uadec.edu.mx (C.M.L.-B.); raul.rodriguez@uadec.edu.mx (R.R.-H.) * Correspondence: eosorio@docentes.uat.edu.mx Received: 4 September 2020; Accepted: 10 October 2020; Published: 14 October 2020 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 di erent 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 di erences were observed for Mn, P, and Zn concentration. Keywords: Huanglongbing (HLB); height; scion; Citrus volkameriana; X-ray fluorescence spectrometry 1. Introduction One of the factors that reduce citrus production is the presence of di erent 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 di erent citrus areas around the world, HLB is caused by di erent strains of Candidatus Liberibacter; however, Agronomy 2020, 10, 1564; doi:10.3390/agronomy10101564 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1564 2 of 14 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 a ected by secretions of the bacteria that consist of dependent e ectors 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 a ects plant growth and development [10]; more specifically, in leaves and fruits it induces changes in secondary metabolites content, a ecting 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 di erent 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 di erent 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 Agronomy 2020, 10, 1564 3 of 14 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, di erent 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 L 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]. Agronomy 2020, 10, 1564 4 of 14 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 di erences 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. 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 di erent patterns. C. limon C. sinensis C. aurantium C. macrophylla C. volkameriana C. aurantium C. volkameriana Plant Cq Value 1 26.28 32.62 35.34 34.42 30.26 2 35.43 35.11 36.58 33.12 30.23 3 33.12 34.67 34.85 32.56 29.71 4 34.32 34.39 36.36 34.34 28.50 5 32.18 34.08 35.29 34.27 30.64 6 33.57 37.25 32.92 32.03 30.15 7 33.75 33.30 34.02 24.05 29.75 8 35.26 34.81 32.56 32.96 29.25 9 35.36 36.20 34.74 33.28 31.57 10 34.02 35.48 33.75 31.9 30.12 11 32.32 37.23 38.11 29.05 29.44 12 34.04 35.29 35.02 34.64 31.14 13 31.59 36.37 34.53 31.23 31.09 14 36.34 37.27 36.28 32.93 21.11 15 34.72 37.64 37.47 35.88 27.65 16 34.66 36.68 36.15 26.30 31.02 17 34.18 36.04 36.11 26.02 30.18 18 32.95 36.82 36.41 25.50 30.12 19 32.55 36.39 34.54 27.07 30.76 20 35.12 36.28 35.06 24.06 31.43 21 32.19 36.95 34.22 24.60 29.74 22 33.96 31.04 34.65 27.11 32.84 23 32.08 37.12 34.63 28.12 29.52 24 35.07 36.07 34.32 28.17 32.02 25 35.47 35.61 34.46 28.91 29.45 26 34.42 37.06 36.46 19.44 18.46 27 34.16 36.06 36.21 23.79 31.26 28 36.4 36.93 36.22 25.46 31.44 29 34.75 36.86 34.42 24.39 31.60 30 34.81 36.91 34.24 26.53 28.39 31 34.90 37.06 36.37 26.97 29.38 32 36.08 36.97 35.87 28.78 30.82 33 35.25 36.95 35.02 27.02 33.52 34 34.81 36.31 34.52 27.01 31.67 35 36.12 36.89 34.44 26.95 32.78 36 36.13 37.55 34.41 26.83 33.12 37 35.32 37.15 34.62 29.58 31.51 38 34.31 36.39 34.67 27.41 33.40 39 36.40 36.57 34.63 23.46 32.63 40 35.28 36.10 34.77 28.34 31.84 Agronomy 2020, 10, 1564 5 of 14 Table 2. Means comparison of quantification cycle (Cq) values of C. limon and C. sinensis developed on di erent rootstocks. Specie Rootstock Cq Values C. aurantium 34.24 b C. limon C. macrophylla 36.06 a C. volkameriana 35.13 ab C. aurantium 28.61 d C. sinensis C. volkameriana 30.23 c Values with the same letter within a column are not statistically di erent as determined by the Tukey test (p 0.05). MSD: Minimum significant di erence. 