Etiology of three recent diseases of citrus in São Paulo State: sudden death, variegated chlorosis and huanglongbing

The state of S?o Paulo (SSP) is the first sweet orange growing region in the world. Yet, the SSP citrus industry has been, and still is, under constant attack from various diseases. In the 1940s, tristeza-quick decline (T-QD) was responsible for the death of 9 million trees in SSP. The causal agent was a new virus, citrus tristeza virus (CTV). The virus was efficiently spread by aphid vectors, and killed most of the trees grafted on sour orange rootstock. Control of the disease resided in replacing sour orange by alternative rootstocks giving tolerant combinations with scions such as sweet orange. Because of its drought resistance, Rangpur lime became the favourite alternative rootstock, and, by 1995, 85% of the SSP sweet orange trees were grafted on this rootstock. Therefore, when in 1999, many trees grafted on Rangpur lime started to decline and suddenly died, the spectre of T-QD seemed to hang over SSP again. By 2003, the total number of dead or affected trees was estimated to be over one million. The new disease, citrus sudden death (CSD), resembles T-QD in several aspects. The two diseases have almost the same symptoms, they spread in time and space in a manner strikingly similar, and the pathological anatomy of the bark at the bud union is alike. Transmission of the CSD agent by graft-inoculation has been obtained with budwood inoculum taken not only on CSD-affected trees (grafted on Rangpur lime), but also on symptomless trees (grafted on Cleopatra mandarin) from the same citrus block. This result shows that symptomless trees on Cleopatra mandarin are tolerant to the CSD agent. Trees on rootstocks such as Sunki mandarin or Swingle citrumelo are also tolerant. Thus, in the CSD-affected region, control consists in replacing Rangpur lime with compatible rootstocks, or in approach-grafting compatible rootstock seedlings to the scions of trees on Rangpur lime (inarching). More than 5 million trees have been inarched in this way. A new disease of sweet orange, citrus variegated chlorosis (CVC), was observed in 1987 in the Triangulo Mineiro of Minas Gerais State and the northern and north-eastern parts of SSP. By 2000, the disease affected already 34% of the 200 million sweet orange trees in SSP. By 2005, the percentage had increased to 43%, and CVC was present in all citrus growing regions of Brazil. Electron microscopy showed that xylem-limited bacteria were present in all symptomatic sweet orange leaves and fruit tissues tested, but not in similar materials from healthy, symptomless trees. Bacteria were consistently cultured from twigs of CVC-affected sweet orange trees but not from twigs of healthy trees. Serological analyses showed the CVC bacterium to be a strain of Xylella fastidiosa. The disease could be reproduced and Koch's postulates fulfilled, by mechanically inoculating a pure culture of X. fastidiosa isolate 8.1.b into sweet orange seedlings. The genome of a CVC strain of X. fastidiosa was sequenced in SSP in the frame of a project supported by FAPESP and Fundecitrus. X. fastidiosa is the first plant pathogenic bacterium, the genome of which has been sequenced. Until recently, America was free of huanglongbing (HLB), but in March 2004 and August 2005, symptoms of the disease were recognized, respectively in the State of S?o Paulo (SSP) and in Florida, USA. HLB was known in China since 1870 and in South Africa since 1928. Because of its destructiveness and its rapid spread by efficient psyllid insect-vectors, HLB is probably the most serious citrus disease. HLB is caused by a phloem sieve tube-restricted Gram negative bacterium, not yet available in culture. In the 1990s, the bacterium was characterized by molecular techniques as a member of the alpha proteobacteria designated Candidatus Liberibacter africanus for the disease in Africa, and Candidatus Liberibacter asiaticus for HLB in Asia. In SSP, Ca. L. asiaticus is also present, but most of the trees are infected with a new species, Candidatus Liberibacter americanus.     

