Research Article, J Plant Physiol Pathol Vol: 7 Issue: 1
A Comparison of Zinc, Phosphorous and Potassium Levels in Leaves and Fruit Pulp of Healthy and Huanglongbing Affected Citrus Cultivars
*Corresponding Author : William Gurley
Department of Microbiology & Cell Science, University of Florida, Gainesville 32611-0700, USA
Tel: +1 352 392-1568
E-mail: [email protected]
Received: November 11, 2018 Accepted: November 28, 2018 Published: January 04, 2019
Citation: Gilani K, Naz S, Aslam F, Gurley W (2019) A Comparison of Zinc, Phosphorous and Potassium Levels in Leaves and Fruit Pulp of Healthy and Huanglongbing Affected Citrus Cultivars. J Plant Physiol Pathol 7:1. doi: 10.4172/2329-955X.1000192
Mineral nutrition plays an important role in the growth and development of plants and is a significant factor in plant disease defense. In order to determine whether the distribution pattern of mineral nutrients is correlated with the Huanglongbing (HLB) disease status, leaf and pulp samples were collected from fifteen healthy and HLB-affected cultivars of citrus from the Sargodha district of Pakistan. Samples were removed from the field-grown trees and their HLB status determined by quantitative PCR. The levels of zinc (Zn), phosphorous (P), and potassium (K) were measured by inductively-coupled plasma mass spectrometry. Pulp tissues contained lower levels of all three minerals regardless of the infection status. No significant change (t-test) was found in K levels associated with the HLB-affected leaves (p=0.7843) or pulp (p=0.0997). Phosphorous decreased 9% (p=0.0437) in leaves and increased 29% (p=0.0120) in pulp in HLB-affected samples compared to healthy. Zn showed a 31% (p<0.0001) decrease in infected leaves but no change in the pulp tissue (p=0.6728). These results indicate that the partitioning of Zn and P between leaves and fruit was differently affected by the HLB-infection status of the tree. However, no relationship between HLB infection status and K was observed.
Keywords: Citrus greening; HLB; Mineral nutrients; Resorption; Citrus sinensis; Citrus reticulate; Candidatus liberibacter asiaticus
Huanglongbing (or citrus greening) has spread worldwide and poses a major threat to global citrus production [1,2]. In Pakistan, Huanglongbing (HLB) is caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), which is transmitted by Diaphorina citri Kuwayama, the Asian citrus psyllid [3-5]. The bacterium from a single infection site can spread throughout the plant within a single year from leaves to roots [6,7]. The major symptoms of HLB include yellowing of leaves and veins, and production of small inedible fruits, which remain green. CLas-infected trees experience accelerated fruit drop; the seeds are usually aborted, and the fruit is bitter . The disease was discovered in southern China in 1919 and named “Yellow dragon disease” (Huanglongbing) [6,9]. HLB was subsequently found in Africa , and in 2005, it was recognized in Florida . HLB has inflicted large economic losses on the citrus industry worldwide due to poor fruit quality and has dramatically reduced yields and shortened the lifespan for the infected trees . The widespread distribution of HLB and the large-scale disruptive effects on global citrus production has increased the need to better understand the causes, symptoms, and spread of this disease.
Punjab province contributes 95% of citrus fruit production in Pakistan due to its favorable soil (pH ≤ 8.2 and Soil Electrical Conductivity EC ≤ 2dS/m) and climatic conditions [12-14]. Sargodha District is the primary area of citrus production within Panjab province . In 2016, citrus exports worldwide were up to $5 billion, with Pakistan contributing $100 million representing 2% of the total . Citrus greening was first reported in 1927 in Pakistan by Hussain and Nath ; however, severe economic losses were not reported until 1999 due to 25-40% infection rates in sweet oranges and 22% in Kinnow (Mandarin) .
The blotchy mottled appearance of HLB-affected leaves results from the non-uniform loss of chlorophyll due to a decrease in the number of palisade parenchyma cells and a reduction of thylakoid membranes in chloroplasts [18,19]. The disruption of chloroplast structure is thought to be caused by an excessive accumulation of starch grains [19,20] and Zn deficiency [18,20-25]. The starch buildup has been attributed to phloem blockage due to CLas-triggered plugging of phloem sieve elements by the synthesis of callose and filamentous phloem protein (PP2), combined with phloem necrosis or collapse [21,25-28].
