Evaluation of Three New Citrus Rootstocks under Boron Toxicity Conditions

Boron (B) toxicity is a common limiting factor both in arid and semiarid regions, such as the Mediterranean Basin. Citrus trees are sensitive to B-toxicity, which generates a negative impact in citrus orchards. In this work, two promising citrus rootstocks (UFR-6 and 2247 x 6070-02-2) have been assessed against B-toxicity and compared with Carrizo citrange, a common commercial citrus rootstock in Mediterranean Basin. Three B concentration treatments (Control, 1 and 2.5 mM H3BO3) were established, irrigating the plants three times per week for 21 days under greenhouse conditions. During the assay, above-ground symptoms, and chlorophyll index (SPAD) were recorded. At the end of the experiment, stomatal conductance, relative water content, and B concentration in leaves and roots were determined. The increasing B concentration in plants generates visual damage in leaves for all citrus rootstocks assayed. Carrizo citrange displayed the greatest visual symptoms, decreased its chlorophyll index (SPAD), and stomatal conductance throughout the B-treatment. However, UFR-6 and 2247 x 6070-02-2 displayed less symptoms than Carrizo citrange and only reduced its parameters under the 2.5 mM H3BO3 treatment. These results can aid citrus grower rootstock planting decisions with under B-toxicity conditions.

Determination of Genome Size Differentiation and Ploidy Levels in Some Citrus Rootstock Populations

In plants, knowing the ploidy level of plant material used in breeding studies, and especially for biotechnology applications, carries great importance. The presence of a rapid variety of dynamics in citrus fruits allows their use as rootstock and varieties ensuring adaptability to various climate and soil conditions with different breeding methods. A variety of appropriate rootstocks are used for commercial citrus species. This study investigated the genome sizes and ploidy levels in citrus rootstocks commonly used around the world with flow cytometry in seedling populations. The study used Gou-Tou, C-35, Troyer Citrange, Taiwanica, Citremon, Yuzu, Sunki mandarin, Flying Dragon, Yuma Citrange, Macrophylla and Chinese orange rootstocks. Fresh leaf tissues were mixed with the triploid Tahiti lime leaf tissue, used as standard species, and cell nuclei were isolated. Cells stained with propidium iodide were read with flow cytometry and histograms and cytograms were obtained. According to the obtained results, all seedlings of species had diploid genome volumes. In terms of genome volume, there were differences found between species. Yuzu seedlings were determined to be the species with largest genome volume (0.808 pg/2C), while Flying Dragon trifoliate had smallest genome volume (0.700 pg/2C).

Determining Seed Viability During Fruit Maturation to Improve Seed Production and Availability of New Citrus Rootstocks

In recent years, the pressure for replanting and resetting huanglongbing (HLB or citrus greening) affected citrus groves has led to an inadequate seed supply for the most popular rootstock cultivars in the State of Florida, United States. Early fruit harvesting of citrus rootstock source trees might reduce fruit losses and enhance seed availability, especially in HLB-endemic and hurricane susceptible areas, if the physiological quality of the seeds is adequate. The effects of fruit maturity on seed quality and seedling performance of US-802, US-897, and US-942 citrus rootstocks were investigated for two consecutive growing seasons. The study included the evaluation of seed germination and nursery performance of the citrus rootstock seedlings. The germination test was performed in vitro, where seeds were hand-peeled, surface-sterilized and placed in culture tubes containing basal Murashige and Skoog medium. For the emergence test, seeds were sown in seedling trays containing sterilized growing substrate in a greenhouse with controlled-environment conditions. Rootstock fruits from all three varieties harvested in August and September had seeds with higher germination potential, as more than 90% of the seeds generated seedlings. US-942 had more % of emergence than US-802 and US-897, resulting in faster seed germination; in contrast, US-802 had the faster shoot growth rate. Assays on fruit abscission response showed that by August, fruit from all three varieties were responsive to ethylene and abscised, although response varied and was higher in US-942, suggesting the seeds were mature enough. Taken together, our findings indicate that fruits these three rootstocks can be harvested as early as August in contrast to the current procedures without losing germination potential. This will result in an increase in available seeds for nurseries in Florida.

