Diversity of Endophytic Bacterial Populations and Their Interaction with Xylella fastidiosa in Citrus Plants

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.     

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

AIMS: 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). METHODS AND RESULTS: 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. CONCLUSIONS: The in vitro interaction experiments indicated that the growth of X. fastidiosa was stimulated by endophytic Methylobacterium extorquens and inhibited by endophytic Curtobacterium flaccumfaciens. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.     

Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks

Fungi and bacteria were isolated from surface disinfected leaf tissues of several citrus rootstocks. The principal bacterial species isolated were Alcaligenes sp., Bacillus spp. (including B. cereus, B. lentus, B. megaterium, B. pumilus, and B. subtilis), Burkholderia cepacia, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium extorquens, and Pantoea agglomerans, with P. agglomerans and B. pumilus being the most frequently isolated species. The most abundant fungal species were Colletotrichum gloeosporioides, Guignardia citricarpa, and Cladosporium sp. Genetic variability between 36 endophytic bacterial isolates was analysed by the random amplified polymorphic DNA (RAPD) technique, which indicated that B. pumilus isolates were more diverse than P. agglomerans isolates, although genetic diversity was not related to the host plants. In vitro interaction studies between G. citricarpa isolates and the most frequently isolated endophytic bacteria showed that metabolites secreted by G. citricarpa have an inhibitory growth effect on some Bacillus species, and a stimulatory growth effect on P. agglomerans.     

The endophyte Curtobacterium flaccumfaciens reduces symptoms caused by Xylella fastidiosa in Catharanthus roseus

Citrus variegated chlorosis (CVC) is a disease of the sweet orange [Citrus sinensis (L.)], which is caused by Xylella fastidiosa subsp. pauca, a phytopathogenic bacterium that has been shown to infect all sweet orange cultivars. Sweet orange trees have been occasionally observed to be infected by Xylella fastidiosa without evidencing severe disease symptoms, whereas other trees in the same grove may exhibit severe disease symptoms. The principal endophytic bacterial species isolated from such CVC-asymptomatic citrus plants is Curtobacterium flaccumfaciens. The Madagascar periwinkle [Citrus sinensis (L.)] is a model plant which has been used to study X. fastidiosa in greenhouse environments. In order to characterize the interactions of X. fastidiosa and C. flaccumfaciens, periwinkle plants were inoculated separately with C. flaccumfaciens, X. fastidiosa, and both bacteria together. The number of flowers produced by the plants, the heights of the plants, and the exhibited disease symptoms were evaluated. PCR-primers for C. flaccumfaciens were designed in order to verify the presence of this endophytic bacterium in plant tissue, and to complement an existing assay for X. fastidiosa. These primers were capable of detecting C. flaccumfaciens in the periwinkle in the presence of X. fastidiosa. X. fastidiosa induced stunting and reduced the number of flowers produced by the periwinkle. When C. flaccumfaciens was inoculated together with X. fastidiosa, no stunting was observed. The number of flowers produced by our doubly- inoculated plants was an intermediate between the number produced by the plants inoculated with either of the bacteria separately. Our data indicate that C. flaccumfaciens interacted with X. fastidiosa in C. roseus, and reduced the severity of the disease symptoms induced by X. fastidiosa. Periwinkle is considered to be an excellent experimental system by which the interaction of C. flaccumfaciens and other endophytic bacteria with X. fastidiosa can be studied.     

Evaluation of endophytic colonization of Citrus sinensis and Catharanthus roseus seedlings by endophytic bacteria

Over the last few years, the endophytic bacterial community associated with citrus has been studied as an important component interacting with Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC). This bacterium may also colonize some model plants, such as Catharanthus roseus and Nicotiana clevelandii. In the present study, we compared the endophytic colonization of Citrus sinensis and Catharanthus roseus using the endophytic bacteria Klebsiella pneumoniae. We chose an appropriate strain, K. pneumoniae 342 (Kp342), labeled with the GFP gene. This strain was inoculated onto seedlings of C. sinensis and C. roseus. The isolation frequency was determined one week after the inoculation and the endophytic colonization of K. pneumoniae was observed using fluorescence microscopy. Although the endophytic bacterium was more frequently isolated from C. roseus than from C. sinensis, the colonization profiles for both host plants were similar, suggesting that C. roseus could be used as a model plant to study the interaction between endophytic bacteria and X. fastidiosa.     

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.     

