Abstract
The devastating citrus disease huanglongbing (HLB) associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) has caused a more than 70% reduction in citrus production since its discovery in Florida in 2005. Most citrus scion cultivars are sensitive to HLB, whereas some cultivars used as rootstocks are tolerant. Using such tolerant rootstocks can help trees to cope better with the disease’s impact. Evaluating rootstock effects on a grafted scion in the field takes many years, but shorter-term evaluation is imperative to aid in rootstock selection for an HLB-endemic production environment. In this study, we investigated grafted healthy and CLas-infected citrus trees under controlled greenhouse conditions. The objectives were to identify traits suitable for assessing grafted tree tolerance in advance of longer-term field studies and aiding in the selection of superior rootstock cultivars. We assessed 10 commercially important rootstocks grafted with ‘Valencia’ sweet orange scion and with known field performance. At 6, 9, 15, and 21 months after graft inoculation (mai), leaf CLas titers were determined and canopy health was evaluated. Plants were destructively sampled at 21 mai to assess plant biomasses and other physiological and horticultural variables. There was little influence of the rootstock cultivar on CLas titers. Surprisingly, few HLB foliar disease symptoms and no differences in soluble and nonsoluble carbohydrate concentrations were measured in infected compared with healthy plants, despite high CLas titers and significant reductions in plant biomasses. Most trees on rootstocks with trifoliate orange parentage were less damaged by HLB than other rootstocks, although results did not always agree with reported field performance. Among the different variables measured, leaf size appeared to be most predictive for grafted tree assessment of HLB sensitivity. The results of this study provide a better understanding of the strengths and weaknesses of assessing rootstock influence on grafted tree performance in a controlled greenhouse environment. Although such studies provide valuable information for cultivar tolerance to HLB, other rootstock traits will ultimately contribute to field survival and productivity in an HLB endemic production environment.
Citrus huanglongbing (HLB), also known as “citrus greening,” is a destructive disease of citrus which was discovered in Florida in 2005 and has since reduced citrus production by more than 70% (Rosson, 2020; Singerman et al., 2018). HLB is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium (Garnier et al., 1984), which has not yet been successfully grown in pure culture (Merfa et al., 2019), and so remains elusive as the undisputed causal agent. Typical HLB symptoms include yellowing and asymmetric blotchy mottling of leaves, often resembling zinc and other nutrient deficiencies, followed by canopy die back, and decline of growth and production (Bové, 2006; da Graça, 2016). In Florida, CLas is vectored by the Asian citrus psyllid Diaphorina citri (Hall et al., 2013). Grafting a healthy tree with CLas-infected budwood is another possible way of transmission (Halbert and Manjunath, 2004). For experimental purposes, graft inoculation in the controlled environment of a greenhouse is faster and more effective compared with controlled vector inoculation or natural inoculation in an open field environment (Albrecht et al., 2014; Lopes et al., 2009).
Most citrus species, cultivars, hybrids, and citrus relatives are highly susceptible to HLB (McClean and Schwarz, 1970). HLB tolerance was identified in some citrus relatives such as trifoliate orange (Poncirus trifoliata) and several of its hybrids (Albrecht and Bowman, 2012; Folimonova et al., 2009; Ramadugu et al., 2016). Selecting a tolerant rootstock can help infected trees to cope better with the negative impacts of the disease. This was demonstrated in several field studies where some rootstock cultivars performed considerably better under HLB pressure than others (Boava et al., 2015; Bowman and McCollum, 2015; Bowman et al., 2016a, 2016b; Kunwar et al., 2021). To determine the influence of a selected rootstock on the grafted tree tolerance to HLB under open field conditions, measures of tree size, canopy volume, fruit yield, and fruit quality are standard practice and often complemented by foliar disease ratings and CLas titer determinations (Bowman et al., 2016a; Kunwar et al., 2021; Stover et al., 2016). For citrus trees to reach maturity and become productive takes several years. Greenhouse evaluation of new cultivars is therefore highly advantageous to select the most promising cultivars before long-term field evaluation.
To date, no cure exists for HLB. Many strategies, including vector control (Qureshi et al., 2008), thermal therapy (Hoffman et al., 2013), and chemotherapy (Zhang et al., 2021), have been tried to reduce the spread of the disease and minimize CLas titers, but have had limited or no success. HLB has been considered endemic in Florida since 2013 (Graham et al., 2020). Vector management combined with nutritional therapies and other cultural practices (Ferrarezi et al., 2019; Stansly et al., 2014) are now being employed to keep trees in production but are costly or difficult to implement on a large scale and harmful to the environment. In contrast, using tolerant and otherwise superior rootstock cultivars requires no additional investment and is environmentally sustainable.
