Joseph M. Bové

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

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.

Mycoplasma genitalium, a New Species from the Human Urogenital Tract

Two mycoplasmas recovered from human urogenital tracts were similar in their biochemical and serological properties. These organisms possessed a unique terminal structure that appeared to be associated with attachment to tissue cells and erythrocytes. The organisms fermented glucose but did not hydrolyze urea or arginine. Growth occurred at 30 to 37°. Cholesterol was required for growth. Unlike most other mycoplasmas, both strains were susceptible to thallium acetate. These two organisms were serologically distinct from other Mycoplasma species and from a group of unclassified serotypes of mycoplasmas. On the basis of these findings and other morphological, biological, and serological properties of the microorganisms, we propose that mycoplasmas with these characteristics belong to a new species, Mycoplasma genitalium. Strain G-37 (= ATCC 33530) is the type strain.

Distribution and quantification of Candidatus Liberibacter americanus, agent of huanglongbing disease of citrus in São Paulo State, Brasil, in leaves of an affected sweet orange tree as determined by PCR.

Huanglongbing (HLB), an insect-transmitted disease of citrus, known for many years in Asia and Africa, has appeared in the state of São Paulo State (SSP), Brazil, in 2004, and the state of Florida, USA, in 2005. HLB endangers the very existence of citrus, as trees infected with the bacterial pathogen, irrevocably decline. In the absence of curative procedures, control of HLB is difficult and only based on prevention. Even though not available in culture, the HLB bacterium could be shown to be Gram-negative and to represent a new candidate genus, Candidatus Liberibacter, in the alpha subdivision of the Proteobacteria. Three Candidatus (Ca.) L. species occur: Ca. L. africanus in Africa, Ca. L. asiaticus in Asia, SSP, and Florida, and Ca. L. americanus in SSP. The liberibacters occur exclusively in the phloem sieve tubes. On affected trees, HLB symptoms are often seen on certain branches only, suggesting an uneven distribution of the Liberibacter. Occurrence of Ca. L. americanus, the major HLB agent in SSP, has been examined in 822 leaf samples from an affected sweet orange tree by two conventional PCR techniques and a newly developed real time (RTi) PCR, also used for quantification of the Liberibacter in the leaves. Even though RTi-PCR was able to detect as few as 10 liberibacters per gram of leaf tissue (l/g), no liberibacters could be detected in any of the many leaf samples from a symptomless branch, while in blotchy mottle leaves from symptomatic branches of the same tree, the Liberibacter titer reached values as high as 10(7)l/g. These results demonstrate the uneven distribution of the Liberibacter in HLB-affected trees.

Etiology of Three Recent Diseases of Citrus in Sao Paulo State: Sudden Death, Variegated Chlorosis and Huanglongbing

The state of São 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 Kochs 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 São 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. IUBMB Life, 59: 346‐354, 2007

Spiroplasma melliferum, a New Species from the Honeybee (Apis mellifera)

