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...

Background Epicoccum nigrum Link (syn. E. purpurascens Ehrenb. ex Schlecht) is a saprophytic ascomycete distributed worldwide which colonizes a myriad of substrates. This fungus has been known as a biological control agent for plant pathogens and produces a variety of secondary metabolites with important biological activities as well as biotechnological application. E. nigrum produces darkly pigmented muriform conidia on short conidiophores on sporodochia and is a genotypically and phenotypically highly variable species. Since different isolates identified as E. nigrum have been evaluated as biological control agents and used for biocompound production, it is highly desirable that this species name refers to only one lineage. However, according to morphological and genetic variation, E. nigrum present two genotypes that may comprise more than one species.Methodology/Principal FindingsWe report the application of combined molecular (ITS and β-tubulin gene sequence analysis, PCR-RFLP and AFLP techniques), morphometric, physiological, genetic compatibility and recombination analysis to study the taxonomic relationships within an endophytic population that has been identified as E. nigrum. This combined analysis established two genotypes showing morphological, physiological and genetic divergence as well as genetic incompatibility characterized by colony inhibition, strongly indicating that these genotypes correspond to different species. Genotype 1 corresponds to E. nigrum while genotype 2 represents a new species, referred to in this study as Epicoccum sp.Conclusions/SignificanceThis research contributes to the knowledge of the Epicoccum genus and asserts that the classification of E. nigrum as a single variable species should be reassessed. In fact, based on the polyphasic approach we suggest the occurrence of cryptic species within E. nigrum and also that many of the sequences deposited as E. nigrum in GenBank and culture collection of microbial strains should be reclassified, including the reference strain CBS 161.73 sequenced in this work. In addition, this study provides valuable tools for differentiation of Epicoccum species.

During some phases of of their life-cycle endophytic fungi colonize plants asymptomatically being found most frequently inside the aerial part of plant tissues. After surface disinfection of apparently healthy leaves from three varieties of mandarin orange and one tangor, and after incubation on appropriate culture medium, 407 fungal isolates were obtained, giving a total infection frequency of 81%. No fungal growth was observed from disinfected seeds, indicating that fungi are probably not transmitted via seeds. Of the fungal isolates, 27% belonged to the genus Guignardia, with 12 isolates being identified as Guignardia citricarpa Kiely, which is described as a citrus pathogen. The isolates were variable in respect to the presence of sexual structures and growth rates. Most of the isolates produces mature asci, supporting the hypothesis that they are nonpathogenic endophytes, which recently were identified as G. mangiferae. High intraspecific genetic variability (an average similarity coefficient of 0.6) was detected using random amplified polymorphic DNA (RAPD) markers generated by seven different primers. The highest similarity coefficient (0.9) was between isolates P15 and M86 and the smallest (0.22) between isolates P15 and C145. These results did not allow us to establish an association between genetic similarity of the fungal isolates and the citrus varieties from which they were obtained.

Resumo Marcadores moleculares RAPD foram utilizados para avaliar a diversidade genética em uma população de 94 híbridos de tangerina Cravo (Citrus reticulata Blanco) com laranja Pêra (C. sinensis (L.) Osbeck), obtidos por polinização controlada. Nas reações de amplificação foram usados 102 primers decâmeros de seqüência arbitrária, que amplificaram 640 fragmentos, sendo 77,2% monomórficos entre os parentais. Utilizou-se o coeficiente de Jaccard para estimar a similaridade genética entre os híbridos, o método UPGMA para gerar o fenograma (NTSYS 1,7) e o software BOOD para determinar a consistência de cada agrupamento. Verificou-se que a laranja Pêra apresenta maior heterozigosidade que a tangerina Cravo, sendo, respectivamente, 180 e 126 os números observados de loci em heterozigose. Houve alta similaridade genética entre os parentais, caracterizada pelo baixo polimorfismo de marcadores RAPD. Não houve a formação de agrupamentos estatisticamente significativos dos híbridos entre si e com os parentais, demonstrando que a constituição genética dos híbridos foi originada pela segregação independente das marcas RAPD, sendo quebrado o efeito de ligação em função do tamanho amostral em estudo. Híbridos com maior similaridade genética em relação à tangerina Cravo e laranja Pêra podem ser facilmente selecionados a partir de marcadores RAPD.Termos para indexação: frutas cítricas, marcadores genéticos, heterozigoto, variação genética. Genetic diversity among hybrids of Cravo mandarin and Pêra sweet orangeAbstract RAPD molecular markers were used to evaluate the genetic diversity in a progeny of 94 hybrids between Cravo mandarin (Citrus reticulata Blanco) and Pêra sweet orange (C. sinensis (L.) Osbeck), obtained by controlled pollination. One hundred two random decamer primers were screened, amplifying 640 fragments, 77.2% monomorphics between the parents. The Jaccard coefficient was used to estimate the genetic similarity among the hybrids, the UPGMA method to establish the phenogram (NTSYS 1.7) and the software BOOD to determine the significance of each cluster. The results indicated that Pêra sweet orange heterozygosity degree is higher than Cravo mandarin, with 180 and 126 loci in heterozygose, respectively. It was observed high genetic similarity between the parents, characterized by the low degree of RAPD polymorphism. Significant statistic groups were not established among the hybrids and the parents, showing that the hybrid genetic constitution was originated by independent RAPD markers segregation. Then, the ligation effect was broken because of the sample size. Hybrids with high genetic similarity degree in relation to Cravo mandarin and Pêra sweet orange could be easily selected using RAPD markers.