Instituto Ludwig de Pesquisa sobre o Câncer, Rua Prof. Antonio Prudente, 109-4 andar, 01509-010, Sao Paulo-SP

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The genome sequence of the plant pathogen Xylella fastidiosa

Using the PCR, we amplified the 16S ribosomal DNAs (rDNAs) of an Asian strain and an African strain of the uncultured, gram-negative, walled, phloem-limited bacterium-like organism (BLO) associated with citrus greening disease. We evaded coamplification of chloroplast 16S rDNA by using restriction enzymes; the chloroplast 16S rDNA was sensitive to Bcl I digestion and resistant to Eco RI digestion, while the 16S rDNA of the BLO was resistant to Bcl I digestion and sensitive to Eco RI digestion. The 16S rDNA of the African BLO strain was amplified from Bcl I-digested DNA extracted from infected periwinkle leaf midribs. The Asian strain was isolated from plant extract by using a specific monoclonal antibody coated onto the surface of a PCR tube. The 16S rDNAs of the two BLO strains were cloned and sequenced. Comparisons with sequences of 16S rDNAs obtained from the GenBank data base revealed that the two citrus greening disease BLOs belong to the alpha subdivision of the class Proteobacteria. Even though their closest relatives are members of the alpha-2 subgroup, these BLOs are distinct from this subgroup as we observed only 87.5% homology between the 16S rDNAs examined. Therefore, the two BLOs which we studied probably are members of a new lineage in the α subdivision of the Proteobacteria. We propose the trivial name “liberobacter” for this new group of bacteria and will wait until additional characteristics have been determined before we propose a formal name.

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The phloem-limited bacterium of greening disease of citrus is a member of the α subdivision of the Proteobacteria

Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.

Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants.

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Xylella fastidiosa: Cause of Pierce's Disease of Grapevine and Other Emergent Diseases.

SUMMARY Phytoplasmas for which 16S rDNA sequences are available have been classified into 20 major phylogenetic groups or subclades. Further phytoplasmas have been assigned to these groups, according to other molecular data such as RFLP analysis of PCR-amplified ribosomal DNA, nucleic acid hybridization, and serological comparison. A total of 75 phytoplasmas were distinguishable among the molecularly characterized phytopathogenic mollicutes.

Invited review current status of molecular classification of the phytoplasmas

A xylem-limited bacterium resemblingXylella fastidiosa has been shown previously by electron mmcroscopy to be associated with citrus variegated chlorosis (CVC), a new disease of sweet organe tress in Brazil. A bacterium was consistently cultured from plant tissues from CVC twigs of sweet orange trees but not from tissues of healthy trees on several cell-free media known to support the growth ofXylella fastidiosa. Bacterial colonies typical ofX. fastidiosa became visible on PW, CS20, and PD2 agar media after 5 and 7–10 days of incubation, respectively. The cells of the CVC bacterium were rod-shaped, 1.4–3 μm in length, and 0.2–0.4 μm in diameter, with rippled walls. An antiserum against an isolate (8.1.b) of the bacterium gave strong positive reactions to double-antibody-sandwich (DAS), enzyme-linked immunosorbent assay (ELISA) with other cultured isolates from CVC citrus, as well as with several type strains ofX. fastidiosa. This result indicates that the CVC bacterium is a strain ofX. fastidiosa. ELISA was also highly positive with all leaves tested from CVC-affected shoots. Leaves from symptomless tress reacted negatively. Sweet organe seedlings inoculated with a pure culture of the CVC bacterium supported multiplication of the bacterium, which became systemic with 6 months after inoculation and could be reisolated from the inoculated seedlings. Symptoms characteristic of CVC developed 9 months post inoculation.

Culture and serological detection of the xylem-limited bacterium causing citrus variegated chlorosis and its identification as a strain ofXylella fastidiosa.

