Host suitability and feeding preference of the African citrus triozid Trioza erytreae Del Guercio (Hemiptera: Triozidae), natural vector of “Candidatus Liberibacter africanus”

African citrus greening (ACGD) and huanglongbing (HLB) diseases are the most damaging diseases of citrus worldwide. Currently, the disease has no cure and has been attributed to the collapse of the citrus industry in several countries. In Africa, the causative agent “Candidatus” Liberibacter africanus is vectored by African citrus triozid (ACT) Trioza erytreae Del Guercio (Hemiptera: Triozidae). African citrus triozid is native to Africa but has been recently reported in Asia and Europe. Apart from citrus, Murraya koenigii (L.) and Clausena anisata (Willd) Hook. F. ex Benth. are also considered as preferred host plants. At present, there is scant information on host plant suitability and preference of T. erytreae. Also, there are contradictory reports on its reproduction and survival on rutaceous and non‐rutaceous host plants. In the present study, we tested the suitability and preference of rutaceous and non‐rutaceous trees and shrubs as potential ACT host plants in choice and no‐choice bioassays. The development from egg to the adult stage was longest on Calodendrum capense (Wright & Arn.) Engl. Host plants of superior quality accordingly to several ACT's biological parameters measured also revealed significantly higher morphometric characteristics. Our findings on the host status of the five rutaceous plants imply that these plants can greatly influence the population dynamics of ACT as well as the epidemiology of ACGD, and these can be a useful guide in the area‐wide management of the pest in Kenya.     

Non-Enzymatic Reduction of Nitrite by Means of Ascorbic Acid in Citrus and Other Higher Plant Tissues

It has been proved that the nitrite reduction in the leaves and other plant tissues of citrus and other green plants is partly or mainly a non-enzymatic chemical process, and a heat-stable factor present in these tissues is responsible for this reduction. It is suggested that ascorbic acid plays a major role in this chemical reaction since the reduction is inhibited by ascorbic acid oxidase. A significant association was also found between the ascorbic acid content and the nitrite reduction capacity of citrus leaves. Evidence has been presented that this non-enzymatic chemical reduction of nitrite occurs also in vivo as undetached citrus leaves on branches placed in NaNO₂ solution have shown diminution of their ascorbic acid content along with the absorption of nitrite. Stronger accumulation of nitrite in these leaf tissues was observed under dark conditions, apparently due to the inhibition of the biosynthesis of the ascorbic acid.     

Plant biotechnology for lignocellulosic biofuel production

Lignocelluloses from plant cell walls are attractive resources for sustainable biofuel production. However, conversion of lignocellulose to biofuel is more expensive than other current technologies, due to the costs of chemical pretreatment and enzyme hydrolysis for cell wall deconstruction. Recalcitrance of cell walls to deconstruction has been reduced in many plant species by modifying plant cell walls through biotechnology. These results have been achieved by reducing lignin content and altering its composition and structure. Reduction of recalcitrance has also been achieved by manipulating hemicellulose biosynthesis and by overexpression of bacterial enzymes in plants to disrupt linkages in the lignin–carbohydrate complexes. These modified plants often have improved saccharification yield and higher ethanol production. Cell wall‐degrading (CWD) enzymes from bacteria and fungi have been expressed at high levels in plants to increase the efficiency of saccharification compared with exogenous addition of cellulolytic enzymes. In planta expression of heat‐stable CWD enzymes from bacterial thermophiles has made autohydrolysis possible. Transgenic plants can be engineered to reduce recalcitrance without any yield penalty, indicating that successful cell wall modification can be achieved without impacting cell wall integrity or plant development. A more complete understanding of cell wall formation and structure should greatly improve lignocellulosic feedstocks and reduce the cost of biofuel production.     

