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Botrytis is a necrotrophic fungal genus of great economic importance worldwide. Together, the Botrytis species are able to infect over one thousand host plant species, including dicotyledons and monocotyledons. As the identification of Botrytis species in Brazil has mostly been based only on morphological characterization and comparisons of the rDNA ITS region, which is not informative in the genus, its diversity remains unknown. Thus, in this study we determined the diversity and prevalence of Botrytis spp. in Brazil by multilocus phylogeny. Phylogenetic reconstruction of the genus was performed using the nuclear genes glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60) and RNA polymerase II second largest subunit (RPB2). From analyses of 56 Botrytis isolates obtained from different hosts and geographical regions in Brazil, we found that Botrytis cinerea is the most prevalent species with considerable intraspecific genetic diversity detected by nuclear genes. Two new hosts to B. cinerea and eight host never previously reported in Brazil were found. We also reported for the first time the occurrence of Botrytis pseudocinerea associated with Acca sellowiana (Myrtaceae). Due to the new phylogenetic positioning of Botrytis pelargonii and Botrytis eucalypti, a taxonomic review of these species was suggested.

Dieback and wilt caused by Erwinia psidii is an emerging disease that has been causing considerable damage in eucalypt plantations. Because it is a recently emerged disease, several aspects of the bacterial interaction with its host still remain to be elucidated. In this work, we studied the E. psidii colonization and biofilm formation in eucalypt tissues by specific detection using PCR and scanning electron microscopy (SEM). The results indicate that the bacterium is able to translocate in stem tissue mainly acropetally, although movement in the basipetal direction was also observed to a lesser extent, always through the xylem. No colonization of phloem tissues was observed. In addition to colonizing the xylem, E. psidii colonized the parenchymatous tissue. The bacterium formed cell aggregates enveloped by fibrillar material that evolved into complex, well‐structured biofilms in stem and leaf tissues. In contrast, no biofilm formation was observed on abiotic surfaces. These observations suggest that biofilm formation plays an important role in the elicitation of dieback and wilt symptoms caused by E. psidii on eucalypt plants. This study not only shows ultrastructural aspects of the E. psidii communities but also tissue damage in eucalypt plants that was associated with the presence of bacterial aggregates and formation of tyloses.

Calonectria leaf blight, caused by Calonectria pteridis, is currently one of the main foliar diseases in eucalypt plantations in Brazil. In warm and high rainfall regions, the disease can be a limiting factor for eucalypt production when planting susceptible genotypes. The most effective method for controlling this disease in the field is the use of resistant genotypes, which requires knowledge of the genetic variability and aggressiveness of the pathogen population for effective deployment of plant resistance. This work evaluated the genetic diversity and aggressiveness of C. pteridis populations obtained from infected eucalypt plants in Monte Dourado (Para state) and Imperatriz (Maranhao state), Brazil. To study the genetic diversity, 16 ISSR primers were tested, five of which amplified polymorphic, reproducible and informative bands. Thirty‐one closely related genotypes were identified from 84 isolates studied, indicating that the population has a low genetic diversity. The aggressiveness of seven isolates, selected according to geographic origin and their clustering in the ISSR‐based dendogram, was determined by inoculation of a hybrid Eucalyptus grandis × E. urophylla clone under controlled conditions. Disease severity was assessed by both measuring the percentage of plant defoliation and assigning a score according to a diagrammatic scale of symptoms. A high correlation between the two evaluation methods was observed, which revealed significant differences in aggressiveness among the isolates. The diagrammatic scale is recommended for disease evaluation because results are obtained much faster, before the occurrence of severe defoliation. No correlation between clustering in the ISSR‐based phylogenetic analysis and aggressiveness was observed.

Dieback, caused by Erwinia psidii is currently one of the most severe emerging diseases of Eucalyptus spp. in Brazil. Because of its recent report, little is known about the mechanisms underlying bacterial infection. We studied the colonization and movement of E. psidii in host tissue using a strain labeled with green fluorescent protein. We were able to transform E. psidii with pGreen-TIR and to demonstrate plasmid stability in the absence of antibiotic selection both in vitro and in vivo. We demonstrate that tissue colonization by E. psidii is not restricted to the inoculation point (leaf axil). E. psidii colonizes the xylem vessels, sclerenchyma and parenchyma of leaves and stems of eucalypt. At 35 days after inoculation, the bacterium was found at 5 cm above the inoculation point, indicating that it colonizes the plant acropetally. Confocal microscopy analysis revealed that when root-inoculated the bacterium penetrates the primary and secondary roots and reaches the xylem, but it was never found in the root crown or stem, irrespective of the evaluation time. Our results provide insights into the biology of the E. psidii-eucalypt interaction, which requires a better understanding in order to design efficient strategies for pathogen control and disease management.

