Phylogenetic Diversity of Trichoderma Strains and Their Antagonistic Potential against Soil-Borne Pathogens under Stress Conditions.

Trichoderma species are known as excellent biocontrol agents against soil-borne pathogens that cause considerable crop losses. Eight strains of Trichoderma were isolated from five Egyptian regions. They identified based on translation elongation factor-1α (TEF1) sequencing as four different Trichoderma species: Trichoderma asperellum, Trichoderma harzianum, Trichoderma viride, and Trichoderma longibrachiatum. Optimal growth conditions (temperature and media), and the phosphate solubilization capability of Trichoderma strains were evaluated in vitro. Further, the ability of these strains to antagonize Fusarium solani, Macrophomina phaseolina, and Fusarium graminearum was also evaluated. The results revealed that Trichoderma harzianum (Th6) exhibited the highest antagonistic ability against F. solani, M. phaseolina and F. graminearum with inhibition rates of 71.42%, 72.97%, and 84.61%, respectively. Trichoderma viride (Tv8) exhibited the lowest antagonism against the same pathogens with inhibition rates of 50%, 64% and 69.23%, respectively. Simple-sequence repeats (SSRs) and random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic variability of the Trichoderma strains. The results revealed that of 45 RAPD amplified bands, 36 bands (80%) were polymorphic and of SSRs amplified 36 bands, 31 bands (86.11%) were polymorphic. The amplification of calmodulin and ß-1,3-endoglucanase was noted at 500 bp and 230 bp, respectively. Data indicated that T. viride (Tv8) had the highest phosphate solubilization index (10.0 mm), while T. harzianum (Th6) had the lowest phosphate solubilization index (4.0 mm). In conclusion, T. harzianum (Th6) had the highest antagonistic activity in dual culture assay along with the growth rate; while T. viride (Tv8) had the highest phosphate solubilization activity. There are still gaps in obtaining new formulations, selecting potent Trichoderma strains to confirm disease control in planta. For improving Trichoderma recommendation in the organic agricultural system and sustaining the fertility of the soil, the field application of highly antagonistic biocontrol agents in different types of soil and plant species will be the first approach toward bio-pesticide treatments along with bio-fertilizer inoculation. Furthermore, secondary metabolites will be investigated for the most promising strains with the combination of different pathogens and application timing.

Genetic diversity and biocontrol efficacy of indigenous Trichoderma isolates against Fusarium wilt of pepper.

Trichoderma species are recognized as biocontrol agents with great potential in inhibiting fungal pathogens that cause significant crop losses. In this study, 15 Trichoderma isolates were collected from various Egyptian locations. Internal transcribed spacer sequencing revealed four different Trichoderma species; Trichoderma harzianum, Trichoderma asperellum, Trichoderma longibrachiatum, and Trichoderma viride. The antagonistic activity of Trichoderma isolates against Fusarium oxysporum f. sp. capsici was evaluated in vitro. The effect of Trichoderma isolates on pepper growth plants in the presence of F. oxysporum was studied in planta. The inhibition of pathogen mycelial growth in vitro ranged between 35.71% and 85.75%. The isolates Ta3 and Tl had the highest antagonistic ability in vitro against F. oxysporum f. sp. capsici. However, Th7 and Th6 of T. harzianum isolates showed the highest values of disease severity reduction under greenhouse conditions. The genetic diversity of the Trichoderma isolates (Ta1, Ta2, Th1, Th2, Th3, Th4, Th5, and Tv) was investigated on the basis of ISSR and SCoT markers. SCoT primers generated a total of 28 bands, out of which 14 (50%) were polymorphic. ISSR primers gave 32 bands, and 11 of these bands (34.37%) were polymorphic.

Isolation and molecular characterization of Frankia strains resistant to some heavy metals.