To reliably test new cultivars in the greenhouse, concise evaluation criteria need to be established. Aside from plant biomass assessments, disease symptom ratings, and CLas titer determinations, analysis of carbohydrates and leaf macro- and micronutrient concentrations may provide valuable information. CLas resides in the phloem of infected plants where it causes major metabolic disturbances, especially of carbohydrate metabolism (Albrecht and Bowman, 2008; Fan et al., 2010; Kim et al., 2009). One of the characteristic symptoms of HLB are massive starch accumulations (Etxeberria et al., 2009), which lead to the typical blotchy mottled appearance of affected leaves. Metabolic disturbances, phloem dysfunction (Brodersen et al., 2014), root starvation (Etxeberria et al., 2009), and root decline (Johnson et al., 2014) may further lead to nutrient deficiencies (Pustika et al., 2008). Consequently, nutritional applications have increased in frequency in Florida (Rouse et al., 2017; Zambon et al., 2019), although beneficial effects are variable.
Deciphering the factors that are involved in HLB tolerance are important for citrus breeder to develop selectable traits or markers for resistance breeding and to control or at least mitigate the destructive consequences of the disease. In the absence of HLB-tolerant commercially relevant scion cultivars, the use of HLB tolerant rootstocks is one of the best strategies to diminish HLB impacts.
The only way to study rootstock effects on the grafted tree tolerance to HLB under controlled conditions (by directly comparing healthy and infected plants) is in the greenhouse. However, HLB tolerance–associated plant traits in the greenhouse that correlate well with tree open-field performance remain elusive. One objective of this study was to compare the performance of different commercially important rootstock cultivars grafted with ‘Valencia’ scion in the healthy state and HLB-affected state. Greenhouse studies that investigated cultivars in the seedling state or as scions (Albrecht and Bowman, 2011, 2012; Alves et al., 2021; Folimonova et al., 2009) have provided precise information on cultivar tolerance; whereas early information derived from studies investigating rootstock effects on the grafted tree tolerance has been less clear (Stover et al., 2018). Our previous greenhouse study provided relatively strong indication of greater HLB tolerance for some rootstocks than others in grafted combination with sweet orange, and those results correlated well with observed field performance for those rootstocks (Bowman and Albrecht, 2020). From this prior greenhouse study, scion leaf area, scion leaf number, scion leaf color, and rootstock bacterial titer demonstrated the best positive association with what has been observed as the field tolerance of the rootstocks to HLB. With this background, our second objective was to define plant traits best suited for the greenhouse assessment of the grafted tree tolerance by using rootstock cultivars with known field performance and sensitivity to HLB.
This study is the first part of a larger study; while this part examines rootstock effects on the grafted scion, the second part examines HLB effects on the belowground portion of the grafted tree (Bodaghi et al., 2022).
Materials and Methods
Plant material
Ten commercial citrus rootstock cultivars with different sensitivity to CLas were used in this study (Table 1); all these rootstocks are known to produce primarily nucellar seedlings that are genetically identical to the seed parent. Plants were grown from seed by planting one seed each into 3.8 cm × 21 cm cone cells (Containers; Stuewe and Sons, Tangent, OR) containing premoistened soilless potting mix (Pro Mix BX, Premier Horticulture, Inc., Quakertown, PA). After germination, plants were irrigated as needed and fertilized biweekly using a water-soluble fertilizer (20N–10P–20K; Peters Professional, The Scotts Company, Marysville, OH) at a dose of 400 mg N·L−1. Insecticides were applied as needed. Any off-types arising from zygotic embryos were identified based on their different leaf morphology and growth habit and discarded.
Rootstocks, parentage, and previously reported huanglongbing (HLB) response.
After 6 months, plants were transplanted into 19.7 cm × 31.8 cm plastic tree pots (Stuewe and Sons, Tangent, OR) and were grafted with FDACS DPI (Florida Department of Agriculture and Consumer Services, Division of Plant Industry)–certified ‘Valencia’ (Citrus sinensis) budwood (clone 1–14–19) using the inverted-T method. All plants were grown under DPI-certified disease-free conditions in the U.S. Horticultural Research Laboratory greenhouses (USDA, Fort Pierce, FL).