The genetic variability and aggressiveness of Brazilian Erwinia psidii isolates from Eucalyptus spp. was studied and compared with reference isolates from guava (Psidium guajava). Repetitive element sequence (rep)‐based PCR markers of 101 isolates from Eucalyptus spp. and five from guava showed that the populations of E. psidii displayed a relatively low genetic variability. No correlation of genetic clustering based on rep‐PCR analysis with geographic origin or host of origin was observed, indicating that genome rearrangements associated with adaptation to a particular host were not detected by these molecular markers. A higher genotypic richness was detected in the Mato Grosso do Sul population, probably reflecting a pathogen dissemination associated with the recent expansion in eucalypt plantations. Wilcoxon and ANOVA tests of disease severity data indicated differences in aggressiveness among isolates and an isolate × clone interaction. The area under the disease progress curve (AUDPC) and disease severity for some isolates were significantly different between two susceptible clones tested. Notably, isolate LPF681 from guava was not able to cause disease on a susceptible Eucalyptus urophylla clone, suggesting that some co‐evolution between pathogen and host has taken place. The variability in aggressiveness and virulence among isolates of E. psidii observed in this study will be important for the establishment of appropriate screening approaches to select for disease resistance.

Bacterial leaf blight is a major disease of eucalypt, especially under nursery conditions. Different bacterial species have been associated with the disease in several countries, and despite its importance worldwide, it is not clear to date whether similar disease symptoms are caused by the same or by different etiological agents. In this study, 43 bacterial strains were isolated from blighted eucalypt leaves collected in different geographic areas of Brazil and inoculated onto a susceptible eucalypt clone. Polyphasic taxonomy, including morphological, physiological, biochemical, molecular, and pathogenicity tests showed that only certain strains of Xanthomonas axonopodis caused symptoms of the disease. Strains varied in their aggressiveness, but no correlation with geographic origin was observed. MLSA-based phylogenetic analysis using concatenated dnaK, fyuA, gyrB and rpoD gene sequences allocated the strains in a well-defined clade, corresponding to Rademarker?s group RG 9.6. Inoculation of nineteen plant species belonging to seven botanical families with representative strain LPF 602 showed it to be pathogenic only on Eucalyptus spp, and Corymbia spp. Based on distinct biochemical and pathogenic characteristics that differentiate the eucalypt strains from other pathovars of the X. axonopodis species, here we propose their allocation into the new pathovar X. axonopodis pv. eucalyptorum pv. nov. Made available in DSpace on 2019-01-02T23:39:23Z (GMT). No. of bitstreams: 1 26755.pdf: 1921182 bytes, checksum: 5475bd39cdda97a1767406d95440d333 (MD5) Previous issue date: 2019-01-02

Ceratocystis fimbriata Ellis & Halsted recently was recorded causing seed and seedling blight on Carapa guianensis Aubl. (andiroba), a tree species native to the Amazon Rainforest and prized for its valuable timber and medicinal seed oil. C. fimbriata more commonly causes wilt type diseases in woody hosts, especially on non-native host trees. However, on andiroba the disease occurs on seedlings and seeds, affecting the species regeneration. We studied 73 isolates of C. fimbriata on andiroba from three regions of the Amazon Basin to see if they represented natural or introduced populations. Analysis of ITS rDNA sequences and phylogenetic analysis of mating type genes revealed new haplotypes of C. fimbriata from the Latin American Clade that were closely related to other Brazilian populations of the fungus. In mating experiments, andiroba isolates were inter-fertile with tester strains of C. fimbriata from Brazil and elsewhere, confirming that they belong to a single biological species. Using microsatellite markers, 14 genotypes and populations with intermediate levels of genetic variability were found, suggesting that the fungus is indigenous to the Amazon Basin. Inoculation tests indicated that the andiroba isolates are host-specialized on andiroba, supporting the proposition of the special form C. fimbriata f. sp. carapa. Made available in DSpace on 2019-03-20T00:43:38Z (GMT). No. of bitstreams: 1 26788.pdf: 3260751 bytes, checksum: e216b037faec9a95d9551042e8f53907 (MD5) Previous issue date: 2019

Schinus terebinthifolius and Mabea fistulifera have been used for forest repositioning and urban forestry in Brazil. In October 2012, in a routine inspection at the research nursery of the Forestry Department of the Universidade Federal de Vic ß osa, in Minas Gerais, Brazil, a mortality of approximately 40% of the seedlings was observed as a result of diseases characterized by leaf blight and intense defoliation, which culminated in the death of the plants. Microscopy observations revealed oozing from the infected tissue and isolations revealed a bacterial aetiology for both diseases. Bacterial cells that formed bright yellow mucoid colonies with round edges were routinely isolated from lesion margins. Inoculation of isolated strains into healthy seedlings reproduced the symptoms observed under natural conditions. Bacterial cells showing the same morphological, biochemical and molecular characteristics as those originally isolated from naturally infected plants were reisolated from inoculated plants. Morphological, physiological and biochemical tests as well as 16S rDNA sequencing and multilocus sequence analysis using four housekeeping genes, dnaK, fyuA, gyrB and rpoD, confirmed the newly isolated strains belong to Xanthomonas axonopodis. Plant cross-inoculations showed the strains did not belong to any known phylogenetically related pathovar. Pathovars X. axonopodis pv. schini pv. nov. and X. axonopodis pv. mabeae pv. nov. are proposed as the causal agents of bacterial leaf blight on S. terebinthifolius and M. fistulifera, respectively

