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Sisal is an economically and socially significant perennial crop for the semiarid region of the world. Bole rot disease, caused by black aspergilli, mainly by Aspergillus welwitschiae, is responsible for great losses due to the ability of the pathogen to kill the plant. The health status of plants that harbor endophytic biocontrol agents (BCAs) is being investigated in this study. We conducted experiments with endophytic bacteria from different parts of healthy and diseased sisal plants to select potential BCAs to control the disease. Studies on the populational densities showed that leaves have less bacteria when compared to roots and stems, and that diseased plants tended to have higher populations of bacterial endophytes. These results were obtained with both conventional plate counting and qPCR. A total of 497 isolates were screened as potential biocontrol agents on sisal discs and nine were selected as having direct antagonistic activity. Five isolates were from healthy and four from diseased plant parts. All these nine isolates significantly decreased the bole rot disease incidence in two field experiments and were identified by sequencing of the 16S rDNA as species belonging in the genera Bacillus, Brevibacterium, Burkholderia (2 isolates), Paenibacillus, Pseudomonas and Serratia (3 isolates). Isolate 466 of Burkholderia lowered 80% of disease incidence in both field experiments, which was not significantly different from the non-inoculated, negative control. We discussed these findings under an ecological-physiological interaction standpoint, as well as the possibilities of including diseased plants in bioprospection strategies and applying the selected isolates in the development of bioproducts.

BackgroundIntragenomic variability in 16S rDNA is a limiting factor for taxonomic and diversity characterization of Bacteria, and studies on its occurrence in natural/environmental populations are scarce. In this work, direct DNA amplicon sequencing coupled with frequent-cutter restriction analysis allowed detection of intragenomic 16S rDNA variation in culturable endophytic bacteria from cacao seeds in a fast and attractive manner.MethodsTotal genomic DNA from 65 bacterial strains was extracted and the 16S rDNA hyper variable V5–V9 regions were amplified for enzyme digestion and direct Sanger-type sequencing. The resulting electropherograms were visually inspected and compared to the correspondingAluI-restriction profiles, as well as to complete genome sequences in databases. Restriction analysis were employed to substitute the need of amplicon cloning and re-sequencing. A specifically improved polyacrylamide-gradient electrophoresis allowed to resolve 5-bp differences in restriction fragment sizes. Chi-square analysis on 2 × 2 contingency table tested for the independence between the ‘number ofAluI bands’ and ‘type of eletropherogram’.ResultsTwo types of electropherograms were obtained: unique template, with single peaks per base (clean chromatograms), and heterogeneous template, with various levels of multiple peaks per base (mixed chromatograms). Statistics revealed significant interaction between number of restriction fragments and type of electropherogram for the same amplicons: clean or mixed ones associated to ≤5 or ≥6 bands, respectively. The mixed-template pattern combined with theAluI-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity.ConclusionThe approach presented here was useful for a rapid, first-tier detection of intragenomic variation in culturable isolates, which can be applied in studies of other natural populations; a preliminary view of intragenomic heterogeneity levels can complement culture-dependent and -independent methods. Consequences of these findings in taxonomic and diversity studies in complex bacterial communities are discussed.

Made available in DSpace on 2022-04-28T17:20:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-10-18 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Objective Trichoderma species are found in soil and in association with plants. They can act directly or indirectly in the biological control of plant diseases and in the promotion of plant growth, being among the most used fungi in the formulation of bioproducts applied to agricultural systems. The main objective of this study was to characterize at a first-tier level a collection of 67 Trichoderma isolates from various tropical sources, based solely on sequencing of the internal transcribed spacer (ITS) region of the rRNA genes. Our goal was to provide a preliminary idea of the baseline diversity in this collection, to combine this information later with an array of other isolate-specific physiological data. This study provides a required knowledge at molecular level for assessment of this germplasm potential as a source of biotechnological products for beneficial effects in plants. Results Sequencing of the ITS region showed that the 67 Trichoderma isolates belonged in 11 species: T. asperellum, T. atroviride, T. brevicompactum, T. harzianum, T. koningiopsis, T. longibrachiatum, T. pleuroticola, T. reesei, T. spirale, T. stromaticum and T. virens. A total of 40.3% of the isolates were very closely related to each other and similar to T. harzianum. The baseline genetic diversity found indicates that the collection has different genotypes, which can be exploited further as a source of bioproducts, aiming at providing beneficial effects to plants of interest to cope with biotic and abiotic stresses. State Univ Sao Paulo USP, Ctr Nucl Energy Agr CENA, BR-13416000 Piracicaba, SP, Brazil State Univ Santa Cruz UESC, Dept Biol Sci DCB, BR-45662900 Ilheus, BA, Brazil Fed Univ Lavras UFLA, Dept Phytopathol DFP, BR-37200000 Lavras, MG, Brazil State Univ Sao Paulo USP, Ctr Nucl Energy Agr CENA, BR-13416000 Piracicaba, SP, Brazil CNPq: 402973/2012-7 CAPES: 001

