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
, 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 Δ
, Δ
and Δ
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. Δ
and Δ
significantly reduced the antagonistic activity of
against
and
in direct confrontation assays, but Δ
showed similar activity to the WT. The Δ
, Δ
, and Δ
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
seedlings were determined in closed-chamber experiments. The increase in root and shoot biomass induced by
VOCs was significantly lowered by Δ
and Δ
, but not by Δ
In terms of fungistatic activity at a distance, Δ
had a significant reduction in this trait against
and
, while fungistasis was highly increased by Δ
and Δ
. 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
either as positive or negative modulators. Nox2 may also have a role in regulating production of VOCs with fungistatic activity.
•Ssk1 may function as a hub-like RR in T. atroviride, as growth, and differentiation (conidiation).•Skn7 has a significant role in the production of volatile organic compounds.•Ssk1 and Rim15 have a ...role in mycoparasitism and both are negative regulators of volatile organic compounds. Rim15 seems to be a negative regulator of either the accumulation or biosynthesis of terpenes.
The Skn7, Ssk1 and Rim15 proteins are response regulators involved in osmotic, oxidative and nutritional stress in fungi. In order to verify the involvement of these genes in Trichoderma atroviride IMI206040’s growth, conidiation, direct antagonism against plant pathogens (Rhizoctonia solani and Sclerotinia sclerotiorum), production of volatile organic compounds (VOCs) with fungistatic effect, and interaction with plants (growth promotion), single mutants were generated, and the phenotypic patterns were analysed in comparison to the wild-type (wt) strain. The mutants were submitted to osmotic, oxidative, membrane and cell wall stress conditions in vitro. The Δskn7 and Δrim15 mutants did not show either significant differences at morphological level, or marked decreases in mycelial growth and conidiation in relation to wt, whereas Δssk1 had altered phenotypes in most conditions tested. The plant-growth promotion of Arabidopsis thaliana seedlings induced by VOCs was not quantitatively modified by any of the mutants in relation to the wt strain, although possible differences in secondary root hairs was noticed for Δrim15. The fungistatic activity was significantly altered for Δssk1 and Δrim15. Overall, the Δssk1 strain showed remarkable morphological differences, with decrease in mycelial growth and conidiation, being also affected in the antagonistic capacity against plant pathogens. The impacts demonstrated by the deletion of ssk1 suggest this gene has a relevant participation in the signalling response to different stresses in T. atroviride and in the interactive metabolism with phytopathogens and plants. On the other hand, unlike other fungal models, Skn7 did not appear to have a critical participation in the above-mentioned processes; Rim15 seemed to confirm its involvement in modulating cellular responses to nutritional status, although with a possible cross-talk with other cellular processes. Our results suggest that Ssk1 likely plays a key regulatory role, not only in basic metabolisms of T. atroviride, but also in biocontrol-related characteristics.
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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.
Bacillus thuringiensis (Bt) is known for the biological control of important insect pests, but scientific advances have revealed several interesting characteristics, in addition to this classical ...function as a bioinsecticide. To investigate the current knowledge about these non-insecticidal activities, a systematic research on primary data in the scientific literature was conducted on alternative functions of Bt with biotechnological potential. Out of a total of 140 articles selected, 15 non-insecticidal Bt activities were found. Publications related to this topic are available since 1971, and different metadata were reported, such as biomolecules and genes involved in Bt performances in non-insecticidal bioactivities. A total of 11 Bt activities with different effect measures (response variables) were identified, with an average of 48 distinct Bt strains evaluated per activity. Approximately 81.2% of all identified experiments/tests deal with the direct effects of Bt on target cells/organisms, with 36.3% of the strains within these studies tested for antibacterial action; of all microbial targets tested, 92.8% are bacteria, which led to 75.2% of the experimental conditions for all direct activities being performed in vitro. Regarding indirect Bt activities, 67.6% of these studies reported tritrophic Bt-plant-pathogen interactions. Bioremediation also appears as a relevant Bt activity being investigated in-depth. Alternative Bt activities offer innovative ways of developing biotechnology for different areas of anthropic interest; hence, we also focus on the possibility of finding multifunctional strains of Bt, as this may be advantageous from a bioeconomic point of view. Our findings are discussed in terms of research trends, aspects, details and depth of the current knowledge on alternative non-insecticidal Bt traits. We also discuss the potential application of this science for useful technological developments, aiming at solving issues related to human health, sustainable agriculture and environmental preservation/restoration.