Small-fruited acid limes (Citrus auranr(foSolia (L.) Swingle) have been grown in the Sultanate of Oman along the northern coastal plain, or Batinah. (Fig. 1 ) for many generations. The people of Oman say !hat Sinbad the Sailor came from the Batinah coast and that the presence o f sun-dried lime fruits on his boat night have been one of the reasons for the success OF his voyages. I n many Arabic countries, limes are preferred to lemons, Limes as an export crop, after being second to dates for a long time, became by the early 1970s the mast important export commodity apart from oil. In 1971, limes were worth 333,300 Omani rials ( I rial = $2.5 U.S. in 1990) while dates were worth only I 18,000 rials. En the late 1979s. lime growers witnessed a severe decline of lime trees. kcause the presence of witches'-brooms on affected trees was the most characteristic sympmrn, the disease was named witches'-broom disease of time (WBDL). From the time the first witches'-broom appears. only 4-5 years elapse before the trcc dies. By 1986. several orchards were completety destroyed and Farmers began to grow banana plants instead. Two surveys by one of us (J. M.B.) in April 1986 and April 1987 showed WBDL to spread rapidly to unaffected areas in the Ratinah, In 1989, the disease was seen for the first time in the neighboring United Arab Emirates (UAE). This paper summarizes for the first time the work that has been done on the geographical distribution of WBDL, the association of rnycopIasmalike organisms (MLOs) with thc disease. transmis-

Witches' broom, a lethal mycoplasmal disease of lime trees in the sultanate of Oman and the United Arab Emirates.

A monoclonal antibody (MA 2A10), specific for the mycoplasmalike organism (MLO) associated with tomato stolbur, was used to detect the MLO in plants and insects by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and/or immunofluorescence. All solanaceous plants showing stolbur symptoms reacted with the monoclonal antibody; some nonsolanaceous plants, such as celery, strawberry, bindweed, and periwinkle, that were naturally infected with MLOs also reacted with MA 2A10. Eleven hopper species tested positive by DAS-ELISA with MA 2A10 (.)

Use of a monoclonal antibody to detect the stolbur mycoplasmalike organism in plants and insects and to identify a vector in France.

Marginal chlorosis has affected strawberry production in France for about 15 years. A phloem-restricted uncultured bacterium, "Candidatus Phlomobacter fragariae," is associated with the disease. A large-scale survey for marginal chlorosis in French strawberry production fields and nurseries by polymerase chain reaction amplification of "Ca. P. fragariae" 16S rDNA revealed that symptoms of marginal chlorosis were not always induced by "Ca. P. fragariae" and that the stolbur phytoplasma could induce identical symptoms. "Ca. P. fragariae" was found to be predominant in strawberry production fields, whereas the stolbur phytoplasma was predominantly detected in nurseries. Two transmission periods of the disease, one in spring and the other from late summer to early fall, were evident. Cixius wagneri planthoppers captured on infected strawberry plants were demonstrated to be efficient vectors of "Ca. P. fragariae." The involvement in natural disease spread of the whitefly Trialeurodes vaporariorum, previously shown to acquire and multiply "Ca. P. fragariae" under greenhouse conditions, remains uncertain.

“Candidatus Phlomobacter fragariae” Is the Prevalent Agent of Marginal Chlorosis of Strawberry in French Production Fields and Is Transmitted by the Planthopper Cixius wagneri (China)

Xylella fastidiosa isolate 8.1.b obtained from a sweet orange tree affected by citrus variegated chlorosis in the state of Sao 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.

L. Zreik

Papers

Culture and serological detection of the xylem-limited bacterium causing citrus variegated chlorosis and its identification as a strain ofXylella fastidiosa.

Witches' broom, a lethal mycoplasmal disease of lime trees in the sultanate of Oman and the United Arab Emirates.

Use of a monoclonal antibody to detect the stolbur mycoplasmalike organism in plants and insects and to identify a vector in France.

“Candidatus Phlomobacter fragariae” Is the Prevalent Agent of Marginal Chlorosis of Strawberry in French Production Fields and Is Transmitted by the Planthopper Cixius wagneri (China)

A triply cloned strain of xylella fastidiosa multiplies and induces symptoms of citrus variegated chlorosis in sweet orange