3.2. Plant Height and Stem Diameter No significant di erences (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 di erent rootstocks in each of the species, it was observed that uninfected Italian lemon and orange plants did not show significant di erences in plant height and stem diameter between each of the rootstocks (Table 4). Table 3. Plant height and stem diameter of uninfected and infected C. limon and C. sinensis plants. Species Presence Height (cm) Stem (mm) Not infected 167.66 a 16.44 a C. limon Infected 179.93 a 16.81 a MSD 13.14 1.25 Not infected 149.40 a 15.26 a Infected 154.43 a 15.74 a C. sinensis MSD 19.83 2.08 Values with the same letter within a column are not statistically di erent as determined by the Tukey test (p 0.05). MSD: Minimum significant di erence. Table 4. Plant height and rootstock stem diameter of uninfected and infected C. limon and C. sinensis plants. C. limon Not Infected Infected Rootstock Height (cm) Stem (mm) Height (cm) Stem (mm) C. volkameriana 170.60 a 17.82 a 196.88 a 19.35 a C. macrophylla 162.40 a 16.86 a 176.27 b 16.32 b C. aurantium 167.0 a 15.18 a 166.63 b 14.76 c MSD 51.95 4.31 10.70 0.67 C. sinensis Not Infected Infected Rootstock Height (cm) Stem (mm) Height (cm) Stem (mm) C. volkameriana 149.20 a 18.02 a 169.45 a 17.48 a C. aurantium 149.60 a 13.46 b 131.61 b 13.04 b MSD 22.05 2.73 12.17 0.99 Values with the same letter within a column are not statistically di erent as determined by the Tukey test (p 0.05). MSD: Minimum significant di erence. 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 a ected by HLB. With C. sinensis, none of the rootstocks showed Agronomy 2020, 10, 1564 6 of 14 significant di erences 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 di erences (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%). Table 5. Means comparison of mineral concentration (ppm) in Citrus limon and Citrus sinensis. C. limon (ppm) Mg Al Si P S Cl K Ca NI 7.40 a 1.02 a 0.32 a 1.38 a 3.97 a 6.15 a 27.89 a 50.33 a I 8.47 a 1.04 a 0.56 a 1.40 a 3.27 b 7.63 a 24.56 a 51.47 a MSD 1.23 0.34 0.96 0.49 0.32 2.01 4.98 6.7 Sc Ti V Cr Mn Fe Cu Zn NI 0.11 a 0.0009 a 0.00002 a 0.00001 a 0.08 a 0.23 a 0.08 b 0.03 a I 0.11 a 0.00009 a 0.00002 a 0.00001 a 0.08 a 0.24 a 0.01 a 0.03 a MSD 0.03 0.01 0.0000005 0.0000002 0.01 0.12 0.02 0.02 C. sinensis (ppm) Mg Al Si P S Cl K Ca NI 10.18 a 0.98 a 0.56 a 1.51 a 2.99 a 9.88 a 20.52 a 51.79 a I 9.8 a 0.99 a 0.46 a 1.48 a 3.63 a 8.38 a 17.42 a 56.22 a MSD 8.75 1.10 0.60 0.38 6.51 10.30 54.12 27.70 Sc Ti V Cr Mn Fe Cu Zn NI 0.11 a 0.0050 a 0.00003 a 0.00002 a 0.08 a 0.20 b 0.10 a 0.03 a I 0.07 a 0.00008 a 0.00003 a 0.000009 a 0.07 a 0.25 a 0.08 a 0.03 a MSD 0.98 0.06 0.0001 0.0000006 0.29 0.03 0.04 0.06 Values with the same letter within a column are not statistically di erent as determined by the Tukey test (p 0.05). MSD: Minimum significant di erence. Status: NI (not infected), I (Infected). 3.4. Standardized Canonical (Can) Nutrient Coecients, 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. Table 6. Standardized canonical (Can) nutrient coecients, plant height, and rootstock stem diameter. Variable Can1 Can2 Can3 Cl 89.39737621 2.96360649 0.78333747 K 57.79235462 3.53450965 1.92557449 Ca 90.20094892 2.60397644 2.05580445 Height 28.91402537 1.13598256 0.16235885 Stem 15.01864614 0.50627275 0.78861065 Agronomy 2020, 10, x FOR PEER REVIEW 7 of 14 Table 6. Standardized canonical (Can) nutrient coefficients, plant height, and rootstock stem diameter. Variable Can1 Can2 Can3 Cl 89.39737621 2.96360649 −0.78333747 K 57.79235462 3.53450965 −1.92557449 Agronomy 2020, 10, 1564 7 of 14 Ca 90.20094892 2.60397644 −2.05580445 Height 28.91402537 1.13598256 −0.16235885 In order of importance, the variable that shapes the component and achieves a separation