Six months after grafting (Nov. 2017), 12 plants of each graft combination were inoculated with buds from infected greenhouse-grown ‘Valencia’ orange plants that were polymerase chain reaction (PCR) positive for CLas and symptomatic for HLB. Three 1–1.5 cm long bud pieces were used to inoculate each plant using the inverted-T method. Buds were inserted on different sides in the trunk at 4–6 cm above the graft union. Six plants of each genotype were mock inoculated with disease-free bud pieces obtained from healthy greenhouse-grown ‘Valencia’ orange plants for use as noninfected controls.
Nine months after inoculation (Aug. 2018), plants were transplanted into 37 cm × 45 cm (48 L) plastic tree containers (Nursery Supplies, Chambersburg, PA) containing Pro Mix BX potting medium. Of the 12 CLas-inoculated plants, only those with a cycle threshold (Ct) value of 32 or lower were included for a total of six replications.
Plants were arranged in a completely randomized design on the greenhouse benches and kept under natural light conditions at a temperature of 21 to 28 °C. Plants were irrigated and fertilized as described previously, and insecticides were applied as needed. Tree size was managed by removing ≈30% of shoot growth every 6 months. The experiment was ended 21 months after inoculation (mai).
Determination of CLas titers
Four fully expanded leaves from the most recent growth were collected from each tree 6, 9, 15, and 21 mai. One hundred milligrams of ground tissue were used for DNA extraction. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Real-time PCR assays were performed using primers HLBas and HLBr and probe HLBp (Li et al., 2006). Primers COXf and COXr and probe COXp were used for normalization. Amplifications were performed using an Applied Biosystems QuantStudio 3 Real-Time PCR system and the iTaq Universal Probes Supermix (Bio-Rad, Hercules, CA). All reactions were carried out in duplicate in a 20-μL reaction volume using 5 μL DNA.
Plant biometric measurements
Canopy volume, scion and rootstock trunk diameters, leaf area, and the number of leaves were assessed at the end of the study, 21 mai. Canopy volume was determined based on the formula V = 0.524 × height × width2 (Forner-Giner et al., 2014). Canopy height was measured from the lowest point of branching to the highest point of the canopy. Canopy width was measured in two perpendicular directions. Trunk diameters of scion and rootstock were measured with a digital caliper at 5 cm above and 5 cm below the graft union (avoiding the inoculation area), respectively, and in two perpendicular directions.
For leaf area determination, all leaves were removed from each plant. Leaves were counted and the total leaf area was measured using an LI-3000C Portable Area Meter in combination with the LI-3050C Transparent Belt Conveyer Accessory leaf area meter (LI-COR Biosciences, Lincoln, NE).
Canopy health assessment
Canopy health was assessed 6, 9, 15, and 21 mai by visually rating HLB disease symptoms and canopy thickness. To assess foliar HLB disease symptoms, trees were examined for chlorosis, blotchy mottle, green islands, and other foliar abnormalities presumably associated with HLB, and scored on a scale of 0 to 5 with 0 = no foliar disease/nutritional symptoms, 1 = <10% of leaves with foliar symptoms, 2 = 10% to 25% of leaves with foliar symptoms, 3 = 25% to 50% of leaves with foliar symptoms, 4 = 50% to 75% of leaves with foliar symptoms, 5 = >75% of leaves with foliar symptoms.
Canopy thickness was rated on a scale of 1 to 5 with 1 = very thin canopy, 2 = thin canopy, 3 = moderately dense canopy, 4 = dense canopy, and 5 = very dense canopy. If plants died during experimentation, they were assigned a value of 0.
Leaf nutrient concentrations
Ten fully expanded leaves were collected from each tree at the end of the experiment. Leaf macro (N, P, K, Ca, Mg, S) and micronutrients (B, Zn, Mn, Fe, Cu) were analyzed by Waters Agricultural Laboratories Inc. (Camilla, GA). The total nitrogen concentration was measured by combustion (Sweeney, 1989). The other nutrients were analyzed by inductively coupled argon plasma atomic emission spectroscopy (ICAP) after acid digestion (Huang and Schulte, 1985).
Leaf carbohydrate concentrations
Four fully expanded leaves were collected randomly from each quadrant of the plant canopy 21 mai. One hundred and fifty milligrams of ground tissue were used for carbohydrate extraction. Each sample was extracted in 1 mL of 80% ethanol for 1 h at 70 °C and centrifuged for 5 min at 20,000 gn. The pellet was re-extracted as described above and supernatants were combined for analysis of soluble carbohydrates. Insoluble pellets were used for starch determination.