Twenty-eight strains of spiroplasma subgroup I-2 isolated from insects and flower surfaces were similar in their serological properties. Strain BC-3T (T = type strain), which was isolated from the honeybee, was chosen as a representative of this cluster and was characterized according to accepted standards. This strain and other strains of the cluster entered the hemocoel of their insect hosts after per os acquisition, caused pathology in various tissues, and reduced adult longevity. Growth in SM-1 or M1D medium occurred at 20 to 37°, with optimum growth at about 32 to 35°. Cholesterol was required for growth. Glucose, fructose, and other carbohydrates were fermented, and arginine was catabolized. Seventeen strains, including strain BC-3T, reacted with considerable homogeneity in deformation tests and were completely separable from strains of subgroup I-1 (Spiroplasma citri) and subgroup I-3 (corn stunt spiroplasma). A group of five subgroup I-2 strains showed homogeneity upon one-dimensional polyacrylamide gel electrophoresis of cell proteins. Strain BC-3T was also serologically distinct from subgroups I-4 through I-8; from Spiroplasma floricola, Spiroplasma apis, and Spiroplasma mirum; and from representative strains of spiroplasma groups II and VI through XI. Previously published studies on strain BC-3T and related strains demonstrated that (i) these organisms comprise a unique subgroup of the S. citri complex (group I); (ii) deoxyribonucleic acid-deoxyribonucleic acid homologies between strain BC-3T and strains of other group I subgroups do not exceed 70%; (iii) the patterns of protein sharing among group I strains revealed by two-dimensional polyacrylamide gel electrophoresis support molecular genetic indications of partial relatedness; (iv) the EcoRI restriction endonuclease patterns of deoxyribonucleic acids from strain BC-3T and serologically related strains show close relatedness; (v) sequencing of 5S ribosomal ribonucleic acid suggests some degree of relatedness with all organisms now classified in the Mollicutes; (vi) strain BC-3T is capable of viscotactic and chemotactic responses; (vii) strain BC-3T possesses fibrils that may mediate various types of motility; and (viii) a lytic virus (SpV4) isolated from Spiroplasma sp. strain B63 (a representative of subgroup I-2) is morphologically and genomically distinct from other spiroplasma viruses and forms plaques only on lawns of subgroup I-2 spiroplasmas. Previous work on strain AS 576, another member of subgroup I-2, demonstrated (i) a viscotactic response, (ii) moderate sensitivity to osmotic environments, (iii) susceptibility to tetracycline and aminoglycoside antibiotics, (iv) growth in a relatively simple, chemically defined medium, (v) nutritional utilization patterns in defined medium, and (vi) a genome molecular weight of 109. On the basis of our new findings and the previously described properties of strain BC-3T and related subgroup I-2 strains, we propose that spiroplasma strains with the characteristics described here be classified as a new species, Spiroplasma melliferum. Strain BC-3, the type strain, has been deposited in the American Type Culture Collection as strain ATCC 33219.

Phloem-and xylem-restricted plant pathogenic bacteria

1. Introduction: an overview This review concerns plant pathogenic bacteria, which are strictly restricted either to the sieve tubes in the phloem or to the vessels in the xylem. These bacteria are endogenous as opposed to exogenous bacteria such as species of Erwinia , Pseudomonas , Ralstonia or Xanthomonas , which colonize the apoplast (intercellular spaces) of plant tissues, even though some of the exogenous bacteria, such as Xylophilus ampelina , can also induce vascular infections. Due to their vascular habitat, the endogenous bacteria have a systemic distribution throughout the plant, they are transmitted from plant to plant by graft inoculation, and most of them are vectored by insects which feed in the phloem (leafhoppers, psyllids), or the xylem (sharpshooters). Because of these virus-like properties, the diseases caused by endogenous bacteria have long been taken for virus diseases. The principal conducting cells of the phloem are the sieve tube elements [37]. These elements are joined end to end into sieve tubes, and are associated with parenchymatic, nucleated cells, the companion cells, an important role of which is to load sucrose into the sieve tubes. The sieve tube elements are living cells, which become enucleated at maturity. The sieve plates are lateral wall areas between two adjacent sieve elements. The sieve plates are clustered with pores, resembling giant plasmodesmata and interconnecting two adjacent sieve elements through their cytoplasms. The diameter of a pore ranges from a fraction of a micron (m )t o 15m and more, and is large enough to allow passage of sieve tube-restricted bacteria. The principal conducting elements of the xylem are tracheids and vessel members [37]. Both are dead cells, contain no cytoplasm, and have lignified secondary walls. The vessel elements are joined end to end into vessels, and the adjoining ends have open perforation plates. These openings allow relatively unimpeded longitudinal spread of the xylem-restricted bacteria within

Cloning and sequencing of the replication origin (oriC) of the Spiroplasma citri chromosome and construction of autonomously replicating artificial plasmids.

A 5.6-kbp fragment ofSpiroplasma citri DNA containing thednaA gene has been cloned and sequenced. Nucleotide sequence analysis shows that this fragment harbors the genes for the replication initiator protein (dnaA), the beta subunit of DNA polymerase III (dnaN), and the DNA gyrase subunits A and B (gyrA andgyrB). The arrangement of these genes,dnaA-dnaN-gyrB-gyrA, is similar to that found in all Gram-positive bacterial genomes studied so far, except that norecF gene was found betweendnaN andgyrB. Several DnaA-box consensus sequences were found upstream ofdnaA and in thednaA-dnaN intergenic region. ThednaA region with the flanking DnaA-boxes and the tetracycline resistance determinant,tetM, were linked into a circular recombinant DNA. This DNA was able to replicate autonomously when introduced by electroporation intoS. citri cells. These experiments show that thednaA region with the DnaA-boxes is the origin of replication ofS. citri and can be used to construct gene vectors.