between Stem −15.01864614 −0.50627275 0.78861065 the groups is Ca (90.20), then Cl (89.39), and in a smaller proportion K (57.59) and height (28.91). On 3.5. the Signif contrary icance of t , stem he Ma diameter halanobi ( s Distan 15.01) c does es bet not ween Eac contribute h of thanything e Groups and to the Canoni model cal Disper (Table sio 1).ns of Uninfected and Infected Species 3.5. Significance of the Mahalanobis Distances between Each of the Groups and Canonical Dispersions of Table 7 shows the significance of the Mahalanobis distances between each of the groups. That Uninfected and Infected Species is, it shows the separation between uninfected and infected plants of Italian lemon and Valencia Table 7 shows the significance of the Mahalanobis distances between each of the groups. That is, orange cv. Valencia, respectively. The distances of uninfected lemon with the remaining groups, it shows the separation between uninfected and infected plants of Italian lemon and Valencia orange cv. infected lemon (5695), uninfected orange (7398), and infected orange (15914), were significant (p < Valencia, respectively. The distances of uninfected lemon with the remaining groups, infected lemon 0.05). However, there was no significant distance between infected lemon (group 1) and uninfected (5695), uninfected orange (7398), and infected orange (15914), were significant (p < 0.05). However, orange (group 2); therefore, the distance between these two groups is equal to 0. On the other hand, there was no significant distance between infected lemon (group 1) and uninfected orange (group 2); there is a significant distance of 2573 between the uninfected lemon and infected oranges. Finally, the therefore, the distance between these two groups is equal to 0. On the other hand, there is a significant distance of 1614 between the groups of uninfected and infected orange (2 and 3, respectively) is distance of 2573 between the uninfected lemon and infected oranges. Finally, the distance of 1614 significant, that is, the distance is non-zero. Therefore, Figure 1 shows the separation between each between the groups of uninfected and infected orange (2 and 3, respectively) is significant, that is, of the evaluated groups (uninfected lemon, infected lemon, uninfected orange, and infected orange) the distance is non-zero. Therefore, Figure 1 shows the separation between each of the evaluated plants. groups (uninfected lemon, infected lemon, uninfected orange, and infected orange) plants. Table 7. The significance of Mahalanobis distances between uninfected and diseased Italian lemon Table 7. The significance of Mahalanobis distances between uninfected and diseased Italian lemon and and Valencia orange plants. Valencia orange plants. Presence 0 1 2 3 Presence 0 1 2 3 0 0 5695 7398 15914 0 0 5695 7398 15914 1 5695 * 0 114.04 2573 1 5695 * 0 114.04 2573 2 7398 * 114.04 § 0 1614 2 7398 * 114.04 § 0 1614 3 15914 * 2573 * 1614 * 0 3 15914 * 2573 * 1614 * 0 *: p ≤ 0.05, §: p > 0.05. 0 = not infected Italian lemon, 1 = infected Italian lemon, 2 = not infected Valencia *: 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. orange and 3 = infected Valencia orange. Figure 1. Canonical dispersions of uninfected Italian lemon (A), infected Italian lemon Figure 1. Canonical dispersions of uninfected Italian lemon (A), infected Italian lemon (B), uninfected (B), uninfected Valencia orange (B’), and infected Valencia orange (C). The ovals represent each Valencia orange (B’), and infected Valencia orange (C). The ovals represent each of the of the aforementioned groups. Likewise, figures represent the values of the canonical dispersions, aforementioned groups. Likewise, figures represent the values of the canonical dispersions, the white the white figures are the centroids and in the case of group C, the centroid is located in the larger X. figures are the centroids and in the case of group C, the centroid is located in the larger X. 