In eukaryotic systems, membrane-bound NADPH oxidases (Nox) generate reactive oxygen species (ROS) as a part of normal physiological functions. In the soil-borne mycoparasitic and plant facultative symbiont Trichoderma atroviride, Nox1 and the regulator NoxR are involved in differentiation induced by mechanical damage, while the role of Nox2 has not been determined. The knock-out strains Δnox1, ΔnoxR and Δnox2 were compared to the parental strain (WT) in their ability to grow and conidiate under a series of stress conditions (osmotic, oxidative, membrane, and cell-wall stresses). All three genes were differentially involved in the stress-response phenotypes. In addition, several interactive experiments with biotic factors (plant seedlings and other fungi) were performed comparing the mutant phenotypes with the WT, which was used as the reference strain. Δnox1 and ΔnoxR significantly reduced the antagonistic activity of T. atroviride against Rhizoctonia solani and Sclerotinia sclerotiorum in direct confrontation assays, but Δnox2 showed similar activity to the WT. The Δnox1, ΔnoxR, and Δnox2 mutants showed quantitative differences in the emission of several volatile organic compounds (VOCs). The effects of a blend of these volatiles on plant-growth promotion of Arabidopsis thaliana seedlings were determined in closed-chamber experiments. The increase in root and shoot biomass induced by T. atroviride VOCs was significantly lowered by ΔnoxR and Δnox1, but not by Δnox2. In terms of fungistatic activity at a distance, Δnox2 had a significant reduction in this trait against R. solani and S. sclerotiorum, while fungistasis was highly increased by ΔnoxR and Δnox1. Identification and quantification of individual VOCs in the blends emitted by the strains was performed by GC-MS and the patterns of variation observed for individual volatiles, such as 6-Pentyl-2H-pyran-2-one (6PP-1) and (E)-6-Pent-1-enylpyran-2-one (6PP-2) were consistent with their negative effects in plant-growth promotion and positive effects in fungistasis at a distance. Nox1 and NoxR appear to have a ubiquitous regulatory role of in a variety of developmental and interactive processes in T. atroviride either as positive or negative modulators. Nox2 may also have a role in regulating production of VOCs with fungistatic activity.

Enterobacter and Bacillus are common endophytes in many plants. Endophytic Enterobacter cloacae 344 and Bacillus amyloliquefaciens 629 from cacao had their colonization patterns assessed in different plants and organs. The 344’s colonization in all plants was ~12x higher than those observed for 629. CFU countings were 16 and 3 times lower for 344 and 629, respectively, in non-sterile than in sterile conditions. The results suggested that 344 is a better endophytic colonizer than 629, but this seems a better competitor than 344. Indigenous endophytic fungi interfered with the colonization levels of both strains in all plants. Plant species, tissue and competing microbes appear to define the levels of endophytic colonization by these bacteria. Key words: Cacao, endophytic bacteria, population densities.

Background Subunits of ribosomal RNA genes (rDNAs) characterized by PCR-based protocols have been the proxy for studies in microbial taxonomy, phylogenetics, evolution and ecology. However, relevant factors have shown to interfere in the experimental outputs in a variety of systems. In this work, a ‘theoretical’ to ‘actual’ delta approach was applied to data on culturable mock bacterial communities (MBCs) to study the levels of losses in operational taxonomic units (OTUs) detectability. Computational and lab-bench strategies based on 16S rDNA amplification by 799F and U1492R primers were employed, using a fingerprinting method with highly improved detectability of fragments as a case-study tool. MBCs were of two major types: in silico MBCs, assembled with database-retrieved sequences, and in vitro MBCs, with AluI digestions of PCR data generated from culturable endophytes isolated from cacao trees. Results Interfering factors for the 16 s rDNA amplifications, such as the type of template, direct and nested PCR, proportion of chloroplast DNA from a tropical plant source (Virola officinalis), and biased-amplification by the primers resulted in altered bacterial 16S rDNA amplification, both on MBCs and V. officinalis leaf-extracted DNA. For the theoretical data, the maximum number of fragments for in silico and in vitro cuts were not significantly different from each other. Primers’ preferences for certain sequences were detected, depending on the MBCs’ composition prior to PCR. The results indicated overall losses from 2.3 up to 8.2 times in the number of OTUs detected from actual AluI digestions of MBCs when compared to in silico and in vitro theoretical data. Conclusions Due to all those effects, the final amplification profile of the bacterial community assembled was remarkably simplified when compared to the expected number of detectable fragments known to be present in the MBC. From these findings, the scope of hypotheses generation and conclusions from experiments based on PCR amplifications of bacterial communities was discussed. Electronic supplementary material The online version of this article (10.1186/s12866-019-1446-2) contains supplementary material, which is available to authorized users.

Bacillus thuringiensis (Bt) has been used successfully as a biopesticide for more than 60 years. More recently, genes encoding their toxins have been used to transform plants and other organisms. Despite the large amount of research on this bacterium, its true ecology is still a matter of debate, with two major viewpoints dominating: while some understand Bt as an insect pathogen, others see it as a saprophytic bacteria from soil. In this context, Bt's pathogenicity to other taxa and the possibility that insects may not be the primary targets of Bt are also ideas that further complicate this scenario. The existence of conflicting research results, the difficulty in developing broader ecological and genetics studies, and the great genetic plasticity of this species has cluttered a definitive concept. In this review, we gathered information on the aspects of Bt ecology that are often ignored, in the attempt to clarify the lifestyle, mechanisms of transmission and target host range of this bacterial species. As a result, we propose an integrated view to account for Bt ecology. Although Bt is indeed a pathogenic bacterium that possesses a broad arsenal for virulence and defense mechanisms, as well as a wide range of target hosts, this seems to be an adaptation to specific ecological changes acting on a versatile and cosmopolitan environmental bacterium. Bt pathogenicity and host-specificity was favored evolutionarily by increased populations of certain insect species (or other host animals), whose availability for colonization were mostly caused by anthropogenic activities. These have generated the conditions for ecological imbalances that favored dominance of specific populations of insects, arachnids, nematodes, etc., in certain areas, with narrower genetic backgrounds. These conditions provided the selective pressure for development of new hosts for pathogenic interactions, and so, host specificity of certain strains.