Micronutrients play a major role in the physiology of plants. The mineral nutritional status of plants substantially affects plant resistance and tolerance to pathogens . Several reports have indicated that seasonal application of mineral fertilizers alleviated the symptoms of HLB-affected trees [30-33]. For example, the Chinese citrus industry has survived and lived with HLB for the past 60 years, which is partially attributed to nutritional management .
The nutrient status of each of the essential minerals has been shown to have the potential to exert a significant impact on the outcome of plant-pathogen interactions. However, whether levels increase, decrease, or have no effect, the severity or incidence of disease varies depending on the pathogen and the plant [35,36]. Pathogenic organisms often cause disruptions in the levels or distribution of mineral nutrition that are deleterious to the plant . Alterations in the nutritional state of the infected plant can be categorized as follows: 1-immobilization of, or competition for, specific minerals within the rhizosphere, 2-reduction in uptake due to root destruction, 3-disruption or redirection of vascular transport, and 4-impaired utilization, or concentration, of mineral nutrients around infection sites [35,37]. Since some of the symptoms of HLB closely resemble those of mineral deficiency, it is important to know whether changes in levels of these minerals are associated explicitly with HLB.
The present study compared the levels of three minerals between healthy and infected trees. Zn, P, and K were quantified in leaves and pulp collected from field grown citrus from the Citrus Research Institute in Sargodha, Pakistan . The goal of this study was to determine the impact of HLB on the nutritional status of citrus, specifically within Pakistan. Previous reports indicate that Zn levels often vary between healthy and infected leaves, but no consistent trend has emerged. For Example, Masaoka et al.  and Nwugo et al.  showed increased Zn in infected leaves (50%-128%), while Tian  and Zhao  showed either a reduction (90%) or no significant change. In order to develop a fuller picture of the potential impact of the disease, the current study was expanded from previously reported studies to include fruit tissues (pulp) in addition to leaves.
Material and Methods
Thirteen different cultivars of Citrus sinensis (L.) var. Osbeck and two cultivars of Citrus reticulate (L.) var. Blanco were included in this study: Kozan, Kinnow (Mandarin), Jaffa, Emby Gold, Hinckley, Mars Early, Salustiana, Cassa Grande, Hamlin, Tarocco-Rose, Tarocco- Nucellar, Blood Red, Ruby Blood, Frost Rose, and New Hall. Plant samples were taken from healthy and CLas-infected cultivars that were selected from the Citrus Research Institute (Latitude 32°07´41” N and Longitude 72°40´38” E), Sargodha District, Pakistan. Twenty to thirty years old trees from plots that were subjected to identical fertilization regimes since planting were used for this study. The maximum height of these trees was 3.88 m. Sample replicates were taken from five uniform diseased and healthy trees for each cultivar. Fruit and leaves were harvested from each quadrant of the tree canopy and the samples combined according to HLB-infection status and cultivar.
Collection and leaf sampling: Medium sized leaves (5-7 cm) and fruits (130-175 g) were selected for sampling. From each tree, a total of 200 to 300 leaves and one to two fruits were taken. fruits were taken. Leaves and fruit from infected trees showed clear visible symptoms, while samples from healthy trees exhibited no apparent insect or other physical damage. Both leaves and fruit were collected and placed separately in sealable plastic bags and stored on ice for transport from the orchards to the laboratory.
The leaf samples were taken from three different time periods from 2014 to 2015: July 2014, December 2014, and February 2015. Healthy and HLB-Infected trees were located in adjacent blocks of a single field. Five trees for each of 15 cultivars were chosen for this study, and 100 to 200 leaves per tree were collected during each of the three sampling periods. Fruits (5-10/cultivar) were collected in Dec 2014 and February 2015. Plant material was surface sterilized with dilute detergent, washed extensively with tap water, and then rinsed with autoclaved distilled water. Whole leaves and sections of fruit pulp were reduced to a fine powder using a coffee grinder or a mortar and pestle. The HLB-infection status of the sample material was confirmed by PCR analysis after each collection period. Once the infection status was confirmed, the samples were freeze-dried and stored at -20°C. All freeze-dried samples from the same cultivar were combined according to their respective infection status before conducting nutrient analyses. After the combined samples arrived at the University of Florida, their HLB infection status was re-confirmed by qPCR analysis. Mineral analysis was conducted by the University of Florida, Department of Soil and Water Science (Dr. Lena Q. Ma Laboratory).
