Lime Plants Research Articles

Uptake of rare earth elements by citrus plants from phosphate fertilizers

Rare earth elements (REE) are a group of the periodic table formed by 17 chemical elements (lanthanoids plus yttrium and scandium). They have been used in different field applications. In agriculture, they can be found in some phosphate fertilizers at levels one or two orders of magnitude higher than those found in normal agricultural soils. Citrus plants are known to present high levels of REE when compared to most other species, however, there is little information about bioavailability of REE in phosphate fertilizers for citrus plants. This work focuses on the study of REE behavior by the application of increasing doses of single superphosphate fertilizer in Rangpur lime (Citrus limonia Osbeck) plants in a greenhouse study. The technique used was instrumental neutron activation analysis (INAA). The results showed that the fertilizer has caused significant increases in the content of REE in the citrus plant tissues, with higher concentrations in leaves than in branches. The highest substrate-leaf transfer factor was observed for La (0.0047), though the concentrations in the plants followed the same order found in the substrate, i.e. Ce > La > Sm > Eu > Sc. There was an increase of rare earth elements concentrations in Rangpur lime plants by superphosphate fertilizer application.

Endophytic Bacteria Isolated from Citrus Plants for Biological Control of Citrus Canker in Lime Plants.

Citrus canker caused by Xanthomonas citri subsp. citri is a disease affecting the yield and fruit quality of lime (Citrus aurantiifolia). This research investigated endophytic bacteria obtained from six healthy Citrus spp. to inhibit the pathogen and to control citrus canker on lime plants. Numbers of the endophytic bacteria isolated from C. aurantifolia, C. hystrix, C. maxima, C. nobilis, C. reticulata and C. sinensis were 28, 25, 29, 42, 12 and 34 isolates, respectively. The selected endophytic bacteria that were effective against X. citri subsp. citri were Bacillus amyloliquefaciens LE109, B. subtilis LE24 and B. tequilensis PO80. The optimum culture medium for an antagonistic effect on the pathogen in B. amyloliquefaciens LE109 and B. tequilensis PO80 was yeast extract peptone dextrose broth, and in B. subtilis LE24 was modified soluble starch broth. To control citrus canker in lime, young expanded leaves of lime plants were aseptically punctured and inoculated with 30 μl of bacterial suspension of the pathogen (108 CFU/ml in 0.85% NaCl) per punctured location. After the pathogenic inoculation for 24 h, the leaves were then inoculated with 30 μl of the selected endophytic bacteria (108 CFU/ml in 0.85% NaCl), and treated with 30 μl of the culture media containing bioactive compounds produced by the selected endophytic bacteria. The leaves inoculated with cell suspensions of B. amyloliquefaciens LE109 or B. subtilis LE24 could completely control citrus canker. However, the leaves inoculated with B. tequilensis PO80 displayed 10% disease incidence. Additionally, the leaves treated with the crude bioactive compounds of B. amyloliquefaciens LE109 or B. subtilis LE24 could completely control citrus canker. Notably, the leaves treated with the crude bioactive compounds of B. tequilensis PO80 displayed 5% disease incidence. The results of this study showed that the Bacillus strains play important roles in the biocontrol of citrus canker in lime.

Aluminum-induced high IAA concentration may explain the Al susceptibility in Citrus limonia

In acidic soils (pH < 5.0), aluminum (Al) occurs as Al3+, which is phytotoxic and reduces the root growth by hormonal imbalance and/or cell wall rigidity. However, the explanations for the decrease in root growth are not clear. A 60-day study was held with ‘Rangpur’ lime (Citrus limonia) plants grown in nutrient solution containing 0 and 1480 µM Al. We measured plant biometric data and used root apices to analyze auxin (IAA), the expression of some Al-responsive genes that had been differentially expressed in a transcriptome analysis and anatomical profiles. We found up-regulated expression of multidrug and toxic compound exudation (Cl-MATE channel), citrate synthase (Cl-CS) and pectin methylesterase inhibitor (Cl-PMEI) genes, but while Cl-PMEI expression was increased after 7 days, Cl-CS and Cl-MATE were up-regulated only after 60 days, suggesting that the timing of these events was ineffective against Al. These results could be associated with the Al-induced decrease in root growth and anatomical root damage. In addition, genes related to IAA cell transport were not differentially expressed in the transcriptome analysis. Therefore, high IAA and up-regulation of auxin-related small RNAs suggest Al-induced high IAA concentration rather than Al-induced disruption in IAA distribution in root cells of this species.