Repeated measures are widely used in a variety of aspects related to plant research, such as the assessment of genotypes subjected to stressing agents. Analysis of variance is routinely employed in split-plot experimental designs with repeated measurements on the subplots. However, this is not the best approach, as measurements cannot be randomly taken and so the H-F condition is rarely met. In this study, procedures of multivariate analysis of profiles (MAP) are presented as statistically advantageous options for solving similar analytical problems. We used data from trials with 10 clonal genotypes of cacao (Theobroma cacao) in which their seedlings’ resistance against ceratocystis wilt was assessed on 11 repeated measures that did not comply with the H-F condition. Multivariate analyses based on the disease-response profiles of all clones allowed us to understand the genotype-specific patterns of disease growth, identify when profiles reached the plateau, verify the levels of damage at this point, and a...

A maioria dos fungos tem a função de decompor moléculas orgânicas complexas possibilitando o aporte de nutrientes para o solo pelo processo de ciclagem e fertilização. Este estudo objetiva analisar a riqueza, freqüência, constância e a similaridade entre os fungos decompositores da serapilheira de três plantas endêmicas da Mata Atlântica do Sul da Bahia e em três áreas. Folhas em decomposição foram coletadas no período de dezembro/2007 a junho/2008, lavadas, colocadas em câmaras-úmidas e incubadas na temperatura ambiente. Obteve-se uma riqueza total de 52 espécies e 39 gêneros. A riqueza obtida nas áreas foi: Parque Estadual Serra do Conduru-PESC (78,8%), Ecoparque (71,2%) e Reserva Capitão (61,5%). Em relação às plantas, Harleyodendron unifoliolatum Cowan apresentou uma riqueza de 75,0%, Manilkara maxima Pennington 57,7% e Parinari alvimii Prance 42,3%. Houve similaridade fúngica entre as plantas e entre as áreas. A maioria dos fungos apresentou frequência esporádica e constância acidental. Este estudo, pioneiro na Mata Atlântica do Sul da Bahia e em relação às plantas estudadas, contribui para o conhecimento dos fungos anamorfos existentes neste bioma, riquíssimo em biodiversidade. Most fungi have the function of breaking down complex organic molecules, enabling the supply of nutrients to the soil through the process of cycling and fertilization. This paper aimed to analyze the richness, frequency, constancy and similarity among decomposing fungi on the litter of three endemic trees in three areas of the Atlantic Forest in southern Bahia. Decaying leaves were collected from December 2007 to June 2008, washed, placed in wet chambers and incubated at room temperature. Fifty-two fungal species belonging to 39 genera were found. The richness of each area was the following: Parque Estadual Serra do Conduru (78,8%), Ecoparque (71.2%) and Reserva Capitão (61.5%). In relation to plants, the richness of Harleyodendron unifoliolatum Cowan was 75.0%, Manilkara maxima Pennington 57.7% and Parinari alvimii Prance 42.3%. There was similarity between plants and between areas. The frequency of most fungi was sporadic. This pioneer work on these trees of the Atlantic Forest of southern Bahia contributes to the knowledge of the anamorphic fungi existing in this biologically rich biome.

BACKGROUND: Supernatant-secreted proteins in Bacillus thuringiensis (Berliner) (Bt) with insecticidal activity provide an important source of information for discovery of new useful strains and/or entomotoxins. However, physiological variation among isolates might interfere in the detection efficiency of screening procedures on Bt collections. The aim of this study was to assess the magnitude of this variation in a sample of isolates from a tropical Bt collection, which was gauged through the assessment of their temporal patterns of growth and protein secretion in culture supernatants (SNs), as well as of the corresponding toxicity against fall armyworm (Spodoptera frugiperda, JE Smith). Feeding bioassays were performed, with larvae being treated with heated and non-heated total protein extracted from SNs collected at different culture times. Larva mortality and reduction in pupa formation were observed. RESULTS: Intra- and interisolate variations were observed in the temporal patterns of growth, quality and quantity of protein secreted, as well as in insecticidal activity of these SNs, based on larvae mortality and pupation rates. These results suggest that the insecticidal potential of certain isolates can be hidden if comparisons are done on the basis of the same number of cells in the culture and/or the same culturing time. CONCLUSIONS: Methods of screening Bt collections on the basis of feeding bioassays can be misleading with regards to identifying more promising isolates for biocontrol purposes if physiological differences are not considered. The consequences and implications of these findings for the development of experimental systems that depend on toxicity bioassays to identify alternative Bt strains and entomotoxins with practical applicability have been discussed. Copyright © 2011 Society of Chemical Industry

The endophytic niches of plants are a rich source of microbes that can directly and indi- rectly promote plant protection, growth and development. The diversity of culturable en- dophytic fungi from stems and branches of Theobroma cacao (cacao) and Theobroma grandiflorum (cupuacu) trees growing in the Amazon region of Brazil was assessed. The col- lection of fungal endophytic isolates obtained was applied in field experiments to evaluate their potential as biocontrol agents against Phytophthora palmivora, the causal agent of the black-pod rot disease of cacao, one of the most important pathogens in cocoa-producing regions worldwide. The isolated endophytic fungi from 60 traditional, farmer-planted, healthy cacao and 10 cupuacu plants were cultured in PDA under conditions inducing spor- ulation. Isolates were classified based upon the morphological characteristics of their cul- tures and reproductive structures. Spore suspensions from a total of 103 isolates that could be classified at least up to genus level were tested against P. palmivora in pods attached to cacao trees in the field. Results indicated that w70 % of isolates showed biocontrol effects to a certain extent, suggesting that culturable endophytic fungal biodiversity in this system is of a mostly mutualistic type of interaction with the host. Eight isolates from genera Tri- choderma (reference isolate), Pestalotiopsis, Curvularia, Tolypocladium and Fusarium showed the highest level of activity against the pathogen, and were further characterized. All dem- onstrated their endophytic nature by colonizing axenic cacao plantlets, and confirmed their biocontrol activity on attached pods trials by showing significant decrease in disease severity in relation to the positive control. None, however, showed detectable growth-pro- motion effects. Aspects related to endophytic biodiversity and hostepathogeneendophyte interactions in the environment of this study were discussed on the context of developing sustainable strategies for biological control of black-pod rot of cacao.