DNA Extraction from leaves tissues: A DNeasy® Plant Mini Kit (Qiagen) was used to extract the DNA from ≤ 200 mg of lyophilized leaf tissues. The plant material was initially disrupted using a mortar and pestle, and the powder placed into 2 ml sterilized screw cap vials with two 2.3 mm steel grinding balls (Fisher Scientific Company). The samples were further disrupted by mechanical shaking at 1,500 strokes/min for two 30 sec bursts using an automated tissue homogenizer (2000 Geno/Grinder, Spex CertiPrep). The removal of RNA with RNase A and DNA extraction were according to the Qiagen protocol supplied with the kit.
Analysis of mineral ions by inductively coupled plasma spectroscopy (ICP): Mineral analysis was performed on leaves and pulp from healthy and CLas-infected citrus samples. Oven-dried ground plant tissue material (1.00 g) was placed in a 50 mL porcelain crucible and ashed in a muffle furnace at 500°C. The residue was then dissolved in 6 M HCl and analyzed by ICP for quantification of Zn and K levels. Inductively Colorimetric Analysis was used for determination of P levels. The mineral results are reported in ppm plant dry weight. Statistical analysis of the data was performed using the Student’s t-test (Excel: two samples, unequal variance) and the SAS one-way ANOVA test, which included the Tukey HSD post hoc test.
Determination of HLB infection status using quantitative real-time PCR
Uniformly prepared DNA extracts from plant samples were used for PCR analysis. The resulting Ct values were plotted to determine the copy number of CLas DNA applying a calibration curve prepared using serial dilutions of a gel-purified amplicon. The range of Ct values for healthy samples ranged from 36.3 to no Ct detectable. These values corresponded to hypothetical copy numbers of CLas amplicon from 0-139. A copy number of 139 or less was considered negative since the corresponding Ct was greater than the 30 Ct threshold reported by others [43-47]. For infected leaves, most copy number values were in the millions, with the highest being 4.50 × 106 (Ct=16.50). There were 3 exceptions ranging in copy number from 1.39 × 103 (Ct=28.2) to 1.87 × 105 (Ct=21.1). The HLB infection status was designated either positive or negative based on these results.
Measurement of minerals in leaf and pulp samples
The levels of Zn, P, and K in leaves are shown in Table 1 and Figure 1. The average values for Zn dropped from 20.94 ppm in healthy leaves to 14.53 ppm in CLas-infected leaves. The average K levels were approximately 1,000-fold higher than Zn and showed considerable variation in infected leaves, ranging from 18,151 to 5,751 ppm. For P, quantities ranged from 1,562 ppm in healthy to 1,000 ppm in CLasinfected leaf samples.
|Cultivar name||Zn Ppm||Zn % change||P Ppm||P %
|K Ppm||K %
Table 1: Mineral analysis of Zn, P and K in healthy and HLB-affected leaves.
Although average K levels showed no statistically significant change between healthy and CLas-infected leaves as determined by one-way ANOVA (F(1,13)=0.08, Pr>F=0.7836), both Zn and P showed statistically decreased means in infected leaf samples compared to healthy (Zn F(1,13)=60.33, Pr>F <0.0001 and P F(1,13)=4.87, Pr>F <0.0437) (Table 1). This trend was most apparent with Zn where all but one cultivar (Frost Rose) showed substantially lower levels in HLB-affected leaves. The largest decreases in Zn content (~50%) in leaves were for Torocco-Rose and New Hall cultivars. On average, Zn decreased by 31%. P levels were also found to decrease in the infected leaves compared with healthy, but to a lesser extent (-9%). As with Zn, for a given cultivar, there was a consistent pattern of decreased levels of P in the HLB-affected leaf samples.
The mineral levels found in pulp samples are presented in Table 2 and Figure 2. In general, the quantities of all three minerals varied widely. The concentrations of P and K in healthy leaves and pulp were similar and in general agreement with levels reported by others. Concentrations of Zn, however, were roughly twice as high in leaves than in fruit which is similar to the findings of Mattos et al. . For pulp, values for Zn ranged from 5.0 ppm in HLB-affected to 13.0 ppm in healthy samples. The P in pulp varied from 341 ppm in fruit from healthy to 1,331 ppm in HLB-affected trees, and K levels ranged from 5,090 ppm in healthy to 10,899 ppm in samples from HLB-positive trees.
|Cultivar Name||Zn ppm||Zn %
|P ppm||P %
|K ppm||K %
Table 2: Mineral analysis of Zn, P and K in pulp from healthy and HLB-affected trees.