Effect of Potassium Silicate Application on Populations of Asian Citrus Psyllid in Tahiti Lime

Asian citrus psyllid [ACP (Diaphorina citri)] is one of the most serious threats to the global citrus (Citrus sp.) culture, and management of ACP has depended primarily on the application of chemical insecticides. The expression of resistance mechanisms to herbivory is a key component in integrated pest management in crop production in which silicon (Si) applications can play an important role in plant–insect relationships. The objective of the current study was to evaluate the application of Si to tahiti lime (Citrus latifolia) plants under natural infestations of ACP. Two experiments were conducted using 15-month-old seedlings and 2-year-old trees, respectively. Treatments were 1) foliar Si sprays (potassium silicate) at a dose of 2 mL·L–1, 2) soil Si application at a dose of 1 kg commercial product per plant, 3) combined soil and foliar applications of Si at the doses just listed, and 4) untreated plants (control). The application of Si treatments to both seedlings and trees affected ACP oviposition, causing a reduction of 60%. Applications of Si did not affect the nutritional status (macronutrients and micronutrients) of plants in either test, except that the foliar concentration of Si tended to be greater in the soil and soil + foliar treatments than in the other treatment in both seedlings and trees. Based on these results, we suggest that Si can be added as a component of ACP integrated pest management programs.

Aluminum prevents stomatal conductance from responding to vapor pressure deficit in Citrus limonia

Stomatal aperture generally increases in response to low vapor pressure deficit (VPD) and decreases at high VPD. Aluminum (Al) inhibits root growth, indirectly exposing the roots to low water availability, which may decrease leaf hydration and, consequently, the stomatal conductance (gs). In this study, Citrus limonia (‘Rangpur’ lime) was grown in nutrient solution with 1480 μM Al for 90 days, and we expected that the presence of Al could prevent gs from responding to VPD. As expected, gs did not respond to the increase in VPD in plants exposed to Al. Aluminum also reduced the relative water content and midday leaf water potential (Ψmd) after 60 and 90 days. The CO2 assimilation rate (A) followed the same response pattern exhibited by gs, the estimation of the carboxylation efficiency was not reduced in plants exposed to Al and measured under drier air, while photochemical responses were slightly reduced in plants exposed to Al, indicating that the Al-induced decrease in A was dependent on gs and less ascribed to low photochemical performance. Like in drought conditions, the long-term exposure to Al reduces leaf hydration and compromises gs responses to the atmosphere, eventually impairing A in ‘Rangpur’ lime plants.

Biological and molecular characterization of Uruguayan citrus tristeza virus field isolates

Citrus tristeza virus (CTV) is the causal agent of the most important viral disease of citrus. Symptoms that may affect the productive potential of citrus plants are observed in Uruguayan orchards even though resistant rootstocks are used. CTV is fully eliminated in propagative materials by the National Sanitation and Certification Program, but since the virus and its vector are widespread in the country, the risk of infection in the field persists. In this situation, using mild CTV strains in a cross-protection program would be a useful alternative to attempt to increase yield and quality of the local citrus industry. To this aim, this study assessed the biological and molecular characteristics of 32 local CTV isolates. Bioassays were conducted in a greenhouse with controlled conditions. Each isolate was graft-inoculated on Mexican lime, sweet orange, sour orange and Duncan grapefruit indicator plants. Symptoms and their intensity were evaluated. Molecular characterization was carried out by RT-PCR amplification, using primers for the p25, p20 and p23 genes. PCR products were sequenced, nucleotide sequences were aligned with international reference strains and phylogenetic trees were constructed. Results of the biological and molecular analysis showed the prevalence of severe CTV isolates with a high genetic variability. Two out of 32 characterized isolates were selected as mild CTV isolates to be tested as candidates for future cross-protection experiments. The survey showed a complex scenario for the management of CTV in Uruguay.