The collective impact of several environmental factors on the biocontrol activity of Trichoderma stromaticum (Ts )a gainst Moniliophthora perniciosa (Mp), the cause of cacao witches’ broom disease, was assessed under field conditions of shaded cacao (Theobroma cacao) in south-eastern Bahia, Brazil. Biocontrol experiments were performed adjacent to an automated weather station, with sensors and Ts-treated brooms placed at different canopy heights. Sporulation occurred at the same dates for all Ts isolates, but in different quantities. Broom moisture >30%, air temperature of approximately 23 ± 3� C, relative humidity >90%, solar radiation intensities

Witches’ broom is the most devastating disease of cacao in Brazil, and losses to it entail serious socio-economical and environmental problems. Biological control of the causal agent Moniliophthora perniciosa ( Mp ) using the antagonistic fungus Trichoderma stromaticum ( Ts ) is promising, although the identification of superior isolates is necessary. Here, we report a study on the selection of more effective Ts isolates based on field experiments. Sixty-three Ts isolates from a local collection were applied on brooms and placed under typical conditions of shaded-cacao plantations in southeastern Bahia State (Brazil), during two periods of three months each. The percentages of Ts sporulation and incidence and severity of Mp were the parameters used for biocontrol assessments. The results from both experiments were very distinct, indicating a high phenotypic variation in this collection and suggesting a significant effect of the environment in the Ts – Mp interaction. Ts- sporulation rates were negatively correlated with the presence of Mp in the brooms and a number of isolates reduced Mp incidence more efficiently than the reference isolate. Contrasting isolates in their efficiency of reducing Mp incidence were selected and further tested in four subsequent field trials for validation purposes. The results partially confirmed their biocontrol phenotypes but also suggested isolate-specific responses to environmental variations. Inhibition of Mp -basidiospore germination by total protein secreted in culture supernatants of Ts isolates correlated well with field results and revealed a potentially useful procedure for pre-screening of large collections towards selection of better biological control isolates. The characteristics and efficiency of the method as a reliable protocol for identification of superior BCAs in the witches’ broom—cacao pathosystem is discussed.

The Black-Pod Rot of cacao (Theobroma cacao) caused by Phytophthora species is one of the most important diseases affecting this crop worldwide, with average annual losses above 30%. The aim of this work was to assess the potential for the endophytic Trichoderma martiale strain ALF 247 to control Phytophthora palmivora. When ALF 247 was applied at concentrations ranging from 1 × 104 to 5 × 107 conidia per milliliter, the disease severity decreased proportionally. Addition of vegetable oil and/or sucrose in the formulations did not affect the biocontrol results. Fungicides such as copper hydroxide and fosetyl-Al had no effect on conidial germination of this T. martiale strain, with the germination percentage maintained above 90%. Once sprayed, the population of T. martiale tended to decrease progressively on the surface of cacao pods (∼30–40 days post-application), with a concomitant increase in the severity of pod disease. Production of 1.02 × 108 conidia g−1 was obtained after culturing ALF 247 on solid substrate (rice grains) supplemented with calcium carbonate. The results indicate a clear-cut potential of the T. martiale ALF 247 to be used for control of Black-Pod Rot of cacao, although further studies are required to render this isolate technically and economically efficient as a biocontrol agent on agronomic scale.

Ceratocystis wilt of cacao (caused by Ceratocystis cacaofunesta) is a dangerous disease and results in the death of the plant. This fungus was recently identified in the major cacao-producing regions of Brazil, and was observed to be more aggressive than isolates from other geographical locations. The objective of this study was to develop and test a consistent method to assess cacao genotype response to C. cacaofunesta, based on young plants (seedlings or cuttings). The fungus was inoculated by the deposition of propagule suspensions on cut stems. The parameters to assess disease progress were (a) disease incidence, (b) differences in mortality between the most contrasting cacao genotypes for resistance and susceptibility, (c) disease index, (d) consistency of response over time and (e) relative lesion heights. When seedlings were used for the analyses, the ICS-1 and TSH-1188 genotypes proved to be useful as genetic standards for susceptibility and resistance to C. cacaofunesta, respectively. Inoculum concentrations between 104 and 105 propagules ml−1 and the moment at which the disease incidence stabilized provided appropriate conditions for genotypic comparison. When ten cacao genotypes propagated by cuttings (clones) were assessed, the results confirmed TSH-1188 as the reference genotype for resistance to C. cacaofunesta, while the remaining clones could be grouped as resistant (CEPEC-2008), moderately resistant (CEPEC-2002, CEPEC-2007) and susceptible (CEPEC-2009, CCN-10, CCN-51, HW-25, PH-16, SJ-02). The analytical concepts and results were discussed in terms of their application in breeding programmes aimed at developing genetic resistance to Ceratocystis wilt of cacao.