Data was analyzed using the one-way ANOVA. Both Zn and P show statistically significant differences between the means for healthy and HLB-affected leaf samples. Top panel: distribution of Zn; Middle panel: distribution of P; and Bottom panel: distribution of K. Lower case letters (a and b) designate statistically significant groups.
In pulp, contrary to leaves, neither Zn (Pr>F=0.6728) nor K (Pr>F=0.0997) levels were affected by the HLB status of the tree. Instead, average levels of P were higher in the HLB-affected trees as compared to healthy trees. Most cultivars showed substantially greater levels of P in the HLB-affected samples (on average 29% higher; Pr>F=0.0120) as compared to the healthy samples, with Emby Gold, Kozan and Salustiana cultivars exhibiting large differences of 50%, 55% and 104%, respectively. Differences in K levels between pulp from healthy and HLB-affected trees were not statistically significant (Pr>F=0.0997), even though Kozan, Salustiana and Hamlin cultivars showed substantially higher levels of 35%, 29% and 50%, respectively; while for other cultivars, the increases were not as prominent. With both leaf data and pulp data, the significance of the difference in means was determined using a one-way ANOVA analysis with Welch’s ANOVA being used in cases where variance failed Levene’s test for homogeneity. Welch’s ANOVA was used for both leaves and pulp for P and K samples.
Data was analyzed using the one-way ANOVA. Only P shows statistically significant differences between the means for healthy and HLB-affected leaf samples. Top panel: distribution of Zn; Middle panel: distribution of P; and Bottom panel: distribution of K. Lower case letters (a and b) designate statistically significant groups.
Comparison of minerals between healthy and HLB-affected leaves and pulp
A different view of overall variability may be obtained by displaying the results graphically with respect to individual cultivars, as illustrated in Figures 3 and 4. For example, on an individual cultivar basis, it is apparent that Zn and P were consistently lower in the HLB-affected leaves, and that P was consistently higher in pulp from HLB-affected trees. In pulp, K was consistently higher in infected samples, but the overall means for healthy and infected showed no statistical difference (p=0.0997). In the present study, it was unclear whether differences in mineral levels with regards to the HLB-status were cultivar-specific or simply reflected variability inherent in sweet orange generally.
Figure 3: Histogram representation of Zn, P and K content in healthy and HLB-affected leaves. Both Zn (31% decrease) and P (9% decrease) showed statistically significant decreases in HLB-affected samples between means as determined by one-way ANOVA (Table 1) and the Student’s t-test (Zn p<0.0001; P p=0.0437).
The summarized relationships between average levels (all cultivars combined) of each of the three minerals with respect to leaf and pulp samples and the corresponding HLB-infection status are graphically illustrated in Figure 5. Overall, the levels of all three minerals were less in pulp tissues as compared to leaves, regardless of the HLB status. The partitioning of P to the pulp increased in HLB-affected trees (Figure 5). Average P levels in the pulp of tested citrus cultivars were 29% (t-test p=0.0120) lower in the healthy samples versus HLB-affected. However, this pattern was reversed in leaves, which showed a 9% decrease in P in CLas-infected versus healthy samples (t-test p=0.0437). The average Zn levels markedly declined by 31% in infected leaves (t-test p<0.0001). However, despite the relatively large decrease in Zn content in infected leaves, no corresponding change was found to occur in the pulp.
In Figure 5, the histogram bars represent the cumulative means for all cultivars and summarizes the total data set for leaves and fruit pulp. Analysis by one-way ANOVA indicated that only the significant differences in means were for Zn and P in leaves, and P in the pulp. Of the three minerals evaluated, only P showed significant changes with respect to HLB-status in both leaves and fruit pulp. It is interesting that P changes in opposite manner between leaves and pulp: It increased in HLB-affected leaves but increased in the pulp from HLB positive-trees.