Screening of ‘King’ Mandarin Hybrids as Tolerant Citrus Rootstocks to Flooding Stress

This work compares the tolerance to long-term anoxia conditions (35 days) of five new citrus ‘King’ mandarin (Citrus nobilis L. Lour) × Poncirus trifoliata ((L.) Raf.) hybrids (named 0501XX) and Carrizo citrange (CC, Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.), the widely used citrus rootstock in Spain. Growth parameters, chlorophyll concentration, gas exchange and fluorescence parameters, water relations in leaves, abscisic acid (ABA) concentration, and PIP1 and PIP2 gene expressions were assessed. With a waterlogging treatment, the root system biomass of most hybrids went down, and the chlorophyll a and b concentrations substantially dropped. The net CO2 assimilation rates (An) and stomatal conductance (gs) lowered significantly due to flooding, and the transpiration rate (E) closely paralleled the changes in gs. The leaf water and osmotic potentials significantly increased in most 0501 hybrids. As a trend, flooding stress lowered the ABA concentration in roots from most hybrids, but increased in the leaves of CC, 05019 and 050110. Under the control treatment (Ct) conditions, most 0501 hybrids showed higher PIP1 and PIP2 expressions than the control rootstock CC, but were impaired due to the flooding conditions in 05019 and 050110. From this study, we conclude that 0501 genotypes develop some adaptive responses in plants against flooding stress such as (1) stomata closure to prevent water loss likely mediated by ABA levels, and (2) enhanced water and osmotic potentials and the downregulation of those genes regulating aquaporin channels to maintain water relations in plants. Although these traits seemed especially relevant in hybrids 050110 and 050125, further experiments must be done to determine their behavior under field conditions, particularly their influence on commercial varieties and their suitability as flooding-tolerant hybrids for replacing CC, one of the main genotypes that is widely used as a citrus rootstock in Spain, under these conditions.

Water-Stress Influences on Three New Promising HLB-Tolerant Citrus Rootstocks

Drought and flooding conditions are increasingly common abiotic factors that affect citrus crops in both the Mediterranean Basin and Florida. Furthermore, emerging diseases, such as Huanglongbing (HLB), are a potential risk for these crops in those producing areas. This study aimed to evaluate the behavior under water-stress treatments of three new citrus rootstocks (UFR-6, B11R5T60, and 2247 x 6070-02-2) with reported tolerance of HLB, comparing them with a common commercial citrus rootstock (Carrizo citrange). Four water conditions were established: Control, Medium Water Stress (MWS), Drought, and Flooding. Chlorophyll index (SPAD), growth in height, relative growth rate, biomass (fresh and dry weight) and plant water status were evaluated. Citru rootstock response were different for each genotype; Carrizo citrange was negatively affected by all water treatments in the chlorophyll index (SPAD) and biomass production. By contrast, UFR-6 showed a positive response in SPAD and growth under MWS and Drought, B11R5T60 displayed similar behavior to Control under all water stresses, and the response of 2247 x 6070-02-2 under MWS treatment was adequate but was not under Drought or Flooding conditions. Our study describes the behavior of these promising new citrus rootstocks against water stress; B11R5T60 exhibiting the best performance. These results can be useful for the citrus industry to address water-stress problems in these crops.

Novel Plantibodies Show Promise to Protect Citrus from Greening Disease

Candidatus Liberibacter asiaticus (CLas), the bacteria responsible for citrus greening disease [huanglongbing (HLB)], has become a worldwide threat to citrus (Citrus sp.) production. HLB has proven difficult to study and treat because of the complex interactions between CLas, the citrus host, and insect vectors. We have selected for single chain fragment variable (scFv) antibodies from a specialized bacteriophage library for binding activity against CLas proteins InvA and TolC. Portions of each protein were chosen as antigens based on predicted binding availability and theorized necessary functions in pathogenicity. Binding affinity for individual scFv-expressing clones was confirmed by phage enzyme-linked immunosorbent assay (ELISA). The scFv sequences were stably transformed under the control of a tandem Cauliflower mosaic virus 35S (CaMV 2x35S) promoter by Agrobacterium tumefacien–mediated transformation into ‘Carrizo’ citrange (Citrus sinensis × Poncirus trifoliate), a citrus rootstock cultivar. Replicated plants of single transformations were inoculated by infestation with CLas positive asian citrus psyllid (Diaphorina citri), a CLas vector. Inoculation and disease progression was monitored through quantitative real-time polymerase chain reaction. Inoculated transgenic plants showed significantly reduced CLas titer compared with wild types. A subpopulation of transgenic plants displayed no measurable surviving bacteria after 12 months. Interestingly, individual replicated plants from the same transgenic events strongly segregated into two populations by resistance phenotype: a minority that were indistinguishable from wild-type plants and a majority that were highly resistant. Our results are the first step in developing a novel protection strategy for HLB.

