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

Multiple gene analyses identify distinct “bois noir” phytoplasma genotypes in the Republic of Macedonia

“Bois noir” (BN) is a grapevine yellows disease, associated with phytoplasma strains related to ‘Candidatus Phytoplasma solani’, that causes severe losses to viticulture in the Euro-Mediterranean basin. Due to the complex ecological cycle of its etiological agent, BN epidemiology is only partially known, and no effective control strategies have been developed. Numerous studies have focused on molecular characterization of BN phytoplasma strains, to identify molecular markers useful to accurately describe their genetic diversity, geographic distribution and host range. In the present study, a multiple gene analysess were carried out on 16S rRNA, tuf, vmp1, and stamp genes to study the genetic variability among 18 BN phytoplasma strains detected in diverse regions of the Republic of Macedonia. Restriction fragment length polymorphism (RFLP) assays showed the presence of one 16S rRNA (16SrXII-A), two tuf (tuf-type a, tuf-type b), five vmp1 (V2-TA, V3, V4, V14, V18), and three stamp (S1, S2, S3) gene patterns among the examined strains. Based on the collective RFLP patterns, seven genotypes (Mac1 to Mac7) were described as evidence for genetic heterogeneity, and highlighting their prevalence and distribution in the investigated regions. Phylogenetic analyses on vmp1 and stamp genes underlined the affiliation of Macedonian BN phytoplasma strains to clusters associated with distinct ecologies

Green Technology: Bacteria-Based Approach Could Lead to Unsuspected Microbe–Plant–Animal Interactions

The recent and massive revival of green strategies to control plant diseases, mainly as a consequence of the Integrated Pest Management (IPM) rules issued in 2009 by the European Community and the increased consumer awareness of organic products, poses new challenges for human health and food security that need to be addressed in the near future. One of the most important green technologies is biocontrol. This approach is based on living organisms and how these biocontrol agents (BCAs) directly or indirectly interact as a community to control plant pathogens and pest. Although most BCAs have been isolated from plant microbiomes, they share some genomic features, virulence factors, and trans-kingdom infection abilities with human pathogenic microorganisms, thus, their potential impact on human health should be addressed. This evidence, in combination with the outbreaks of human infections associated with consumption of raw fruits and vegetables, opens new questions regarding the role of plants in the human pathogen infection cycle. Moreover, whether BCAs could alter the endophytic bacterial community, thereby leading to the development of new potential human pathogens, is still unclear. In this review, all these issues are debated, highlighting that the research on BCAs and their formulation should include these possible long-lasting consequences of their massive spread in the environment

Pathogenic potential of bacteria isolated from commercial biostimulants

Microbial-based products are a promising alternative to agrochemicals in sustainable agriculture. However, little is known about their impact on human health even if some of them, i.e., Bacillus and Paenibacillus species, have been increasingly implicated in different human diseases. In this study, 18 bacteria were isolated from 2 commercial biostimulants, and they were genotypically and phenotypically characterized to highlight specific virulence properties. Some isolated bacteria were identified as belonging to the genus Bacillus by BLAST and RDP analyses, a genus in-depth studied for plant growth-promot- ing ability. Moreover, 16S rRNA phylogenetic analysis showed that seven isolates grouped with Bacillus species while two and four clustered, respectively, with Neobacillus and Peribacillus. Unusually, bacterial strains belonging to Franconibacter and Stenotrophomonas were isolated from biostimulants. Although Bacillus species are generally considered nonpathogenic, most of the species have shown to swim, swarm, and produced biofilms, that can be related to bacterial virulence. The evalu- ation of toxins encoding genes revealed that five isolates had the potential ability to produce the enterotoxin T. In conclusion, the pathogenic potential of microorganisms included in commercial products should be deeply verified, in our opinion. The approach proposed in this study could help in this crucial step

First Report of Grapevine virus A (GVA) and Grapevine fleck virus (GFkV) in the Former Yugoslav Republic of Macedonia

Grapevine (Vitis vinifera L.) is an economically important crop and can host several different viruses, including those ones that have to be excluded from the certified propagating material in Europe. Among these, the Vitivirus Grapevine virus A (GVA) and the Maculavirus Grapevine fleck virus (GFkV) are phloem-limited viruses that are associated with two different grapevine diseases, Kober stem grooving, belonging to the rugose wood complex, and Fleck diseases, respectively. During a survey conducted in the season 2012 in the Former Republic of Macedonia, symptomatic plants with reddening of leaves were collected for laboratory analyses. In this study, grapevine red varieties (Vranec, Francovka and Pinot noir) from four different localities (Stip, Kavadarci, Valandovo, and Gevgelija) in Macedonia were examined