Overview of Strain Characterization in Relation to Serological and Molecular Detection of Citrus tristeza <i>Closterovirus</i>

Tristeza is a devastating viral disease in all the citrus growing countries throughout the world and has killed millions of citrus trees in severely affected orchards. The citrus species grafted on sour orange rootstock are affected by this disease. Predominantly, the sweet orange, grapefruit and lime trees grafted on sour orange exhibit severe symptoms like quick decline, vein clearing, pin holing, bark scaling and degeneration leading to variable symptoms. Symptomatic expression of Citrus tristeza virus (CTV) in different hosts has been attributed to virus isolates which are from severe to mild. Different serological and molecular assays have been deployed to differentiate the strains of CTV. Citrus tristeza virus is diversified towards its strains on the basis of biological, serological and molecular characterization. Phenotypic expression is due to genetic alteration and different molecular basis have now been adopted for strain differentiation. This review will give a brief idea about the different CTV isolates, their characterization based on nucleic acid and serological assays. Different methods along with salient features for strain characterization has also been reviewed. This review will also open the new aspects towards formulation of management strategies through different detection techniques.     

Symptomatic expression of tristeza-infected citrus plants in Pakistan

Variable symptoms were recorded during a survey in the citrus trees infected or suspected to be infected with citrus tristeza virus based on ELISA tests. Sweet orange manifested main symptoms like bud union crease, pin holing, bark cracking, incompatibility, yellowing and dropping of leaves. Pin holing was particularly prevalent in Sweet orange (Citrus sinensis Osbeck), Kinnow (Citrus reticulata Blanco) and Grapefruit (Citrus paradisii Mcaf.), while incompatibility was only in Sweet orange and Grapefruit.     

The resistance of sour orange to Citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways

Citrus tristeza virus (CTV) induces in the field the decline and death of citrus varieties grafted on sour orange (SO) rootstock, which has forced the use of alternative decline‐tolerant rootstocks in affected countries, despite the highly desirable agronomic features of the SO rootstock. Declining citrus plants display phloem necrosis below the bud union. In addition, SO is minimally susceptible to CTV compared with other citrus varieties, suggesting partial resistance of SO to CTV. Here, by silencing different citrus genes with a Citrus leaf blotch virus‐based vector, we have examined the implication of the RNA silencing and salicylic acid (SA) defence pathways in the resistance of SO to CTV. Silencing of the genes RDR1, NPR1 and DCL2/DCL4, associated with these defence pathways, enhanced virus spread and accumulation in SO plants in comparison with non‐silenced controls, whereas silencing of the genes NPR3/NPR4, associated with the hypersensitive response, produced a slight decrease in CTV accumulation and reduced stunting of SO grafted on CTV‐infected rough lemon plants. We also found that the CTV RNA silencing suppressors p20 and p23 also suppress the SA signalling defence, with the suppressor activity being higher in the most virulent isolates.     

Identification and genomic characterization of a new virus (Tymoviridae family) associated with citrus sudden death disease

Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.     

Detection of citrus yellow mosaic virus by PCR and nucleic acid spot hybridisation using non-radioactive probes in commercial citrus species

Citrus yellow mosaic virus (CYMV) was detected by polymerase chain reaction (PCR) in leaf samples of sweet orange, Rangpurlime, Pumello pink and acid lime and also in twig bark, fruit rind, fruit juice except fruit rag of sweet orange cv. Sathgudi, where all the positive samples showed bright amplification of the 726 bp band except in fruit rind. The CYMV could be detected by the biotin labelled probe of CYMV up to 1:100 dilutions by TE and TNE extraction methods in sweet orange cv. Sathgudi as well as Rangpurlime.     