Citrus x aurantiifolia, a new host report of Macrophomina phaseolina in Iran

Charcoal root rot-like symptoms were observed on Mexican lime (Citrus x aurantiifolia) plants in a nursery located in Hormozgan province, southern Iran. The fungus was identified as Macrophomina phaseolina based on morphological and molecular characteristics. Pathogenicity tests revealed the association of six M. phaseolina isolates with disease. Reisolation from roots of inoculated plants yielded isolates of M. phaseolina with morphological characteristics identical to those of the original isolates used for inoculations, thus fulfilling Koch’s postulates. This is the first record of charcoal rot caused by M. phaseolina on citrus in Iran.

Biochemical and physiological responses to long‐term Citrus tristeza virus infection in Mexican lime plants

Reactions that occur when a plant is subjected to Citrus tristeza virus (CTV) infection often result in triggering of numerous defence mechanisms to fight the infection. The reactions vary according to virus strain, host genotype, time of exposure to the infection and environmental conditions. To date, no study has examined in detail the consequences of 10‐year exposure to CTV infection on the biochemical and physiological status of susceptible Mexican lime plants (Citrus aurantifolia). To understand the reaction of such plants, changes in nutrient status, total proteins, enzyme activity involved in scavenging of reactive oxygen species, photosynthetic and transpiration rates, chlorophyll content, membrane permeability and water content were analysed in plants infected with different CTV isolates and in healthy plants. The activity of superoxide dismutase and polyphenol oxidase significantly decreased in the infected leaves, and membrane permeability was lower in the infected plants. Macro‐ and micronutrient elements were significantly changed: concentrations of leaf nitrogen, zinc, magnesium and iron were elevated but potassium concentration depressed in comparison to noninfected control leaves. Levels of other analysed nutrient elements, enzymes, photosynthesis and stomatal conductance, chlorophyll content and relative water content were unchanged. Clear physiological changes were found among infected and noninfected control plants but none between plants infected with different CTV isolates. The data suggest that some of the defence mechanisms investigated here were suppressed due to the continuous and long‐term pressure of biotic stress.

Emerging citrus diseases in Europe caused by species of Diaporthe

Species of Diaporthe are considered important plant pathogens, saprobes, and endophytes on a wide range of plant hosts. Several species are well-known on citrus, either as agents of pre- or post-harvest infections, such as dieback, melanose and stem-end rot on fruit. In this study we explored the occurrence, diversity and pathogenicity of Diaporthe species associated with Citrus and allied genera in European orchards, nurseries, and gardens. Surveys were carried out during 2015 and 2016 in Greece, Italy, Malta, Portugal, and Spain. A total of 79 Diaporthe strains were isolated from symptomatic twigs, branches and trunks. A multi-locus phylogeny was established based on five genomic loci (ITS, tef1, cal, his3 and tub2), and the morphological characters of the isolates determined. Preliminary pathogenicity tests were performed on lemon, lime, and orange plants with representative isolates. The most commonly isolated species were D. foeniculina and D. baccae, while only four isolates of D. novem were collected. Two new Diaporthe species, described here as D. limonicola and D. melitensis spp. nov. were found associated with a new devastating dieback disease of lemon plants. Furthermore, one cluster of sterile Diaporthe isolates was renamed as D. infertilis. Pathogenicity tests revealed most of the Citrus species as susceptible to D. baccae, D. foeniculina, and D. novem. Moreover, D. limonicola and D. melitensis caused serious cankers affecting all the Citrus species tested. This study is the first report of D. baccae and D. novem on citrus in Europe, and the first detection of a new Diaporthe canker disease of citrus in Europe. However, no isolates of D. citri were found. The study improves our understanding of the species associated with several disease symptoms on citrus plants, and provides useful information for effective disease management.

Management of Asiatic Citrus Canker Under Field Conditions in Saudi Arabia Using Bacteriophages and Acibenzolar-S-Methyl.

