Jorge Teodoro de Souza

Isolation of actinomycetes and endospore-forming bacteria from the cacao pod surface and their antagonistic activity against the witches’ broom and black pod pathogens

In this study, actinomycetes and endospore-forming bacteria were isolated from the surface of cacao pods. The activity of these microorganisms againstCrinipellis perniciosa andPhytophthora palmivora, causal agents of witches’ broom and black pod diseases of cacao, respectively, was investigated. A total of 336 isolates of actinomycetes and endosporeforming bacteria were tested on a detached pod assay againstC. perniciosa. The screening procedure used proved to be fast and inexpensive, allowing the selection of five actinomycetes as the most promising isolates for the biocontrol ofC. perniciosa. Under laboratory conditions the actinomycetes were able to inhibit 100% ofC. perniciosa basidiospore germination. However, under field conditions the selected actinomycetes were unable to protect cacao pods against both pathogens. In these experiments, inhibition ofC. perniciosa ranged from 6% to 21% in relation to the control, whereas there was no inhibition of black pod caused byP. palmivora. Formulations need to be improved in order to enhance the activity of the actinomycetes against cacao pathogens in the field. Molecular identification of the selected isolates showed that they are species of the genusStreptomyces.

Trichoderma stromaticum and its overseas relatives

Trichoderma stromaticum, T. rossicum and newly discovered species form a unique lineage in Trichoderma. Phylogenetic and phenotypic diversity in Trichoderma stromaticum are examined in the light of reported differences in ecological parameters and AFLP patterns. Multilocus phylogenetic analysis using 4 genes (tef1, rbp2, cal, chi18-5) did not reveal phylogenetic basis for the two reported divergent AFLP patterns or for ecological parameters; however, this analysis does indicate incomplete speciation with one supported clade derived from within T. stromaticum that corresponds to AFLP Group 2 of de Souza et al. (2006, Phytopathology 96:61–67). Trichoderma stromaticum is known only from tropical America and is typically found in association with Theobroma cacao infected with Moniliophthora perniciosa. It is reported here for the first time on pseudostromata of M. roreri in Peru. Strains of T. stromaticum also have been isolated as endophytes from stems of Theo. cacao. There are no recognized close relatives of T. stromaticum in tropical America. The closest relatives of T. stromaticum are collected in Africa and Thailand; somewhat more distantly related are T. rossicum and T. barbatum, both found in north temperate regions.

Development of molecular markers based on retrotransposons for the analysis of genetic variability in Moniliophthora perniciosa

Moniliophthora perniciosa is a fungus that causes witches’ broom disease (WBD) in the cacao tree (Theobroma cacao). The M. perniciosa genome contains different transposable elements; this prompted an evaluation of the use of its retrotransposons as molecular markers for population studies. The inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) techniques were used to study the variability of 70 M. perniciosa isolates from different geographic origins and biotypes. A total of 43 loci was amplified. Cluster analysis of different geographical regions of C biotype revealed two large groups in the state of Bahia, Brazil. Techniques using retrotransposon-based molecular markers showed advantages over previously used molecular techniques for the study of genetic variability in M. perniciosa.

Aumento da eficiência nutricional de tomateiros inoculados com bactérias endofíticas promotoras de crescimento

Plant growth-promoting endophytic bacteria can increase plant nutritional efficiency thus favouring its yield. With the purpose of evaluating the influence of 10 previously selected isolates of growth-promoting endophytic bacteria on the uptake, utilization and transport of nutrients by tomato plants, greenhouse experiments were installed. The hypocotyl was cut in order to apply the endophytic bacteria to tomato seedlings cultivar Santa Clara. Fifty five days after transplanting the upper portion of the cut seedlings, the plants were collected to determine the dry matter of the aerial parts and concentration of macro and micro nutrients. The concentration of N, P, K, Ca, Mg, Cu and Zn in the shoot and N, P, Mg and Mn in roots of inoculated plants differed from non-inoculated controls. Endophytic bacteria Micrococcus sp. (UFLA 11-LS) and Brevundimonas sp. (UFV-E49) were identified by sequencing of the 16S ribosomal DNA. The P uptake in plants inoculated with these isolates was higher than in the non-inoculated controls. Plants treated with the first isolate were more efficient in the use of N, P, K, Ca, Mg, S, Cu, Fe, and Zn. The highest concentration of N, P, K, Mg, and Zn were found in the shoot of plants inoculated with Brevundimonas sp. The results of this study indicate that these endophytic bacteria isolates may be employed to increase the nutritional efficiency of tomato plants.