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 di erent 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 di erent combinations of scion/rootstock, it was observed that inoculation with Ca. Liberibacter asiaticus a ected the mineral content since two di erent combinations of scion/rootstock did not have Agronomy 2020, 10, x FOR PEER REVIEW 8 of 14 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 Agronomy 2020, 10, 1564 8 of 14 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 the same content. Branches of di erent lengths were observed, the smallest branches corresponded grouped with treatment 7 (uninfected C. sinensis/C. aurantium). On the other hand, the combination to C. limon/C. volkameriana (infected and uninfected), which suggests that there were fewer changes C. limon/C. aurantium (in its two versions) had a great disparity in concentration and different in mineral content due to inoculation with the bacteria. minerals. Figure 2. Dendrogram of treatments in Citrus limon and Citrus sinensis (graft/rootstock) plants. 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 Uninfected (−) and infected (+) graft/rootstock, 1: −C. limon/C. aurantium), 2: +C. limon/C. aurantium 3: 3: C. limon/C. macrophylla, 4: +C. limon/C. macrophylla, 5: C. limon/C. volkameriana, 6: +C. limon/C. −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 volkameriana, 7: −C. sinensis/C. aurantium, 8: +C. sinensis/C. aurantium, 9: −C. sinensis/C. volkameriana and and 10: +C. sinensis/C. volkameriana. 10: +C. sinensis/C. volkameriana. The next least a ected treatments were infected and uninfected C. sinensis/C. volkameriana due to 4. Discussion being grouped, although they had larger branches than those of C. limon/C. volkameriana. The mineral concentration of the other combinations was greatly a ected since the infected and uninfected versions 4.1. Detection of Ca. Liberibacter Asiaticus were not grouped. For example, according to its mineral content, the uninfected C. limon/C. macrophylla The number of PCR cycles ranged from 18 to 38, indicating abundant to moderate target nucleic forms a group with treatment 1 (uninfected C. limon/C. aurantium) and then is grouped with treatment acid in the sample, respectively, and samples are accurately identified as infected or uninfected, as 7 (uninfected C. sinensis/C. aurantium). On the other hand, the combination C. limon/C. aurantium (in its they are considered positive for Ca. Liberibacter asiaticus when the cycle quantification values (Cq) two versions) had a great disparity in concentration and di erent minerals. <40 [28]. Likewise, there is a high correlation between bacterial concentration and the severity of HLB. 4. ThDiscussion at 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 4.1. Detection of Ca. Liberibacter Asiaticus bacteria inoculum. On the contrary, C. macropylla and C. volkameriana present lower Cq values. 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% Agronomy 2020, 10, 1564 9 of 14 to 28% significantly lower in infected plants in comparison to uninfected plants [31]. In the same way, another study [32] evaluated the e ects 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 di erences 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 di erent rootstocks was evaluated to determine if rootstocks have an e ect 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 e ects 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 e ect 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 