Ascorbate Peroxidase (APX) is a heme-containing, non-glycosylated enzyme that destroys harmful hydrogen peroxide via the ascorbate glutathione pathway in plants. This enzyme is considered to be an indispensable part of the electron-scavenging pathway and is involved in preventing oxidative damage in plants. Using differential display RT-PCR and 5' RACE a full length c-DNA clone was isolated, from citrus, with very high similarity at the nucleotide and amino acid level, to ascorbate peroxidases from several plant species. It is well known that APXs have highly conserved motifs like the Arg-38, Ars-71, Glu-65 and Asp-208 residues around the distal Hist-42 and proximal His-163. These residues are essential for binding the ligand heme. Additionally, Trp-179 is conserved in most APXs and is the third participant in hydrogen bonding network, together with His-163 and Asp-208. All these conserved motifs were present in the putative APX from citrus in addition of the presence of the peroxidase active site motif residues (APITLRLAWHSA) and the peroxidase heme-ligand motif (DIVVLSGGHTL). Expression analysis in E. Coli reviewed a recombinant protein of 27 Kda.
Reactive oxygen species (ROS) are continuously produced during the normal aerobic metabolism and also under environmental stress conditions. They are the major damaging factors to the photosynthetic machinery under stress conditions and need to be scavenged for the normal growth of the plant. Ascorbate peroxidase (APX) is the key enzyme in detoxifying H2O2, one of ROS from chloroplast and cytosol. A cDNA encoding a putative APXcit was isolated from mature `Dancy' tangerine (Citrus reticulata Blanco) juice vesicles using differential display reverse transcription-polymerase chain reaction (RT-PCR). Subsequently, full-length APXcit cDNA clone and genomic clone were obtained and sequenced. The full-length APXcit sequence is composed by 1082-bp nucleotides, including an open reading frame (ORF) of 753 bp, encoding a protein of 250 amino acids (27 kDa). The 5' un-translated region (UTR) of the APXcit gene consisted of 91 nucleotides and the 3' UTR consisted of 238 nucleotides. Homology search for APXcit at GenBank database showed high similarity to APX from several plant species.
Viroids are graft- or mechanically transmissible agents, disseminated through budding. Biological indexing of commercially important citrus cultivars grown in the Lower Rio Grande Valley of Texas showed that many are infected with citrus viroids. Most of these trees carried more than one viroid. In most cases, the infected trees are asymptomatic carriers because sour orange, the predominant rootstock used in Texas, does not show symptoms of viroid infection. Detection of viroids through biological indexing on sensitive indicator plants followed by sequential polyacrylamide gel electrophoresis (sPAGE) is the gold standard but is time-consuming and requires plants to be kept at optimum conditions. A conditional use of reverse transcriptase–polymerase chain reaction (RT-PCR) provides an efficient and alternative detection of viroids for use in the Texas virus-free citrus budwood certification program. RT-PCR could be useful in Texas to help expedite the evaluation for the presence of viroids before conducting the final biologic indexing. Using RT-PCR, we could detect, clone, and sequence full-length viroids of Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) (both cachexia and noncachexia variants), Citrus viroid-III (Citrus dwarfing viroid), and Citrus viroid-IV (Citrus bark cracking viroid) from a collection of viroid-inoculated grapefruit plants. The source plants were previously shown to be viroid-infected by biological indexing on Etrog citron plants. Based on our results, RT-PCR can be a conditional substitute for biological indexing of mother trees in foundation blocks and shoot tip-grafted trees in the virus-free budwood program. A positive RT-PCR result has a serendipitous value because those trees can be discarded from the pool before expensive biological indexing.
Citrus Huanglongbing (HLB, also known as “citrus greening”), an important disease worldwide, is associated with three species of phloem-limited Candidatus liberibacter, of which Candidatus L. asiaticus (CLas) is the predominant one that has severely affected citrus production. TaqMan real-time polymerase chain reaction (PCR) (TM) has been the standard and very efficient method to diagnose several strains of Candidatus Liberibacter in citrus; however, it detects total bacteria and is unable to differentiate dead from live Liberibacter. The detection of only live bacteria is essential for testing methods of control for this important citrus disease. It is well known that ethidium monoazide and propidium monoazide (PMA) are compounds that supposedly enter only dead or membrane-damaged bacteria, intercalate the DNA strand, and make the DNA unavailable for amplification by PCR. These compounds are widely used when extracting the plant DNA to detect only live bacteria. In this research, we tested primers amplifying products from 79 to 1160 bp in TM and SYBR Green real-time PCR (SG) and PMA as DNA intercalating compound. Specifically, primers amplifying a 500-bp amplicon in SG provided the most reliable live-only detection, whereas those producing a smaller amplicon were unable to distinguish between live and dead. This is the first report of testing primers amplifying various amplicon sizes for the detection of only live CLas cells in citrus.