Feeding and Oviposition Behaviour of Trioza erytreae (Hemiptera: Triozidae) on Different Citrus Rootstock Material Available in Europe

Trioza erytreae (Del Guercio, 1918) (Hemiptera: Triozidae) is a vector of Candidatus Liberibacter spp., the causal agent of Huanglongbing disease (HLB). This study evaluates the preference of T. erytreae in different citrus seedlings. Thus, six different non-grafted citrus rootstocks were used for these experiments: (a) Carrizo citrange; (b) Citrus macrophylla; (c) ‘Cleopatra’ mandarin; (d) Forner-Alcaide No. 5; (e) Forner-Alcaide No. 517, and (f) Poncirus trifoliata (‘Flying Dragon’). The behaviour and survival of this psyllid was evaluated through the feeding preference of T. erytreae adults for different rootstocks (in a choice trial under greenhouse conditions) and oviposition and survival of T. erytreae adults on the different citrus material (in a no-choice trial under laboratory conditions). Trioza erytreae showed a clear preference for hosting and feeding on C. macrophylla, and Carrizo citrange was the most suitable rootstock for insect reproduction and survival followed by C. macrophylla. Conversely, Poncirus trifoliata was the least attractive rootstock to T. erytreae adults in the greenhouse trial and led to significantly lower T. erytreae survival. Our results suggest that conventional citrus rootstocks, such as Carrizo citrange and C. macrophylla, could increase T. erytreae populations.

Citrus rootstocks modify scion antioxidant system under drought and heat stress combination

Key message The activation of the antioxidant system under stress combination is a transmissible trait from the rootstock to the scion. Therefore, rootstock selection is key to improve crop performance and a sustainable production under changing climate conditions. Abstract Climate change is altering weather conditions such as mean temperatures and precipitation patterns. Rising temperatures, especially in certain regions, accelerates soil water depletion and increases drought risk, which affects agriculture yield. Previously, our research demonstrated that the citrus rootstock Carrizo citrange (Citrus sinensis × Poncirus trifoliata) is more tolerant than Cleopatra mandarin (C. reshni) to drought and heat stress combination, in part, due to a higher activation of the antioxidant system that alleviated damage produced by oxidative stress. Here, by using reciprocal grafts of both genotypes, we studied the importance of the rootstock on scion performance and antioxidant response under this stress combination. Carrizo rootstock, under stress combination, positively influenced Cleopatra scion by reducing H2O2 accumulation, increasing superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzymatic activities and inducing SOD1, APX2 and catalase (CAT) protein accumulations. On the contrary, Cleopatra rootstock induced decreases in APX2 expression, CAT activity and SOD1, APX2 and CAT contents on Carrizo scion. Taken together, our findings indicate that the activation of the antioxidant system under stress combination is a transmissible trait from the rootstock to the scion and highlight the importance of the rootstock selection to improve crop performance and maintain citrus yield under the current scenario of climate change.

Effect of the Rare Earth Element Lanthanum (La) on the Growth and Development of Citrus Rootstock Seedlings

Rare earth elements (REEs) can affect the growth and development of plants. However, few studies have been carried out on the effects of REEs on citrus seedlings. In this study, the growth parameters, toxicity symptoms, chlorophyll content, and La content of three citrus rootstocks are analyzed under different concentrations of La, a representative REE. The results show that the growth of citrus rootstock seedlings was stimulated at La ≤ 0.5 mmol·L−1 and inhibited at concentrations above 1 mmol·L−1. The chlorophyll and carotenoid contents of trifoliate orange (Poncirus trifoliata L. Raf.) and Ziyang Xiangcheng (C. junos Sieb. ex Tanaka) leaves of plants grown at low concentrations of La (≤1.5 mmol·L−1) were similar to those of the control but were significantly reduced at 4 mmol·L−1 La. Toxic symptoms gradually appeared with increasing La concentrations, with yellowed leaves and burst veins appearing at 4 mmol·L−1 La. The symptoms of toxicity were most severe in trifoliate orange, followed by Shatian Pomelo (Citrus grandis var. shatinyu Hort) and then Ziyang Xiangcheng. Moreover, in leaves, the Ca content was significantly negatively correlated with La content (p < 0.01). These results indicate that La has a hormesis effect on the growth of citrus rootstocks. Of the studied citrus seedlings, Ziyang Xiangcheng is the most resistant to La.