di erences 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]. Agronomy 2020, 10, 1564 10 of 14 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, a ecting chloroplasts and functions related to sugar accumulation and metabolic activities [47]. Since there were no significant di erences 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 e ects 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 e ects of HLB for its antibacterial activity [56,57]. Some of the results of this study di er with those reported in the literature, this is because di erent 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 di erences 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 a ect 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 di erences 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. Agronomy 2020, 10, 1564 11 of 14 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. References 1. Wang, N.; Pierson, E.A.; Setubal, J.A.; Xu, J.; Levy, J.G.; Zhang, Y.; Li, J.; Rangel, L.T.; Martins, J. The Candidatus Liberibacter–host interface: Insights into pathogenesis mechanisms and disease control. Ann. Rev. Phytopathol. 2017, 55, 451–482. [CrossRef] 2. Robles-González, M.M.; Orozco-Santos, M.; Manzanilla-Ramírez, M.A.; Velázquez-Monreal, J.J.; Medina-Urrutia, V.M.; Sanches-Stuchi, E. Experiencias con Huanglongbing en limón mexicano, en el estado de Colima, México. Citrus Res. Technol. 2018, 39, 2–12. [CrossRef] 3. Rao, M.J.; Ding, F.; Wang, N.; Deng, X. Metabolic mechanisms of host species against citrus Huanglongbing (Greening Disease). Crit. Rev. Plant Sci. 2019, 37, 1–16. [CrossRef] 4. Salcedo-Baca, D.; Hinojosa, R.A.; Mora-Aguilera, G.; Covarrubias-Gutierrez, I.; DePaolis, F.J.; Mora-Flores, J.S.; Cíntora, C. Evaluación del impacto económico de la enfermedad de los cítricos Huanglongbing (hlb) en la cadena citrícola mexicana. IICA, SAGARPA, SENASICA. México. 2011, pp. 40–47. Available online: http://repiica.iica.int/docs/b2146e/b2146e.pdf (accessed on 20 June 2020). 5. Hall, D.G. Incidence of “Candidatus Liberibacter asiaticus” in a Florida population of Asian citrus psyllid. J. Appl. Entomol. 2018, 142, 97–103. [CrossRef] 6. Johnson, E.G.; Wu, J.; Bright, D.B.; Graham, J.H. Association of ‘Candidatus Liberibacter asiaticus’ root infection, but not phloem plugging with root loss on huanglongbing-a ected trees prior to appearance of foliar symptoms. Plant Pathol. 2014, 63, 290–298. [CrossRef] 7. Achor, D.; Welker, S.; Mahmoud, S.B.; Wang, C.; Folimonova, S.Y.; Dutt, M.; Gowda, S.; Levy, A. Dynamics of Candidatus Liberibacter asiaticus Movement and Sieve-Pore Plugging in Citrus Sink Cells. Plant Physiol. 2020, 182, 882–891. [CrossRef] [PubMed] 8. Louzada, E.S.; Vasquez, O.E.; Braswell, W.E.; Yanev, G.; Devanaboina, M.; Kunta, M. Distribution of ‘Candidatus Liberibacter asiaticus’ Above and Below Ground in Texas Citrus. Phytopathology 2016, 106, 702–709. [CrossRef] [PubMed] 9. Deng, H.; Achor, D.; Extberria, E.; Yu, Q.; Du, D.; Stanton, D.; Liang, G.; Gmitter, F.G., Jr. Phloem Regeneration Is a Mechanism for Huanglongbing-Tolerance of “Bearss” Lemon and “LB8-9” Sugar Belle Mandarin. Plant Sci. 2019, 10, 1–13. [CrossRef] [PubMed] 10. Da-Graca, J.V.; Douhan, G.W.; Halbert, S.E.; Keremane, M.L.; Lee, R.F.; Vidalakis, G. Huanglongbing: An overview of a complex athosystem ravaging the world’s citrus. J. Integr. Plant Biol. 2016, 58, 373–387. [CrossRef] [PubMed] 11. Suh, J.H.; Niu, Y.S.; Wang, Z.; Gmitter, F.G.; Wang, Y. Metabolic analysis reveals altered long-chain fatty acid metabolism in the host by Huanglongbing disease. J. Agric. Food. Chem. 2018, 66, 1296–1304. [CrossRef] [PubMed] 12. Dala-Paula, B.M.; Plotto, A.; Bai, J.; Manthey, J.A.; Baldwin, E.A.; Ferrarezi, R.S. E