Citrus Variegated Chlorosis Disease Research Articles

Leafhoppers1 Associated with Citrus Orchards on the Yucatan Peninsula, Mexico

In the order Hemiptera, leafhoppers are the most diverse family, with more than 25,000 species distributed worldwide. Leafhoppers (Auchenorrhyncha: Cicadellidae) are of phytosanitary importance, because they are efficient vectors of plant pathogens such as phytoplasmas and other bacteria and viruses that cause diseases in a wide range of plants. Citrus variegated chlorosis disease is caused by the bacteria Xyllela fastidiosa (Wells et al. 1987), currently distributed and causing problems in citrus crops in Argentina, Brazil, Paraguay, and Costa Rica. However, there are only a few studies of Mexican leafhoppers associated with disease and relationship with a host plant. This research focused on taxonomy of Cicadellidae mainly on species related to citrus plants (Rutaceae). As a result, 25 leafhopper species were identified on the Peninsula of Yucatan. But, only four species, according to the literature, were associated with citrus plants: Homalodisca sp., Oncometopia clarior (Walker851), Phera obtusifrons Fowler, 1899, and Hortensia similis (Walker,1851). The rest of the leafhopper species collected, apparently used citrus plants only as temporary habitat, although some might be important pests of other crops.

Two new superior primer pairs for universal detection of Xylella spp. in conventional PCR and TaqMan quantitative real-time PCR

Xylella fastidiosa causes many economically important plant diseases such as Pierce's disease of grapevine, citrus variegated chlorosis disease, and olive quick decline syndrome. Another species in the same genus, Xylella taiwanensis, causes pear leaf scorch. Here, to enable an initial screening of plants suspected of being infected with Xylella spp. by conventional polymerase chain reaction (cPCR), new primer pairs—X67S1/XL2r and XrDf1/XLr4—were designed to target the 16S ribosomal DNA (rDNA) of not only X. fastidosa but also X. taiwanensis. In cPCR to detect both species, X67S1/XL2r showed features superior to those of other primer pairs, such as fewer false negatives and false positives, whereas XrDf1/XLr4 seemed to be unsuitable because of abundant non-specific amplification. However, when XrDf1/XLr4 was combined with a probe in a TaqMan quantitative real-time PCR (qPCR), the assay detected no false positives and was more useful in the universal detection of Xylella spp. than TaqMan qPCR assays reported previously.

Scion Substitution: A New Strategy to Control Citrus Variegated Chlorosis Disease.

Citrus variegated chlorosis (CVC) disease, caused by the xylem-limited and insect-transmitted bacterium Xylella fastidiosa, has caused severe losses in orange production in Brazil. Disease control requires insecticide applications, tree removal, and pruning of symptomatic branches. Pruning success has been erratic, especially in areas of high disease incidence. In this work, in planta X. fastidiosa distribution and the effectiveness of severe pruning procedures for curing diseased adult trees were investigated. Most sampled upper parts of the trees contained X. fastidiosa, but at higher frequencies in symptomatic branches. Removal of all main branches (decapitation) was not effective and revealed a 20 to 30% incidence of latent infections. Trunk decapitation resulted in a higher number of healthy scions but killed 10 to 30% of the remaining trunks. Removal of all scion and grafting the newly sprouted shoots of 'Rangpur' lime (Citrus limonia Osbeck) or 'Cleopatra' (Citrus reshni Hort. ex Tan.) rootstocks with healthy buds allowed production of fast-growing and productive new scions that remained free from CVC for at least 2 years in four locations. With this method, highly affected trees do not need to be fully removed and the costs involved in this practice and in young tree acquisition and plantings are circumvented; therefore, it is a feasible option for less technically inclined small growers in Brazil.

The diversity of citrus endophytic bacteria and their interactions with Xylella fastidiosa and host plants.

The bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC) and has been associated with important losses in commercial orchards of all sweet orange [Citrus sinensis (L.)] cultivars. The development of this disease depends on the environmental conditions, including the endophytic microbial community associated with the host plant. Previous studies have shown that X. fastidiosa interacts with the endophytic community in xylem vessels as well as in the insect vector, resulting in a lower bacterial population and reduced CVC symptoms. The citrus endophytic bacterium Methylobacterium mesophilicum can trigger X. fastidiosa response in vitro, which results in reduced growth and induction of genes associated with energy production, stress, transport, and motility, indicating that X. fastidiosa has an adaptive response to M. mesophilicum. Although this response may result in reduced CVC symptoms, the colonization rate of the endophytic bacteria should be considered in studies that intend to use this endophyte to suppress CVC disease. Symbiotic control is a new strategy that uses symbiotic endophytes as biological control agents to antagonize or displace pathogens. Candidate endophytes for symbiotic control of CVC must occupy the xylem of host plants and attach to the precibarium of sharpshooter insects to access the pathogen. In the present review, we focus on interactions between endophytic bacteria from sweet orange plants and X. fastidiosa, especially those that may be candidates for control of CVC.