Chromium is a highly toxic heavy metal for microorganisms, animals and plants. Due to its widespread industrial use, it has become a serious pollutant in a diverse array of environments. Genipa americana L. (Rubiaceae) is a neotropical wood plant that shows adaptive capabilities to adverse conditions, such as soil flooding. We conducted an experiment under greenhouse conditions in order to test the hypothesis that G. americana can be used as a phytoremediator tree in Cr3+-contaminated watersheds. In this way, approximately 2-month-old seedlings were subjected to increased concentrations of Cr3+ in nutritive solution to analyse the effects of the metal on the leaf gas exchange, growth, carbon allocation and mineral composition in the above and belowground components of plant dry mass. The seedlings were submitted to seven increasing concentrations of Cr3+ in nutrient solution. At 34 days, leaf gas exchange parameters in the maximal tested dose of Cr3+ (30 mg L−1) remained below the control; the net values of photosynthetic rate (A) and stomatal conductance to water vapor (gs) were lowered approximately 80 and 90%, respectively. There were decreases of 36% in total leaf area (LA) between 0 and 30 mg L−1 Cr3+ and reductions in the roots, stems, leaves and total biomasses of 29, 39, 30 and 31%, respectively. The largest values of A to gs ratios were obtained for controls. The Cr3+ accumulated preferentially in the roots and low levels of its transport to the aerial parts was detected. Notwithstanding, Cu and Na concentrations in the seedlings increased with increment of Cr3+ in the nutrient solution. On the other hand, Fe and K contents presented a negative proportional increment to the increase of the concentration of Cr3+. There was little effect of Cr3+ on the contents of Mn, N and P in the seedlings. Despite some stress symptoms (root-apex necrosis, leaf chlorosis, and decreases in A and gs, growth and absorption of some mineral elements), the G. americana seedlings showed a great amount of Cr3+ uptake from the solution, immobilizing and storing it in high concentrations in the roots. Therefore, this woody species presents a great potential as a phytoremediator, mainly as a rhizofilterer and phytostabilizer of Cr3+.

The witches' broom disease caused by the fungus Crinipellis perniciosa is the main limiting factor for cocoa production in South America and the Caribbean. In Brazil, this disease affects almost all cocoa-growing regions, causing serious economic, social and ecological damage. The aim of this study was to map genomic regions associated with resistance to C. perniciosa using an F 2 population derived from a cross between 'Scavina-6' (resistant) and 'ICS-1' (susceptible). The phenotypic index was determined as the average number of vegetative witches' brooms per canopy area of each plant, the witches' brooms were counted and eliminated during six field evaluations between May 1998 and August 1999. A total of 124 random amplified polymorphic DNA (RAPD) and 69 amplified fragment length polymorphism (AFLP) markers were mapped along 25 linkage groups covering 1713 cM of cocoa genome. After employing single factor and composite interval mapping analyses, a major quantitative trait loci (QTL) flanked by the marker AV14.940 was identified in the linkage group 11, explaining almost 35% of the resistance to witches' broom. The present result suggests that this QTL acts as a major dominant component of resistance to this pathogen, with great potential for use in marker-assisted selection procedures in cocoa breeding programmes.

The objective of this study was to investigate the effect of aluminum (Al) on NO3 − and NH4 + uptake and on activity/expression of the N‐assimilating enzymes glutamine synthetase (GS1, GS2, and GSr isoforms, EC 6.3.1.2), glutamate synthase (NADH‐GOGAT, EC 1.4.7.14; ferredoxin‐GOGAT, EC 1.4.7.1), and glutamate dehydrogenase (GDH, EC 1.4.1.2) of maize (Zea mays L.) genotypes contrasting in tolerance to Al toxicity. Seeds were germinated on paper towels, screened for uniformity and allowed to grow for 5–6 d in a modified Steinberg nutrient solution, pH 5.5, in a glasshouse, under natural light. Immediately after transference to a growth chamber (12 h photoperiod, 26°C ± 0.5/18°C ± 0.9 day/night temperature, 72% RH and 540 µ Em−2 s−1 quantum flux density) Al stress was imposed by lowering pH to 4.5 and addition of 166 µmoles L−1 AlCl3. Control plants were grown in the same nutrient solution without Al, pH 4.5. Aluminum inhibited NO3 − and NH4 + uptake and decreased shoot dry matter yield in all genot...

Bioinsecticides with lower concentrations of endospores/crystals and without loss of efficiency are economically advantageous for pest biocontrol. In addition to Cry proteins, other Bacillus thuringiensis (Bt) toxins in culture supernatants (SN) have biocontrol potential (e.g., Vip3A, Cry1I, Sip1), whereas others are unwanted (β-exotoxins), as they display widespread toxicity across taxa. A strain simultaneously providing distinct toxin activities in crystals and SN would be desirable for bioinsecticides development; however, strains secreting β-exotoxins should be discarded, independently of other useful entomotoxins. Entomotoxicity of crystals and SN from a Brazilian Bt tolworthi strain (Btt01) was tested against Spodoptera frugiperda to assess the potential for biocontrol-product development based on more than one type of toxin/activity. Tests showed that 107 endospores mL−1 caused >80% of larvae mortality, suggesting Btt01 may be used in similar concentrations as those of other Bt-based biopesticides. When it was applied to cornfields, a significant 60% reduction of larvae infestation was observed. However, bioassays with Btt01 SN revealed a thermostable toxic activity. Physicochemical characterization strongly suggests the presence of unwanted β-exotoxins, with isolate-specific temporal variation in its secretion. Knowledge of the temporal pattern of secretion/activity in culture for all forms of toxins produced by a single strain is required to both detect useful activities and avoid the potential lack of identification of undesirable toxins. These findings are discussed in the contexts of commercial Bt product development, advantages of multiple-activity strains, and care and handling recommended for large-scale fermentation systems.

Hematoxylin staining is an early indicator of Aluminum (Al) toxicity effects on the apices of young, developing roots grown in nutrient solution. In this work, the potential of this technique as a reliable and reproducible phenotypic index for Al tolerance in tropical maize genotypes was assessed, with its performance systematically compared to two other parameters widely used in breeding programs – relative seminal-root length (RSRL) and net seminal-root length (NSRL). Seeding roots from contrasting genotypes for Al sensitivity stained remarkably different after 24- and 48-h and 7-day exposures to 222 μM Al in nutrient solution, with the Al-dye complex being detected in both the outer (epidermis) and inner (cortex) portions of the roots from the sensitive cultivar. Hematoxylin staining was compared to the RSRL and NSRL parameters using 20 families from the third generation of selfing (S3) following the cross between two contrasting inbred lines that had been previously classified by the RSRL index in an independent procedure. The coloration technique showed the highest capacity to discriminate among tolerant and sensitive genotypes and displayed significant correlation coefficients to the other two indexes. Evaluation of the results from diallel crosses involving nine inbred lines proved that hematoxylin staining was also particularly adequate for identifying expressive hybrid vigor, as demonstrated by the general (GCA) and specific (SCA) combining ability estimates obtained by using the three indexes simultaneously. Hence, hematoxylin staining of Al-stressed root apices appears to be a powerful tool to assist in Al-tolerance selection in tropical maize breeding programs.

