The aster yellows controversy: current status

Evidence for and against the spiroplasmal etiology of aster yellows (AY) disease is examined. A spiroplasma, serologically identical to Spiroplasma citri, was cultivated by some workers from lettuce (Lactuca sativa L.) plants claimed to be naturally infected with AY. The isolated spiroplasma was shown to be infectious by injecting Macrosteles fascifrons with the cultured organisms and then confining the injected leafhoppers on healthy plants. The reports claiming that a spiroplasma is the etiological agent of AY, however, exist only in astract form, and several essential questions still need to be answered to substantiate the claim. Evidence against the claim is based on significant differences that have been observed between the behavior of S. citri and the AY agent in the leafhoppers as well as in the plant. Also, helical organisms could not be found in AY-infected plants by either scanning or immunosorbent electron microscopy, and S. citri is serologically unrelated to the mycoplasma-like organisms found in AY-infected plants. These results strongly support the conclusion that the classical AY disease is not caused by a variant of S. citri.     

Transmission of Spiroplasma citri by the aster leafhopper Macrosteles fascifrons (Homoptera: Cicadellidae)

The aster leafhopper (Macrosteles fascifrons), injected with an isolate of Spiroplasma citri obtained from brittle root-diseased horseradish (Armoracia rusticana), transmitted the spiroplasma to horseradish and China aster (Callistephus chinensis.) After feeding on plants infected with S. citri, M. fascifrons transmitted the spiroplasma from aster to aster and horseradish, from yellow rocket (Barbarea vulgaris) to aster, and from turnip (Brassica rapa) to turnip. Symptoms in infected horseradish were chlorosis and stunting of newly formed leaves, discoloration of root phloem, and reduced plant growth typical of brittle root disease. Chlorosis, stunting, and asymmetry of young leaves occurred in affected aster and turnip. Flowers of infected aster were small and pale in colour and occasionally showed other symptoms including asymmetry, petal distortion, or light green petals. Spiroplasmas were isolated from all plants showing symptoms. Transmission rates by M. fascifrons which acquired S. citri by feeding on infected plants were very low, but injected leafhoppers transmitted more frequently. This is the first report of the transmission of S. citri from diseased to healthy plants by M. fascifrons.     

Disruption of a gene predicted to encode a solute binding protein of an ABC transporter reduces transmission of Spiroplasma citri by the leafhopper Circulifer haematoceps

Spiroplasma citri is transmitted from plant to plant by phloem-feeding leafhoppers. In an attempt to identify mechanisms involved in transmission, mutants of S. citri affected in their transmission must be available. For this purpose, transposon (Tn4001) mutagenesis was used to produce mutants which have been screened for their ability to be transmitted by the leafhopper vector Circulifer haematoceps to periwinkle plants. With one mutant (G76) which multiplied in leafhoppers as efficiently as S. citri wild-type (wt) strain GII-3, the plants showed symptoms 4 to 5 weeks later than those infected with wt GII-3. Thirty to fifty percent of plants exposed to leafhoppers injected with G76 remained symptomless, whereas for wt GII-3, all plants exposed to the transmission showed severe symptoms. This suggests that the mutant G76 was injected into plants by the leafhoppers less efficiently than wt GII-3. To check this possibility, the number of spiroplasma cells injected by a leafhopper through a Parafilm membrane into SP4 medium was determined. Thirty times less mutant G76 than wt GII-3 was transmitted through the membrane. These results suggest that mutant G76 was affected either in its capacity to penetrate the salivary glands and/or to multiply within them. In mutant G76, transposon Tn4001 was shown to be inserted into a gene encoding a putative lipoprotein (Sc76) In the ABCdb database Sc76 protein was noted as a solute binding protein of an ABC transporter of the family S1_b. Functional complementation of the G76 mutant with the Sc76 gene restored the wild phenotype, showing that Sc76 protein is involved in S. citri transmission by the leafhopper vector C. haematoceps.     

