Histidine kinase two-component response regulators Ssk1, Skn7 and Rim15 differentially control growth, developmental and volatile organic compounds emissions as stress responses in Trichoderma atroviride.

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.

Altered physiology, cell structure, and gene expression of Theobroma cacao seedlings subjected to Cu toxicity.

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.

Growth variation among Bacillus thuringiensis strains can affect screening procedures for supernatant-secreted toxins against insect pests.

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.

Endophytic fungal diversity in Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) trees and their potential for growth promotion and biocontrol of black-pod disease.

The endophytic niches of plants are a rich source of microbes that can directly and indirectly promote plant protection, growth and development. The diversity of culturable endophytic fungi from stems and branches of Theobroma cacao (cacao) and Theobroma grandiflorum (cupuaçu) trees growing in the Amazon region of Brazil was assessed. The collection 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 cupuaçu plants were cultured in PDA under conditions inducing sporulation. Isolates were classified based upon the morphological characteristics of their cultures 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 ∼70% 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 Trichoderma (reference isolate), Pestalotiopsis, Curvularia, Tolypocladium and Fusarium showed the highest level of activity against the pathogen, and were further characterized. All demonstrated 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-promotion effects. Aspects related to endophytic biodiversity and host-pathogen-endophyte 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.

Rhythmic conidiation in the blue-light fungus Trichoderma pleuroticola.

Trichoderma species conidiate in response to blue light, however, unlike in the blue-light model fungus Neurospora crassa, conidiation in Trichoderma spp. has been considered to be non-circadian. In this study we uncovered evidence for circadian conidiation in Trichoderma pleuroticola and identified orthologues of the key N. crassa clock components, wc-1 (blr-1) and frq.

Genetic and Biological Diversity of Trichoderma stromaticum, a Mycoparasite of the Cacao Witches'-Broom Pathogen.

ABSTRACT 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.