Eucalypt powdery mildew is an important disease in greenhouses and clonal hedges of Eucalyptus spp. in Brazil, which can cause leaf and shoot distortion, shoot discoloration, and growth reduction that results in production losses. Because reliable information regarding the causal agent of the disease is lacking, this study used ITS and 28S rDNA sequencing and morphological analyses to identify the powdery mildew pathogen that occurrs in eucalypt nurseries within different regions in Brazil. Based on the results of morphological characteristics and phylogenetic analyses, the pathogen isolates were identified as Podosphaera pannosa, also known as the rose powdery mildew pathogen. Cross inoculations with pathogen isolates from rose and eucalypt demonstrated that P. pannosa can infect both host species. The ITS sequence-based phylogeny showed that 42 sequences generated in this study were comprised within a single clade containing P. pannosa, which was supported by a posterior probability of 88%. Identical ITS sequences were obtained from all 42 pathogen isolates, which suggests a clonal population.

Key Message A resistant E. grandis genotype showed a constitutive overexpression of genes related to resistance to myrtle rust caused by A. psidii . Abstract Myrtle rust caused by Austropuccinia psidii is considered one of the most important fungal diseases affecting Eucalyptus spp. plantations in Brazil. Although the selection and planting of resistant eucalypt genotypes have been the major strategies to manage the disease in Brazil, the molecular mechanisms involved in resistance are still unclear. In this study, we evaluated the gene expression profile of two contrasting Eucalyptus grandis genotypes in resistance level to rust by RNA-Seq. The two genotypes showed a very different background gene expression level even without A. psidii infection. The resistant genotype had a constitutive overexpression of a large number of protein-coding genes compared to the susceptible genotype. These genes were mainly associated with signal transduction, photosynthesis, regulation and response to salicylic acid (SA), and protein kinase leucine-rich receptors (PK-LRR). PK-LRR and SA mediated disease resistance are well known to be effective against obligate biotroph pathogens, such as A. psidii. In addition, at 24 h after infection, the susceptible genotype was able to activate some response, however, several resistance-related proteins had their expression level reduced with A. psidii infection. Here, we present the first analysis of E. grandis genotypes transcriptomes infected by A. psidii and it reveals a constitutive overexpression of several resistance-related genes in the resistant genotype compared to the susceptible one. Our findings have the potential to be used as candidate molecular markers for resistance to myrtle rust.

Ceratocystis fimbriata is an important plant pathogen known to cause Ceratocystis Wilt (CW), a prevalent fungal disease known to affect Eucalyptus spp. plantations in Brazil. To better understand the molecular mechanisms related to pathogenicity in eucalyptus, we generated a high-quality assembly and annotation of the Ce. fimbriata LPF1912 isolate (LPF1912) genome, as well as the first transcriptome of LPF1912 from 16 eucalyptus clones at three infection incubation periods (12, 18, and 24 h). The LPF1912 genome assembly contains 805 scaffolds, totaling 31.8 Mb, with 43% of the genome estimated to be coding sequence comprised of 7,390 protein-coding genes of which 626 (8.5%) were classified as secreted proteins, 120 ribosomal RNAs, and 532 transfer RNAs. Comparative genomic analysis among three eucalyptus fungal pathogens (Ce. fimbriata, Ce. eucalypticola, and Calonectria pseudoreteaudii), showed high similarity in the proteome (21.81%) and secretome (52.01%) of LPF1912 and Ce. eucalypticola. GO annotation of pathogenicity-related genes of LPF1912 and Ce. eucalypticola, revealed enrichment in cell wall degrading enzymes (CWDEs), and lipid/cutin metabolism for Ca. pseudoreteaudii. Additionally, a transcriptome analysis between resistant and susceptible eucalyptus clones to CW infection indicated that a majority (11) of LPF1912 differentially expressed genes had GO terms associated with enzymatic functions, such as the polygalacturonase gene family, confirming the crucial role of CWDEs for Ce. fimbriata pathogenicity. Finally, our genomic and transcriptomic analysis approach provides a better understanding of the mechanisms involved in Ce. fimbriata pathogenesis, as well as a framework for further studies.

Here, we report the annotated draft genome sequence of Xanthomonas axonopodis pv. eucalyptorum pathotype strain LPF602 (synonym Xanthomonas axonopodis BSC45a), isolated from eucalypt leaves showing bacterial blight symptoms in Brazil. The availability of these genomic data will help improve the understanding of the evolution and molecular mechanisms involved in the pathogenesis of this microorganism.