Effect of host‐plant and infection with ‘Candidatus Liberibacter asiaticus’ on honeydew chemical composition of the Asian citrus psyllid,Diaphorina citri

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits the citrus greening pathogen ‘Candidatus Liberibacter asiaticus’ (CLas) by feeding on citrus phloem sap. Because phloem sap is rich in sugars but low in amino acids, ACP sucks large quantities and excretes most of it as honeydew. We studied the chemical composition of ACP honeydew on various host plants. Honeydew samples were analyzed with gas chromatography–mass spectrometry. Fourteen sugars, 13 amino acids, and six organic acids were detected in the honeydew of ACP. Sugars composed about 95% of the total compounds. Sucrose and trehalose were the predominant sugars, composing about 58 and 23% of the total sugars, respectively. Proline, asparagine, aspartic acid, and glutamic acid were the most abundant amino acids in ACP honeydew. The host plant and its infection with CLas had some effect on the honeydew composition. Glucose, chiro‐inositol, myo‐inositol, inositol, maltose, and turanose were lower in honeydew collected from CLas‐infected citrus compared to that collected from non‐infected trees. In CLas‐infected citrus (pineapple sweet orange, Citrus sinensis L. Osbeck) and Bergera koenigii (L.) Spreng. [curry leaf tree (both Rutaceae)] honeydews, valine, alanine, serine, glutamine, glycine, and the organic acids were lower than in honeydew from healthy citrus. Mannose, galactose, inositol, mannitol, an unknown disaccharide, and proline were higher in the honeydew collected from B. koenigii than in honeydew collected from healthy citrus (pineapple sweet orange), whereas fructose, chiro‐inositol, myo‐inositol, trehalose, and lactic acid were lower. The findings of this study help us understand the metabolism and the nutrient needs of ACP that transmits CLas, the pathogen of huanglongbing in citrus.     

DNA probes in detection of spiroplasmas and mycoplasma-like organisms in plants and insects

Abstract DNA probes were applied to detect spiroplasmas and uncultivable mycoplasma-like organisms (MLOs) in infected plants and insects. The probes consisted of pMC5, a plasmid carrying the RNA genes of Mycoplasma capricolum and pRA1, a plasmid recovered from Spiroplasma citri . Southern blot hybridization of pMC5 with digested DNAs of periwinkle plants infected with S. citri , or with various MLOs, yielded 2 heavy and several weaker bands. The heavy hybridization bands were shown to represent rRNA genes of the plant chloroplasts, indicating significant nucleotide sequence homology between the mycoplasmal rRNA genes and those of plant chloroplasts. Some of the weaker hybridization bands, not revealed in DNA of healthy plants, appeared to represent rRNA gene sequences of the infectious agent. Use of the spiroplasma plasmid as a probe enabled the detection of S. citri in infected plant material and in hemolymph of infected leafhoppers at a high sensitivity level.     

Suppression of citrus canker disease mediated by flagellin perception

Citrus cancer, caused by strains of Xanthomonas citri (Xc) and Xanthomonas aurantifolii (Xa), is one of the most economically important citrus diseases. Although our understanding of the molecular mechanisms underlying citrus canker development has advanced remarkably in recent years, exactly how citrus plants fight against these pathogens remains largely unclear. Using a Xa pathotype C strain that infects Mexican lime only and sweet oranges as a pathosystem to study the immune response triggered by this bacterium in these hosts, we herein report that the Xa flagellin C protein (XaFliC) acts as a potent defence elicitor in sweet oranges. Just as Xa blocked canker formation when coinfiltrated with Xc in sweet orange leaves, two polymorphic XaFliC peptides designated flgIII-20 and flgIII-27, not related to flg22 or flgII-28 but found in many Xanthomonas species, were sufficient to protect sweet orange plants from Xc infection. Accordingly, ectopic expression of XaFliC in a Xc FliC-defective mutant completely abolished the ability of this mutant to grow and cause canker in sweet orange but not Mexican lime plants. Because XaFliC and flgIII-27 also specifically induced the expression of several defence-related genes, our data suggest that XaFliC acts as a main immune response determinant in sweet orange plants.     

Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination

The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In citrus, selection using the selectable marker gene nptII, that confers resistance to the antibiotic kanamycin, is in general very effective. An attractive alternative is offered by the MAT system (Multi-Auto-Transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with a MAT vector has been attempted in two citrus genotypes, Pineapple sweet orange (Citrus sinensis L. Osb.) and Carrizo citrange (C. sinensis L. Osb. × Poncirus trifoliata L. Raf.). Results indicated that the IPT phenotype was clearly distinguishable in sweet orange but not in citrange, and that excision was not always efficient and precise. Nevertheless, the easy visual detection of the IPT phenotype combined with the higher transformation efficiency achieved in sweet orange using this system open interesting perspectives for the generation of marker-free transgenic citrus plants.     