Alteration of Bark Proteins Associated with Citrus Tristeza Virus (CTV) Infection on Susceptible Citrus Species and Scion-Rootstock Combinations

The effect of citrus tristeza virus (CTV) on bark protems of susceptible citrus species and scion-rootstock combinations was studied by polyaerylamide gel electrophoresis (PAGE). Protein pattern of sour orange bark from CTV-infected trees on this rootstock showed reduced intensity in a protein band, about 20,000 daltons molecular weight, as compared with similar CTV-free trees. This protein modification appears specifically associated with decline induced by tristeza since it was observed on trees of different ages and scion-rootstock combinations, grown in various locations and infected with several CTV isolates, but not on trees exhibiting decline from other causes. The observed protein alteration was localized in the ribosomic fraction. No protein alteration, associated with CTV infection could be found on lemon bark, although this citrus species also behaves as a CTV-susceptible rootstock. Electrophoretic profiles obtained from CTV infected Mexican lime and Etrog citron seedlings also showed reduced intensity in a protein band with the same electrophoretic mobility as the tnodified band of sour orange.     

Improved Transformation Efficiency in Citrus by Plasmolysis Treatment

Procedures for high efficiency production of transgenic citrus plants using an Agrobacterium tumefaciens system with plasmolysis treatment were developed. Longitudinally cut epicotyl segments of eight different citrus species [’Milam’ Rough lemon (Citrus jambhiri Lush), ‘Volkamer’ lemon (Citrus volkameriana L), Rangpur lime (Citrus limonia L), ‘Hamlin’ sweet orange (Citrus sinensis L Osbeck), ‘Duncan’ grapefruit (’Citrus paradisi’ Macf), Sour orange (Citrus aurantium L), ‘Cleopatra’ mandarin (Citrus reticulata Blanco) and Carrizo citrange (Citrus sinensis L Osbeck x Poncirus trifoliata L Raf) ] were plasmolyzed in different concentrations of sucrose and maltose [0, 3, 6, 8, 9, 10, 12 % (w/v) ] prior to Agrobacterium inoculation. Plasmolyzed epicotyl explants were cocultivated with either the hypervirulent Agrobacterium tumefaciens strain, the EHA-101 (harboring a binary vector pGA482GG) or Agl-1 (carrying pCAMBIA1303 vector). Both binary vectors contained neomycin phosphotransferase II (NPT II) and β-glucuronidase (GUS) genes. The binary vector, pCAMBIA1303 also contained a fused mGFP5 gene at the 3’ end of GUS gene as a reporter. Epicotyl explants of Rangpur lime, Rough and ‘Volkamer’ lemons plasmolyzed in 9–12 % maltose showed transient GUS gene expression comprising up to 95 % of the cut surface of explants, while Carrizo citrange showed 80 % expression when they were plasmolyzed in 6–10 % sucrose. On the other hand, epicotyl explants of ‘Hamlin’ sweet orange, Grapefruit, Sour orange and ‘Cleopatra’ mandarin showed transient GUS expession in 80–90 % of explants with 6–10 % sucrose. Basal portions of the regenerated putative transgenic shoots harvested from the cut surface of epicotyl explants within 2–3 months, were assayed for GUS, and apical portions were shoot-tip grafted in vivo for the production of whole plants. The transformation efficiencies in different species obtained are the highest so far reported for citrus.     

Almond brown line and decline: a new disease probably caused by a mycoplasma-like organism

Young almond (Prunus dulcis, cvs Carmel, Peerless and Price) orchards established on the plum rootstock Marianna 2624 (P. cerasifera×P. munsoniana) contained trees that exhibited poor terminal shoot growth and wilted, chlorotic leaves. The scion/rootstock graft union showed an external splitting of the bark and an internal line of necrotic bark tissues that extended into the woody cylinder of the union, which was deeply pitted. Affected trees declined. The disease was named almond brown line and decline (ABLD). Incidence of ABLD ranged up to 55% per cultivar in some orchards. Numerous attempts to graft-transmit orchard collections of ABLD to healthy almond/Marianna 2624 indicators failed. Also, ABLD does not appear to be soil-borne. However, ABLD was serendipitiously determined to be bud-perpetuated when infected scion buds from an apparently healthy appearing Peerless almond/peach tree located in a foundation orchard were grafted onto healthy rooted cuttings of Marianna 2624 to produce yearling trees. Also, graft-inoculations on the almond scion portion of healthy trees, but not the plum rootstock portion, with the peach yellow leafroll mycoplasma-like organism (PYLR-MLO) caused symptoms resembling ABLD. Laboratory and glasshouse assays of several symptomatic trees did not detect tomato ringspot virus and two ilarviruses. These results suggest that an MLO, possibly PYLR-MLO, may be the causal agent of ABLD and that Marianna 2624 is probably resistant to the PYLR-MLO.     