Table 3 shows the relationship between CLas DNA copy numbers and mineral contents. The DNA copy number was determined by q-PCR using primers annealing to the integrated prophage, and the mineral content is given for those leaf or pulp samples showing significant differences in the means between healthy and infected groups. As shown, no correlation exists between the DNA copy number and mineral content, even though significant differences exist between healthy and infected samples.
|Blood red||No Ct||1.86E+06||16.74||1455||1000|
|Casa grande||No Ct||1.71E+06||15.26||1355||718|
|Emby gold||No Ct||1.35E+06||16.11||1431||1092|
|Frost rose||No Ct||2.47E+06||20.19||1601||880|
|Mars early||No Ct||2.89E+06||13.16||1520||713|
Table 3: Comparison of DNA copy number and levels of Zn and P in leaves and pulp.
Discussion and Conclusion
The distribution pattern of three nutrients was determined in healthy and CLas-infected tissues in citrus trees from Pakistan. Our study provides the first determination of mineral content in a fruit tissue from CLas-infected trees. In general, we found average levels of Zn, P, and K all to be higher in the leaves as compared to the pulp, regardless of the CLas-infection status. Phosphorous showed significantly different means between healthy and infected samples for both leaves and pulp; however, levels decreased in HLB-positive leaves and increased in pulp from HLB-positive trees. Zn showed a statistically significant decrease in HLB-positive leaves, while K levels were not altered by the infection status in either the leaf or pulp samples. The lack of correlation between the copy number of CLas DNA and mineral content is not easily accounted for, but may be related to the large range in copy number variation routinely observed between leaf samples (1-5 leaves).
These findings are consistent with several previous studies; however, there is no uniform agreement in the literature, since reports can be found that show both increases and decreases of these minerals in CLas-infected leaves [39-42,49]. There is no simple way to reconcile these divergent findings; however, the results of Cao et al.  suggest that other parameters (that are yet poorly understood) may influence the distribution of minerals since their study showed both seasonal and varietal influences on mineral levels in CLas-infected leaves.
It is well established in plants that mineral nutrients are transferred from senescent leaves to sinks such as new leaves, roots, and developing fruit through a process known as “resorption” [50,51]. A major portion of this transfer of resources occurs via the phloem and is driven by the difference in turgor pressure at the source and sink [9,52]. It seems reasonable that pathogens that inhabit or affect the phloem, such as Liberibacter species, may alter the partitioning of minerals between leaves and sinks, such as fruit, over the progression of the disease. When the results of the current study are viewed with respect to resorption, only changes in P levels meet expectations for changes in reallocation between leaves and fruit. For example, a decrease in P was observed in HLB-affected leaves (-9%) along with an increase in pulp from HLB-positive trees (29%) which is consistent with a change in the partitioning of P from leaves to pulp in infected trees. However, if resorption in responsible for this change in partitioning, a disruption in phloem function by Liberibacter may be expected to decrease resorption efficiency in the transport of P from leaves to fruit, not increase the accumulation of P to fruit as our results suggest. Cao et al.  obtained a similar nonintuitive result where P resorption efficiency in leaves was increased in HLB infected plants. They also found that in C. reticulata, P removed from senescent leaves was not efficiency transported to live leaves, perhaps leaving open the possibility that the P was reallocated to the fruit instead.
Alternatively, the term “resorption” may not accurately describe the processes responsible for the HLB-specific distribution patterns observed for the nutrients in this study. Resorption implies that a loss has occurred from the healthy leaves, and the nutrients have been subsequently transported to growing centers such as fruit. However, most symptomatic leaves were probably infected as new flushes by the feeding psyllid adults and nymphs [34,53]. Hence, the mature leaves may have never been healthy and, as a consequence, exhibited reduced uptake of Zn during growth. Under such circumstances, leaves did not lose Zn or P but simply experienced reduced uptake of these minerals. A change in mineral import rates and partitioning, not resorption, may provide a better explanation of the observations. If this is accurate, then there are still questions regarding the underlying causes for the altered transport or import of nutrients in leaves and fruit tissues.
Further research is needed to develop a detailed understanding of the processes involved in macro- and micronutrient transport, especially regarding situations where the phloem is blocked or damaged. Those studies may provide critical insights into the pathology of the disease process which may suggest novel strategies for reducing or eliminating the deleterious effects resulting from CLas-infection in citrus.
This research was supported by a gift from the University of Florida Foundation to Khadija Gilani and funding from the International Support Initiative Program (IRSIP) of the Higher Education Commission of Pakistan. We thank Lance Verner for assistance with quantitative PCR assays and Dr. Eva Czarnecka-Verner for help with data analysis and careful reading of this manuscript.
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