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.
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.
Bud necrosis (BN) is a common disorder that affects Vitis vinifera L. and reduces its potential yield. To minimize the losses caused by BN, the double pruning management was applied in Brazilian ...Southeast vineyards. In this management strategy plants are pruned at the winter to promote a vegetative cycle and then, at summer, to promote the reproductive cycle at optimal environmental conditions. To investigate the relationship of BN and the double pruning management RNA-seq libraries were sequenced from healthy and necrotic tissues at four different stages of the year. The comparison of differentially expressed genes in necrotic and non-necrotic tissues showed an enhanced expression of genes related to cell death possibly induced by endophytic microorganisms in the necrotic tissues. The de novo assembly, characterization and quantification of transcripts within the RNA-seq libraries showed that genes from the endophytic fungus Alternaria alternata, responsible for the production of toxic compounds were highly expressed under BN. Here we propose a model in which unfavorable conditions and reduced carbohydrate levels in buds can promote the switch from a biotrophic lifestyle to a necrotrophic lifestyle in the endophytic fungi, which seems to be involved in the development of BN.
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.
Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacteria, designated CBAS 719
T
, CBAS 732 and CBAS 720 were isolated from leaf litter samples, collected in Espírito Santo State, ...Brazil, in 2008. Sequences of the 16S rRNA,
gyrB
,
lepA
and
recA
genes showed that these strains grouped with
Burkholderia plantarii
LMG 9035
T
,
Burkholderia gladioli
LMG 2216
T
and
Burkholderia glumae
LMG 2196
T
in a clade of phytopathogenic
Burkholderia
species. Digital DNA-DNA hybridization experiments and ANI analyses demonstrated that strain CBAS 719
T
represents a novel species in this lineage that is very closely related with
B. plantarii
. The genome sequence of the type strain is 7.57 Mbp and its G + C content is 69.01 mol%. The absence of growth on TSA medium supplemented with 3% (w/v) NaCl, citrate assimilation, β-galactosidase (PNPG) activity, and of lipase C14 activity differentiated strain CBAS 719
T
from
B. plantarii
LMG 9035
T
, its nearest phylogenetic neighbor. Its predominant fatty acid components were C
16:0
, C
18:1
ω7c, cyclo-C
17:0
and summed feature 3 (C
16:1
ω7c and/or C
15:0
iso
2-OH). Based on these genotypic and phenotypic characteristics, the strains CBAS 719
T
, CBAS 732 and CBAS 720 are classified in a novel
Burkholderia
species, for which the name
Burkholderia perseverans
sp. nov. is proposed. The type strain is CBAS 719
T
(= LMG 31557
T
= INN12
T
).
Serratia marcescens
are gram-negative bacteria found in several environmental niches, including the plant rhizosphere and patients in hospitals. Here, we present the genome of
Serratia marcescens
...strain N4–5 (=NRRL B-65519), which has a size of 5,074,473 bp (664-fold coverage) and contains 4840 protein coding genes, 21 RNA genes, and an average G + C content of 59.7%. N4–5 harbours a plasmid of 11,089 bp and 43.5% G + C content that encodes six unique CDS repeated 2.5× times totalling 13 CDS. Our genome assembly and manual curation uncovered the insertion of two extra copies of the 5S rRNA gene in the assembled sequence, which was confirmed by PCR and Sanger sequencing to be a misassembly. This artefact was subsequently removed from the final assembly. The occurrence of extra copies of the 5S rRNA gene was also observed in most complete genomes of
Serratia
spp. deposited in public databases in our comparative analysis. These elements, which also occur naturally, can easily be confused with true genetic variation. Efforts to discover and correct assembly artefacts should be made in order to generate genome sequences that represent the biological truth underlying the studied organism. We present the genome of N4–5 and discuss genes potentially involved in biological control activity against plant pathogens and also the possible mechanisms responsible for the artefact we observed in our initial assembly. This report raises awareness about the extra copies of the 5S rRNA gene in sequenced bacterial genomes as they may represent misassemblies and therefore should be verified experimentally.