Plant glutathione peroxidases

Oxidative stress in plants causes the induction of several enzymes, including superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2). The first two are responsible for converting superoxide to H₂O₂ and its subsequent reduction to H₂O, and the third is involved in recycling of ascorbate. Glutathione peroxidases (GPXs, EC 1.11.1.9) are a family of key enzymes involved in scavenging oxyradicals in animals. Only recently, indications for the existence of this enzyme in plants were reported. Genes with significant sequence homology to one member of the animal GPX family, namely phospholipid hydroperoxide glutathione peroxidase (PHGPX), were isolated from several plants. Cit-SAP, the protein product encoded by the citrus csa gene, which is induced by salt-stress, is so far the only plant PHGPX that has been isolated and characterized. This protein differs from the animal PHGPX in its rate of enzymatic activity and in containing a Cys instead of selenocysteine (Sec) as its presumed catalytic residue. The physiological role of Cit-SAP and its homologs in other plants is not yet known.     

Plant Growth Regulators in Citriculture: Factors Regulating Endogenous Levels in Citrus Tissues

Since the review on endogenous growth substances of citrus tissues by Goldschmidt in 1976 (HortScience, 11: 95-99), much information regarding this topic has been published in a wide array of journals. The present review provides a comprehensive overview of published information on endogenous levels of the five classes of plant growth substances (i.e., auxins, cytokinins, gibberellins, ethylene, and abscisic acid), plus polyamines and other endogenous substances that appear to have a role in regulating citrus growth and development. It is the first in a three-part series that next examines hormonal regulation of physiological processes in citrus followed by review of current uses and commercial applications of plant growth regulators in citrus production. In this article, a brief history of the detection and characterization of each class of plant growth substances is given. Following this, variation in endogenous levels associated with different organs (and/or tissues), stages of development, species, cultivars, cultural practices, and environmental factors is reviewed. For each class, current knowledge regarding biosynthesis, metabolism and transport in citrus tissues is summarized. The concluding section deals with future research directions.     

Purification of the bacterium-like organism associated with greening disease of citrus by immunoaffinity chromatography and monoclonal antibodies

An immunoaffinity chromatographic procedure with monoclonal antibodies (MA) has been developed for purification of the uncultivable, bacterium-like organism associated with greening disease of citrus. The greening organism (GO) was partially purified from leaf midribs of infected periwinkle plants by differential centrifugation. The GO present in such preparations was retained on an affinity matrix consisting of CNBr-activated Sepharose 4B on which GO-specific MA had been covalently linked. The unbound plant material was washed from the matrix, and the GOs were eluted with 0.1M glycine (pH 11.5). Purified GOs were compared with organisms observed in the initial plant preparation by both immunofluorescence and electron microscopic techniques. The morphology and serological characteristics of the GO were retained following purification procedures.     

Increased tolerance to Phytophthora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related protein PR-5

Phytophthora citrophthora is the most widely spread oomycete plant pathogen over all the citrus growing areas and represents one of the major causes of crop losses. Constitutive over-expression of genes encoding proteins involved in plant defence mechanisms to disease is one of the strategies proposed to increase plant tolerance to oomycete and fungal pathogens. P23 (PR-5), a 23-kDa pathogenesis-related protein similar to osmotins, is induced in tomato (Lycopersicon esculentum Mill. cv. Rutgers) plants when they are infected with citrus exocortis viroid, and its antifungal activity has been demonstrated in in vitro assays. We have successfully produced transgenic orange (Citrus sinensis L. Obs. cv. Pineapple) plants bearing a chimeric gene construct consisting of the cauliflower mosaic virus 35S promoter and the coding region of the tomato pathogenesis-related PR-5. Nine regenerated transgenic lines constitutively expressed the PR protein. They were challenged with Phytophthora citrophthora using a detached bark assay. A significant reduction in lesion development was consistently observed in one transgenic line in comparison to the control plants. This same line achieved plant survival rates higher than control plants when transgenic trees were inoculated with oomycete cultures. These results provide evidence for the in vivo activity of the tomato PR-5 protein against Phytophthora citrophthora, and suggest that this may be employed as a strategy aimed at engineering Phytophthora disease resistance in citrus.     

Plant transformation technology

Plant transformation has its roots in the research on Agrobacterium that was being undertaken in the early 1980s. The last two decades have seen significant developments in plant transformation technology, such that a large number of transgenic crop plants have now been released for commercial production. Advances in the technology have been due to development of a range of Agrobacterium-mediated and direct DNA delivery techniques, along with appropriate tissue culture techniques for regenerating whole plants from plant cells or tissues in a large number of species. In addition, parallel developments in molecular biology have greatly extended the range of investigations to which plant transformation technology can be applied. Research in plant transformation is concentrating now not so much on the introduction of DNA into plant cells, but rather more on the problems associated with stable integration and reliable expression of the DNA once it has been integrated.     

Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding

The long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus leaf blotch virus‐based vector (clbvINpr‐AtFT and clbvINpr‐CiFT, respectively). Citrus plants of different genotypes graft inoculated with either of these vectors started flowering within 4–6 months, with no alteration of the plant architecture, leaf, flower or fruit morphology in comparison with noninoculated adult plants. The vector did not integrate in or recombine with the plant genome nor was it pollen or vector transmissible, albeit seed transmission at low rate was detected. The clbvINpr‐AtFT is very stable, and flowering was observed over a period of at least 5 years. Precocious flowering of juvenile citrus plants after vector infection provides a helpful and safe tool to dramatically speed up genetic studies and breeding programmes.     

Characteristics of the Uptake Mechanism of Chloride Ions in Excised Roots of a Woody Plant (Citrus)

The characteristics of the uptake mechanism of chloride ions in citrus (Citrus spp.) were studied in excised, high-salt, roots as to the nature of the isotherm at a wide range of uptake durations and Cl^? concentrations. In addition, the effects of metabolic inhibitors, low temperature and various treatments were studied, and compared with Cl^? uptake in excised roots of wheat (Triticum vlgare) under the same conditions. It was found that the uptake mechanism in excised roots of citrus differs considerably from that in wheat: (1) the rate of active uptake from 10 mM NaCl in citrus is 2.0 to 4.3 umol Cl^? per g dry weight and h (vs. 35.5 umol in wheat); (2) there is no saturation of the system even at high external concentrations (up to 90 mM), and uptake is continuous; (3) uptake in citrus is less sensitive to KCN and dinitro-phenol. In addition, it was noted that the age of citrus seedlings and the initial chloride content of both citrus and wheat roots markedly affected Cl^? uptake. The data were analyzed in the light of the dual mechanism hypothesis of ion uptake, and it was concluded that only system 2 (high Km), is operating in the excised citrus roots studied. This is in accordance with the conditions prevailing in the field (whence the plant material was collected): high concentration of the soil solution in contact with the roots, and high salt status of the tissue. It is further suggested that the uptake of ions in citrus (and presumably in other perennial woody plants), is related to its growth habits and to the size and morphology of the root systems.     

An Effective and Low-Cost Culture Medium for Isolation and Growth of Xylella fastidiosa from Citrus and Coffee Plants

Buffered charcoal–yeast extract medium (BCYE) has been used for isolation of Xylella fastidiosa from citrus (Citrus sinensis) and coffee (Coffea arabica) plants affected by citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). BCYE is composed of ACES (2-[2-amino-2oxoethyl) amino]-ethanesulfonic acid) buffer, activated charcoal, yeast extract, L-cysteine, ferric pyrophosphate, and agar. ACES buffer is costly and not always commercially available in Brazil, and the L-cysteine and ferric pyrophosphate need to be filter sterilized in 0.22-μm pore membranes before inclusion in the medium. Omission of L-cysteine, addition of magnesium sulfate, and replacements of ACES and ferric pyrophosphate for potassium phosphate and ferrous sulfate resulted in an effective, less expensive, and entirely autoclavable medium, named phosphate buffered charcoal-yeast extract medium (PCYE). The final cost of PCYE was approximately one tenth that of BCYE. Its effectiveness was tested for the isolation of X. fastidiosa from symptomatic leaves collected from 52 citrus plants affected by CVC and 43 coffee plants affected by CLS. PCYE was as effective as BCYE and has been used routinely in our and other laboratories for isolation, growth, and quantification of X. fastidiosa from plant tissues.     