Soluble carbohydrate determination.
Supernatants were dried in a DNA 110 Speed Vac (Vacufuge Plus, Eppendorf, Hamburg, Germany), resuspended in 500 μL of ultrapure water, and centrifuged for 5 min at 20,000 gn. Supernatants were used for soluble carbohydrate determination. Glucose and fructose were measured sequentially by an enzymatic assay as described in Gomez et al. (2007). In brief, glucose determination was performed by phosphorylation of glucose to form glucose-6-phosphate (G6P) by hexokinase (HK), followed by the conversion of G6P and NAD to gluconate-6-phosphate (6PGlcU) and NADH by glucose-6-phosphate dehydrogenase (G6PDH). Fructose determination was performed by phosphorylation of fructose to form fructose-6-phosphate (F6P) by HK, the conversion of F6P to G6P by phosphoglucose isomerase, and the conversion of G6P and NAD to 6PGlcU and NADH. NADH production was measured at 340 nm using a SpectraMax 190 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). Sucrose was determined by measuring glucose as described above, followed by cleavage into glucose and fructose by invertase. All enzymatic reactions were carried out in 50 mm triethanolamine-HCl, 5 mm MgSO4, 0.02% bovine serum albumin, and 0.5 mm dithiothreitol (Campbell et al., 1999). Assays were performed at least in duplicate.
Starch determination.
Pellets remaining from soluble sugar extraction were dried in a DNA 110 Speed Vac and resuspended in 900 μL of ultrapure water. Starch was dispersed by autoclaving for 1 h at 121 °C and 19 psi. An equal volume of sodium acetate buffer (0.1 M, pH 4.65) was added together with 5 μL (14 units) of amyloglucosidase (Sigma-Aldrich, St. Louis, MO) and samples were incubated for 100 min at 56 °C. After centrifugation for 5 min at 20,000 gn, supernatants were used for starch determination. Starch was measured indirectly by enzymatic assay of released glucose as described for soluble carbohydrates.
Statistical analysis
Two-way analysis of variance (ANOVA) was conducted with disease state and rootstock as fixed effects. Comparison of rootstock effects for each trait was by one-way ANOVA. Mean separation was performed by Tukey’s honestly significant difference test. For ordinal data (canopy health assessments), Kruskal–Wallis ANOVA was used, and separation of mean ranks was performed by Dunn’s test. Differences were defined as statistically significant when the P value was <0.05. Data were analyzed using R 3.6.0 (The R Foundation for Statistical Computing).
Results
Leaf CLas titers.
CLas titers were expressed as Ct values with lower Ct values indicating higher CLas titers and vice versa. The average Ct values of leaves of infected plants were significantly higher at 6 mai and 8 mai (28.0 and 28.4) than at 15 mai and 21 mai (26.3 and 24.7), indicating that CLas titer levels increased over time (Table 2). A significant influence of rootstock cultivar on leaf Ct values was observed only at 6 mai when trees on ‘Carrizo’ had the lowest Ct value (26.0) and therefore the highest level of bacteria and ‘US-1516’ had the highest Ct value (31.0) and therefore the lowest level of bacteria.
Leaf cycle threshold values of infected ‘Valencia’ trees grafted on different rootstocks.
Canopy health.
Few plants showed foliar HLB symptoms at 6 mai (Table 3). At 9 mai, more plants displayed blotchy mottling symptoms, but foliar disease symptoms remained indistinct throughout the study with average foliar disease indices not exceeding 0.5. Although there was a significant rootstock effect at 15 mai, separation of means did not identify significant differences among rootstocks. Only at 21 mai were significant differences detected among rootstocks, and trees grafted on ‘Cleopatra’ had more foliar disease symptom compared with trees grafted on US-942 and US-1516.
Foliar disease symptom and canopy thickness of infected ‘Valencia’ trees grafted on different rootstocks.
Canopy thickness indices were highest at 6 mai (5.0) and lowest at 15 mai and 21 mai (3.7 and 3.9) (Table 3). There were no significant differences among trees grafted on the different rootstocks.
Mock-inoculated trees did not display any foliar abnormalities or canopy thinning throughout the experiment (data not shown).
Plant biometric variables.