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.     

A Triply Cloned Strain of Xylella fastidiosa Multiplies and Induces Symptoms of Citrus Variegated Chlorosis in Sweet Orange

Xylella fastidiosa isolate 8.1.b obtained from a sweet orange tree affected by citrus variegated chlorosis in the state of S?o Paulo, Brazil, and shown in 1993 to be the causal agent of the disease, was cloned by repeated culture in liquid and on solid PW medium, yielding triply cloned strain 9a5c. The eighth and the 16th passages of strain 9a5c were mechanically inoculated into sweet orange plants. Presence of X. fastidiosa in sweet orange leaves of shoots having grown after inoculation (first-flush shoots) was detected by DAS-ELISA and PCR. Thirty-eight days after inoculation, 70% of the 20 inoculated plants tested positive, and all plants gave strong positive reactions 90 days after inoculation. Symptoms first appeared after 3 months and were conspicuous after 5 months. X. fastidiosa was reisolated from sweet orange leaves, 44 days after inoculation. These results indicate that X. fastidiosa strain 9a5c, derived from pathogenic isolate 8.1.b by triply cloning, is also pathogenic. Strain 9a5c is now used for the X. fastidiosa genome sequencing project undertaken on a large scale in Brazil. Received: 1 February 1999 / Accepted: 1 April 1999     

Multiplication and morphology of Spiroplasma citri in the leafhopper Euscelis plebejus

Spiroplasma citri multiplied in all Euscelis plebejus leaf hoppers injected and sometimes reached titres of over 1 × 10⁷ colony forming units per insect. Spiroplasmas could be isolated from the haemolymph at all times although helices were only apparent for a few days after injection. The salivary glands of injected insects contained membrane bound pockets densely packed with mycoplasma-like bodies. These bodies were frequently infected with virus-like particles similar to those found in cultures of S. citri. Spiroplasmas had little effect on the longevity of the leafhoppers.     

Etiology and Transmission of Sesame Phyllody in Iran

Phyllody is a destructive disease of sesame (Sesamum indicum L.) in Iran. The major symptoms of the disease are floral virescence, phyllody and proliferation. Other symptoms which sometimes accompany the disease are yellowing, cracking of seed capsules, germination of seeds in the capsules and formation of dark exudates on the foliage. Light microscopy of hand-cut sections of sesame and colza (Brassica napus L. cv. Oro) stems treated with Dienes' stain showed blue areas in the phloem region of phyllody infected plants. Mycoplasma-like bodies were found in the sieve cells of infected sesame stems when thin sections were examined m an electron microscope. Sesame phyllody was successfully transmitted from sesame to sesame by grafting. Among various leafhoppers collected in sesame fields only Neoaliturus haematoceps transmitted the disease. This is the first report on the identification of a Mycoplasma-like organism (MLO) as the cause of sesame phyllody and N. haematoceps as an MLO vector in Iran. In host range studies using the leafhopper vector, only B. napus cv. Oro, Lepidium sativum, Catharanthus roseus, Lactuca sp. and Portulaca oleracea, but not 17 other species, developed symptoms. The species of vector and host range of MLO indicate that sesame phyllody in Iran is different from that reported in India and Upper Volta.     

Effects of Oxamyl on the Citrus Nematode,Tylenchulus semipenetrans,and on Infection of Sweet Orange

Foliar sprays of 4 μg/ml oxamyl on sweet orange trees in a greenhouse slightly depressed the number of Tylenchulus semipenetrans larvae obtained from roots and soil, but similar treatments were not effective in two orchards. Soil drench treatments decreased the number of citrus nematode larvae obtained from roots or soil of citrus plants grown itt a greenhouse and in orchards. Exposure to 5-10 μg/ml of oxamyl in water was lethal to only a few second-stage larvae treated 10 days, and many second-stage larvae in 2.0 μg/ml oxamyl recovered motility when transferred to fresh water. Aqueous solutions of 50 and 100 μg/ml of oxamyl were toxic to citrus nematode larvae. Additional observations indicate that oxamyl interfered with hatch of citrus nematode larvae and was nematistatic and/or protected sweet orange roots from infection. Oxamyl degraded at different rates in two soils. The number of citrus nematode larvae that infected and developed on sweet orange roots was increased by an undetermined product of the degradation of oxamyl in soil, water, and possibly within plants. This product apparently was translocated in roots.     