Citrus Responses to Xylella fastidiosa Infection.

The xylem-limited bacterium Xylella fastidosa causes the widely disseminated citrus variegated chlorosis disease (CVC). In Brazil, CVC has been known for more than 20 years and affects only sweet orange. Lime and mandarin have remained free of symptoms despite the high inoculum pressure. Knowing the mechanisms underlying this apparent resistance is important to devise new disease control strategies. The reaction of commercial sweet orange ('Caipira', 'Natal', 'Pêra', and 'Valencia'), lime (Mexican and Persian), mandarin ('Cleopatra', 'Cravo', 'Ponkan', and 'Sunki'), and the acid citrus species Rangpur to X. fastidiosa was compared in artificially inoculated seedlings, which were assessed for symptom expression, pathogen isolation, and the amount of apparently occluded xylem vessels in cross-sectioned leaf petioles. Only the sweet orange expressed typical CVC symptoms, varying from 53.8% in Valencia to 63.0% in Caipira. Average percentages of positive isolations (pi) and occluded vessels (ov) were higher in sweet orange (pi= 59.2 to 75.0; ov = 10.7 to 25.8) than mandarin (pi = 2.3 to 16.3; ov = 1.4 to 4.0), lime (pi = 0 to 5.4; ov = 0 to 2.1), or Rangpur (pi = 1,9; ov = 1.1). There were no obvious differences in xylem anatomy among all citrus varieties, suggesting that the mechanisms of resistance to CVC are not related to any physical variation in xylem structure.

Endophytic Methylobacterium extorquens expresses a heterologous β-1,4-endoglucanase A (EglA) in Catharanthus roseus seedlings, a model host plant for Xylella fastidiosa

Based on the premise of symbiotic control, we genetically modified the citrus endophytic bacterium Methylobacterium extorquens, strain AR1.6/2, and evaluated its capacity to colonize a model plant and its interaction with Xylella fastidiosa, the causative agent of Citrus Variegated Chlorosis (CVC). AR1.6/2 was genetically transformed to express heterologous GFP (Green Fluorescent Protein) and an endoglucanase A (EglA), generating the strains ARGFP and AREglA, respectively. By fluorescence microscopy, it was shown that ARGFP was able to colonize xylem vessels of the Catharanthus roseus seedlings. Using scanning electron microscopy, it was observed that AREglA and X. fastidiosa may co-inhabit the C. roseus vessels. M. extorquens was observed in the xylem with the phytopathogen X. fastidiosa, and appeared to cause a decrease in biofilm formation. AREglA stimulated the production of resistance protein, catalase, in the inoculated plants. This paper reports the successful transformation of AR1.6/2 to generate two different strains with a different gene each, and also indicates that AREglA and X. fastidiosa could interact inside the host plant, suggesting a possible strategy for the symbiotic control of CVC disease. Our results provide an enhanced understanding of the M. extorquens-X. fastidiosa interaction, suggesting the application of AR1.6/2 as an agent of symbiotic control.

Nutritional deficiency in citrus with symptoms of citrus variegated chlorosis disease

It is well known that citrus plants that have been infected by Xylella fastidiosa display nutritional deficiencies, probably caused by production of extracellular polymers by the bacteria that block normal nutrient flow through the xylem. The aim of this work was to study the mineral composition of specific foliar areas in different stages of infection in citrus. Thus, the concentrations of macro and micronutrients in leaves of citrus infected by X. fastidiosa were measured. Samples from four infected citrus orchards in the State of São Paulo, Brazil, were respectively collected from Santa Rita do Passa Quatro, Neves Paulista, Gavião Peixoto and Paraíso counties. The presence of X. fastidiosa in leaves was confirmed by polymerase chain reaction (PCR) using specific PCR primers. To understand the variation in leaf-nutrient content in citrus plants, we used foliar nutrient values from control (non-symptomatic) plants as a reference. Chemometric analysis showed that the deficiency of P and K in symptomatic trees for all orchards and high concentrations of Fe, Mn and Zn were observed in chlorotic areas, although other studies revealed deficiency of zinc in leaves. This is the first report showing that a correlation between chlorotic citrus leaf and higher concentrations of Fe, Mn and Zn are observed when infected and healthy plants were compared.