Drought stress affects response of phytopathogen vectors and their parasitoids to infection‐ and damage‐induced plant volatile cues

1. The response of a phytopathogen vector to pathogen‐induced plant volatiles was investigated, as well as the response of the phytopathogen vector's parasitoid to herbivore‐induced plant volatiles released from plants with and without drought stress. 2. These experiments were performed with Asian citrus psyllid (Diaphorina citri), vector of the plant pathogen Candidatus Liberibacter asiaticus (CLas) and its parasitoid Tamarixia radiata as models. Candidatus Liberibacter asiaticus is the presumed causal pathogen of huanglongbing (HLB), also called citrus greening disease. 3. Diaphorina citri vectors were attracted to headspace volatiles of CLas‐infected citrus plants at 95% of their water‐holding capacity (WHC); such attraction to infected plants was much lower under drought stress. Attraction of the vector to infected and non‐stressed plants was correlated with greater release of methyl salicylate (MeSA) as compared with uninfected and non‐stressed control citrus plants. Drought stress decreased MeSA release from CLas‐infected plants as compared with non‐stressed and infected plants. 4. Similarly, T. radiata was attracted to headspace volatiles released from D. citri‐infested citrus plants at 95% of their WHC. However, wasps did not show preference between headspace volatiles of psyllid‐infested and uninfested plants when they were at 35% WHC, suggesting that herbivore‐induced defences did not activate to recruit this natural enemy under drought stress. 5. Our results demonstrate that herbivore‐ and pathogen‐induced responses are environmentally dependent and do not occur systematically following damage. Drought stress affected both pathogen‐ and herbivore‐induced plant volatile release, resulting in concomitant decreases in behavioural response of both the pathogen's vector and the vector's primary parasitoid.     

Phosphate and Sulfate Activate the Phosphoenolpyruvate Carboxylase from the C4 Plant Cynodon dactylon L.

The effect of phosphate, sulfate and other inorganic ions on the activity of phosphoenolpyruvate carboxylase (PEPC) from the C₄ plant Cynodon dactylon were investigated for the first time, as well as their interaction with Clc-6-P, AMP and ma-late. Activation of PEPC by phosphate and sulfate ions was demonstrated and it was not dependent on the accompanying cations, something that was not clarified for PEPCs from other plant sources. No activation of this enzyme was observed by nitrate. PEPC activation was found to be competitive with glucoses-phosphate (Clc-6-P) and AMP stimulation and less sensitive to malate inhibition. This work showed that PEPC from C₄plants could exhibit similar activation properties with the enzyme from CAM plants and different activation properties in plants of the same type, rendering the study of this enzyme from different plant sources necessary.     

Plant selection principle based on xylose isomerase

Summary The xylose isomerase genes (xylA) from Thermoanaerobacterium thermosulfurogenes and Streptomyces rubiginosus were introduced and expressed in three plant species (potato, tobacco and tomato) and transgenic plants were selected on xylose-containing medium. The xylose isomerase genes were transferred to explants of the target plant by Agrobacterium-mediated transformation. The xylose isomerase genes were expressed under the control of the enhanced cauliflower mosaic virus 35S promoter and the Ω′ translation enhancer sequence from tobacco mosaic virus. In potato and tomato, xylose isomerase selection was more efficient than the established kanamycin selection. The level of enzyme activity in the regenerated transgenic plants selected on xylose was 5–25-fold higher than the enzyme activity in control plants selected on kanamycin. The xylose isomerase system enables transgenic cells to utilize xylose as a carbohydrate source. In contrast to antibiotic or herbicide resistance-based system where transgenic cells survive on a selective medium but nontransgenic cells are killed, the xylose system is an example of a positive selection system where transgenic cells proliferate while non-transgenic cells are starved but still survive. The results show that a new selection method, is established. The xylose system is devoid of the disadvantages of antibiotic or herbicide selection, and depends on an enzyme which is already being widely utilized in specific food processes and that is generally recognized as safe for use in the starch industry.     

Mitochondrial DNA and RAPD polymorphisms in the haploid mite Brevipalpus phoenicis (Acari: Tenuipalpidae)

Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) is recognized as the vector of citrus leprosis virus that is a significant problem in several South American countries. Citrus leprosis has been reported from Florida in the past but no longer occurs on citrus in North America. The disease was recently reported in Central America, suggesting that B. phoenicis constitutes a potential threat to the citrus industries of North America and the Caribbean. Besides B. phoenicis, B. obovatus Donnadieu, and B. californicus (Banks) have been incriminated as vectors of citrus leprosis virus and each species has hundreds of host plants. In this study, Brevipalpus mite specimens were collected from different plants, especially citrus, in the States of Florida (USA) and São Paulo (Brazil), and reared on citrus fruit under standard laboratory conditions. Mites were taken from these colonies for DNA extraction and for morphological species identification. One hundred and two Random Amplified Polymorphic DNA (RAPD) markers were scored along with amplification and sequencing of a mitochondrial cytochrome oxidase subunit I gene fragment (374 bp). Variability among the colonies was detected with consistent congruence between both molecular data sets. The mites from the Florida and Brazilian colonies were morphologically identified as belonging to B. phoenicis, and comprise a monophyletic group. These colonies could be further diagnosed and subdivided geographically by mitochondrial DNA analysis.     

Xanthan is not essential for pathogenicity in citrus canker but contributes to<Emphasis Type="Italic"> Xanthomonas</Emphasis> epiphytic survival

Xanthan-deficient mutants of Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, were generated by deletion and marker exchange of the region encoding the carboxy-terminal end of the first glycosyltransferase, GumD. Mutants of gumD did not produce xanthan and remained pathogenic in citrus plants to the same extent as wild-type bacteria. The kinetics of appearance of initial symptoms, areas of plant material affected, and growth of bacteria inside plant tissue throughout the disease process were similar for both wild-type and mutant inoculations. Moreover, exopolysaccharide deficiency did not impair the ability of the bacteria to induce hypersensitive response on non-host plants. Apart from variations in phenotypic aspects, no differences in growth or survival under different stress conditions were observed between the xanthan-deficient mutant and wild-type bacteria. However, gumD mutants displayed impaired survival under oxidative stress during stationary phase as well as impaired epiphytic survival on citrus leaves. Our results suggest that xanthan does not play an essential role in citrus canker at the initial stages of infection or in the incompatible interactions between X. axonopodis pv. citri and non-host plants, but facilitates the maintenance of bacteria on the host plant, possibly improving the efficiency of colonization of distant tissue.     

Citrus abscission and Arabidopsis plant decline in response to 5-chloro-3-methyl-4-nitro-1H-pyrazole are mediated by lipid signalling

The compound 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP) is a pyrazole-derivative that induces abscission selectively in mature citrus (Citrus sinensis) fruit when applied to the canopy and has herbicidal activity on plants when applied to roots. Despite the favourable efficacy of this compound, the mode of action remains unknown. To gain information about the mode of action of CMNP, the effect of application to mature citrus fruit and Arabidopsis thaliana roots was explored. Peel contact was essential for mature fruit abscission in citrus, whereas root drenching was essential for symptom development and plant decline in Arabidopsis. CMNP was identified as an uncoupler in isolated soybean (Glycine max) mitochondria and pea (Pisum sativum) chloroplasts and an inhibitor of alcohol dehydrogenase in citrus peel, but not an inhibitor of protoporphyrinogen IX oxidase. CMNP treatment reduced ATP content in citrus peel and Arabidopsis leaves. Phospholipase A₂ (PLA₂) and lipoxygenase (LOX) activities, and lipid hydroperoxide (LPO) levels increased in flavedo of citrus fruit peel and leaves of Arabidopsis plants treated with CMNP. An inhibitor of PLA₂ activity, aristolochic acid (AT), reduced CMNP-induced increases in PLA₂ and LOX activities and LPO levels in citrus flavedo and Arabidopsis leaves and greatly reduced abscission in citrus and delayed symptoms of plant decline in Arabidopsis. However, AT treatment failed to halt the reduction in ATP content. Reduction in ATP content preceded the increase in PLA₂ and LOX activities, LPO content and the biological response. The results indicate a link between lipid signalling, abscission in citrus and herbicidal damage in Arabidopsis.     

Plant mitochondrial RNase P

Molecular investigations in mitochondria of higher plants have to take in account the complicated genomic structure of these organelles and their complex mode of gene expression. Recently tRNA processing activities and particulary RNase P-like activities have been described for mitochondria of mono- and dicot plants. The determined biochemical characteristics of these plant mitochondrial tRNA processing enzymes now allow a comparison to the bacterial prototype from which they evolved. The substrate specifity of the plant mitochondrial RNase P in particular has unique selection parameters distinct from theE. coli RNase P.