Applications of formulated bacteriophages with skim milk and sucrose or nonformulated bacteriophages combined with acibenzolar-S-methyl (ASM) were compared with copper bactericides applications for suppressing Asiatic citrus canker (ACC) caused by Xanthomonas citri subsp. citri (Xcc) on leaves under greenhouse and field conditions in Saudi Arabia. Bacteriophages were applied one day prior to inoculation of Mexican lime (Citrus aurantifolia) plants with Xcc, then twice a week until the end of the trials. Copper hydroxide was applied once prior to inoculation and then every seven days afterward, whereas ASM was applied one week prior to inoculation and then every 21 days afterward. Under greenhouse conditions, the incidence of ACC on leaves was reduced significantly from 75.2 to 12.8% or 18.3% for plants treated with copper hydroxide or bacteriophages in combination with ASM, respectively. Applications of formulated phages in combination with ASM as soil drench under field conditions significantly decreased disease incidence by 14.8% (Trial 1) and 16.8% (Trial 2) compared with untreated control plants. Overall, the Xcc-inoculated plants treated with bacteriophages + ASM combination showed significant ACC reduction under greenhouse and field conditions. The bacteriophages + ASM combination tested in these trials can be an effective tool in the integrated management programs of Asiatic citrus canker disease.

Aluminum-induced decrease in CO2 assimilation in ‘Rangpur’ lime is associated with low stomatal conductance rather than low photochemical performances

Aluminum (Al) is toxic to most plants. Although inhibition of root elongation can occur even under 10μM Al, above 1000μM damage to photochemical performances has been reported, reducing the CO2 assimilation rate (A). However, Al is retained in the roots of most plants, including Citrus limonia (‘Rangpur’ lime) with no direct explanation for the low A. In this study, ‘Rangpur’ lime plants were cultivated hydroponically in a nutrient solution with 1480μM Al, and we expected to determine the time range within which Al-induced decrease in A starts. To our surprise, low stomatal conductance (gs) rather than low photochemical performances was evident 45days after exposing the roots to Al. Aluminum was mostly retained in the roots and histochemically observed in the stele, suggesting reduction in water uptake/transport in the xylem that became fibrous. We also found a 35% reduction in the midday leaf water potential in plants exposed to Al, indicating association between the fibrous xylem vessels and low gs, which could explain the Al-induced decrease in A in ‘Rangpur’ lime plants.

MONITORING 'CANDIDATUS LIBERIBACTER ASIATICUS' IN A COMMERCIAL ORCHARD OF SWEET ORANGE AND TAHITI LIME IN THE NORTHWEST OF PARANÁ STATE, BRAZIL

Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide, affecting all species of Citrus. Early characteristics of infected trees are individual branches with yellowish leaves, followed eventually by tree decline and death. The objective of this study was to examine seasonal dynamics of within-tree accumulation of ‘Candidatus Liberibacter asiaticus’, the bacterium associated with HLB, in a commercial orchard. Sweet orange and Tahiti lime plants naturally infected with HLB (confirmed by conventional PCR) were monitored for bacterial population using real-time PCR. Total DNA was extracted from the main veins of symptomatic leaves collected monthly over a period of twenty months. The sample from each plant comprised 10 leaves per quadrant, totaling 40 leaves per plant. The 10-leaves sample from each quadrant was analyzed separately. Erratic bacterial accumulation was observed in both sweet orange and Tahiti lime. After detection in one quadrant of the plant, the continued presence of the bacterium in the same quadrant was not verifiable a few months later. In sweet orange trees, the quadrants expressing more severe symptoms had higher bacterial titer, with Ct values between 18 and 33. Similar results were found for Tahiti lime. These results contribute to our knowledge of HLB epidemiology and might help in the search for effective control measures.

Limited success of heat treatments for curing HLB affected trees

A series of assays was conducted in an attempt to eliminate Ca. L. asiaticus (Las) from HLB affected plants using hot air (HA) or steam (ST). HA assays were conducted with 2-y-old greenhouse (GH) potted Valencia/Rangpur lime plants and 4-y-old Hamlin/Sunki field trees. GH plants were exposed to 38 to 44°C for 24 to 192 h in a growth chamber (GC). Field trees were exposed to sunlight under plastic cover sheets (≥40°C) for 24 to 96 h after the branches were pruned 1.5 m above ground. ST assays involved root-uncovered GH plants and 5-y-old pruned Hamlin/Swingle and Pera/Cravo field trees. GH plants were exposed to 45 to 60°C for 5 to 30 min and the trees to 55°C for 5 to 20 min and 60°C for 5 or 10 min. Plant and Las responses to heat were temperature/time dependent. All plants exposed to HA for ≥96 h at 44°C in GC and ca. 1/3 of the trees treated for ≥24 h at ≥40°C in the field died. Around 1/3 of the plants exposed to ST for 15 min at 50°C or 5 min at 55°C, and all plants at 60°C also died. In the field ST also damaged or killed the trees at 55°C for ≥30 min or 60°C for ≥20min. Most of the plants or trees showed typical HLB symptoms and were PCR+ 3 to 5 months after treatment. Under the trunk bark of field trees the temperature took over 10 min to reach maximum of 50°C, which was apparently not enough to kill Las. Las survival in the trunk and roots may explain the lack of success of the heat treatments against HLB.