In the summer of 2011, in a nursery located in Viçosa City, Minas Gerais State, brownish, necrotic, irregular spots were observed on leaves of Mabea fistulifera Mart. (Euphorbiaceae), an indigenous forest species commonly found in Brazil. Around 6,300 seedlings were evaluated and as many as 60% of them showed disease symptoms, including severe defoliation and plant death. Leaves with coalescing lesions turned papery in texture and had a blighted appearance. Bacterial colonies were isolated from these symptomatic leaves on King B's medium and identified based on biochemical and molecular analysis, as a member of the Enterobacteriaceae family. Like other members of the Enterobacteriaceae family, the bacteria were facultative anaerobic, gram-negative, cream-colored on YDC medium, urease and oxidase negative, as well as catalase and asparagine positive. Bacterial DNA was extracted from pure culture grown overnight in liquid 523 medium at 28°C using the Wizard Genomic DNA Purification kit (Promega) and conserved sequences in 16S rDNA (3) and rpoB (1) were amplified by PCR. The sequence of the 1,300-bp 16S rDNA fragment and the 750-bp rpoB gene were analyzed by NCBI BLAST. Related sequences were aligned and analyzed by ClustalW in MEGA 5 software. Phylogenetic analysis by maximum likelihood, using PAUP version 4.0 and TBR algorithm with 1,000 bootstrap replications, grouped the isolate in a clade with Enterobacter cowanii and the result showed 99% and 98% identity to the 16s rDNA and rpoB, respectively. The isolate clustered closely with the type strain of E. cowanii in both phylogenetic trees constructed. Pathogenicity tests were carried out by inoculating leaves of healthy seedlings either by spraying or cutting with a scissor previously dipped into a 108 CFU/ml bacterial suspension. The experiment was in a completely randomized design, with six replications. A pot with one plant was considered one experimental unit. Control seedlings were sprayed or cut with a scissor treated with saline solution. Prior to and after inoculation, plants were kept in a humid chamber for 24 h at 26°C in the dark and at room temperature. Subsequently, plants were transferred to growth chamber at 26°C, under a 12-h photoperiod (40 μmol/s/m2). Consistent with the symptoms observed originally, 7 days after inoculation, all seedlings developed leaf spots. No characteristic symptoms could be observed in the negative control. Furthermore, Koch's postulates were confirmed by reisolation of the bacterium from symptomatic tissues. In summary, the phenotypic, biochemical, and molecular tests identified the pathogen as E. cowanii. Recently, E. cowanii was isolated from Eucalyptus trees with symptoms of bacterial blight, although its pathogenicity was not demonstrated (2). To the best of our knowledge, this is the first report of a member of the Enterobacteriaceae family causing disease in M. fistulifera. The result has a great importance to better understand the role of E. cowanii as a pathogen-causing disease on a forest species. References: (1) C. L. Brady et al. Syst. Appl. Microbiol. 31:447, 2008. (2) C. L. Brady et al. Lett. Appl. Microbiol. 49:461, 2009. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

Here, we present a draft genome sequence of the type strain IBSBF 435 of Erwinia psidii (Enterobacteriaceae), a phytopathogen that causes bacterial blight on guava (Psidium guava) and dieback and wilt on eucalypt (Eucalyptus spp.), both of which are important emerging diseases.

Ceratocystis wilt, caused by Ceratocystis fimbriata is one of the most serious limiting factors for mango production in Brazil. Despite efforts in the selection and the identification of mango cultivars resistant to Ceratocystis wilt, the genetic basis of the resistance remains unknown. Therefore, the objective of this study was to understand the inheritance of resistance to C. fimbriata by artificial inoculations of the pathogen in progenies of six commercial varieties of mango using ?Tommy Atkins? as the male parent. The cultivars ?Keitt?, ?Palmer?, ?Tommy Atkins? and ?Van Dyke? were confirmed as moderately resistant, whereas ?Coquinho?, ?Espada? and ?Haden? were susceptible. The results of the inoculation on the progenies of these cultivars revealed that the resistance in mango is polygenic with a prevalence of genes expressing the effects of dominance and epistasis. The genetic gain with the selection of the 10 more resistant plants was 46%, which indicated a 46% reduction in disease severity. In general, a low frequency of the alleles favorable to disease resistance was observed in the population studied, which suggests the need for the introduction of new sources of genetic materials carrying the genes responsible for resistance. Made available in DSpace on 2017-02-16T22:51:58Z (GMT). No. of bitstreams: 1 PinheiroNeto.pdf: 800399 bytes, checksum: 50acdc416bc0895b33665c458658346d (MD5) Previous issue date: 2017-01-19

Mango is amongst the most exported fruits worldwide and the mango tree has been cultivated in many countries with tropical and subtropical climates. Currently, Ceratocystis wilt caused by Ceratocystis fimbriata is among the most important diseases on mango tree. Planting of resistant genotypes is the best strategy for disease control. In the selection of resistant plants a highly aggressive isolate should be used in inoculations under controlled conditions. Thus, the objective of this study was to evaluate whether there are differences in aggressiveness among isolates of C. fimbriata obtained from diseased mango trees in Brazil. Before inoculation, five randomly selected isolates were tested for sporulation and mycelial growth in different culture media in order to select the best conditions for inoculum production. Twenty-four isolates from the States of São Paulo, Rio de Janeiro, Minas Gerais and Mato Grosso do Sul were inoculated in the susceptible mango cultivar Espada. Subsequently, the four most aggressive isolates were inoculated in other five different mango cultivars. The culture media MEA provided the best levels of sporulation and mycelial growth for four of the five tested isolates. Isolates SEMG1-1 from Minas Gerais and SESP8-3 from São Paulo were the most aggressive. The response of cultivars to the most aggressive isolates tested in the present work varied from highly resistant to highly susceptible, ‘Ubá’ being the most resistant regardless of the isolate tested. These results demonstrate that there are significant differences in aggressiveness among C. fimbriata isolates from mango in Brazil, as well as cultivar × isolate interaction.