CLas infection reduced all measured variables significantly (Table 4). Canopy volumes were reduced from 0.614 m3 for healthy plants to 0.319 m3 for infected plants (48%), but there was no significant rootstock effect nor interaction between disease state and rootstock. Scion trunk diameters were significantly reduced by 21% upon infection but there was a significant interaction with the rootstock cultivar (Supplemental Table 1). The largest trunk diameters were measured for healthy trees on ‘Ridge’, ‘Cleopatra’, sour orange, ‘Carrizo’, ‘US-802’, and ‘US-942’ (18.7–21.5 mm) and the smallest were measured for infected trees on ‘US-812’, ‘Swingle’, ‘Ridge’, and ‘Carrizo’ (14.0–14.5 mm). Rootstock trunk diameters were reduced by 22% upon infection and there was a significant rootstock effect but no interaction. ‘Swingle’ and ‘US-802’ had the largest healthy rootstock trunk diameter (25.6 mm and 26.3 mm) and sour orange had the smallest (20.3 mm).
Disease state and rootstock effects on plant biometric variables of grafted ‘Valencia’ trees 21 months after CLas inoculation.
The largest reductions of canopy volumes in response to CLas infection were measured for trees grafted on ‘Cleopatra’ and ‘Ridge’ (65% and 66%), followed by trees on ‘Carrizo’ and sour orange (58% and 56%) (Table 5). Reductions for trees on the other rootstocks were 31% to 49%, but reductions for ‘Swingle’ and ‘US-1516’ were significant only at levels of 5.8% and 6.3%, respectively. The largest reductions of scion and rootstock trunk diameters were measured for ‘Ridge’ (34% and 33%). ‘US-897’ and ‘US-802’ showed no significant reductions of either scion or rootstock trunk diameters. Canopy volume and trunk diameter reductions are illustrated in Fig. 1.
Reductions (%) in canopy volume, and scion and rootstock trunk diameters of ‘Valencia’ trees grafted on different rootstocks 21 months after Candidatus Liberibacter asiaticus (CLas) inoculation.
Citation: HortScience 57, 1; 10.21273/HORTSCI16205-21
Canopy volume and trunk diameters of healthy and infected ‘Valencia’ trees grafted on different rootstocks 21 months after inoculation.
The total leaf area, number of leaves, and leaf area per leaf were significantly reduced in infected compared with healthy plants (Table 6). The total leaf area and area per leaf were reduced by 52% and 24%, respectively, upon infection whereas the number of leaves was reduced by 38%. There was no significant rootstock effect and no interaction between disease state and rootstock.
Disease state and rootstock effects on leaf area and number of leaves of grafted ‘Valencia’ trees 21 months after CLas inoculation.
Reductions in total leaf area in response to CLas infection were largest for trees on ‘Ridge’, ‘Cleopatra’, and sour orange (60% to 76%) compared with the other rootstocks, which experienced reductions of 34% to 55% (Table 7). The largest reductions for the number of leaves were found in trees on ‘Ridge’ (55%) whereas no significant reductions were found for trees on ‘Swingle’, ‘US-897’, and ‘US-1516’. The leaf area per leaf reductions were largest in ‘Cleopatra’ and ‘Ridge’ (46% and 38%) whereas reductions for the other rootstocks did not exceed 26% or were statistically not significant (‘Carrizo’, ‘US-897’). Leaf area and leaf number reductions are illustrated in Fig. 2.
Reductions (%) in leaf area, number of leaves, and leaf area per leaf of ‘Valencia’ trees grafted on different rootstocks 21 months after Candidatus Liberibacter asiaticus (CLas) inoculation.
Citation: HortScience 57, 1; 10.21273/HORTSCI16205-21
Total leaf area, number of leaves, and leaf size of healthy and infected ‘Valencia’ trees grafted on different rootstocks 21 months after CLas inoculation.
Leaf nutrient concentrations.
Among the macronutrients, magnesium (Mg), calcium (Ca), and sulfur (S) were significantly reduced by 6% to 10% in infected compared with healthy plants (Table 8). Rootstock effect was significant for all macronutrients except Mg, and there was no significant interaction between disease state and rootstock.
Disease state and rootstock effect on leaf macronutrient concentrations of healthy and infected ‘Valencia’ trees grafted on different rootstocks 21 months after CLas inoculation.