Effect of amino acid application on induced resistance against citrus canker disease in lime plants

Abstract Citrus bacterial canker, caused by Xanthomonas citri subsp. citri (Xcc), is a destructive disease. So far, the chemicals used to control this pathogen are either ineffective or harmful to the environment. To improve control of this disease, lime (Citrus aurantifolia) were treated with L-arginine, L-methionine, L-ornithine, and distilled water. Plants were inoculated with Xcc, 48 hours post treatment. Lesion diameters of inoculated leaves were evaluated four weeks after inoculation with a bacterial suspension. Changes in β-1,3-glucanase transcript levels and activity of antioxidant enzymes, catalase, peroxidase, and phenylalanine ammonia-lyase were investigated at 48 hours post treatment and 24, 48, and 72 hours post inoculation. Based on the results of phenotypic, antioxidant enzyme activity and a molecular study of the stressed plants, it was found that those plants treated with the amino acid methionine significantly increased the plant induced resistance as well as decreased the severity of disease by reducing necrotic lesion size.

In Vitro and In Vivo Antibacterial Activity of Some Organic and Inorganic Salts Against Asiatic Citrus Canker Agent Xanthomonas Citri Subsp. Citri

Asiatic citrus canker caused by Xanthomonas citri subsp. citri is becoming a disease of high economic impact, affecting all types of important citrus crops. In this study, the potential antibacterial activity of ten organic and inorganic salts on X. citri subsp. citri and on citrus canker disease development was evaluated. Among the salt compounds, copper, iron and zinc inorganic salts particularly zinc (with the highest diameter of inhibition, the lowest MIC and MBC values and the highest bacterial growth inhibitory effect) had direct antibacterial activity and strongly reduced the development of canker disease and bacterial population of lime plants.

Citrus leaf blotch virus invades meristematic regions in Nicotiana benthamiana and citrus

To invade systemically host plants, viruses need to replicate in the infected cells, spread to neighbouring cells through plasmodesmata and move to distal parts of the plant via sieve tubes to start new infection foci. To monitor the infection of Nicotiana benthamiana plants by Citrus leaf blotch virus (CLBV), leaves were agroinoculated with an infectious cDNA clone of the CLBV genomic RNA expressing green fluorescent protein (GFP) under the transcriptional control of a duplicate promoter of the coat protein subgenomic RNA. Fluorescent spots first appeared in agroinfiltrated leaves 11-12 days after infiltration, indicating CLBV replication. Then, after entering the phloem vascular system, CLBV was unloaded in the upper parts of the plant and invaded all tissues, including flower organs and meristems. GFP fluorescence was not visible in citrus plants infected with CLBV-GFP. Therefore, to detect CLBV in meristematic regions, Mexican lime (Citrus aurantifolia) plants were graft inoculated with CLBV, with Citrus tristeza virus (CTV), a virus readily eliminated by shoot-tip grafting in vitro, or with both simultaneously. Although CLBV was detected by hybridization and real-time reverse transcription-polymerase chain reaction (RT-PCR) in 0.2-mm shoot tips in all CLBV-inoculated plants, CTV was not detected. These results explain the difficulty in eliminating CLBV by shoot-tip grafting in vitro.

Differentially expressed genes and temporal and spatial expression of genes during interactions between Mexican lime (Citrus aurantifolia) and a severe Citrus tristeza virus isolate

Determining transcriptional responses of host plants to virus infection will help understand mechanisms involved in host–virus interactions. In this study, 430 genes differentially regulated by a severe CTV isolate in lime plants were identified by using SSH. The differential expression levels of 25 ESTs in CTV-N21 infected lime were validated by qRT-PCR. The largest set of those genes involved in primary metabolism (26.0%), and then in protein metabolism (15.1%). Our results revealed a large set of genes (51 ESTs) were commonly up-regulated by two severe strains CTV-N21 and CTV-T305 in lime. The quantitative analysis for temporal and spatial transcriptional levels of six genes in lime plants infected by CTV-N21 indicated that the transcriptional levels of those genes fluctuated during CTV infection and differed in different citrus leaves.