A PCR-based strategy was employed to identify myb-related genes potentially involved in the differentiation and development of cotton seed trichomes. cDNA clones representing six newly identified cotton myb-domain genes (GhMYB) of the R2R3-MYB family were characterized in the allotetraploid species Gossypium hirsutum L. (2n = 4x = 52; AADD). Several interesting motifs and domains in the transregulatory region (TRR) were identified as potential candidates for modulating GhMYB activity. One such structural feature is a basic 40-amino acid stretch (TRR1) located immediately downstream of the DNA-binding domain (DBD) in five of the GhMYBs. Furthermore, the conserved motif GIDxxH identified in a subset of plant MYBs is also present in the same position in the TRR1 domains of GhMYB1 and GhMYB6, exactly 12 amino acid residues downstream of the last tryptophan in the R3 repeat of the DBD. At least two of the GhMYBs (GhMYB4 and GhMYB5) contain unidentified ORFS in the 5' leader sequence (5'-uORFs) that may serve to regulate the synthesis of these particular GhMYB proteins at the translational level. Multiple alignment of DBD sequences indicated that GhMYBs show structural similarity to plant R2R3-MYB factors implicated in phenylpropanoid biosynthesis. GhMYB5 is the most distantly related cotton R2R3-MYB and is found in an isolated cluster that includes the drought-inducible AtMYB2. Sequence comparisons of DBD and TRR domains from GhMYBs, MIXTA (AmMYBMx) and G11 (AtMYBG11) did not reveal any striking similarity beyond conserved motifs. However, based on earlier phylogenetic analysis, GhMYB2, GhMYB3, and GHMYB4 are members of a cluster that contains GLABROUS1, while GhMYB1 and GhMYB6 belong to a closely related cluster. Semi-quantitative RT-PCR analysis revealed two discrete patterns of GhMYB gene expression. Type I cotton MYB (GhMYB-1, -2, and -3) transcripts were found in all tissue-types examined and were relatively more abundant than those derived from type II GhMYB genes (GhMYB-4, -5, and -6), which showed distinct, tissue-specific expression patterns. The developmental regulation of GhMYBs is consistent with a role for these DNA-binding factors in the differentiation and expansion of cotton seed trichomes.

Since its first agronomical use in 1938, Bacillus thuringiensis (Berliner) (Bt) has been the major representative of microbes with a fruitful biocontrol application in agriculture. Its bioproducts – based on spores/entomotoxins – comprise ∼1.8% of today's world market of agrochemicals [1]. Despite the vast knowledge about mechanisms of insecticidal action and specificity of Bt, its largely unresolved ecology has been a matter of hot debate due to its occurrence in an astounding variety of niches.

Structural analysis of hmg-coA reductase (hmgr) genes in the allotetraploid cotton species Gossypium hirsutum L. revealed the first-known existence of a pseudogene, psihmg5, for this important enzyme. Complete sequencing of the genomic clone hmg5 unveiled several deleterious lesions, resulting in an organization that departed significantly from the linear canonical hmgr gene structure. Although analysis of the 5' flanking region indicated a promoter-like composition based on comparison with other known plant hmgr genes, the precise loss of intron 3, and putative poly-(A) signals, small poly-(A) tracts, and terminal repeats (TRs) found in the 3'-flanking region are characteristic features of retro-pseudogenes. DNA-blot analysis indicated that a psihmg5-related subfamily exists within a larger hmgr gene family in cotton. Several mechanisms are proposed to account for the formation of this partially intronless pseudogene, including intragenic homologous-replacement recombination and gene conversion involving a cDNA. Alignments of psihmg5 with functional cotton homologs also raised interesting possibilities for the formation of 'chimeric' gene structures, or differential intragenic mutation rates, as potential evolutionary mechanisms involved in shaping the hmgr gene family in cotton.

Seedlings of Theobroma cacao CCN 51 genotype were grown under greenhouse conditions and exposed to increasing concentrations of Cu (0.005, 1, 2, 4, 8, 16, and 32 mg Cu L(-1)) in nutrient solution. When doses were equal or higher than 8 mg Cu L(-1), after 24 h of treatment application, leaf gas exchange was highly affected and changes in chloroplasts thylakoids of leaf mesophyll cells and plasmolysis of cells from the root cortical region were observed. In addition, cell membranes of roots and leaves were damaged. In leaves, 96 h after treatments started, increases in the percentage of electrolyte leakage through membranes were observed with increases of Cu in the nutrient solution. Moreover, there was an increase in the concentration of thiobarbituric acid-reactive substances in roots due to lipid peroxidation of membranes. Chemical analysis showed that increases in Cu concentrations in vegetative organs of T. cacao increased with the increase of the metal in the nutrient solution, but there was a greater accumulation of Cu in roots than in shoots. The excess of Cu interfered in the levels of Mn, Zn, Fe, Mg, K, and Ca in different organs of T. cacao. Analysis of gene expression via RTq-PCR showed increased levels of MT2b, SODCyt, and PER-1 expression in roots and of MT2b, PSBA, PSBO, SODCyt, and SODChI in leaves. Hence, it was concluded that Cu in nutrient solution at doses equal or above 8 mg L(-1) significantly affected leaf gas exchange, cell ultrastructure, and transport of mineral nutrients in seedlings of this T. cacao genotype.