Nitrogen concentrations were highest in trees grafted on ‘Swingle’ (3.57%) compared with most of the other rootstocks (2.88% to 3.10%). Phosphorous concentrations were higher in trees grafted on ‘US-802’, ‘US-812’, ‘US-897’, ‘US-942’, and ‘US-1516’ (0.220) compared with the other rootstocks (0.197% to 0.207%). Potassium concentrations were highest in trees on ‘US-1516’ (3.09%) and lowest in trees on ‘Cleopatra’, ‘US-812’, and ‘US-897’ (2.85% to 2.87%). Trees on ‘Carrizo’ and US-897’ had the highest Ca concentrations (2.42% and 2.41%) and trees on sour orange had the lowest (2.07%). Sulfur concentrations were highest in trees grafted on ‘Carrizo’, ‘US-802’, ‘US-812’, ‘US-942’, and ‘US-1516’ (0.353% to 0.373%) and lowest in trees on ‘Cleopatra’ (0.305%).
Although significant reductions in response to CLas infection were measured across all rootstocks for Mg, Ca, and S, within each rootstock cultivar, few significant reductions were found (Table 9). Significant reductions were measured for Ca if trees were grafted on ‘Ridge’, ‘Cleopatra’, ‘Carrizo’, and ‘US-942’, and for S if trees were grafted on ‘US-897’.
Leaf magnesium, calcium, and sulfur concentrations of healthy and infected ‘Valencia’ trees grafted on different rootstocks 21 months after inoculation.
Among the leaf micronutrients, iron (Fe), zinc (Zn), and manganese (Mn) concentrations were significantly reduced by 6% to 11% in response to CLas infection (Table 10). Rootstock effect was significant for all micronutrients and there was a significant interaction between disease state and rootstock for Fe (Supplemental Table 2). Iron concentrations were highest in healthy trees on ‘Carrizo’ and lowest in healthy trees on ‘US-1516’ and infected trees on ‘Ridge’. Boron concentrations were highest in trees grafted on ‘Cleopatra’ and ‘US-802’ (46 ppm and 47 ppm) and lowest in trees on sour orange and ‘Carrizo’ (36 ppm). Zinc concentrations were highest in trees on ‘US-802’ (22 ppm) and lowest in trees on sour orange. The largest Mn concentrations were measured in trees on ‘Ridge’ and ‘Carrizo’ (43 ppm and 42 ppm) and the lowest in trees on ‘US-1516’ (28 ppm). Copper concentrations were highest in trees on ‘US-942’ (3.5 ppm) compared with most of the other rootstocks (2.0–2.3 ppm).
Disease state and rootstock effect on leaf micronutrient concentrations of healthy and infected ‘Valencia’ trees grafted on different rootstocks 21 months after CLas inoculation.
Within rootstocks, iron reductions were only significant for trees on ‘Ridge’, ‘Carrizo’, and ‘US-812’ (Table 11). Zinc reductions were only significant for sour orange and ‘Carrizo’; the same was found for Mn.
Leaf iron, zinc, and manganese concentrations of healthy and infected ‘Valencia’ trees grafted on different rootstocks 21 months after inoculation.
Leaf carbohydrate concentrations.
CLas infection did not have a significant impact on any of the carbohydrates measured, and neither did rootstock (Table 12). Significant differences were observed between root and leaf tissue, with leaves having higher concentrations of fructose, sucrose, and starch and lower concentrations of glucose than roots. There was no significant interaction between disease state, rootstock, and tissue.
Disease state and rootstock effects on carbohydrate concentrations of healthy and infected ‘Valencia’ trees 21 months after inoculation.
Discussion
Rootstock selection has been a successful strategy to alleviate the impacts of stresses and diseases on the grafted tree. Assessing the rootstock response to HLB has become an important component of citrus breeding programs and knowledge of the response before long-term field planting is desirable. Studies with rootstock seedlings can provide valuable information on the cultivar response to HLB; however, this response may change after grafting with a susceptible scion. Here we investigated CLas and rootstock effects on the grafted tree response to identify horticultural or physiological markers predictive of HLB sensitivity that may be used for rootstock screening before field testing.