Molecular detection of Candidatus Phytoplasma spp. causing witches’ broom disease of acid lime (Citrus aurantifolia) in India

Phytoplasma infected acid lime plants in India develop characteristic symptoms like small chlorotic leaves, multiple sprouting and shortened internodes. Leaves drop prematurely and infected branches have distorted twigs resembling witches’ broom appearance which eventually show die-back symptoms. During its first report in 1999, witches’ broom disease identification was made on the basis of symptomatology and electron microscopy. However, molecular techniques have proved to be more accurate and reliable for phytoplasma detection than the conventional methods. During survey in the year 2010 six samples were collected from infected acid lime plants showing typical field symptoms from Vidarbha region of Maharastra. Initially, phytoplasma bodies were observed in phloem tissues of all six symptomatic samples under JEM 100S transmission electron microscope and all these six samples were subsequently screened using different set of phytoplasma specific universal primers by nested PCR, a widely recommended molecular technique for phytoplasma detection. In the present study P1/P7 “universal” phytoplasma-primer set was used for first round of PCR and amplified products were processed separately for nested PCR with three different nested primer pairs viz. R16F2n/R16R2, R16mF2/R16mR1 and fU5/rU3. The presence of phytoplasma was confirmed in all six suspected samples and one representative ~1.2 kb size amplicon was sequenced and deposited in GenBank as Candidatus Phytoplasma species AL-M (JQ808143). This is the first report of PCR based molecular detection of phytoplasma-induced witches’ broom disease of acid lime (WBDL) in India. Further molecular evaluation to determine the identity to the species level is in progress.

Magnetic Susceptibility and Heavy Metal Content in Dust From the Lime Plant and the Cement Plant in Opole Voivodeship

Magnetic Susceptibility and Heavy Metal Content in Dust From the Lime Plant and the Cement Plant in Opole Voivodeship Until now, dust arising from lime manufacture has been considered harmless to the environment so it has been investigated marginally from the standpoint of environmental protection, especially when it came to magnetic properties and heavy metal content. The aim of the research was filling the gap in this area. The research comprised measurements of magnetic susceptibility, the content of heavy metals, reaction (pH) and specific conductivity of lime dust and also raw material and fuel used for lime production. The samples were taken from one of the lime plants located in Opole Province. Similar investigations were also performed for dust taken from the nearby cement plant using dry method of cement production. It was proven that magnetic susceptibility, heavy metal content and conductivity of lime dust was lower in comparison to cement dust, which resulted from the fact that the lime plant used neither low raw materials nor additives. Due to the high atmosphere dust level in the vicinity of the investigated plants, extremely basic reaction of the tested dust and high content of metals, the studied dust cause alkalization of soils and contribute to the increase of heavy metal content in soils, posing a threat to the environment.

Genetic diversity and population differentiation of Guignardia mangiferae from "Tahiti" acid lime.

Among the citrus plants, “Tahiti” acid lime is known as a host of G. mangiferae fungi. This species is considered endophytic for citrus plants and is easily isolated from asymptomatic fruits and leaves. G. mangiferae is genetically related and sometimes confused with G. citricarpa which causes Citrus Black Spot (CBS). “Tahiti” acid lime is one of the few species that means to be resistant to this disease because it does not present symptoms. Despite the fact that it is commonly found in citric plants, little is known about the populations of G. mangiferae associated with these plants. Hence, the objective of this work was to gain insights about the genetic diversity of the G. mangiferae populations that colonize “Tahiti” acid limes by sequencing cistron ITS1-5.8S-ITS2. It was verified that “Tahiti” acid lime plants are hosts of G. mangiferae and also of G. citricarpa, without presenting symptoms of CBS. Populations of G. mangiferae present low-to-moderate genetic diversity and show little-to-moderate levels of population differentiation. As gene flow was detected among the studied populations and they share haplotypes, it is possible that all populations, from citrus plants and also from the other known hosts of this fungus, belong to one great panmictic population.