Model plants for studying the interaction between Methylobacterium mesophilicum and Xylella fastidiosa

Over the last few years, endophytic bacterial communities associated with citrus have been studied as key components interacting with Xylella fastidiosa. In this study, we investigated the possible interaction between the citrus endophyte Methylobacterium mesophilicum SR1.6/6 and X. fastidiosa in model plants such as Catharanthus roseus (Madagaskar periwinkle) and Nicotiana clevelandii (Clevelands tobacco). The aim of this study was to establish the fate of M. mesophilicum SR1.6/6 after inoculation of C. roseus and N. clevelandii plants, using PCR-DGGE (polymerase chain reaction--denaturing gradient gel electrophoresis) and plating techniques. Shifts in the indigenous endophytic bacterial communities were observed in plants inoculated with strain SR1.6/6, using specific primers targeting alpha- and beta-Proteobacteria. Cells of strain SR1.6/6 were observed in a biofilm structure on the root and hypocotyl surfaces of in vitro seedlings inoculated with M. mesophilicum SR1.6/6. This emphasizes the importance of these tissues as main points of entrance for this organism. The results showed that C. roseus and N. clevelandii could be used as model plants to study the interaction between M. mesophilicum and X. fastidiosa.     

Influence of Xylella fastidiosa infection of citrus on host selection by leafhopper vectors

Infection of plants by pathogens can influence their attractiveness and suitability to insect vectors and other herbivores. Here we examined the effects of Citrus sinensis (L.) Osbeck (Rutaceae) infection by the bacterium Xylella fastidiosa, which causes citrus variegated chlorosis (CVC), on the feeding preferences of two sharpshooter vectors, Dilobopterus costalimai Young and Oncometopia facialis (Signoret) (Homoptera: Cicadellidae). Experiments were performed inside observation chambers, in which a healthy plant and an infected one (with or without CVC symptoms) were supplied to a group of 40 sharpshooters. The number of insects that selected each treatment was recorded at several time intervals in 48 h. In another experiment, the ingestion rate on healthy and infected (symptomatic or not) plants was evaluated by measuring the liquid excretion of sharpshooters that were confined on branches of each plant for 72 h. Both sharpshooter species preferred healthy plants to those with CVC symptoms. However, O. facialis did not discriminate between healthy citrus and symptomless infected plants. Feeding by D. costalimai was markedly reduced when confined on CVC‐symptomatic plants, but not on asymptomatic infected ones. The ingestion rate by O. facialis was not affected by the presence of CVC symptoms. The results suggest that citrus trees with early (asymptomatic) infections by X. fastidiosa may be more effective as inoculum sources for CVC spread by insect vectors than those with advanced symptoms.     

Transmission of <Emphasis Type="Italic">Methylobacterium mesophilicum</Emphasis> by <Emphasis Type="Italic">Bucephalogonia xanthophis</Emphasis> for paratransgenic control strategy of Citrus variegated chlorosis

Methylobacterium mesophilicum, originally isolated as an endophytic bacterium from citrus plants, was genetically transformed to express green fluorescent protein (GFP). The GFP-labeled strain of M. mesophilicum was inoculated into Catharanthus roseus (model plant) seedlings and further observed colonizing its xylem vessels. The transmission of this endophyte by Bucephalogonia xanthophis, one of the insect vectors that transmit Xylella fastidiosa subsp. pauca, was verified by insects feeding from fluids containing the GFP bacterium followed by transmission to plants and isolating the endophyte from C. roseus plants. Forty-five days after inoculation, the plants exhibited endophytic colonization by M. mesophilicum, confirming this bacterium as a nonpathogenic, xylem-associated endophyte. Our data demonstrate that M. mesophilicum not only occupy the same niche of X. fastidiosa subsp. pauca inside plants but also may be transmitted by B. xanthophis. The transmission, colonization, and genetic manipulation of M. mesophilicum is a prerequisite to examining the potential use of symbiotic control to interrupt the transmission of X. fastidiosa subsp. pauca, the bacterial pathogen causing Citrus variegated chlorosis by insect vectors.     

Impact of genetically modified Enterobacter cloacae on indigenous endophytic community of Citrus sinensis seedlings

Enterobacter cloacae (strain PR2/7), a genetically modified endophyte (GME) in citrus plants, carrying different plasmids (pEC3.0/18, pCelE, pEglA and pGFP), was inoculated into Citrus sinensis seedlings under greenhouse conditions. The impact of this on the indigenous bacterial endophytic community was studied by analyses of 2 different morphologic groups. The germination rates of inoculated seeds were evaluated in greenhouse, and plasmid stability under in vitro conditions. Results demonstrated a great and diverse endophytic community inside plants, and specialization in tissue colonization by some bacterial groups, in different treatments. Shifts in seed germination rate were observed among treatments: in general, the PR2/7 harboring pEglA bacterial clone significantly reduced seed germination, compared to the PR2/7 harboring pEC3.0/18 clone. This suggests that the presence of the pEglA plasmid changes bacteria-seed interactions. The endophytic community of citrus seedlings changed according to treatment. In seedlings treated with the PR2/7 with pEglA clone, the population of group II decreased significantly, within the context of the total endophytic community. These results indicate that the application of GMEs induces shifts in the endophytic bacterial community of citrus seedlings.     

