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.

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.

No abstract

Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil.Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.

Short sequence repeats (SSRs) with a potential variable number of tandem repeat (VNTR) loci were identified in the genome of the citrus pathogen Xylella fastidiosa and used for typing studies. Although monoand dinucleotide repeats were absent, we found several intermediate-length 7-, 8-, and 9-nucleotide repeats, which we examined for allelic polymorphisms using PCR. Five genuine VNTR loci were highly polymorphic within a set of 27 X. fastidiosa strains from different hosts. The highest average Nei's measure of genetic diversity (H) estimated for VNTR loci was 0.51, compared to 0.17 derived from randomly amplified polymorphic DNA (RAPD) analysis. For citrus X. fastidiosa strains, some specific VNTR loci had a H value of 0.83, while the maximum value given by specific RAPD loci was 0.12. Our approach using VNTR markers provides a high-resolution tool for epidemiological, genetic, and ecological analysis of citrus-specific X. fastidiosa strains.Xylella fastidiosa has been associated with diseases in economically important crops such as grapevine, plum, almond, peach, citrus, and more recently, coffee (13, 17) as well as some diseases in ornamental plants (3). Reciprocal transmission tests including X. fastidiosa from several hosts evidenced the occurrence of host infection groups (for details, see reference 10). In Brazil, X. fastidiosa has been responsible for causing diseases in important crops such as citrus and coffee (5, 13), although it has also been observed in plum (7) and Hibiscus schizopetalus (E. W. Kitajima, H. D. Coletta-Filho, M. A. Machado, and Q. S. Novaes, 33th Congr. Brazil Phytopathol. Soc., abstr. 323, 2000). The major economic damage is in the sweet orange crop (Citrus sinensis Osb.) which has suffered an annual loss of about $100 million. However, the total cost to Brazilian agriculture is probably higher because the economic damage caused to the coffee crop by X. fastidiosa has not been estimated.Methods for distinguishing between bacterial strains are important for detecting disease outbreaks for epidemiological analysis, and for understanding the genetic structure of microbial populations. Molecular techniques of DNA profiling based on PCR of randomly amplified polymorphic DNA (RAPD) and repetitive element polymorphism PCR (rep-PCR) have been used with great success for studies of genetic variation and the relationships between X. fastidiosa strains (2,6,8,16). However, the data produced by these methods are biallelic, which limits the amount of genetic information per locus and thus the use of these methods in estimating genetic diversity. Interlaboratory reproducibility is also a weakness of RAPD and rep-PCR analysis (20). Short sequence repeats (SSRs) with a potential variable number of repeats (VNTR) within prokaryotic DNA have been used as markers for discrimination between strains (1, 9, 12, 23). Some of these repetitive regions are located within or near DNA coding regions and so could have the potential to affect gene expression (22).The complete sequencing of the genome of the pat...