Exploiting RNA interference (RNAi) in disease control through non-transformative methods that overcome the hurdle of producing transgenic plants has attracted much attention over the last years. ...Here, we explored such a method and used non-pathogenic bacteria as a versatile system for delivering RNAi to fungi. Specifically, the RNaseIII-null mutant strain of
HT115(DE3) was transformed with two plasmid vectors that enabled the constitutive or IPTG-inducible production of double-stranded RNAs (dsRNAs) against genes involved in aflatoxins production in
(
) or virulence of
(
). To facilitate the release of the dsRNAs, the bacterial cells were further genetically engineered to undergo a bacteriophage endolysin R-mediated autolysis, following a freeze-thaw cycle. Exposure under in vitro conditions of
or
to living bacteria or their whole-cell autolysates induced silencing of
and
in a bacteria concentration-dependent manner, and instigated a reduction in aflatoxins production and mycelial growth, respectively. In planta applications of the living bacteria or their crude whole-cell autolysates produced similar results, thus creating a basis for translational research. These results demonstrate that bacteria can produce biologically active dsRNA against target genes in fungi and that bacteria-mediated RNAi can be used to control fungal pathogens.
Intragenomic 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.
Total 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 corresponding
I-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 of
I bands' and 'type of eletropherogram'.
Two 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 the
I-restriction profiles indicated a high proportion of 49% of the culturable endophytes from a tropical environment showing evidence of intragenomic 16S rDNA heterogeneity.
The 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.
AIMS: In 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. METHODS: Burk’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. RESULTS: A 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. CONCLUSIONS: This study showed that sisal-associated diazotrophic bacteria are diverse and may be further exploited to promote plant growth.
The aim of this study was to develop a diagrammatic scale to evaluate black rot (Xanthomonas campestris pv. compestris) severity on kale (Brassica oleraceae var. acephala) leaves. The diagrammatic ...scale was developed and validated with eight levels of severity, ranging from 0.19 to 48.8%. More than 95% of the leaves collected from the field showed severity levels ranging from 0.1 to 21%, and 5% of the leaves showed severities higher than 22%. The validation of the scale was performed by 10 inexperienced evaluators, and the data were analysed with two methods: linear regression and Lin's statistics. Without the scale, most evaluators overestimated disease severity, whereas the use of the scale resulted in increased precision, accuracy, repeatability, and reproducibility of the estimates according to both validation methods. In conclusion, the proposed diagrammatic scale proved to be useful for assessments of black rot severity in kale leaves. The scale may be of interest to researches performing studies on epidemiology or breeding for resistance.
The leaf surface combines biochemical substances and pre-existing morphological structures, as well as the presence of microorganisms. This dynamic environment constitutes a plant's initial defense, ...as well as the first contact of phytopathogens during invasion. Spore germination starts on the phylloplane and is a fundamental process for fungal development, and hence the establishment of disease. In this review, we address the phylloplane's innate defense mechanisms and biochemical reactions involved in the early stage of phytopathogenic fungal development. The focus is present the pre-infection molecular and biochemical processes of the interaction between
Theobroma cacao
and
Moniliophthora perniciosa
, showing how the defense mechanisms of the phylloplane can act to inhibit proteins involved at the beginning of fungal spore germination. We conclude that the phylloplane of the cocoa resistant genotype to
M. perniciosa
has performed chemical compounds, pre-existing morphological structures and the presence of microorganisms that participate in the pre-infection defense of the plant. Also, the inhibition of proteins involved in the germination mechanism of
M. perniciosa
basidiospores by chemical and structural compounds present in the cocoa phylloplane may decrease the disease index. Therefore, understanding how the phylloplane defense acts in the fungal spore germination process is essential to develop pre-infection control strategies for cacao plants against witches' broom.
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