Culture-Independent Assessment of Rhizobiales-Related Alphaproteobacteria and the Diversity of <Emphasis Type="Italic">Methylobacterium</Emphasis> in the Rhizosphere and Rhizoplane of Transgenic Eucalyptus

The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism–plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR–denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR–DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.     

Isolation and Characterization of Beneficial Bacteria Associated with Citrus Roots in Florida

Cultivable diversity of bacteria associated with citrus was investigated as part of a larger study to understand the roles of beneficial bacteria and utilize them to increase the productive capacity and sustainability of agro-ecosystems. Citrus roots from Huanglongbing (HLB) diseased symptomatic and asymptomatic citrus were used in this study. A total of 227 and 125 morphologically distinct colonies were isolated and characterized from HLB asymptomatic and symptomatic trees, respectively. We observed that the frequency of bacterial isolates possessing various plant beneficial properties was significantly higher in the asymptomatic samples. A total of 39 bacterial isolates showing a minimum of five beneficial traits related to mineral nutrition [phosphate (P) solubilization, siderophore production, nitrogen (N) fixation], development [indole acetic acid (IAA) synthesis], health [production of antibiotic and lytic enzymes (chitinase)], induction of systemic resistance [salicylic acid (SA) production], stress relief [production of 1-amino-cyclopropane-1-carboxylate deaminase] and production of quorum sensing [N-acyl homoserine lactones] signals were characterized. A bioassay using ethidium monoazide (EMA)-qPCR was developed to select bacteria antagonistic to Candidatus Liberibacter asiaticus. Using the modified EMA-qPCR assay, we found six bacterial isolates showing maximum similarity to Paenibacillus validus, Lysinibacillus fusiformis, Bacillus licheniformis, Pseudomonas putida, Microbacterium oleivorans, and Serratia plymutica could significantly reduce the population of viable Ca. L. asiaticus in HLB symptomatic leaf samples. In conclusion, we have isolated and characterized multiple beneficial bacterial strains from citrus roots which have the potential to enhance plant growth and suppress diseases.     

Transmission efficiency of Xylella fastidiosa by sharpshooters (Hemiptera: Cicadellidae) in coffee and citrus

Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner) is a bacterial pathogen transmitted by several sharpshooters in two tribes of Cicadellinae (Proconiini and Cicadellini). Here, we compared the transmission efficiency of X. fastidiosa in coffee (Coffea arabica L.) and citrus [Citrus sinensis (L.) Osbeck] by Cicadellini [Bucephalogonia xanthophis (Berg) and Dilobopterus costalimai Young] and Proconiini [Homalodisca ignorata Melichar and Oncometopia facialis (Signoret)] sharpshooters that occur in both crops. At different seasons, healthy adults of each species were submitted to a 48-h acquisition access period on citrus or coffee source plants infected with X. fastidiosa isolates that cause Citrus variegated chlorosis (CVC) and Coffee leaf scorch (CLS), respectively, and then confined on healthy seedlings of the corresponding host plant for a 48-h inoculation access period. No significant effect of inoculation season was observed when comparing infection rates of citrus or coffee plants inoculated by vectors at different times of the year. In citrus, the transmission rate by single insects was significantly higher for H. ignorata (30%) in relation to B. xanthophis (5%) and O. facialis (1.1%), but there was no difference among vector species in coffee, whose transmission rates ranged from 1.2 to 7.2%. Comparing host plants, H. ignorata was more effective in transmitting X. fastidiosa to citrus (30%) in relation to coffee (2.2%), whereas the other vectors transmitted the bacterium to both hosts with similar efficiencies. Despite these variations, vector efficiency in coffee and citrus is lower than that reported in other hosts.     

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.     

Characterisation and pathogenicity of bacteria from shoot tips of the globe artichoke (Cynara scolymus L.)

Bacteria have been isolated from shoot tips of symptomless globe artichoke plants. These were identified as Pseudomonas fluorescens, Pseudomonas marginalis, Pseudomonas spp., Serratia liquefaciens, Enterobacter agglomerans/Erwinia, Agrobacterium radiobacter, an unidentified member of Rhizobiaceae and another classified in the “corynebacteria” group. The most frequently isolated species was P. fluorescens, biovars II and III. The endogenous character of these bacteria was studied in plants growing in vitro and in the open field. P. fluorescens, P. marginalis, S. liquefaciens and E. agglomerans/Erwinia caused symptoms in plants growing in vitro, but only P. fluorescens biovar II and P. marginalis produced symptoms in plants growing in open fields. Differences in pathogenicity were observed on inoculated plants growing in vitro or in the open field. This suggests that several endophytic bacterial species may be responsible for the high levels of contaminants found during the micropropagation of globe artichoke.     