Summary The dieback and wilting caused by Erwinia psidii are emerging eucalypt diseases that have been observed since 2014 in the south and central-south regions of Brazil. Field observations have shown variability in disease severity resistance among Eucalyptus spp. clones and species. It is hypothesized that this variability is due to genetic resistance. To confirm this hypothesis, inoculations in genetically distinct eucalypt plants are necessary. However, lack of an inoculation method and disease assessment makes difficult to select resistant genotypes for use in commercial plantations or genetic breeding programmes. Three inoculation methods were tested on eight clones of Eucalyptus spp. Among them, inoculum deposition with bacteria-impregnated toothpick on the axillary buds was the simplest and most effective, capable to reproduce the disease symptoms observed under conditions of natural infection. We also developed a rating scale for disease assessment. Among eight clones tested, only Clone 1 (Eucalyptus saligna) and Clone 2 (Eucalyptus urophylla) were resistant.

The variation in disease incidence of bacterial wilt caused by Ralstonia solanacearum among eucalypt clones (Eucalyptus spp.) in the field indicates that the disease may be controlled by planting resistant material. However, efficient inoculation methods for bacterial wilt on eucalypt are scarce and have low replicability. In this work, we developed an effective protocol for inoculation of R. solanacearum, which was subsequently validated on different eucalypt clones. Three methods were tested: (i) soil infestation with bacterial cell suspension; (ii) immersion of wounded roots in the bacterial cell suspension; and (iii) injection of bacterial cell suspension in the base of the stem. The injection method proved to be the most efficient for inoculating eucalypt with R. solanacearum. Differentiation between resistant and susceptible clones was observed 30 days after inoculation in independent assays. Base stem inoculation of 21 eucalypt clones showed that four clones, classified as resistant, did not exhibit wilt symptoms or bacterial ooze at the end of the experiment. Although no wilting symptoms were observed, four other clones were considered susceptible because at least one plant showed bacterial ooze from the inoculated tissue. The remaining 13 clones were highly susceptible, presenting typical wilt symptoms and bacterial ooze.

Rust caused by Puccinia psidii is a serious disease of Eucalyptus and other Myrtaceae. Here we evaluated the pre-penetration processes in leaves at different phenological stages of a susceptible clone (3918) of Eucalyptus grandis. The first ten leaves, from the tip to the base, on the apical branch were detached and inoculated with urediniospores. We found a gradual reduction in fungal germination (88 to 39 %), apressorium formation (78 to 18 %), penetration (69 to 0 %) and sporulation (90 to 0 number of sorus /cm2) with the increase in leaf age. In addition, thickness and amount of wax was higher in the older leaves. The results of this study suggest that resistance to P. psidii infection in older Eucalyptus leaves occurs at the pre-penetration stage.

Summary Powdery mildew symptoms and signs were observed on seedlings of Lagerstroemia speciosa within an ornamental plant nursery in Vicosa, State of Minas Gerais, Brazil. Signs were most prominent as intense mycelial growth and sporulation on the shoots, new branches and leaves, which led to tissue necrosis. Based on morphological characteristics and phylogenetic analyses of ITS and 28S rDNA sequences, the powdery mildew pathogen of L. speciosa was identified as Erysiphe australiana, a well-known powdery mildew pathogen infecting Lagerstroemia spp. worldwide. To our knowledge, this is the first report of E. australiana causing powdery mildew on L. speciosa in Brazil.

Summary A new disease of unknown bacterial aetiology has been observed in eucalyptus stands since 2009. It is characterized by die-back, wilting and lesions on the branches, petiole and midrib in association with macroscopic and microscopic bacterial ooze. To date, this disease has been observed in stands of clonal Eucalyptus saligna, E. grandis and E. urophylla x E. grandis hybrids and in E. dunnii seedling plantations in the states of Sao Paulo, Rio Grande do Sul and Mato Grosso do Sul. Considering the economic importance of eucalyptus plantations and the potential losses caused by this disease, this study aimed to identify and characterize the causal agent. Thirty-four strains were obtained from infected plants, which were collected in the field from four locations. The inoculation of detached leaves and intact rooted cuttings supported pathogenicity in eucalyptus. The phylogenetic analysis of four housekeeping genes (16S rDNA, gapA, recA and rpoB) as well as biochemical tests confirmed the identity of strains belonging to the species Erwinia psidii. This is the first report of E. psidii as the cause of wilt and die-back in Eucalyptus spp. in Brazil.