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•Bacillus thuringiensis isolates offer the possibility of valuable multifunctional bioproducts for a bioeconomy world;•The non-insecticidal action of Bt can be divided into direct and indirect activities, with the latter related to plant hosts;•The most frequent non-insecticidal activities of Bt have been related to antimicrobial and bio/phytoremediation activities;•Relevant findings are related to Bt activities against cancer cells;•Multifunctional Bt isolates can include insecticidal and non-insecticidal actions simultaneously.
•E. nigrum reduced the rice blast disease incidence and severity significantly.•E. nigrum was able to improve the numbers of tillers, heart, and rice productivity.•E. nigrum colonized rice plant ...tissues confirming its endophytic ability.•E. nigrum could be use as novel biofertilizer and bio-fungicide against P. oryzae.
Rice (Oryza sativa L.) feeds around half of humanity mainly in Asia, South America, and Africa. The filamentous, ascomycete fungus Pyricularia oryzae Cavara (syn: Magnaporthe oryzae B. Couch) hemibiotrophic phytopathogen is the causal agent of the most devastating disease, blast on rice. The fungus E. nigrum is ubiquitous resistant in adverse conditions and used as a biological control agent (BCA) against numerous phytopathogenic fungi in the function of its ability to produce many secondary metabolites. This study aimed to evaluate the efficiency of an isolate of E. nigrum on rice blast disease suppression, as well as rice growth promotion and productivity of the cultivar BRS.MG Caçula, in pot assay under greenhouse conditions, and evaluate the interaction between plants and microorganisms, using microscopic approaching. Rice leaf blast disease incidence and severity were evaluated on seeds and plants, which were treated with E. nigrum and P. oryzae isolate IA25, and compared with the control treatment. Foliar spraying and coating of the rice seed with E. nigrum significantly reduced the incidence rate of rice blast from 31.25% to 41.76%, respectively, compared to the control treatment. Likewise, the severity rate was reduced from 34.92% to 54.51%. In-plant growth, it was found that seed-soaking with Epicoccum provided plant fitness and increases the number of tillers to 8.34 against 5.95 for the control situation. The Epifluorescence Microscopy (EFM) and Scanning Electron Microscopy (SEM) showed the colonization of rice root by endophytic fungus E. nigrum. The greenhouse experiments confirmed that E. nigrum significantly decreased rice leaf blast disease incidence and severity rate while improving plant tillering.
Plant–microbe interactions are critical for the sustainability of agricultural production. In this study, our aims were to characterize the genetic and functional diversity of the culturable ...bacterial community associated with the cacao rhizosphere and access their potential for growth promotion of cacao seedling. Culture-dependent and molecular methods were used to characterize the population densities and diversity of bacterial communities from soil and cacao plants at two locations and two plant ages. A total of 63 strains were identified through
hsp60
sequencing.
Pseudomonas
and
Enterobacter
were the most abundant genera in association with the cacao rhizosphere, whereas
Bacillus
was more numerous in soil. Parameters of seedling growth promotion were evaluated 60 days after inoculation of seeds, with partition of the assessments into root and shoot weight. Each isolate showed beneficial, neutral or deleterious effects on plant growth, depending on the isolate and on the parts of plant assessed. Interestingly, although an apparent overall decrease in total biomass of seedlings (roots + shoots dry matters) was observed for the majority of isolates (89%), 94% of all isolates, in fact, revealed an increase in plant roots/shoots dry biomass ratio. Despite that part of the isolates (35%) appeared to significantly decrease plant height, and that 65% did not influence plant height (neutral effect), 18 had significantly increased root dry biomass; nevertheless, seven of these root growth-increasing isolates simultaneously decreased shoots-related growth parameters. The results of this study evidentiated the functional diversity of culturable cacao rhizobacteria and how the partitioning of roots and shoots in the assessment of plant growth parameters could reveal the biotechnological potential of these isolates for promoting growth of clones for rehabilitation of commercial cacao plantations.
Graphical Abstract
Key Points
• The most common culturable bacteria in cacao roots were Pseudomonas and Enterobacter
• Most culturable bacteria from cacao roots increased the root/shoot ratio
• Roots and shoots should be examined separately to detect cacao beneficial bacteria
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•Diseased plants tend to show higher population densities of bacterial endophytes.•No association was found between plant health status and bacterial diversity.•Less than 2% of the ...endophytes showed direct antagonism against the pathogen in vitro.•The efficacy of the biocontrol agents is independent of the health status of the host plant.
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.
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.
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