Six months after graft inoculation, the rootstock cultivar influenced CLas titers in the scion, but effects were significant only for ‘Carrizo’ and ‘US-1516’ and without any clear association to the known field performance. No differences for CLas titers were found during the remaining study period, suggesting that none of the rootstocks examined here were able to reduce CLas titers in the leaves, regardless of their presumed level of tolerance. The same was found in a previous greenhouse study that was conducted over a shorter period than this study (Bowman and Albrecht, 2020). In contrast, Stover et al. (2018) found differences in CLas titers of the sweet orange scion induced by the rootstock cultivar, but differences were inconsistent among experiments and time of evaluation. It has been documented that bacterial DNA can persist in plant tissues even after cell death, which can lead to misinterpretation of the results (Etxeberria et al., 2019) and may contribute to the reported differences among studies. CLas titers in the leaves, therefore, do not appear to be a useful measure for assessing grafted tree tolerance, at least not under controlled greenhouse conditions and in graft combinations with a susceptible scion.
Only mild foliar HLB disease symptoms were discernible throughout the evaluation period although symptom severity increased during the later stages of the study. Differences among rootstocks were only evident at the end of the experiment but were not meaningful to distinguish between superior and inferior rootstocks. Lee et al. (2015) provided evidence that plants become infected within 15 d after receiving CLas inoculum, but HLB foliar disease symptoms may not manifest until several months after inoculation (Albrecht et al., 2012, 2014; Shokrollah et al., 2011). Stover et al. (2018) did not identify typical foliar blotchy mottle symptoms until 1 year after pathogen exposure, regardless of whether plants were graft inoculated or psyllid inoculated. A field study investigating young mandarin trees with different combinations of rootstocks and interstocks detected HLB symptoms in nearly two thirds of the trees at 6 mai (Shokrollah et al., 2011). Although some of the differences among studies might be due to the different inoculation methods and other differences in experimentation, it is evident that even under controlled conditions infected trees often do not exhibit HLB disease symptoms until later stages of the disease.
Ramadugu et al. (2016) evaluated different citrus species and citrus relatives as seedlings and under natural field conditions and high HLB pressure. The trial was conducted over 6 years to clearly define the disease response based on CLas titers, and foliar disease symptoms. Folimonova et al. (2009), who assessed different citrus cultivars as seedlings or grafted scions for their HLB response, reduced the time for foliar disease symptoms to become discernible and more severe by subjecting plants to continuous light conditions. Although different genotypes expressed different responses to HLB, the authors did not find any strict correlation between bacterial titer and severity of the disease. The absence of severe foliar disease symptoms found in our study could be related to the lower light conditions in the greenhouse compared with natural field conditions in combination with shading due to the proximity of plants on the greenhouse benches. The inconsistencies and difficulties associated with inducing a uniform foliar disease symptom response in seedling as well as grafted trees suggest the limited value of this measure.
CLas infection reduced canopy volumes substantially in most graft combinations. Although the largest reductions were found for trees grafted on the most susceptible cultivars (Ridge and Cleopatra), reductions were substantial even on tolerant rootstocks such as ‘US-942’. Similar results were found for the scion and trunk diameter reductions, which were largest for trees on ‘Ridge’ but showed no clear trend for the remaining rootstock cultivars. Regardless of infection, the largest rootstock trunk diameters were found for ‘Swingle’ and ‘US-802’, which are both known for this trait (Bowman, 2007; Bowman and Joubert, 2020; Tazima et al., 2013)
Like the canopy volume, the leaf area and number of leaves exhibited large reductions in response to CLas infection regardless of the rootstock on which trees were grafted. The largest canopy volume reductions occurred in trees on ‘Ridge’, ‘Cleopatra’, and sour orange, but reductions were also large in trees on ‘US-942’. These results are different from those of a recent greenhouse study where the grafted ‘Valencia’ scion generally performed better on ‘US-942’ than most of the other rootstocks in the study (Bowman and Albrecht, 2020). In that study, which was conducted over 50 weeks, it was suggested that longer evaluation and larger pots may be necessary for grafted tree evaluations. The study presented here was nearly twice as long and trees were transplanted into large pots, but this did not appear to have eliminated some of the inconsistencies between greenhouse and field observations. ‘US-942’ has been the number-one propagated rootstock in Florida since the 2018–19 production season (Rosson, 2020) because of its superior performance under high HLB pressure. The good field performance of this rootstock may therefore be associated with other rootstock traits, such as a better ability to absorb water and nutrients and to cope with stresses other than HLB, which are not evaluated under controlled greenhouse conditions optimized for good growth.
The leaf size or area per individual leaf was reduced by 24% on average but reductions were larger than average for the two most susceptible cultivars, Ridge and Cleopatra, and lower than average for the tolerant rootstocks. This suggests that leaf size might serve as a more suitable marker for HLB sensitivity than the canopy volume or the total leaf area.
