Solid-State Fermentation of Trichoderma spp.: A New Way to Valorize the Agricultural Digestate and Produce Value-Added Bioproducts.

In this study, the agricultural digestate from anaerobic biogas production mixed with food wastes was used as a substrate to grow Trichoderma reesei RUT-C30 and Trichoderma atroviride Ta13 in solid-state fermentation (SSF) and produce high-value bioproducts, such as bioactive molecules to be used as ingredients for biostimulants. The Trichoderma spp. reached their maximum growth after 6 and 3 SSF days, respectively. Both Trichoderma species were able to produce cellulase, esterase, and citric and malic acids, while T. atroviride also produced gibberellins and oxylipins as shown by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) profiling. Experimental evaluation of germination parameters highlighted a significant promotion of tomato seed germination and root elongation induced by T. atroviride crude extracts from SSF. This study suggests an innovative sustainable use of the whole digestate mixed with agro-food waste as a valuable substrate in fungal biorefineries. Here, it has been applied to produce plant growth-promoting fungi and bioactive molecules for sustainable agriculture.

Diversity of Nematode Microbial Antagonists from Algeria Shows Occurrence of Nematotoxic Trichoderma spp.

Fungi and bacteria associated to phytoparasitic nematodes Globodera rostochiensis and Meloidogyne spp. in Algeria were identified and characterized. Trichoderma spp. showed the highest prevalence in the cysts of G. rostochiensis. A number of isolates were identified through PCR amplification and the sequencing of the internal transcribed spacer (ITS)1-2 and Rpb2 gene regions. The most represented species were T. harzianum and T. afroharzianum. The latter and T. hirsutum were reported for the first time in Algeria. Fusarium spp., including F. oxysporum and F. solani, comprised a second group of fungi found in cysts. Taxa associated to females of Meloidogyne spp. included T. harzianum, Fusarium spp. and other hyphomycetes. To assess the efficacy of Trichoderma spp., two assays were carried out in vitro with the culture filtrates of two T. afroharzianum and T. harzianum isolates, to check their toxicity versus the second stage juveniles of M. incognita. After 24-48 h exposure, a mortality significantly higher than the control was observed for both filtrates at 1% dilutions. The TRI genes involved in the production of trichothecenes were also amplified with the PCR from some Trichoderma spp. isolates and sequenced, supporting a putative role in nematode toxicity. Bacteria isolated from the cysts of G. rostochiensis included Brucella, Rhizobium, Stenotrophomonas and Bacillus spp., identified through 16S rRNA gene sequencing. The potential of the microbial isolates identified and their mechanisms of action are discussed, as part of a sustainable nematode management strategy.

Distribution and Genetic Variability of Fusarium oxysporum Associated with Tomato Diseases in Algeria and a Biocontrol Strategy with Indigenous Trichoderma spp.

Fifty fungal isolates were sampled from diseased tomato plants as result of a survey conducted in seven tomato crop areas in Algeria from 2012 to 2015. Morphological criteria and PCR-based identification, using the primers PF02 and PF03, assigned 29 out of 50 isolates to Fusarium oxysporum (Fo). The banding patterns amplified for genes SIX1, SIX3 and SIX4 served to identify races 2 and 3 of Fo f. sp. lycopersici (FOL), and Fo f. sp. radicis lycopersici (FORL) among the Algerian isolates. All FOL isolates showed pathogenicity on the susceptible tomato cv. "Super Marmande," while nine of out 10 Algerian FORL isolates were pathogenic on tomato cv. "Rio Grande." Inter simple sequence repeat (ISSR) fingerprints showed high genetic diversity among Algerian Fo isolates. Seventeen Algerian Trichoderma isolates were also obtained and assigned to the species T. asperellum (12 isolates), T. harzianum (four isolates) and T. ghanense (one isolate) based on ITS and tef1α gene sequences. Different in vitro tests identified the antagonistic potential of native Trichoderma isolates against FORL and FOL. Greenhouse biocontrol assays performed on "SM" tomato plants with T. ghanense T8 and T. asperellum T9 and T17, and three Fo isolates showed that isolate T8 performed well against FORL and FOL. This finding was based on an incidence reduction of crown and root rot and Fusarium wilt diseases by 53.1 and 48.3%, respectively.

Population genetic structure and mycotoxin potential of the wheat crown rot and head blight pathogen Fusarium culmorum in Algeria.

Surveys for crown rot (FCR) and head blight (FHB) of Algerian wheat conducted during 2014 and 2015 revealed that Fusarium culmorum strains producing 3-acetyl-deoxynivalenol (3ADON) or nivalenol (NIV) were the causal agents of these important diseases. Morphological identification of the isolates (n FCR=110, n FHB=30) was confirmed by sequencing a portion of TEF1. To assess mating type idiomorph, trichothecene chemotype potential and global population structure, the Algerian strains were compared with preliminary sample of F. culmorum from Italy (n=27), Australia (n=30) and the United States (n=28). A PCR assay for MAT idiomorph revealed that MAT1-1 and MAT1-2 strains were segregating in nearly equal proportions, except within Algeria where two-thirds of the strains were MAT1-2. An allele-specific PCR assay indicated that the 3ADON trichothecene genotype was predominant globally (83.8% 3ADON) and in each of the four countries sampled. In vitro toxin analyses confirmed trichothecene genotype PCR data and demonstrated that most of the strains tested (77%) produced culmorin. Global population genetic structure of 191 strains was assessed using nine microsatellite markers (SSRs). AMOVA of the clone corrected data indicated that 89% of the variation was within populations. Bayesian analysis of the SSR data identified two globally distributed, sympatric populations within which both trichothecene chemotypes and mating types were represented.

Fusarium algeriense, sp. nov., a novel toxigenic crown rot pathogen of durum wheat from Algeria is nested in the Fusarium burgessii species complex.

A novel crown rot pathogen of wheat discovered during pathogen surveys in Algeria in 2014 and 2015 is formally described as Fusarium algeriense. Multilocus molecular phylogenetic data resolved the eight isolates of this pathogen as a genealogically exclusive species lineage in the F. burgessii species complex. The previously described species of this complex, F. burgessii and F. beomiforme, produce abundant chlamydospores in culture, and their optimal temperature for growth is 30 C. In comparison, F. algeriense did not produce chlamydospores under the conditions tested and its optimal temperature for growth is 25 C. Furthermore, F. algeriense differs from F. burgessii because it does not produce polyphialides and F. beomiforme, because it does not produce globose-to-napiform conidia in the aerial mycelium. Isolates of F. algeriense induced moderate crown rot on the susceptible spring wheat cultivar Norm in a temperature-controlled incubator. Fusarium burgessii and F. beomiforme, in contrast, only induced mild symptoms of this disease. BLASTn searches of the whole-genome sequence of F. algeriense strains NRRL 66647 and 66648, using homologs of genes that are responsible for synthesis of toxic secondary metabolites, indicated that they have the potential to produce several polyketide and non-ribosomal peptide-derived mycotoxins. However, moniliformin and 2-AOD-ol (2-amino-14,16-dimethyloctadecan-3-ol) were the only mycotoxins detected by liquid chromatography-mass spectrometry (LC-MS) analyses of strains cultivated in vitro on a solid medium. A polymerase chain reaction (PCR) assay for MAT idiomorph revealed that MAT1-1 and MAT1-2 strains of F. algeriense were present in Algeria, which suggests this pathogen might possess a heterothallic sexual reproductive mode.