The Asian citrus psyllid, Diaphorina citri Kuwayama, one of the known vectors for citrus greening disease or Huanglongbing (HLB) pathogens, has been present in Texas for over a decade, but the detection of the disease is recent. HLB has been confirmed in only two adjacent commercial citrus groves of grapefruit and sweet orange. A study was conducted to compare the population of Candidatus Liberibacter asiaticus (CLas) cells in different plant parts including peduncle, columella, leaves, seeds, young shoots, flower buds, flowers, and bark of 6-year-old known infected grapefruit and sweet orange trees. The bacterial population was estimated using a previously described grand universal regression equation Y = 13.82 – 0.2866X, where Y is the log of the target copy number and X is the Ct (threshold cycle) of the assay. Except for bark tissue, there was no significant difference in the concentration of CLas cells in other plant parts between the two cultivars. Within the cultivar, the bacterial concentration also varied with the plant part, with peduncle, columella, midrib having significantly higher titer of CLas compared with other plant parts. The obtained results here are in agreement with previous studies conducted on Florida samples, but the consistently lowest bacterial titer recorded in young shoots, leaf blade, and especially leaf margins relative to the midrib has never been previously reported.
Citrus viroid-induced resistance to Phytophthora infection in citrus was measured by the number of Phytophthora sporangia in ‘Rio Red’ grapefruit (Citrus paradisi Macf.) bait tissue infected with citrus viroids compared with non-inoculated controls. Different viroid isolates containing mixtures of viroids [Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), Citrus viroid IV (CVd-IV)] were designated by plant numbers and sources. Source 13E was associated with the lowest number of sporangia in bark, leaves, and roots used as baits, whereas CEVd E9, a known severe CEVd isolate, significantly reduced the number of sporangia in leaves and bark. Sources 1A, 2E, 3E, 4D, and 6E showed a significantly reduced number of sporangia on bark, leaves, and roots compared with healthy plants and 44A; however, their effect was not as pronounced as that of E9 and 13E. Sources 12E and 44A did not suppress sporangia production. Previous reverse transcriptase–polymerase chain reaction analysis showed that all source plants had mixed infections with several viroids, whereas 12E and 44A contained no viroids. In addition to confirming the earlier reports on the suppression of Phytophthora infection in general, our study showed significantly reduced Phytophthora sporangia development resulting from a number of viroids in mixed infection, but there did not appear to be any effect related to viroid species. To determine if concentration affected resistance to Phytophthora, phenolic acids were extracted. Extraction of phenolic acids with 80% ethanol was more efficient compared with 100% methanol and an acetonitrile–water mixture. High-performance liquid chromatography revealed no notable detection of salicylic acid in healthy and viroid-infected plants, but there was a small peak corresponding to salicylic acid in Phytophthora-infected and both viroid and Phytophthora-infected plants. Flavone was detected in all the source plants with a slight increase in Phytophthora-infected and both viroid and Phytophthora-infected plants. A peak corresponding to quercetin dehydrate was detected in Phytophthora-infected plants. Efficient use of the right viroid isolate(s) can result in suppression of Phytophthora infection of citrus.
Seeds from four citrus rootstocks including sour orange, Bitters-C22 citrandarin, Sarawak pummelo Rio Red grapefruit, and Sarawak pummelo Bower mandarin were exposed to high inoculum levels of Phytophthora nicotianae to screen for tolerance. Inoculation of pregerminated seeds (PGIS) and non-PGIS was carried out. The average P. nicotianae propagule counts from the soil samples where these seedlings were raised ranged from 424 to 1361 colony forming units/cm3. The proportion of live to dead plants was recorded at 11 months postinoculation, which showed that Sarawak Bower performed significantly better than other rootstocks. Evaluation of the rootstocks 18 months postinoculation resulted in only one surviving sour orange plant, which suggests potential rootstock resistance.