柑橘内生真菌的分离鉴定及其发酵产物对柑橘溃疡病菌的抑制活性

本研究从柑橘抗病品种的健康植株不同组织中分离纯化和鉴定内生真菌,并测定其发酵产物对柑橘溃疡病菌的抑制活性,以明确柑橘抗病品种中内生真菌的组成及其产抗柑橘溃疡病菌活性代谢产物的潜力,为柑橘溃疡病抗菌剂的开发奠定基础。该研究通过组织培养法分离内生真菌,采用形态学和分子生物学方法对其进行鉴定; 基于前期的拮抗预试验结果,选取代表性菌株进行发酵培养,通过乙酸乙酯浸提、真空抽滤、旋转蒸发浓缩制备粗提物; 采用带毒平板涂布法测定不同菌株发酵产物乙酸乙酯提取物对柑橘溃疡病菌的抑制活性。结果表明:(1)共分离得到72株内生真菌,归为2门(Ascomycota、Basidiomycota)、14个属,其中优势属为刺盘孢属(Colletotrichum)、球座菌属(Guignardia)、链格孢属(Alternaria)和镰刀菌属(Fusarium)。(2)不同柑橘品种中内生真菌多样性指数为温州蜜柑(桂林)>沙糖桔(桂林)>沙糖桔(梧州)。(3)不同组织中内生真菌多样性变化因地理位置差异而有所不同,采自桂林的温州蜜柑和沙糖桔均为叶片中的内生真菌的多样性高于枝条,而采自梧州的沙糖桔为叶片中的多样性低于枝条,并且采自梧州的柑橘样品与采自桂林的柑橘样品中的内生真菌相似性低。(4)测定了30株内生真菌乙酸乙酯提取物对柑橘溃疡病菌的抑制活性,其中29株菌株表现出不同程度抑制活性。不同柑橘品种中的优势属的MIC介于0.312 5～10 mg·mL^-1之间,特有属的MIC介于0.156～5 mg·mL^-1,共有属镰刀菌属的MIC介于0.312 5～2.5 mg·mL^-1之间。研究结果表明柑橘抗病品种中内生真菌具有丰富多样性,并且其发酵提取物普遍对柑橘溃疡病菌具有抑制作用。特有属抑菌活性总体优于优势属,共有属镰刀菌属在不同柑橘抗病品种中均具有显著抑菌效果。     

Construction of EGFP-Labeling System for Visualizing the Infection Process of Xanthomonas axonopodis pv. citri in planta

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker, an economically important disease to world citrus industry. To monitor the infection process of Xac in different citrus plants, the enhanced green florescent protein (EGFP) visualizing system was constructed to visualize the propagation and localization in planta. First, the wild-type Xac was isolated from the diseased leaves of susceptible 'Bingtang' sweet orange, and then the isolated Xac was labeled with EGFP by triparental mating. After PCR identification, the growth kinetics and pathogenicity of the transformants were analyzed in comparison with the wild-type Xac. The EGFP-labeled bacteria were inoculated by spraying on the surface and infiltration in the mesophyll of 'Bingtang' sweet orange leaves. The bacterial cell multiplication and diffusion processes were observed directly under confocal laser scanning microscope at different intervals after inoculation. The results indicated that the EGFP-labeled Xac releasing clear green fluorescence light under fluorescent microscope showed the infection process and had the same pathogenicity as the wild type to citrus. Consequently, the labeled Xac demonstrated the ability as an efficient tool to monitor the pathogen infection.     