Relationship between hydraulic conductance and citrus dwarfing by the Flying Dragon rootstock (Poncirus trifoliata L. Raft var. monstruosa)

This work studied the hydraulic characteristics and physiological behavior of two trifoliate orange (Poncirus trifoliata L. Raft) varieties—Flying Dragon (FD) and Rubidoux (RT)—with contrasting size-controlling potential when used as rootstocks for citrus trees. Thus, Valencia orange scions growing on RT root system develop about 40 % more biomass than scions on FD. The anatomical study of xylem root tissue of both rootstocks showed that the number of vessels per cross-sectional area in RT almost doubled that found in FD, whereas diameter distribution did not vary significantly. Hydraulic resistance determined in rootstocks, and bud union segments were, respectively, 2- and 3.4-fold higher in trees on FD than in trees on RT. Root systems accounted for 46.5 and 55.2 % of whole-plant hydraulic resistance, whereas bud union segments represented 7.5 and 14.6 % of this parameter, the dwarfing rootstock (FD) having the highest values. Reduced hydraulic conductance in plants on FD rootstock diminished water potential in high evaporative demand periods, causing a reduction in stomatal conductance with respect to plants on RT. This leads to lower net photosynthetic CO₂ assimilation, which may affect biomass production. Translocation of ¹³C-labeled photoassimilates from leaves to roots was lower in plants on FD than in plants on RT, indicating that in the dwarfing rootstock (FD) there may be a vascular resistance to sucrose transport at the budding union level. Findings show that reduced hydraulic conductance may be the main cause of rootstock-induced dwarfing in citrus grafted onto FD.     

Tetraploid Rangpur lime rootstock increases drought tolerance via enhanced constitutive root abscisic acid production

Whole‐genome duplication, or polyploidy, is common in many plant species and often leads to better adaptation to adverse environmental condition. However, little is known about the physiological and molecular determinants underlying adaptation. We examined the drought tolerance in diploid (2x) and autotetraploid (4x) clones of Rangpur lime (Citrus limonia) rootstocks grafted with 2x Valencia Delta sweet orange (Citrus sinensis) scions, named V/2xRL and V/4xRL, respectively. Physiological experiments to study root–shoot communication associated with gene expression studies in roots and leaves were performed. V/4xRL was much more tolerant to water deficit than V/2xRL. Gene expression analysis in leaves and roots showed that more genes related to the response to water stress were differentially expressed in V/2xRL than in V/4xRL. Prior to the stress, when comparing V/4xRL to V/2xRL, V/4xRL leaves had lower stomatal conductance and greater abscisic acid (ABA) content. In roots, ABA content was higher in V/4xRL and was associated to a greater expression of drought responsive genes, including CsNCED1, a pivotal regulatory gene of ABA biosynthesis. We conclude that tetraploidy modifies the expression of genes in Rangpur lime citrus roots to regulate long‐distance ABA signalling and adaptation to stress.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

Rootstock-induced physiological and biochemical mechanisms of drought tolerance in sweet orange