Clonal genotypes resistant to fungal diseases are an important component of the cocoa production system in southeastern Bahia state (Brazil), so that technologies for faster production of stronger and healthier plantlets are highly desirable. In this study, the effects of inoculated bacterial endophytes isolated from healthy adult cacao plants on seedlings, and aspects related to inoculation methods, colonization patterns, and photosynthesis were investigated. Sequencing of 16S rRNA, hsp-60, and rpo-B genes placed the wild-type isolates within the species Enterobacter cloacae (isolates 341 and 344) and Bacillus subtilis (isolate 629). Spontaneous rifampicin-resistant (rif(R)) variants for 344 were also produced and tested. Endophytic application was either by immersion of surface sterilized seeds in bacterial suspensions or direct inoculation into soil, 20 days after planting non-inoculated seeds into pots. Results from in vitro recovery of inoculated isolates showed that the wild-type endophytes and rif(R) variants systemically colonized the entire cacao seedlings in 15-20 days, regardless of the inoculation method. Some endophytic treatments showed significant increases in seedlings' height, number of leaves, and dry matter. Inoculation methods affected the combined application of endophytes, which maintained the growth-promotion effects, but not in the same manner as in single applications. Interestingly, the 344-3.2 rif(R) variant showed improved performance in relation to both the wild type and another related variant. Photosynthetic rates and stomatal conductance increased significantly for some endophytic treatments, being partially associated with effects on growth and affected by the inoculation method. The results suggest that E. cloacae and B. subtilis endophytes from healthy adult plants (not transmitted by seeds) were able to promote vegetative growth on cacao seedlings. The development of products for large-scale use in seedlings/plantlets production systems was discussed.

In soil, anoxia conditions generated by waterlogging induce changes in genetic, morphological and physiological processes, altering the growth and development of plants. Mass propagation of cacao (Theobroma cacao L.) plantlets (clones) is affected by waterlogging caused by heavy rains and irrigation methods used to induce rooting. An experiment was undertaken to assess the effects of a 45-day flooding (anoxia) on physiological and morphological traits of 35 elite cacao genotypes, aiming at potentially identifying those with greater tolerance to flooding of the growth substrate. Eighteen fluorochrome-labeled microsatellite (SSR) primer pairs were used to assess genetic variability among clones, with 248 alleles being amplified and used to calculate similarity coefficients. The resulting dendrogram indicated the presence of four major groups, in which two represented 60% and 31% of the genotypes tested. A general trend toward high levels of heterozygosity was also found for physiological and morphological traits. The survival index (IS) for flood tolerance observed varied from 30 to 96%. Clones TSA-654, TSA-656, TSA-792, CA-1.4, CEPEC-2009 and PH-17 showed an IS value above 94%, whereas CEPEC-2010, CEPEC-2002, CA-7.1 and VB-903 clones were those mostly affected by waterlogging, with IS value below 56%. All genotypes displayed lenticel and adventitious root formation in response to waterlogging, although with different intensities. To determine whether patterns of physiological response could be associated with tolerance to anoxia, a similarity-grouping analysis was performed using the ratio between waterlogged and control values obtained for a series of physiological variables assessed. No specific pattern of physiological and morphological responses to waterlogging was strictly associated with survival of plantlets. However, results revealed by the dendrogram suggest that absence of leaf chlorosis may be a proper trait to indicate cacao clones with higher survival rates under flooding conditions. Consequences of these findings are discussed in the context of developing improved strategies for mass production of clones from elite cacao genotypes.

de Souza, J. T., Pomella, A. W. V., Bowers, J. H., Pirovani, C. P., Loguercio, L. L., and Hebbar, K. P. 2006. Genetic and biological diversity of Trichoderma stromaticum, a mycoparasite of the cacao witches’-broom pathogen. Phytopathology 96:61-67. The witches’-broom disease, caused by the basidiomycete Crinipellis perniciosa, is the most limiting factor for cacao cultivation in Brazil. Trichoderma stromaticum is a mycoparasite of the witches’-broom pathogen of cacao that is currently being applied in the field to manage the disease in Bahia State, Brazil. In this work, molecular and traditional methods were used to study the genetic and biological diversity of this mycoparasite. Ninety-one isolates, mostly collected from farms not sprayed with the fungus, were analyzed by amplified fragment length polymorphisms (AFLP), which showed that two genetic groups (I and II) of T. stromaticum occur in Bahia State. This classification of T. stromaticum into two distinct AFLP groups was also in agreement with several other characteristics, including growth on agar media at different temperatures and sporulation on infected stem segments (broom pieces) and rice grains. Group II favors higher temperatures compared with group I. The genetic and biological differences of the isolates, however, were not evident in field experiments, where sporulation was evaluated on the surface of brooms under natural conditions. Our results show that there is considerable genetic and biological diversity within T. stromaticum in Bahia and other cacao-growing regions of South America that are affected by the witches’-broom disease. This diversity could be explored in the development of efficient biological control agents against the disease. Factors that may affect the application and performance of this biocontrol agent in the field, such as sporulation on rice substrate and on the brooms and growth at various temperatures, are discussed.