Elemental composition changes in citrus affected by the CVC disease

The citrus variegated chlorosis (CVC) disease results in serious economical losses for the Brazilian citriculture. The influence of CVC disease on the elemental composition of citrus plants was investigated. Leaves of sweet orange varieties Hamlin, Pera Rio and Valencia were collected from healthy and CVC-affected trees for chemical characterization by instrumental neutron activation analysis (INAA). Significant differences between healthy and CVC-affected leaves were identified for Ca, Ce, Co, Eu, Fe, K, La, Na, Nd, Rb, Sc and Sm. Rare earth elements presented consistently higher mass fractions in the healthy leaves.

Rapid, specific and quantitative assays for the detection of the endophytic bacterium Methylobacterium mesophilicum in plants

Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis disease in sweet orange. There is evidence that X. fastidiosa interacts with endophytic bacteria present in the xylem of sweet orange, and that these interactions, particularly with Methylobacterium mesophilicum, may affect disease progress. However, these interactions cannot be evaluated in detail until efficient methods for detection and enumeration of these bacteria in planta are developed. We have previously developed standard and quantitative PCR-based assays specific for X. fastidiosa using the LightCycler® system [Li, W.B., Pria Jr., L.P.M.W.D., X. Qin, and J.S. Hartung, 2003.Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings. Phytopathology 93:953-958.], and now report the development of both standard and quantitative PCR assays for M. mesophilicum. The assays are specific for M. mesophilicum and do not amplify DNA from other species of Methylobacterium or other bacteria commonly associated with citrus or plant tissue. Other bacteria tested included Curtobacterium flaccumfaciens, Pantoea agglomerans, Enterobacter cloacae, Bacillus sp., X. fastidiosa, Xanthomonas axonopodis pv. citri, and Candidatus Liberibacter asiaticus. We have demonstrated that with these methods we can quantitatively monitor the colonization of xylem by M. mesophilicum during the course of disease development in plants artificially inoculated with both bacteria.

Interaction between endophytic bacteria from citrus plants and the phytopathogenic bacteria Xylella fastidiosa, causal agent of citrus-variegated chlorosis.

To isolate endophytic bacteria and Xylella fastidiosa and also to evaluate whether the bacterial endophyte community contributes to citrus-variegated chlorosis (CVC) status in sweet orange (Citrus sinensis [L.] Osbeck cv. Pera). The presence of Xylella fastidiosa and the population diversity of culturable endophytic bacteria in the leaves and branches of healthy, CVC-asymptomatic and CVC-symptomatic sweet orange plants and in tangerine (Citrus reticulata cv. Blanco) plants were assessed, and the in vitro interaction between endophytic bacteria and X. fastidiosa was investigated. There were significant differences in endophyte incidence between leaves and branches, and among healthy, CVC-asymptomatic and CVC-symptomatic plants. Bacteria identified as belonging to the genus Methylobacterium were isolated only from branches, mainly from those sampled from healthy and diseased plants, from which were also isolated X. fastidiosa. The in vitro interaction experiments indicated that the growth of X. fastidiosa was stimulated by endophytic Methylobacterium extorquens and inhibited by endophytic Curtobacterium flaccumfaciens. This work provides the first evidence of an interaction between citrus endophytic bacteria and X. fastidiosa and suggests a promising approach that can be used to better understand CVC disease.

Presence of Xylella fastidiosa in Sweet Orange Fruit and Seeds and Its Transmission to Seedlings

ABSTRACT Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.

An evolutionary perspective of Pierce's disease of grapevine, citrus variegated chlorosis, and mulberry leaf scorch diseases.

Xylella fastidiosa causes diseases on a growing list of economically important plants. An understanding of how xylellae diseases originated and evolved is important for disease prevention and management. In this study, we evaluated the phylogenetic relationships of X. fastidiosa strains from citrus, grapevine, and mulberry through the analyses of random amplified polymorphic DNAs (RAPDs) and conserved 16S rDNA genes. RAPD analysis emphasized the vigorous genome-wide divergence of X. fastidiosa and detected three clonal groups of strains that cause Pierce's disease (PD) of grapevine, citrus variegated chlorosis (CVC), and mulberry leaf scorch (MLS). Analysis of 16S rDNA sequences also identified the PD and CVC groups, but with a less stable evolutionary tree. MLS strains were included in the PD group by the 16S rDNA analysis. The Asiatic origins of the major commercial grape and citrus cultivars suggest the recent evolution of both PD and CVC disease in North and South America, respectively, since X. fastidiosa is a New World organism. In order to prevent the development of new diseases caused by X. fastidiosa, it is important to understand the diversity of X. fastidiosa strains, how strains of X. fastidiosa select their hosts, and their ecological roles in the native vegetation.