Genetic structure and biology of Xylella fastidiosa strains causing disease in citrus and coffee in Brazil

Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.     

Fungal endophyte diversity and community patterns in healthy and yellowing leaves of Citrus limon

Fungal endophytes on citrus plants have been little studied, and the effects of citrus diseases on their incidence and diversity have not been addressed. In this study, we examined the foliar fungal endophytes of Citrus limon in the vicinity of Yaoundé, Cameroon, with emphasis on the differences between endophyte communities in healthy and yellowing leaves. From 82.3 % of the 480 leaf fragments, a total of 482 isolates were recovered and analysis of ITS sequences revealed 20 phylotypes. All fungal endophytes were ascomycetes and, except for one species, were common plant pathogens. Mycosphaerella and its anamorphs (34.2 % of all isolates), and Colletotrichum gloeosporioides (50.4 % of all isolates), were isolated most frequently. Mycosphaerellaceous species dominated in healthy leaves, and were absent from yellowing leaves. C. gloeosporioides was isolated significantly more frequently from yellowing than healthy leaves. Yellowing leaves had a significantly higher overall infection frequency but, in contrast, the least species diversity. Difference in the endophyte assemblages of healthy and yellowing leaves suggests that yellowing of leaves may facilitate the incidence of certain endophytes and impose growth inhibition on others.     

Diversity of endophytic enterobacteria associated with different host plants

Fifty-three endophytic enterobacteria isolates from citrus, cocoa, eucalyptus, soybean, and sugar cane were evaluated for susceptibility to the antibiotics ampicillin and kanamycin, and cellulase production. Susceptibility was found on both tested antibiotics. However, in the case of ampicillin susceptibility changed according to the host plant, while all isolates were susceptible to kanamycin. Cellulase production also changed according to host plants. The diversity of these isolates was estimated by employing BOX-PCR genomic fingerprints and 16S rDNA sequencing. In total, twenty-three distinct operational taxonomic units (OTUs) were identified by employing a criterion of 60% fingerprint similarity as a surrogate for an OTU. The 23 OTUs belong to the Pantoea and Enterobacter genera, while their high diversity could be an indication of paraphyletic classification. Isolates representing nine different OTUs belong to Pantoea agglomerans, P. ananatis, P. stewartii, Enterobacter sp., and E. homaechei. The results of this study suggest that plant species may select endophytic bacterial genotypes. It has also become apparent that a review of the Pantoea/Enterobacter genera may be necessary.     

A Suitable <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> CVC Strain for Post-Genome Studies

The genome sequence of the pathogen Xylella fastidiosa Citrus Variegated Chlorosis (CVC) strain 9a5c has revealed many genes related to pathogenicity mechanisms and virulence determinants. However, strain 9a5c is resistant to genetic transformation, impairing mutant production for the analysis of pathogenicity mechanisms and virulence determinants of this fastidious phytopathogen. By screening different strains, we found out that cloned strains J1a12, B111, and S11400, all isolated from citrus trees affected by CVC, are amenable to transformation, and J1a12 has been used as a model strain in a functional genomics program supported by FAPESP (São Paulo State Research Foundation). However, we have found that strain J1a12, unlike strains 9a5c and B111, was incapable of inducing CVC symptoms when inoculated in citrus plants. We have now determined that strain B111 is an appropriate candidate for post-genome studies of the CVC strain of X. fastidiosa.     

Endophytic bacteria in long-term in vitro cultivated “axenic” pineapple microplants revealed by PCR–DGGE

In vitro propagated plants are believed to be free of microbes. However, after 5 years of in vitro culture of pineapple plants, without evidence of microbial contamination, the use of culture-independent molecular approach [classifying heterogeneous nucleic acids amplified via universal and specific 16S rRNA gene by polymerase chain reaction (PCR)], and further analysis by denaturing gradient gel electrophoresis (DGGE) revealed endophytic bacteria in roots, young and mature leaves of such plants. The amplification of 16S rRNA gene (Bacteria domain) with the exclusion of the plant chloroplast DNA interference, confirmed the presence of bacterial DNA, from endophytic microorganisms within microplant tissues. PCR–DGGE analysis revealed clear differences on bacterial communities depending on plant organ. Group-specific DGGE analyses also indicated differences in the structures of Actinobacteria, Alphaproteobacteria and Betaproteobacteria communities in each part of plants. The results suggest the occurrence of a succession of bacterial communities colonizing actively the microplants organs. This study is the first report that brings together evidences that pineapple microplants, previously considered axenic, harbor an endophytic bacterial community encompassing members of Actinobacteria, Alphaproteobacteria and Betaproteobacteria group which is responsive to differences in organs due to plant development.