Novel vegetative insecticidal proteins (Vips) identified in the supernatant of Bacillus thuringiensis (B.t.) cultures have shown to provide adequate control over a wide spectrum of economically important crop pests. To evaluate the potential applicability of these proteins against fall armyworm (Spodoptera frugiperda Smith) larvae, the most important insect pest for tropical maize, the characteristics and mortality effects of culture supernatants from five B.t. strains were investigated. Striking differences among strains were detected, not only in terms of efficiency in killing the insect, but also regarding to mortality effects of heated and non-heated supernatants, which were used to distinguish the heat-sensitive protein-derived insecticidal fraction from a thermostable one, with a non-protein nature (b-exotoxinas). The qualitative, quantitative and temporal patterns of total protein secretion in the medium (supernatant) were assessed through spectrophotometry and polyacrylamide gel electrophoresis. The strains showed remarkably distinct rates of growth and timing for protein secretion relative to cell density in culture. Moreover, the electrophoretic-banding patterns also varied in a strain-specific manner, both in denaturing and non denaturing conditions. Polypeptides displaying a molecular weight that is very close to the expected for previously identified Vip3A proteins were found for the strains with high supernatant-mortality ratios. The data suggest the feasibility and usefulness of searching for protein-derived (Vip-like) insecticidal fractions in B.t. supernatants as a mean of developing especific and efficient alternatives of biological control to be employed in integrated pest management programs of S. frugiperda in tropical maize. Recentemente, proteínas inseticidas vegetativas (Vips), identificadas no sobrenadante da cultura de certas cepas de Bacillus thuringiensis (B.t.), foram capazes de controlar alguns insetos-praga economicamente importantes. Como avaliação do potencial de aplicabilidade destas proteínas contra a lagarta-do-cartucho (Spodoptera frugiperda Smith), o inseto-praga mais importante da cultura do milho, as características e o efeito de mortalidade do sobrenadante da cultura de cinco cepas de B.t. foram investigadas. Notáveis diferenças entre as cepas foram detectadas, não somente em termos de eficiência na mortalidade dos insetos, mas também nas diferenças de mortalidade entre os sobrenadantes aquecido e não-aquecido, tratamentos estes que foram usados para distinguir a fração protéica inseticida termo-sensível de outra fração termo-estável, de natureza não-protéica (b-exotoxinas). Os padrões qualitativo, quantitativo e temporal de secreção da proteína total no meio (sobrenadante) foram avaliados por espectrofotometria e eletroforese em gel de poliacrilamida. As cepas apresentaram distintas taxas de crescimento e tempos de secreção de proteínas em relação a densidade de células na cultura. Além disso, o padrão de bandas eletroforéticas também variou de maneira cepa-específica, em ambas condições desnaturantes e não-desnaturantes. Polipeptídeos que apresentaram peso molecular semelhantes aos esperados para proteínas Vip3A identificadas anteriormente foram encontrados em cepas com alta taxa de mortalidade do sobrenadante. Os dados sugerem a possibilidade e a utilidade de pesquisar frações inseticidas de natureza protéica (do tipo Vip) em sobrenadantes de B.t., como forma para desenvolver alternativas específicas e eficientes de controle biológico que possam ser empregadas em programas de manejo integrado de S. frugiperda em milho tropical.

As a first step towards understanding the biosynthesis of isoprenoids that accumulate in specialized pigment glands of cotton at the molecular level, two full-length genes (hmg1 and hmg2) were characterized encoding hmg-coA reductase (HMGR; EC 1.1.1.34), the enzyme that catalyzes the formation of a key isoprenoid precursor. Cotton hmgr genes exhibited features typical of other plant genes, however, hmg2 encodes the largest of all plant HMGR enzymes described to date. HMG2 contains several novel features that may represent functional specialization of this particular HMGR isoform. Such features include a unique 42 amino acid sequence located in the region separating the N-terminal domain and C-terminal catalytic domain, as well as an N-terminal hydrophobic domain that is not found in HMG1 or other HMGR enzymes. DNA blot analysis revealed that hmg1 and hmg2 belong to small subfamilies that probably include homeologous loci in allotetraploid cotton (Gossypium hirsutum L.). Ribonuclease protection assays revealed that hmg1 and hmg2 are differentially expressed in a developmentally- and spatially-modulated manner during morphogenesis of specialized terpenoid-containing pigment glands in embryos. Induced expression of hmg2 coincided with a possible commitment to sesquiterpenoid biosynthesis in developing embryos, although other developmental processes also requiring HMGR cannot be excluded.

Background The presence of organic sulfur-containing compounds in the environment is harmful to animals and human health. The combustion of these compounds in fossil fuels tends to release sulfur dioxide in the atmosphere, which leads to acid rain, corrosion, damage to crops, and an array of other problems. The process of biodesulfurization rationally exploits the ability of certain microorganisms in the removal of sulfur prior to fuel burning, without loss of calorific value. In this sense, we hypothesized that bacterial isolates from tropical landfarm soils can demonstrate the ability to degrade dibenzothiophene (DBT), the major sulfur-containing compound present in fuels. Results Nine bacterial isolates previously obtained from a tropical landfarm soil were tested for their ability to degrade dibenzothiophene (DBT). An isolate labeled as RR-3 has shown the best performance and was further characterized in the present study. Based on physiological aspects and 16 s rDNA sequencing, this isolate was found to be very closely related to the Bacillus pumillus species. During its growth, high levels of DBT were removed in the first 24 hours, and a rapid DBT degradation within the first hour of incubation was observed when resting cells were used. Detection of 2-hydroxybiphenyl (HBP), a marker for the 4S pathway, suggests this strain has metabolical capability for DBT desulfurization. The presence of MgSO 4 in growth medium as an additional sulfur source has interfered with DBT degradation. Conclusions To our knowledge, this is the first study showing that a Bacillus strain can metabolize DBT via the 4S pathway. However, further evidences suggest RR-3 can also use DBT (and/or its derivative metabolites) as carbon/sulfur source through another type of metabolism. Compared to other reported DBT-degrading strains, the RR-3 isolate showed the highest capacity for DBT degradation ever described in quantitative terms. The potential application of this isolate for the biodesulfurization of this sulfur-containing compound in fuels prior to combustion was discussed.