Spatial and Temporal Analyses of Citrus Sudden Death as a Tool to Generate Hypotheses Concerning Its Etiology

ABSTRACT Citrus sudden death (CSD), a new disease of unknown etiology that affects sweet orange grafted on Rangpur lime, was visually monitored for 14 months in 41 groves in Brazil. Ordinary runs analysis of CSD-symptomatic trees indicated a departure from randomness of symptomatic trees status among immediately adjacent trees mainly within rows. The binomial index of dispersion (D) and the intraclass correlation (k) for various quadrat sizes suggested aggregation of CSD-symptomatic trees for almost all plots within the quadrat sizes tested. Estimated parameters of the binary form of Taylors power law provided an overall measure of aggregation of CSD-symptomatic trees for all quadrat sizes tested. Aggregation in each plot was dependent on disease incidence. Spatial autocorrelation analysis of proximity patterns suggested that aggregation often existed among quadrats of various sizes up to three lag distances; however, significant lag positions discontinuous from main proximity patterns were rare, indicating a lack of spatial association among discrete foci. Some asymmetry was also detected for some spatial autocorrelation proximity patterns, indicating that within-row versus across-row distributions are not necessarily equivalent. These results were interpreted to mean that the cause of the disease was most likely biotic and its dissemination was common within a local area of influence that extended to approximately six trees in all directions, including adjacent trees. Where asymmetry was indicated, this area of influence was somewhat elliptical. Longer-distance patterns were not detected within the confines of the plot sizes tested. Annual rates of CSD progress based on the Gompertz model ranged from 0.37 to 2.02. Numerous similarities were found between the spatial patterns of CSD and Citrus tristeza virus (CTV) described in the literature, both in the presence of the aphid vector, Toxoptera citricida. CSD differs from CTV in that symptoms occur in sweet orange grafted on Rangpur lime. Based on the symptoms of CSD and on its spatial and temporal patterns, our hypothesis is that CSD may be caused by a similar but undescribed pathogen such as a virus and probably vectored by insects such as aphids by similar spatial processes to those affecting CTV.

Revised group classification of the genus Spiroplasma

Significant changes have been made in the systematics of the genus Spiroplasma (class Mollicutes) since it was expanded by revision in 1987 to include 23 groups and eight sub-groups. Since that time, two additional spiroplasmas have been assigned group numbers and species names. More recently, specific epithets have been assigned to nine previously designated groups and three sub-groups. Also, taxonomic descriptions and species names have been published for six previously ungrouped spiroplasmas. These six new organisms are: Spiroplasma alleghenense (strain PLHS-1T) (group XXVI), Spiroplasma lineolae (strain TALS-2T) (group XXVII), Spiroplasma platyhelix (strain PALS-1T) (group XXVIII), Spiroplasma montanense (strain HYOS-1T) (group XXXI), Spiroplasma helicoides (strain TABS-2T) (group XXXII) and Spiroplasma tabanidicola (strain TAUS-1T) (group XXXIII). Also, group XVII, which became vacant when strain DF-1T (Spiroplasma chrysopicola) was transferred to group VIII, has been filled with strain Tab 4c. The discovery of these strains reflects continuing primary search in insect reservoirs, particularly horse flies and deer files (Diptera: Tabanidae). In the current revision, new group designations for 10 spiroplasma strains, including six recently named organisms, are proposed. Three unnamed but newly grouped spiroplasmas are strain TIUS-1 (group XXIX; ATCC 51751) from a typhiid wasp (Hymenoptera: Tiphiidae), strain BIUS-1 (group XXX; ATCC 51750) from floral surfaces of the tickseed sunflower (Bidens sp.) and strain BARC 1901 (group XXXIV; ATCC 700283). Strain BARC 2649 (ATCC 700284) from Tabanus lineola has been proposed as a new sub-group of group VIII. Strains TIUS-1 and BIUS-1 have unusual morphologies, appearing as helices at only certain stages in culture. In this revision, potentially important intergroup serological relationships observed between strain DW-1 (group II) from a neotropical Drosophila species and certain sub-group representatives of group I spiroplasmas are also reported.

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

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 (.)