Optimization of Esterase Production in Solid-State Fermentation of Agricultural Digestate

The continuous increase in biogas production poses the need for innovative applications for its by-products. Solid-state fermentation (SSF) has regained attention in the development of several products because of the possibility to use low-cost and easily available substrates, such as organic wastes. SSF represents a valuable process for agricultural digestate valorization in terms of enzyme production. In the present study, cellulase and esterase were produced by Trichoderma asperellum R on a digestate-based substrate in SSF, with esterase as the highest obtained activity. After assessing the effect of light on it, the esterase production in SSF was optimized using response surface methodology. The influence of substrate composition, temperature and humidity on the enzyme production was evaluated on two sets of data generated based on digestate concentration (50% and 70% w/w). The statistical analyses revealed that these parameters affected esterase production only when Trichoderma asperellum grew on substrate containing 50% w/w of digestate. The best esterase activity (264.6 mU/mg total protein) was achieved with the following optimized SSF parameters: 50% digestate, 50% fruits, 10% sawdust, 30 °C. The current finding of esterase production on digestate-based substrates makes the SSF method presented here a sustainable and completely circular technology

Molecular Characterization of <i>Ciborinia camelliae</i> Kohn Shows Intraspecific Variability and Suggests Transcontinental Movement of the Pathogen

Ciborinia camelliae Kohn is the causal agent of camellia flower blight. The fungus infects only the flowers of camellias. C. camelliae isolates obtained from symptomatic samples, collected in 13 different localities worldwide, were characterized by Multi-Locus Sequence Typing (MLST) using the following: (i) a nuclear ribosomal DNA internal transcribed spacer; (ii) subunit 2 of β-tubulin (β-TUB II), (iii) elongation factor 1-α (EF1α); and (iv) glycerol-3-phosphate dehydrogenase (GPDH). The variability of the strains was assessed using a universally primed–polymerase chain reaction (UP-PCR) with six universal primers. Gene sequence comparison showed high similarity among all the European strains and highlighted the diversity of the New Zealand and Chinese representative strains. The profiles obtained by UP-PCR confirmed the significant diversity of extra-European strains and identified subgroups within the European population. The presence of shared genetic profiles obtained from strains isolated in different countries (New Zealand and France) suggests the movement of strains from one location to another, which is probably due to the exchange of infected plant material. Moreover, our study shows the overall high intraspecific variability of C. camelliae, which is likely due to the sexual reproduction of the fungus, suggesting the risk of emergence of new pathotypes adapting to novel camellia varieties

Multiple gene analyses identify distinct “bois noir” phytoplasma genotypes in the Republic of Macedonia

“Bois noir” (BN) is a grapevine yellows disease, associated with phytoplasma strains related to ‘Candidatus Phytoplasma solani’, that causes severe losses to viticulture in the Euro-Mediterranean basin. Due to the complex ecological cycle of its etiological agent, BN epidemiology is only partially known, and no effective control strategies have been developed. Numerous studies have focused on molecular characterization of BN phytoplasma strains, to identify molecular markers useful to accurately describe their genetic diversity, geographic distribution and host range. In the present study, a multiple gene analysess were carried out on 16S rRNA, tuf, vmp1, and stamp genes to study the genetic variability among 18 BN phytoplasma strains detected in diverse regions of the Republic of Macedonia. Restriction fragment length polymorphism (RFLP) assays showed the presence of one 16S rRNA (16SrXII-A), two tuf (tuf-type a, tuf-type b), five vmp1 (V2-TA, V3, V4, V14, V18), and three stamp (S1, S2, S3) gene patterns among the examined strains. Based on the collective RFLP patterns, seven genotypes (Mac1 to Mac7) were described as evidence for genetic heterogeneity, and highlighting their prevalence and distribution in the investigated regions. Phylogenetic analyses on vmp1 and stamp genes underlined the affiliation of Macedonian BN phytoplasma strains to clusters associated with distinct ecologies

Restructuring of Endophytic Bacterial Communities in Grapevine Yellows-Diseased and Recovered Vitis vinifera L. Plants ▿

Length heterogeneity-PCR assays, combined with statistical analyses, highlighted that the endophytic bacterial community associated with healthy grapevines was characterized by a greater diversity than that present in diseased and recovered plants. The findings suggest that phytoplasmas can restructure the bacterial community by selecting endophytic strains that could elicit a plant defense response