Evidence for host plant preference by Iphiseiodes quadripilis (Acari: Phytoseiidae) on Citrus

In this study, we present field and laboratory evidence on the preference of Iphiseiodes quadripilis (Banks) for grapefruit (Citrus paradisi Macfadyen) leaves compared with sweet orange (Citrus sinensis (L.) Osbeck) leaves. This preference was confirmed in four orchards whether leaf samples were taken from either border trees of contiguous grapefruit or sweet orange or interior row trees with both citrus species in adjacent rows. Iphiseiodes quadripilis was most abundant in grapefruit trees in spite of the greater abundance of the Texas citrus mite, Eutetranychus banksi (McGregor) (Acari: Tetranychidae) in sweet orange trees. Similar preference responses were observed in laboratory tests using a Y-tube olfactometer whether I. quadripilis were collected from sweet orange or grapefruit. Iphiseiodes quadripilis collected from grapefruit trees showed significant preference for grapefruit over sweet orange leaves in contact choice tests using an arena of alternating leaf strips (12 mm long × 2 mm wide) of sweet orange and grapefruit. However, I.␣quadripilis collected from sweet orange trees did not show preference for either grapefruit or sweet orange leaves. Based on these results, grapefruit leaves foster some unknown factor or factors that retain I. quadripilis in greater numbers compared with sweet orange leaves.     

Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion

Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.     

Susceptibility and Resistance of Citrus Genotypes to Alternaria alternata pv. citri

A survey of citrus cultivars in Israel in orchards where Alternaria brown spot was common on Minneola tangelos (mandarin × grapefruit), revealed the occurrence of the disease as typical foliar and fruit lesions on Dancy and Ellendale (mandarins), on Murcott tangor (mandarin × sweet orange), on Nova and Idith (mandarin hybrids), on Calamondin, and on Sunrise and Redblush (grapefruit). Isolates of Alternaria alternata from each of these hosts were proven to be pathogenic to Minneola tangelo.
The host range of A. alternata pv. citri from Israel was assayed by inoculating leaves of diverse citrus genotypes. Several mandarins and their hybrids (Dancy, Kara, King, Wilking, Satsuma, Minneola, Orlando, Mikhal, Idith, Nova, Page, Murcott), grapefruit (Marsh seedless), grapefruit × pummelo (Oroblanco), sweet orange (Shamouti, Valencia, Washington navel) Calamondin, and Volkamer citrus were susceptible. Several mandarins and their hybrids (Clementine, Avana, Yafit, Ortanique), Cleopatra, one sweet orange cultivar (Newhall), pummelo (Chandler), lemon (Eureka), Rough lemon, Rangpur lime, sweet lime, citron, limequat, sour orange, Troyer citrange and Alemow were resistant.     

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Disruption of a Gene Predicted To Encode a Solute Binding Protein of an ABC Transporter Reduces Transmission of Spiroplasma citri by the Leafhopper Circulifer haematoceps

Spiroplasma citri is transmitted from plant to plant by phloem-feeding leafhoppers. In an attempt to identify mechanisms involved in transmission, mutants of S. citri affected in their transmission must be available. For this purpose, transposon (Tn4001) mutagenesis was used to produce mutants which have been screened for their ability to be transmitted by the leafhopper vector Circulifer haematoceps to periwinkle plants. With one mutant (G76) which multiplied in leafhoppers as efficiently as S. citri wild-type (wt) strain GII-3, the plants showed symptoms 4 to 5 weeks later than those infected with wt GII-3. Thirty to fifty percent of plants exposed to leafhoppers injected with G76 remained symptomless, whereas for wt GII-3, all plants exposed to the transmission showed severe symptoms. This suggests that the mutant G76 was injected into plants by the leafhoppers less efficiently than wt GII-3. To check this possibility, the number of spiroplasma cells injected by a leafhopper through a Parafilm membrane into SP4 medium was determined. Thirty times less mutant G76 than wt GII-3 was transmitted through the membrane. These results suggest that mutant G76 was affected either in its capacity to penetrate the salivary glands and/or to multiply within them. In mutant G76, transposon Tn4001 was shown to be inserted into a gene encoding a putative lipoprotein (Sc76) In the ABCdb database Sc76 protein was noted as a solute binding protein of an ABC transporter of the family S1_b. Functional complementation of the G76 mutant with the Sc76 gene restored the wild phenotype, showing that Sc76 protein is involved in S. citri transmission by the leafhopper vector C. haematoceps.