Summary Among the bacterial pathogens of Eucalyptus in Brazil, Ralstonia solanacearum is considered one of the most important because of the characteristics of the pathogen, like the high diversity among the strains related to host range, high virulence, broad geographical distribution and its damage to the crop in recent years. Given its importance and the lack of research on this pathosystem, the present study aimed to perform a molecular characterization of different strains of infected Eucalyptus plants in Brazil. A total of 19 bacterial cultures isolated from Eucalyptus in different regions of Brazil were analysed. A 372-bp product generated by multiplex-PCR amplification using Nmult primers identified all the strains analysed as belonging to phylotype II. Eighteen strains were grouped into subclade IIA and one into subclade IIB. The phylogenetic tree generated from the gene sequences of endoglucanase (egl) confirmed the classification of the strains into phylotype II and separated the strains into sequevars. Strains AMC22, IBSBF2568 and IBSBF2576 were grouped into a single clade, as were strains UFV18 and UFV20, with 89% and 78% a posteriori probability, respectively, forming two new potential sequevars not yet defined. We also identified strains belonging to sequevars 41 (100% probability) and 37 (88% probability). However, most of the strains did not fit into any previously described sequevar and did not form distinct clades. The results of the analysis of fragments amplified using the ERIC-PCR technique indicated the existence of genetic diversity among the strains studied, with a generally high correlation between similarity and the geographical origin of the strains.

Plant growth-promoting rhizobacteria strains from special formulations have been used to optimize eucalyptus cutting production. To undertake quality control for the formulated products, the rhizobacterial strains should be characterized to assess their purity and authentication. In the present study, we characterized nine strains of rhizobacteria, including three Bacillus subtilis (S1, S2 and 3918), two Pseudomonas sp. (MF4 and FL2), P. putida (MF2), P. fulva (Ca), Frateuria aurantia (R1), and Stenotrophomonas maltophilia (CIIb). The strains were differentiated by colony morphology after 24 h of incubation in three different solid state culture media (glucose-nutritive agar, 523 medium and yeast extract-mannitol agar), sensitivity to a panel of 28 antibiotics (expressed according to the formation of inhibition halos of bacterial growth in the presence of antibiotics), and PCR-RFLP profiles of the 16S rDNA gene produced using nine restriction enzymes. It was possible to differentiate all nine strains of rhizobacteria using their morphological characteristics and sensitivity to antibiotics. The molecular analysis allowed us to separate the strains CIIb, FL2 and R1 from the strains belonging to the genera Bacillus and Pseudomonas. By using these three methods concomitantly, we were able to determine strain purity and perform the authentication.

Ceratocystis wilt caused by the fungus Ceratocystis fimbriata, is currently one of the major diseases in commercial plantations of Eucalyptus trees in Brazil. Deployment of resistant genotypes has been the main strategy for effective disease management. The present study aimed at identifying genomic regions underlying the genetic control of resistance to Ceratocystis wilt in Eucalyptus by quantitative trait loci (QTL) mapping in an outbred hybrid progeny derived from a cross between (Eucalyptus dunnii × Eucalyptus grandis) × (Eucalyptus urophylla × Eucalyptus globulus). A segregating population of 127 individuals was phenotyped for resistance to Ceratocystis wilt using controlled inoculation under a completely randomized design with five clonal replicates per individual plant. The phenotypic resistance response followed a continuous variation, enabling us to analyze the trait in a quantitative manner. The population was genotyped with 114 microsatellite markers and 110 were mapped with an average interval of 12.3 cM. Using a sib-pair interval-mapping approach five QTLs were identified for disease resistance, located on linkage groups 1, 3, 5, 8, and 10, and their estimated individual heritability ranged from 0.096 to 0.342. The QTL on linkage group 3 overlaps with other fungal disease-resistance QTLs mapped earlier and is consistent with the annotation of several disease-resistance genes on this chromosome in the E. grandis genome. This is the first study to identify and attempt to quantify the effects of QTLs associated with resistance to Ceratocystis wilt in Eucalyptus.

Eucalypts are susceptible to a wide range of diseases. One of the most important diseases that affect Eucalyptus plantations worldwide is caused by the rust fungus Puccinia psidii. Here, we provide evidence on the complex genetic control of rust resistance in Eucalyptus inter-specific hybrids, by analyzing a number of full-sib families that display different patterns of segregation for rust resistance. These families are totally unrelated to those previously used in other inheritance studies of rust resistance. By using a full genome scan with 114 genetic markers (microsatellites and expressed sequence tag derived microsatellites) we also corroborated the existence and segregation of a resistance locus, explaining 11.5% of the phenotypic variation, on linkage group 3, corresponding to Ppr1. This find represents an additional validation of this locus in totally unrelated pedigree. We have also detected significant additive × additive digenic interactions with LOD >10.0 on several linkage groups. The additive and epistatic QTLs identified explain between 29.8 and 44.8% of the phenotypic variability for rust resistance. The recognition that both additive and non-additive genetic variation (epistasis) are important contributors to rust resistance in eucalypts reveals the complexity of this host-pathogen interaction and helps explain the success that breeding has achieved by selecting rust-resistant clones, where all the additive and non-additive effects are readily captured. The positioning of epistatic QTLs also provides starting points to look for the underlying genes or genomic regions controlling this phenotype on the upcoming E. grandis genome sequence.