ect of Huanglongbing or greening disease on orange juice quality, a Review. Plant Sci. 2019, 9, 1–19. [CrossRef] [PubMed] 13. Slisz, A.M.; Breksa, A.P.; Mishchuk, D.O.; McCollum, G.; Slupsky, C.M. Metabolomic analysis of citrus infection by ‘Candidatus Liberibacter ’ reveals insight into pathogenicity. J. Proteome Res. 2012, 11, 4223–4230. [CrossRef] 14. Gómez-Flores, W.; Garza-Saldaña, J.J.; Varela-Fuentes, S.E. Detection of Huanglongbing disease based on intensity-invariant texture analysis of images in the visible spectrum. Comput. Electron. Agric. 2019, 162, 825–835. [CrossRef] 15. Li, J.; Li, L.; Kolbasov, V.; Ehsani, R.; Carter, E.; Wang, N. Developing citrus Huanglongbing management strategies based on the severity of symptoms in HLB-endemic citrus-producing regions. Phytopathology 2018, 109, 582–592. [CrossRef] [PubMed] 16. Albrecht, U.; Tripathi, I.; Kim, H.; Bowman, K.D. Rootstock e ects on metabolite composition in leaves and roots of young navel orange (Citrus sinensis L. Osbeck) and pummelo (C. grandis L. Osbeck) trees. Trees 2018, 33, 243–265. [CrossRef] Agronomy 2020, 10, 1564 12 of 14 17. Martínez-Ballesta, M.C.; Alcaraz-López, C.; Muries, B.; Mota-Cadenas, C.; Carvajal, M. Physiological aspects of rootstock–scion interactions. Sci. Hortic. 2010, 127, 112–118. [CrossRef] 18. Warschefsky, E.J.; Klein, L.L.; Frank, M.H.; Chitwood, D.H.; Londo, J.P.; Wettberg, V.E.J.B.; Miller, A.J. Rootstocks: Diversity, domestication, and impacts on shoot phenotypes. Plant. Sci. 2016, 21, 418–437. [CrossRef] 19. Graham, J.; Gottwald, T.; Setamou, M. Status of Huanglongbing (HLB) outbreaks in Florida, California and Texas. Trop. Plant Pathol. 2020, 45, 265–278. [CrossRef] 20. Stover, E.; Inch, S.; Richardson, M.L.; Hall, D.G. Conventional Citrus of Some Scion/Rootstock Combinations Show Field Tolerance under High Huanglongbing Disease Pressure. Hort. Sci. 2016, 51, 127–132. [CrossRef] 21. Aksenov, A.A.; Pasamontes, A.; Peirano, D.J.; Zhao, W.; Dandekar, A.M.; Fiehn, O.; Eshani, N.; Davis, C.E. Detection of Huanglongbing Disease Using Di erential Mobility Spectrometry. Anal. Chem. 2014, 86, 2481–2488. [CrossRef] 22. Garza-Saldaña, J.J.; Varela-Fuentes, S.; Gómez-Flores, W. Métodos para la detección presuntiva de Huanglongbing (HLB) en cítricos. Ciencia UAT 2017, 11, 93–104. [CrossRef] 23. Arredondo-Valdés, R.; Delgado-Ortíz, J.C.; Beltrán-Beache, M.; Anguiano-Cabello, J.; Cerna-Chávez, E.; Rodríguez-Pagasa, Y.; Ochoa-Fuentes, Y.M. A review of techniques for detecting Huanglongbing (greening) in citrus. Can. J. Microbiol. 2016, 62, 803–811. [CrossRef] 24. Hitpold, I.; Demarta, L.; Johnson, S.N.; Moore, B.D.; Power, S.A.; Mitchel, C. Silicon and other essential element composition in roots sing X-ray fluorescence spectroscopy: A high throughput approach. In Proceedings of the Ninth ACGIE, 1st ed.; Invertebrate Ecology of Australasian Grasslands: Hawkesbury, Australia, 2017; pp. 191–196. 25. Canteri, M.H.G.; Renard, C.M.G.; Le Bourvellec, C.; Bureau, S. ATR-FTIR spectroscopy to determine cell wall composition: Application on a large diversity of fruits and vegetables. Carbohydr. Polym. 2019, 212, 186–196. [CrossRef] [PubMed] 26. Li, W.; Hartung, J.S.; Lew, L. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus Huanglongbing. J. Microbiol. Meth. 2006, 66, 104–115. [CrossRef] [PubMed] ® ® 27. SAS Institute Inc. The SAS System for Windows (Ver. 9.0); SAS Institute Inc: Cary, NC, USA, 2004. 28. Ammar, E.D.; Hall, D.G.; Alvarez, J.M. E ect of Cyantraniliprole, a Novel Insecticide, on the Inoculation of Candidatus Liberibacter Asiaticus Associated with Citrus Huanglongbing by the Asian Citrus Psyllid (Hemiptera: Liviidae). J. Econ. Entomol. 