The poorly understood physiological and biochemical drought responses induced in sweet orange by citrus rootstocks of contrasting drought tolerance were investigated during a drought/rewatering cycle under controlled conditions. Long-term exposure of the grafted trees to a gradually increasing water deficit and subsequent recovery revealed distinct strategies of drought acclimation that were induced by the different rootstocks. Trees grafted onto the drought-tolerant rootstock ‘Cravo’ rangpur lime were less water conservative, exhibiting an increased cell-wall elasticity that contributes to turgor maintenance and its related processes of growth and photosynthesis over a wider range of soil–water potentials. On the other hand, the drought-tolerant ‘Sunki Tropical’ mandarin and drought-sensitive ‘Flying Dragon’ trifoliate orange rootstocks induced a water conservation strategy by increasing tissue rigidity under drought. ‘Sunki Tropical’ was also able to induce osmotic adjustment, conferring thereby a more efficient water conservation strategy than ‘Flying Dragon’ by allowing for turgor maintenance at lower soil–water potentials while attenuating cell dehydration and shrinkage. In contrast to ‘Cravo’ and ‘Sunki Tropical’, trees grafted onto ‘Flying Dragon’ exhibited a significant photoinhibition of the photosystem II reaction centers, as well as an increased H₂O₂ production and lipid peroxidation under drought treatment. A significantly higher activity of the antioxidant enzyme GPX was also observed in drought stressed trees grafted onto ‘Flying Dragon’. Collectively, these results support the involvement of elastic and osmotic adjustments, as well as the control of oxidative stress, as functional leaf traits associated with the rootstock-induced drought tolerance in sweet orange.     

Influence of virus and virus-like agents on the development of citrus buds cultured in vitro

Tissue culture in vitro was used to determine the effect of six major citrus virus and virus-like agents. Nodal stem segments from inoculated Pineapple sweet orange (Citrus sinensis (L.) Osb.), Mexican lime (C. aurantifolia (Christm.) Swing.) and Arizona Etrog citron 861-Sl (C. medica L.) were cultured in vitro to induce shoots. Some virus and virus-like agents had a marked effect on bud development and further recovery of plantlets. The number and size of the shoots that developed from each bud were affected as a result of infection. The effect depended on the specific virus, the isolate and the host-disease combination. The possible implications of these results are discussed.     

Distribution and biological role of the oligopeptide-binding protein (OppA) in Xanthomonas species

In this study we investigated the prevalence of the oppA gene, encoding the oligopeptide binding protein (OppA) of the major bacterial oligopeptide uptake system (Opp), in different species of the genus Xanthomonas. The oppA gene was detected in two Xanthomonas axonopodis strains among eight tested Xanthomonas species. The generation of an isogenic oppA-knockout derivative of the Xac 306 strain, showed that the OppA protein neither plays a relevant role in oligopeptide uptake nor contributes to the infectivity and multiplication of the bacterial strain in leaves of sweet orange (Citrus sinensis) and Rangpur lime (Citrus limonia). Taken together these results suggest that the oppA gene has a recent evolutionary history in the genus and does not contribute in the physiology or pathogenesis of X. axonopodis.     

Morphogenesis and tissue cultures of three citrus species

Studies were performed to define tissue culture techniques and culture conditions for morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis (L.) Osb.), citron (C. medica L.) and lime (C. aurantifolia) (Christm. Swing). The optimal concentrations of NAA to induce root formation on stem segments were 10 mg l^-1 for sweet orange and lime, and 3 mg l^-1 for citron. The optimal BA concentration for shoot and bud proliferation was 3 mg l^-1 for sweet orange and citron, and 1 mg l^-1 for lime. Callus initiation was accomplished in a culture medium containing 10 mg l^-1 NAA and 0.25 mg l^-1 BA. Callus was maintained by periodical subculture into the same medium supplemented with 10% (v:v) organge juice. In vitro plantlets of the three species were obtained by rooting of shoots developed from bud cultures, and of citron and lime by development of shoots from root cultures. The plants were successfully established on soil.     