Rust caused by Puccinia psidii is one the most destructive diseases of Eucalyptus. Management of the disease is achieved through selection of resistant host genotypes. Recently, eucalypt plants from clone BA6021, resistant to P. psidii isolate race-1, were infected by rust in Brazil. Microsatellite profiles of infected plants confirmed that the host was indeed clone BA6021. In pathogenicity tests, the resistant clones BA6021 and G21 (which carry the resistance gene Ppr-1) were found susceptible to the newly discovered isolate EUBA-1, indicating a new biotype of the pathogen. These results show that the isolate EUBA-1 and other potentially unrecognized pathogen races should be given strong consideration for eucalypt breeding programs aimed rust resistance.

One of the most important diseases of eucalyptus plantations is caused by the rust fungus Puccinia psidii. While the genetic basis of rust resistance has been addressed recently, little is known about the physiological aspects of Eucalyptus–P. psidii interaction. In order to fill this gap, we undertook a study investigating the effects of P. psidii infection on photosynthetic processes of two E. urophylla clones with contrasting resistance to the pathogen. Our results show that gas exchange and chlorophyll a fluorescence parameters were virtually unaffected in the resistant clone. In the susceptible clone, photosynthetic rates were chiefly constrained by biochemical limitations to carbon fixation. Photosynthesis was impaired only in symptomatic tissues since the reductions in photosynthetic rates were proportional to the diseased leaf area. Rust infection provoked chronic photoinhibition to photosynthesis in the susceptible clone. Overall, differences in the ability for light capture, use and dissipation may play a significant role in explaining the clonal differences in Eucalyptus in response to P. psidii infection. To our knowledge, this is the first report of the effect of rust infection on gas exchange and chlorophyll a fluorescence parameters in Eucalyptus.

Ceratocystis wilt, caused by Ceratocystis fimbriata, is one of the most damaging diseases in eucalyptus plantations worldwide. Although there are resistant genotypes, the genetic basis of resistance is still poorly understood. In this paper we studied the resistance level by a stem inoculation experiment of genotypes of Eucalyptus grandis and E. urophylla and estimated the heritability and gains of selection in families derived from controlled interspecific crosses. In both species, highly resistant as well as highly susceptible genotypes to Ceratocystis wilt were found. Out of 21 parents assessed, twelve were resistant and nine susceptible. Estimates of individual narrow (50%) and broad (59%) sense heritability suggested a high degree of genetic control and low allelic dominance of the trait. There was great genetic variation among and within families, a fact that contributes to high heritability and genetic gain. A genetic gain in lesion size of up to -74.4% was obtained from selection of the 50 best clones in the evaluated families, i.e., the mean lesion length in the progeny population can be reduced by 74,4%.

No Brasil, a ferrugem causada por Puccinia psidii Winter destaca-se como a mais importante doença do eucalipto. O uso de genótipos resistentes é o principal método de controle, o que torna a detecção de marcadores ligados a resistência à ferrugem essencial para a seleção de genótipos resistentes. Neste estudo, uma progênie F1 composta por 131 plantas derivadas de um cruzamento interespecífico de Eucalyptus.Um mapa integrado foi construído para o grupo de ligação três com base em marcadores microssatélites. Para detecção de QTL foram utilizadas duas metodologias baseadas em alelos idênticos por descendência (IBD): marca simples de Haseman and Elston e mapeamento por intervalo de Fulker and Cardon. Ambas as metodologias evidenciaram associação mais significativa para o marcador Embra 125. O QTL que explicou 42 % da variação fenotípica foi mapeado a 0,02 cM desse marcador pela metodologia de intervalo de Fulker and Cardon. O marcador Embra125 têm potencial utilização em seleção assistida, com conseqüente aumento na eficiência durante a seleção de genótipos resistentes. In Brazil the rust caused by Puccinia psidii Winter stands out as the most important disease of eucalyptus. The use of resistant genotypes is the main control method, which makes the detection of markers linked to rust resistance essential to the selection of resistant genotypes. In this study, an F1 progeny of 131 plants from interspecific crossings of Eucalyptus was used to identify markers linked to resistance genes for this pathogen. An integrated map was constructed for linkage group three based on microsatellite markers. For QTL mapping two methodologies based on alleles identical-by-descent (IBD) were used: single marker analysis of Haseman and Elston and the interval mapping procedure of Fulker and Cardon. Both methods showed significant association for the Embra 125 marker.The QTL that explained 42 % of the phenotypic variation was mapped to 0.02 cM of this marker by the Fulker and Cardon. Marker Embra 125 has potential use in assisted selection, thus increasing the efficiency of the selection of resistant genotypes.

Ceratocystis fimbriata causes diseases on a wide variety of plants in Brazil, including rubber tree (Hevea brasiliensis), on which it causes gray mold or moldy rot on tapping panels affecting latex yield. However, C. fimbriata isolated from rubber tree have not been critically studied. Phylogenetic analyses of sequences of ITS rDNA and a mating type gene placed rubber tree isolates from Acre and Bahia among Brazilian isolates of C. fimbriata from other hosts. In the analyses of 14 microsatellite loci, the rubber tree isolates from Bahia were identical to each other and had alleles similar to those of Brazilian isolates from mango and eucalyptus. The microsatellite alleles of the Acre rubber tree isolates were identical to each other but distinct from other Brazilian isolates. The rubber tree isolates were morphologically indistinguishable from each other and very similar to the isolates of C. fimbriata on Ipomoea batatas, on which the species was originally described. Based on inoculation experiments results, the Bahia and Acre rubber tree isolates do not appear to be host specialized, which is typical for Brazilian isolates of C. fimbriata sensu stricto.