2015, 108, 399–404. [CrossRef] [PubMed] 29. Flores-Sánchez, J.-L.; Mora-Aguilera, G.; Loaeza-Kuk, E.; López-Arroyo, J.I.; Domínguez-Monge, S.; Acevedo-Sánchez, G.; Robles-García, P. Yield loss caused by Candidatus Liberibacter asiaticus in Persian lime, in Yucatan Mexico. Rev. Mex. Fitopatol. 2015, 33, 195–210. 30. Folimonova, S.Y.; Robertson, C.J.; Garnsey, S.M.; Gowda, S.; Dawson, W.O. Examination of the Responses of Di erent Genotypes of Citrus to Huanglongbing (Citrus Greening) Under Di erent Conditions. Phytopathology 2009, 99, 1346–1354. [CrossRef] 31. Shokrollah, H.; Abdullah, T.L.; Sijam, K.; Abdullah, S.N.A. Potential use of selected citrus rootstocks and interstocks against HLB disease in Malaysia. Crop Prot. 2011, 30, 521–525. [CrossRef] 32. Bowman, K.D.; McCollum, G.; Albrecht, U. Performance of ‘Valencia’ orange (Citrus sinensis [L.] Osbeck) on 17 rootstocks in a trial severely a ected by huanglongbing. Sci. Hortic. 2016, 201, 355–361. [CrossRef] 33. Fan, J.; Chen, C.; Yu, Q.; Khalaf, A.; Achor, D.S.; Brlansky, R.H.; Moore, G.A.; Li, Z.G.; Gmitter, F.G. Comparative Transcriptional and Anatomical Analyses of Tolerant Rough Lemon and Susceptible Sweet Orange in Response to “Candidatus Liberibacter asiaticus” Infection. Mol. Plant-Microbe Interact. 2012, 25, 1396–1407. [CrossRef] 34. Albrecht, U.; McCollum, G.; Bowman, K.D. Influence of rootstock variety on Huanglongbing disease development in field-grown sweet orange (Citrus sinensis [L.] Osbeck) trees. Sci. Hortic. 2012, 138, 210–220. [CrossRef] 35. Song, X.; Bhattarai, K.; Lv, D.; Gao, F.; Ying, X. Can CRISPR Win the Battle against Huanglongbing? J. Plant Pathol. Microbiol. 2017, 8, 1–5. [CrossRef] 36. Widyaningsih, S.; Utami, S.N.H.; Joko, T.; Subandiyah, S. Development of disease and growth on six scion/rootstock combinations of citrus seedlings under Huanglongbing pressure. J. Agric. Sci. 2017, 9, 229–238. [CrossRef] Agronomy 2020, 10, 1564 13 of 14 37. Munir, S.; He, P.; Wu, Y.; He, P.; Khan, S.; Huang, M.; Cui, W.; He, Y. Huanglongbing Control: Perhaps the End of the Beginning. Microb. Ecol. 2017, 76, 192–204. [CrossRef] [PubMed] 38. Albrecht, U.; Fiehn, O.; Bowman, K.D. Metabolic variations in di erent citrus rootstock cultivars associated with di erent responses to Huanglongbing. Int. J. Plant Physiol. Biochem. 2016, 107, 33–44. [CrossRef] 39. Eticha, D.; Kwast, A.; Chiachia, T.R.S.; Horowitz, N.; Stützel, H. Calcium nutrition of orange and its impact on growth, nutrient uptake and leaf cell wall. Citrus Res. Technol. 2017, 38, 62–70. [CrossRef] 40. Killiny, N.; Etxeberria, E.; Ponce Flores, A.; González Blanco, P.; Flores Reyes, T.; Ponce Cabrera, L. Laser-induced breakdown spectroscopy (LIBS) as a novel technique for detecting bacterial infection in insects. Sci. Rep. 2019, 9, 1–7. [CrossRef] 41. Fageria, N.K. The Use of Nutrients in Crop Plants; CRC Press Taylor & Francis Group: Boca Raton, FL, USA, 2009; 448p. 42. Ebel, R.C.; Hamido, S.; Morgan, K.T. Interaction of Huanglongbing and Foliar Applications of Copper on Growth and Nutrient Acquisition of Citrus sinensis cv. Valencia. HortScience 2019, 54, 297–302. [CrossRef] 43. Ranty, B.; Aldon, D.; Cotelle, V.; Galaud, J.P.; Thuleau, P.; Mazars, C. Calcium sensors as key hubs in plant responses to biotic and abiotic stresses. Plant Sci. 2016, 7, 327. [CrossRef] 44. Demidchik, V.; Shabala, S.; Isayenkov, S.; Cuin, T.A.; Pottosin, I. Calcium transport across plant membranes: Mechanisms and functions. New. Phytol. 2018, 220, 49–69. [CrossRef] 45. Chen, H.; McCollum, G.; Baldwin, E.; Bai, J. Impacts of Huanglongbing symptom severity on fruit detachment force and mechanical properties of sweet oranges (Citrus sinensis). Hort. Sci. 2016, 51, 356–361. [CrossRef] 46. Gilani, K.; Naz, S.; Aslam, F.; Gurley, W. A Comparison of Zinc, Phosphorous and Potassium levels in leaves and fruit pulp of healthy and Huanglongbing a ected citrus cultivars. J. Plant Physiol. Pathol. 