Citrus somatic hybridization with potential for improved blight and CTV resistance

Summary The production of five new somatic hybrids with potential for improved disease resistance is reported herein. Protoplast isolation, fusion, and plant regeneration was achieved from Caipira sweet orange (Citrus sinensis L. Osbeck) as an embryogenic parental source and Volkamer lemon (C. volkameriana Pasquale), Cleopatra mandarin (C. reticulata Blanco), and Rough lemon (C. jambhiri Lushington) as non-embryogenic parental sources. Fusion involving Cleopatra mandarin and Rangpur lime (C. limonia L. Osbeck) as embryogenic parental sources with Sour orange (C. aurantium L.) also resulted in somatic hybrid plants. Somatic hybridization was confirmed by leaf morphology evaluation, chromosome counting, and randomly amplified polymorphic DNA (RAPD) analyses. Somatic hybrids may combine complementary characteristics from both parental sources and have potential for tolerance to blight and citrus tristeza virus (CTV).     

Daily Temperature Amplitude Affects the Vegetative Growth and Carbon Metabolism of Orange Trees in a Rootstock-Dependent Manner

Both instantaneous and average growth temperatures affect plant metabolism, and the physiological importance of daily variations in temperature is frequently underestimated. To improve our understanding of the environmental regulation of citrus trees, we hypothesized that vegetative growth would be stimulated in orange plants subjected to large daily temperature variations, even without changes in the average daily air temperature or the amount of energy given by degree-days. This hypothesis was tested with orange plants grafted onto Rangpur lime or Swingle citrumelo rootstocks and grown for 20?days under thermal regimes (day/night) of 25/25°C or 32.5/17.5°C. Such regimes imposed growth conditions with daily temperature variations of 0 and 15°C. Plant growth, photosynthesis, respiration, and carbohydrate availability in leaves, stems, and roots were measured under both thermal conditions. The daily temperature variation affected the carbon metabolism of young citrus trees; plants grown under daily variation of 15°C used more of the carbon stored in mature leaves and roots and the energy generated by respiration for the biosynthesis of vegetative structures, such as leaves and branches. Thus, there was a significant increase in the leaf area of plants subjected to high daily temperature variation. Current photosynthesis was similar in the two thermal regimes; however, the photosynthetic rates increased under the 15°C variation when measurements were normalized to 25°C. In addition to the stimulatory effect of the source?Csink relationship on photosynthesis, we suggest a probable involvement of hormonal regulation of plant growth through gibberellin metabolism. The rootstock affected the response of the canopy to daily temperature amplitude, with the Rangpur lime improving plant growth through higher carbohydrate availability in roots. This is the first report that highlights the importance of daily temperature variations for citrus growth and physiology under nonlimiting conditions.     

Biology of Diaphorina citri (Hem., Psyllidae) on different hosts and at different temperatures

Abstract:  The objective of this work was to study the effect of different hosts and temperatures on Diaphorina citri Kuwayama (Hem., Psyllidae) biology. Citrus limonia (Rangpur lime), Murraya paniculata (orange jessamine) and Citrus sunki (Sunki mandarin) were used as hosts. Measurements included duration and viability of the egg and nymphal stages, sex ratio, fecundity and longevity. In order to verify the effect of temperature on D. citri biology, the duration and viability of its developmental stages and biological cycle were compared at seven different temperature conditions. Durations of the embryonic and nymphal stages were similar for Rangpur lime, orange jessamine and mandarin. However, the nymphal viability obtained on mandarin was lower than the values obtained on the other hosts. Fecundity was higher on orange jessamine and, in all instances, females had greater longevity than males. A fixed number of instars (five) was obtained on the three hosts. Rangpur lime and orange jessamine provided better D. citri development when compared with mandarin. Duration of the egg and nymphal stages varied from 2.6 to 7.7 and from 9.4 to 35.8 days, respectively, at temperatures from 18 to 32°C. Egg viability was higher than 81.6% at the temperature range studied (18–32°C); nymphal viability was higher than 70% at the 18–30°C range, differing from viability at 32°C, which was dramatically reduced. The D. citri lower temperature development threshold (TT) and thermal constant ( K ) values for the egg, nymphal and biological cycle (egg–adult) stages were 12.0°C and 52.6 Degree-Day (DD); 13.9°C and 156.9 DD; and 13.5°C and 210.9 DD respectively.