Nutrient deficiencies have been reported as one of the consequences of HLB (Pustika et al., 2008; Spann and Schumann, 2009), which has led to enhanced nutrient management in affected groves despite some controversy (Gottwald et al., 2012; Rouse et al., 2017; Stansly et al., 2014). The results of our study showed only a moderate effect of CLas infection on leaf nutrient concentrations. Among the macronutrients that were reduced in infected trees were Mg, Ca, and S, but reductions were less than 10% and not evident for all rootstock combinations. Calcium was the most reduced nutrient, but reductions were not related to rootstock sensitivity to HLB. Micronutrient reductions were observed for Fe, Zn, and Mn but like the macronutrients, reductions were small and not related to rootstock tolerance.
Based on the guidelines for interpretation of sweet orange tree leaf nutrient concentrations (Kadyampakeni and Morgan, 2020), trees in our study had optimum to high concentrations of most nutrients except Mg, Ca, Zn, and Cu. This suggests that HLB-induced leaf nutrient deficits are most likely to occur when nutrient concentrations are suboptimal for healthy plants. It also implies that the large plant biomass reductions of infected plants experienced in our study were not a direct consequence of a CLas-induced reduced uptake of nutrients. The overall good nutrient status of our experimental plants may explain the absence of clearly expressed foliar HLB symptoms. This was previously reported by Bowman and Albrecht (2020) who hypothesized that good nutritional management in the greenhouse can correct only a small component of the negative effects from CLas infection, and that this may explain some of the controversies regarding nutrient management for HLB disease (Gottwald et al., 2012). Both Zn and Ca are important components of the plant defense system (Cabot et al., 2019; Lecourieux et al., 2006). Magnesium, a structural component of chlorophyll, plays a fundamental role in the phloem export of photosynthates and carbon partitioning (Cakmak and Kirby, 2008), and its role in plant disease and defense has been described (Huber and Jones, 2013 and references therein). Razi et al. (2011) did not find any relationship between nutritional status and HLB incidence but suggested that nutritional treatments may help in stress relief and sustaining production for some period. Huber and Jones (2013) pointed out that nutrient manipulation is an important part of production management and can provide effective control of many plant diseases when integrated with genetic resistance and other cultural practices.
Disturbance of carbohydrate metabolism resulting in starch accumulation is one of the major consequences of HLB (Albrecht and Bowman, 2008; Fan et al., 2010; Kim et al., 2009). Disease-induced carbohydrate accumulations have also been reported in other host–pathogen systems (Berger et al., 2007; Lepka et al., 1999; Maust et al., 2003) and it was suggested that sugars enhance plant defense responses against pathogen infection (Herbers et al., 2000). However, in this study, although tree growth was clearly reduced upon infection, no differences in carbohydrate concentrations between healthy and infected plants were measured. This was unexpected but is in accordance with the lack of a clearly defined foliar disease response and demonstrates that disturbance of carbohydrate metabolism is not the only reason, or perhaps not even the primary reason, for tree decline. Other metabolic pathways that were found to be disrupted by CLas infection include amino acid, fatty acid, shikimate, and other secondary metabolite pathways (Albrecht et al., 2020; Rao et al., 2018).
Conclusions
The results of this study provide a better understanding of the strengths and limitations of assessing grafted rootstock performance under HLB pressure in the controlled environment of a greenhouse. Among the measured variables, leaf CLas titers were of limited use for identifying rootstock effects and only during the early stages of infection. Similarly, foliar disease symptom assessment provided no meaningful information to discriminate among rootstock effects on the scion, at least under conditions that optimize nutritional availability. CLas-induced changes of leaf nutrient concentrations were moderate and did not suggest any rootstock-related effect on HLB tolerance. The lack of a CLas-induced effect on carbohydrate accumulation was surprising but highlights the importance of other metabolic pathways involved in the HLB disease process. Plant biomass reductions were generally more severe for the most susceptible cultivars, but except for the leaf size, severe reductions were also observed for tolerant rootstocks, which have demonstrated good field tolerance to HLB. Taken together, our results suggest that other rootstock traits besides simple tolerance to CLas contribute to field survival and productivity in an HLB endemic production environment, and that long-term field assessment is indispensable.
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Scion trunk (SC) diameter—disease state and rootstock interaction.
Leaf iron concentration—disease state and rootstock interaction.