Diazotrophic bacteria associated with sisal (Agave sisalana Perrine ex Engelm): potential for plant growth promotion

AimsIn the semi-arid region of Bahia State, Brazil, sisal (Agave sisalana Perrine ex Engelm) has been cultivated for fibre production for several decades without the use of chemical fertilizers. The contribution of biological nitrogen fixation (BNF) to this crop and the occurrence of diazotrophic rhizosphere, epiphytic or endophytic bacteria have not been investigated. The aims of this work were to study the occurrence and diversity of diazotrophic bacteria in rhizosphere soil, root and leaf tissues of sisal plants from this region, as well as to test their potential for plant growth promotion.MethodsBurk’s nitrogen free semi-solid medium was used for isolation and the MPN method was used for quantification of diazotrophic bacteria. BOX-A1R PCR and 16S rRNA sequence analyses were performed to study the diversity of bacterial isolates harboring the nifH-gene. Six isolates were selected for growth promotion of cucumber plants.ResultsA high diversity of BNF bacterial isolates was observed in sisal plants and rhizosphere soil. Bacterial populations were higher in roots, followed by soil and sisal leaves. Burkholderia, Leifsonia and Paenibacillus were the predominant genera. All isolates tested were able to promote cucumber growth.ConclusionsThis study showed that sisal-associated diazotrophic bacteria are diverse and may be further exploited to promote plant growth.

Additional species of Aspergillus causing bole rot disease in Agave sisalana

The production of sisal in Bahia, Brazil, has been declining due to the occurrence of a disease know as bole rot. Aspergillus niger was regarded as the only causal agent. In this study A. brasiliensis and A. tubingensis, in addition to Aspergillus niger, identified on the basis of morphological and molecular analyses, were shown to cause bole rot of sisal. Their pathogenicity was confirmed but their significance for the epidemiology of the disease in the field remains unclear.

Screening of endophytic bacteria isolated from tomato plants as potencial biocontrol agents and growth promotion

Forty isolates of endophytic bacteria obtained from healthy tomato plants were tested for their potential as biocontrol agents of tomato diseases. A massal screening was performed at greenhouse using Pseudomonas syringae pv. tomato and Alternaria solani as challenging pathogens. Based on the average number of lesions per plant, four isolates were selected as potential agents of biocontrol of these tomato diseases caused by fungi and bacteria. These isolates were identified by 16S ribosomal DNA sequence analysis as Acinetobacter johnsonii (UFV-E05), Serratia marcescens (UFV-E13), Sinorhizobium sp. (UFV-E25) and Bacillus megaterium (UFV-E26). The four endophytes selected for biocontrol were also evaluated for their ability of promoting plant growth and only S. marcescens (UFV-E13) presented increase in the height of the plants.

Combination of endophytic bacteria and resistant cultivars improves control of Ralstonia wilt of tomato

The aim of this study was to evaluate the combined effect of endophytic bacteria and tomato cultivars with different resistance levels to Ralstonia solanacearum in the control of tomato bacterial wilt. Endophytic bacteria were successfully introduced into tomato seedlings by three methods: cutting of the hypocotyl, substrate drenching and seed microbiolization. One hundred and fifty endophytic bacterial isolates were screened in greenhouse assays. Two isolates, identified as Bacillus sp. and Serratia marcescens by 16S rDNA sequencing were the most promising. These isolates, when combined with tomato cultivars Drica, Caraíbe, Yoshimatsu, and Santa Clara, reduced the area under the disease progress curve (AUDPC) of bacterial wilt by 16 to 65%. Additionally, it was shown that antibiosis is not involved in the control of R. solanacearum by Bacillus sp. and S. marcescens. This study adds new isolates of endophytic bacteria as potential agents to control Ralstonia wilt in tomato and emphasizes the importance of combining these bacteria with host resistance in the management of the disease.