2018, 7, 1–8. [CrossRef] 47. Hasanuzzaman, M.; Bhuyan, M.; Nahar, K.; Hossain, M.; Mahmud, J.; Hossen, M.; Fujita, M. Potassium: A vital regulator of plant responses and tolerance to abiotic stresses. Agronomy 2018, 8, 31. [CrossRef] 48. Souza-Ferraz, R.L.; De Andrade-Barbosa, M.; Araújo-Wanderley, M.J.; Da Silva-Costa, P.; Dourado-Magalhães, I.; De Souza-Medeiros, A.; Miranda-Faria, H.; Marchioro, V.; Soares-de Melo, A.; Alves-dos Anjos, F. Potassium phosphite reduction of Candidatus Liberibacter spp. population on leaves of Ponkan tangerines tree with Huanglongbing. Afr. J. Microbiol. Res. 2018, 12, 248–253. [CrossRef] 49. Magbalot-Fernández, A.; De Guzman, C.C. Potassium fertilization for higher flowering and fruit yield in ‘Magallanes’ Pummelo (Citrus maxima). AJAHR 2019, 3, 1–8. [CrossRef] 50. Minzanova, S.T.; Mironov, V.F.; Mironova, L.G.; Nizameev, I.R.; Kholin, K.V.; Voloshina, A.D.; Milyukov, V.A. Synthesis, properties, and antimicrobial activity of pectin complexes with cobalt and nickel. Chem. Nat. Compd. 2016, 52, 26–31. [CrossRef] 51. Xiao, J.X.; Hu, C.Y.; Chen, Y.Y.; Yang, B.; Hua, J. E ects of low magnesium and an arbuscular mycorrhizal fungus on the growth, magnesium distribution and photosynthesis of two citrus cultivars. Sci. Hortic. 2014, 177, 14–20. [CrossRef] 52. Li, Q.; Chen, H.H.; Qi, Y.P.; Ye, X.; Yang, L.T.; Huang, Z.R.; Chen, L.S. Excess copper e ects on growth, uptake of water and nutrients, carbohydrates, and PSII photochemistry revealed by OJIP transients in Citrus seedlings. Environ. Sci. Pollut. Res. 2019, 26, 30188–30205. [CrossRef] 53. McGollum, G.; Baldwin, E. Huanglongbing: Devastating Disease of Citrus. In Horticultural Reviews, 1st ed.; Janick, J., Ed.; John Wiley & Sons, Inc: Hoboken, NJ, USA, 2017; Volume 44, pp. 315–362. 54. Nwugo, C.C.; Duan, Y.; Lin, H. Study on Citrus Response to Huanglongbing Highlights a Down-Regulation of Defense-Related Proteins in Lemon Plants Upon ‘Ca. Liberibacter asiaticus’ Infection. PLoS ONE 2013, 8, e67442. [CrossRef] 55. Razi, M.F.D.; Khan, I.A.; Jaskani, M.J. Citrus plant nutritional profile in relation to Huanglongbing prevalence in Pakistan. Pak. J. Agric. Sci. 2011, 48, 299–304. 56. Shahzad, F.; Chun, C.; Schumann, A.; Vashisth, T. Nutrient Uptake in Huanglongbing-a ected Sweet Orange: Transcriptomic and Physiological Analysis. J. Amer. Soc. Hort. Sci. 2020, 1–14. [CrossRef] 57. Duan, Y.; Zhang, M.; Yang, C.; Powell, C.A.; Wang, J.; Huang, Y.; Avery, P.B. Field Evaluation of Integrated Management for Mitigating Citrus Huanglongbing in Florida. Plant Sci. 2019. [CrossRef] 58. Tian, S.; Lu, L.; Labavitch, J.M.; Webb, S.M.; Xiaoe Yang, X.; Brown, P.H.; He, Z. Spatial imaging of Zn and other elements in Huanglongbinga ected grapefruit by synchrotron-based micro X-ray fluorescence investigation. J. Exp. Bot. 2014, 65, 953–964. [CrossRef] Agronomy 2020, 10, 1564 14 of 14 59. Shen, W.; Cevallos-Cevallos, J.M.; Rocha, U.N.; Arevalo, H.A.; Stansly, P.A.; Roberts, P.D.; Bruggen, A.H.C. Relation between plant nutrition, hormones, insecticide applications, bacterial endophytes, and Candidatus Liberibacter Ct values in citrus trees infected with Huanglongbing. Eur. J. Plant Pathol. 2013, 137, 727–742. [CrossRef] 60. Ali, A. Best Practices of Integrated Plant Nutrition. In Best Practices of Integrated Plant Nutrition, System in SAARC Countries, 1st ed.; Jahan, F.N., Gurung, T.R., Eds.; SAARC Agriculture Centre: Dhaka, Bangladesh, 2017; Volume 8, pp. 104–134. 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