Transmission of the Citrus Variegated Chlorosis Bacterium Xylella fastidiosa with the Sharpshooter Oncometopia nigricans.

Citrus variegated chlorosis (CVC) is an economically important, destructive disease in Brazil and is caused by the bacterium Xylella fastidiosa Wells. The bacterium has been found to be transmitted in Brazil by sharpshooter leafhoppers (Cicadellidae). Sharpshooters are present in most citrus growing areas of the United States. The sharpshooter leafhopper, Oncometopia nigricans Walker, frequently is found feeding on citrus in Florida. This sharpshooter transmits the X. fastidiosa strains that cause Pierce's disease of grape and ragweed stunt. Research was initiated to determine if O. nigricans was capable of vectoring the X. fastidiosa that causes CVC. In 59 different transmission tests, using 1 to 57 insects per test, transmission of the bacterium was observed 12 times (20.3%). Symptom development in the greenhouse was not a reliable indicator of transmission. Transmission was verified by specific polymerase chain reaction-based assays. Individual insects were able to transmit the bacterium. This information on sharpshooter transmission of CVC is needed to assess the threat posed by the CVC disease to the citrus industries in the United States.

Coffee Leaf Scorch Caused by a Strain of Xylella fastidiosa from Citrus.

Citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS) are two economically important diseases in Brazil caused by the bacterium Xylella fastidiosa. Strains of the bacterium isolated from the two plant hosts are very closely related, and the two diseases share sharpshooter insect vectors. In order to determine if citrus strains of X. fastidiosa could infect coffee and induce CLS disease, plant inoculations were performed. Plants of coffee, Coffea arabica 'Mundo Novo', grafted on Coffea canephora var. robusta 'Apuatão 2258' were mechanically inoculated with triply cloned strains of X. fastidiosa isolated from diseased coffee and citrus. Three months postinoculation, 5 of the 10 plants inoculated with CLS-X. fastidiosa and 1 of the 10 plants inoculated with CVC-X. fastidiosa gave positive enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR). Eight months postinoculation, another six plants inoculated with CVC-X. fastidiosa gave positive PCR results. The two X. fastidiosa strains were isolated from the inoculated plants and showed the same characteristics as the original clones by microscopy, ELISA, and PCR. None of the plants inoculated with sterile periwinkle wilt (PW) medium as controls gave positive reactions in diagnostic tests, and none developed disease symptoms. Six months postinoculation, seven plants inoculated with CLS-X. fastidiosa and eight inoculated with CVC-X. fastidiosa began to develop characteristic CLS symptoms, including apical and marginal leaf scorch, defoliation, and reductions of internode length, leaf size, and plant height, terminal clusters of small chlorotic and deformed leaves, and lateral shoot dieback. We have demonstrated that X. fastidiosa from citrus plants is pathogenic for coffee plants. This has important consequences for the management of CLS disease and has implications for the origin of citrus variegated chlorosis disease.

Distribution of Xylella fastidiosa in Citrus Rootstocks and Transmission of Citrus Variegated Chlorosis Between Sweet Orange Plants Through Natural Root Grafts.

To study translocation of Xylella fastidiosa to citrus rootstocks, budsticks from citrus variegated chlorosis (CVC)-affected cv. Pera sweet orange (Citrus sinenesis (L.) Osb.) were top grafted on 15 citrus rootstocks. Disease symptoms were conspicuous 3 months later on all 15 rootstocks tested. The presence of X. fastidiosa was confirmed by light microscopy, double-antibody sandwich enzyme-linked immunosorbent assays, and polymerase chain reaction in rootlets and main roots of CVC-symptomatic Pera sweet orange in 11 of the 15 rootstocks tested. These results suggest that bacterial translocation from the aerial plant parts to the root system occurs but is not essential for X. fastidiosa to induce symptoms in the aerial parts. Bacterial translocation to the roots was not correlated with CVC leaf-symptom severity in the Pera scion. To determine if CVC disease could be transmitted by natural root grafts, two matched seedlings of each of four sweet orange cultivars (Pera, Natal, Valencia, and Caipira) were transplanted into single pots. One seedling rootstock of each pair was inoculated by top grafting with a CVC-contaminated budstick while the other seedling rootstock was cut but not graft inoculated. Transmission of X. fastidiosa from an inoculated plant to a noninoculated plant sharing the same pot was observed in all four sweet orange cultivars tested. Transmission was confirmed by observation of natural roots grafts between the two plants, presence of X. fastidiosa in the root grafts, and disease development in the uninoculated plants. This is the first report of transmission of CVC disease through natural root grafts.