Frankia strains isolated from Saudi Arabia, reported for the first time, were identified based on the morphological and molecular tools compared to those isolated from Egypt. All strains displayed typical morphological characterization of Frankia strains represented by branched hyphae, production of vesicles and sporangia. The phylogenetic analysis and relationships among Frankia strains were investigated by comparing 16S rRNA gene sequences. The analysis revealed three genetic groups which formed two clusters. The first cluster was composed of eight Frankia strains subdivided into two genetic groups (one group containing five strains; CgIT3 L2 , CgIS3 N2 , CgIS1 N1, CgIT7N2, and G5; the other group included of three strains: CgIT5L3, CgIS1 N2 , and CcI13). The second cluster was composed of only one genetic group of Frankia strain CgIS3 N1 . The strains in each genetic group exhibited similar genetic distances. All Frankia strains were able to reinfect their host of Casuarina species. For ability of these strains to resist heavy metals, our results proved that all Frankia strains isolated can resist Cu, Co, and Zn at low concentration except Pb which exhibit highly toxic effect at the same concentration used. Frankia strain G5 was proved to be the most resistant strain for heavy metals tested.

Silencing of the chalcone synthase gene in Casuarina glauca highlights the important role of flavonoids during nodulation.

Nitrogen-fixing root nodulation is confined to four plant orders, including > 14,000 Leguminosae, one nonlegume genus Parasponia and c. 200 actinorhizal species that form symbioses with rhizobia and Frankia bacterial species, respectively. Flavonoids have been identified as plant signals and developmental regulators for nodulation in legumes and have long been hypothesized to play a critical role during actinorhizal nodulation. However, direct evidence of their involvement in actinorhizal symbiosis is lacking. Here, we used RNA interference to silence chalcone synthase, which is involved in the first committed step of the flavonoid biosynthetic pathway, in the actinorhizal tropical tree Casuarina glauca. Transformed flavonoid-deficient hairy roots were generated and used to study flavonoid accumulation and further nodulation. Knockdown of chalcone synthase expression reduced the level of specific flavonoids and resulted in severely impaired nodulation. Nodule formation was rescued by supplementing the plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of an important role for flavonoids during the early stages of actinorhizal nodulation.

The role of flavonoids in the establishment of plant roots endosymbioses with arbuscular mycorrhiza fungi, rhizobia and Frankia bacteria.

Flavonoids are a group of secondary metabolites derived from the phenylpropanoid pathway. They are ubiquitous in the plant kingdom and have many diverse functions including key roles at different levels of root endosymbioses. While there is a lot of information on the role of particular flavonoids in the Rhizobium-legume symbiosis, yet their exact role during the establishment of arbuscular mycorrhiza and actinorhizal symbioses still remains unclear. Within the context of the latest data suggesting a common symbiotic signaling pathway for both plant-fungal and plant bacterial endosymbioses between legumes and actinorhiza-forming fagales, this mini-review highlights some of the recent studies on the three major types of root endosymbioses. Implication of the molecular knowledge of endosymbioses signaling and genetic manipulation of flavonoid biosynthetic pathway on the development of strategies for the transfer and optimization of nodulation are also discussed.

Activation of the isoflavonoid pathway in actinorhizal symbioses.

We investigated the involvement of flavonoids in the actinorhizal nodulation process resulting from the interaction between the tropical tree Casuarina glauca Sieb. ex Spreng. and the actinomycete Frankia. Eight C. glauca genes involved in flavonoid biosynthesis: chalcone synthase (CHS), chalcone isomerase (CHI), isoflavone reductase (IFR), flavonoid-3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5' hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR) and flavonol synthase (FLS), were identified from a unigene database and gene expression patterns were monitored by quantitative real-time PCR (qRT-PCR) during the nodulation time course. Results showed that FLS and F3'5'H transcripts accumulated in mature nodules whereas CHI and IFR transcripts accumulated preferentially early after inoculation with Frankia. Comparison of IFR and CHI expression in inoculated plants and in control plants cultivated with or without nitrogen confirmed that early expression of IFR is specifically linked to symbiosis. Taken together, these data suggest for the first time that isoflavonoids are implicated in actinorhizal nodulation.