Rizobactérias no controle da mancha angular do algodoeiro

Avaliou-se o potencial de rizobacterias na inducao de resistencia do algodoeiro a Xanthomonas axonopodis pv. malvacearum. Apos o isolamento das rizobacterias, foram selecionados os isolados capazes de reduzir os sintomas da mancha angular bacteriana em casa de vegetacao, os quais foram aplicados espacialmente separados do patogeno desafiador. Os melhores isolados foram testados quanto a capacidade de reduzir os sintomas da ramulose e da murcha de Verticillium e de inibir diretamente os patogenos in vitro. Do total de 123 isolados de rizobacterias foram selecionados cinco, L2-1 (Bacillus cereus), MT5-6 (Bacillus cereus), L2-2 (Achromobacter xylosoxidans), MT5-5 (Bacillus cereus) e MT5-11 (Brevibacterium sp.), os quais apresentaram controle da mancha angular acima de 40%, em relacao a testemunha. Nenhum isolado reduziu a severidade da ramulose e da murcha de Verticillium em relacao a testemunha, nem apresentou efeito inibitorio direto in vitro a X. axonopodis pv. malvacearum e Colletotrichum gossypii var. cephalosporioides. Para V. dahliae, apenas o isolado L2-1 apresentou efeito inibitorio.

Occurrence of basil leaf spot caused by Pseudomonas cichorii in Bahia State, Brazil

73 Basil (Ocimum basilicum) belongs to the Lamiaceae family and is widely distributed in tropical areas. This herb has medicinal properties and is a source of essential oils (Burgess et al., 1986. http://fshs.org/ proceedings-o/1986-vol-99/249-251%20(BURGESS).pdf), but its main use is for culinary purposes. There are several diseases that may cause damage to basil. Fusarium wilt (Fusarium oxysporum f. sp. basilicum), gray mold (Botrytis cinerea), damping off or root rot (Rhizoctonia solani; Pythium spp.) and downy mildew (Peronospora belbahrii) are caused by fungi and Oomycetes (Tran, 2011. http://plantclinic. cornell.edu/factsheets/basildiseases.pdf). Leaf spot is caused by the bacterium Pseudomonas cichorii (Burgess et al., 1986. http://fshs.org/ proceedings-o/1986-vol-99/249-251%20(BURGESS).pdf). Of these diseases, only two have been reported for Brazil: Fusarium wilt was reported for the Federal District (Reis et al., 2007. http://www.scielo.br/ scielo.php?pid=S0100-54052007000200006&script=sci_arttext) and Pseudomomas cichorii for São Paulo State (Destéfano et al., Revista de Agricultura, v.78, n.1, p.37-48, 2003). Basil samples showing leaf spot symptoms were collected from a garden at Cruz das Almas (12° 39′ 11′′ S39° 7′ 19′′ W), Bahia State, in April 2013. At this time of the year, temperature is mild and humidity is high. Symptoms occurred on all aerial parts of infected plants, including leaves, petioles and stems. Spots were dark brown and had irregular shape and variable size. Isolations were performed on nutrient agar medium (NA), from where glossy, smooth and translucent bacterial colonies were obtained. One strain, named BR02, was chosen for subsequent characterization. A hypersensitivity test was performed on tobacco plants by inoculating leaves with a cell suspension (10 cells.mL). Pathogenicity tests were done by spraying healthy basil plants that were incubated for 48 h at 26°C in a moist chamber. Hypersensitive response (HR) was noticed at 24 h after injection (10 cells.mL) in tobacco leaves (Figure 1A). Typical leaf spots developed on inoculated plants in 36 h (Figure 1B), but not on non-inoculated control plants. The bacterium was re-isolated from diseased plants and was identical to the inoculated strain. For taxonomic characterization, a fragment of the small subunit of the ribosomal RNA (16S rRNA) of strain BR-2 was amplified and sequenced in an ABI PRISM 3100 sequencer (Life Technologies), Occurrence of basil leaf spot caused by Pseudomonas cichorii in Bahia State, Brazil