Currently, Xanthomonas axonopodis is one of the main foliar pathogens for Eucalyptus spp. in Brazil. It induces leaf blight and defoliation of seedlings in the nursery and young plants in the field. However, little is known about thefavorable conditions for infection. The establishment and development of bacterial leaf blight caused by X. axonopodis in eucalyptus was studied for different leaf ages, temperatures and leaf wetness durations. Disease severity increased with leaf age, and the highest severity was observed on the fourth pair of completely expanded leaves (from the apex to the base). A higher level of bacterial colonization was also observed on the fourth pair of leaves quantified as bacterial cells/cm2 of leaf area. Twelve hours of free water on the leaf surface, prior to inoculation, was essential to promote a severe infection. However, with the increase in the leaf wetness duration, a decrease in disease severity was observed. The optimal temperature for disease development was 26-30oC.

Calonectria leaf blight (CLB) caused by Calonectria pteridis is one of the main leaf diseases in Brazilian Eucalyptus plantations in warm climates with prolonged periods of rain. The main symptoms are leaf spots followed by intense defoliation in highly susceptible plants. Exploiting the existing inter- and intraspecific variability for defoliation is the best option to control this disease. Nevertheless, nothing is known about the genetic architecture of resistance to CLB in Eucalyptus. We built microsatellite-based genetic maps for E. urophylla × E. camaldulensis (EU11 × EC06) F1 family of 89 plants. Four or five clonal replicates per individual offspring were clonally propagated, totalling 445 plants which were phenotyped for defoliation in the basal third of the branches at 30 days following controlled inoculation with a single-spore isolate. Genetic mapping was performed using a pseudo-testcross, and QTLs detected using composite interval mapping. Five QTLs were detected for resistance to CLB; of them, only one could be validated in two unrelated pedigrees, and its effect was conservatively estimated as controlling between 5 and 10 % of the phenotypic variation when the bias derived from the limited size of the mapping population was taken into account. This work provides a starting point for future studies of the genetics of resistance to CLB, and adds further evidence to the challenge of ascertaining the effects of QTLs detected in a single biparental background across unrelated families.

A new foliar disease caused by Teratosphaeria pseudoeucalypti on eucalyptus (E. globulus, E. urophylla x E. globulus and E. nitens x E. globulus) in Brazil is described . The disease is characterized by leaf spots of variable sizes and shapes, resulting in leaf blight and premature defoliation. Based on the morphological characteristics and multilocus phylogenetic analysis of the EF-1α, β-T and ITS-2 gene regions of five isolates, the fungus was identified as T. pseudoeucalypti. This is the first report of this pathogen outside Australia and a method for sporulation in culture is described.

In this report the major locus for Puccinia psidii rust resistance, Ppr1, was positioned on the reference genetic map for Eucalyptus. Additionally, its position was validated by association genetics in a related and two unrelated pedigrees involving different Eucalyptus grandis resistant trees crossed to individuals of two other species, Eucalyptus tereticornis and Eucalyptus camaldulensis. These results are consistent with the hypothesis that Ppr1 controls a large proportion of the variation for rust resistance, strengthening its role as a major locus in Eucalyptus and providing its unequivocal genomic position on linkage group 3. A localized map with 19 microsatellite loci was built around Ppr1. Multiallelic profiles were observed at several mapped microsatellites suggesting recent tandem duplications in the genomic landscape surrounding Ppr1. Markers EMBRA125 and EMBRA1071 flank Ppr1 at 9.5% and 7% recombination, respectively, and were found to be in linkage equilibrium in a E. grandis breeding population, consistent with the expectations in outcrossed Eucalyptus. Their potential use for MAS will specifically be directed to identifying resistant offspring of P. psidii resistant parent trees that are heterozygous at Ppr1. In these circumstances, a significant amount of LD is expected to occur between specific alleles at flanking microsatellites and the resistance allele at Ppr1. Moreover, the positional information of Ppr1 paves the way for prospective undertakings in this genomic region with the upcoming availability of a draft genome for E. grandis.

Chrysophorte cubensis induced canker occurs in nearly all tropical and subtropical regions where eucalypts are planted, causing losses in both wood quality and volume productivity, especially so in the warmer and more humid regions of Brazil. The wide inter and intra-specific genetic variability of resistance to canker among Eucalyptus species facilitates the selection of resistant plants. In this study, we evaluated resistance to this pathogen in five Eucalyptus grandis (G) and 15 E. urophylla (U) trees, as well as in 495 individuals from 27 progenies derived from crosses between the trees. In the field, six-months-old test seedlings were inoculated with C. cubensis. Lesion length in the xylem and bark was measured eight months later. The results demonstrated that xylem lesions could preferentially be used for the selection of resistant clones. Eight trees (7 U and 1 G) were susceptible, and the remainder (8 U and 4 G) resistant. Individual narrow and broad sense heritability estimates were 17 and 81%, respectively, thereby suggesting that canker resistance is quantitative and highly dependent on dominance and epistasis.