Genetic Diversity and Pathogenicity of the Fusarium Species Complex on Soybean in Serbia.

Using morphological and cultural characteristics for identification, 36 Fusarium isolates were recovered from diseased roots, stems, and seeds of soybean from several localities throughout Vojvodina Province, Serbia. Based on molecular characterization, 12 Fusarium species were identified: F. acuminatum, F. avenaceum, F. commune, F. equiseti, F. graminearum, F. incarnatum, F. oxysporum, F. proliferatum, F. solani, F. sporotrichioides, F. subglutinans, and F. tricinctum. The elongation factor 1-α-based phylogeny grouped the isolates into 12 well-supported clades, but polymorphisms among sequences in some clades suggested the use of the species complex concept: (i) F. incarnatum-equiseti species complex (FIESC)-F. incarnatum and F. equiseti; (ii) F. oxysporum species complex (FOSC)-F. oxysporum; (iii) F. solani species complex (FSSC)-F. solani; and (iv) F. acuminatum/F. avenaceum/F. tricinctum species complex (FAATSC)-F. acuminatum, F. avenaceum, and F. tricinctum. Pathogenicity tests showed that the most aggressive species causing soybean seed rot were F. sporotrichioides, F. graminearum, FIESC, and F. avenaceum. Furthermore, F. subglutinans, FSSC, and F. proliferatum showed a high percentage of pathogenicity on soybean seeds (80 to 100%), whereas variability in pathogenicity occurred within isolates of F. tricinctum. FOSC, F. commune, and F. acuminatum had the lowest pathogenicity. To our knowledge, this is the first study of the characterization of Fusarium species on soybean in Serbia. This study provides valuable information about the composition of Fusarium species and pathogenicity that will be used in further research on soybean resistance to Fusarium-based diseases.

Rapid molecular assay for the evaluation of clove essential oil antifungal activity against wheat common bunt.

Common bunt of durum wheat (DW), Triticum turgidum L. ssp. durum (Desf.) Husn., is caused by the two closely related fungal species belonging to Tilletia genus (Tilletiales, Exobasidiomycetes, Ustilaginomycotina): Tilletia laevis Kühn (syn. T. foetida (Wallr.) Liro.) and T. caries (DC) Tul. (syn. T. tritici (Bjerk.) G. Winter). This is one of the most devastating diseases in wheat growing areas worldwide, causing considerable yield loss and reduction of wheat grains and flour quality. For these reasons, a fast, specific, sensitive, and cost-effective method for an early diagnosis of common bunt in wheat seedlings is urgent. Several molecular and serological methods were developed for diagnosis of common bunt in wheat seedlings but at late phenological stages (inflorescence) or based on conventional PCR amplification, with low sensitivity. In this study, a TaqMan Real Time PCR-based assay was developed for rapid diagnosis and quantification of T. laevis in young wheat seedlings, before tillering stage. This method, along with phenotypic analysis, was used to study conditions favoring pathogen infection and to evaluate the effectiveness of clove oil-based seed dressing in controlling the disease. The overall results showed that: i) the Real Time PCR assay was able to quantify T. laevis in young wheat seedlings after seed dressing by clove oil in different formulations, greatly reducing times of analysis. It showed high sensitivity, detecting up to 10 fg of pathogen DNA, specificity and robustness, allowing to directly analyze crude plant extracts and representing a useful tool to speed up the tests of genetic breeding for disease resistance; ii) temperature was a critical point for disease development when using wheat seeds contaminated by T. laevis spores; iii) at least one of the clove oil-based formulations tested was able to efficiently control wheat common bunt, suggesting that clove oil dressing could represent a promising tool for managing the disease, especially in sustainable farming.

New-Generation Sequencing Technology in Diagnosis of Fungal Plant Pathogens: A Dream Comes True?

The fast and continued progress of high-throughput sequencing (HTS) and the drastic reduction of its costs have boosted new and unpredictable developments in the field of plant pathology. The cost of whole-genome sequencing, which, until few years ago, was prohibitive for many projects, is now so affordable that a new branch, phylogenomics, is being developed. Fungal taxonomy is being deeply influenced by genome comparison, too. It is now easier to discover new genes as potential targets for an accurate diagnosis of new or emerging pathogens, notably those of quarantine concern. Similarly, with the development of metabarcoding and metagenomics techniques, it is now possible to unravel complex diseases or answer crucial questions, such as "What's in my soil?", to a good approximation, including fungi, bacteria, nematodes, etc. The new technologies allow to redraw the approach for disease control strategies considering the pathogens within their environment and deciphering the complex interactions between microorganisms and the cultivated crops. This kind of analysis usually generates big data that need sophisticated bioinformatic tools (machine learning, artificial intelligence) for their management. Herein, examples of the use of new technologies for research in fungal diversity and diagnosis of some fungal pathogens are reported.

Antibacterial Activity of Essential Oils and Trametes versicolor Extract against Clavibacter michiganensis subsp. michiganensis and Ralstonia solanacearum for Seed Treatment and Development of a Rapid In Vivo Assay.

Clavibacter michiganensis subsp. michiganensis (Smith) Davis et al. (Cmm) and Ralstonia solanacearum Yabuuchi et al. (Smith) (Rs) are important seed-borne bacterial pathogens of tomato (Solanum lycopersicum) listed as A2 pests in the EPPO (European and Mediterranean Plant Protection Organization) region. At present, there are few strategies to control these pathogens, and seed control with eco-compatible approaches is widely encouraged. In this work, the essential oils (EOs) of oregano (Origanum vulgare), garlic (Allium sativum), basil (Ocimum basilicum), cinnamon (Cinnamomum zeylanicum), clove buds (Syzygium aromaticum), thyme (Thymus vulgaris), and Trametes versicolor extract (Tve) were tested in vitro for their antimicrobial activity against Cmm and Rs (broth microdilution method). The tested EOs and the Tve extract caused a significant inhibition of bacterial growth, with very promising MBC (minimum bactericidal concentration) and MIC90 (minimum inhibitory concentration causing a 90% growth inhibition) values. Moreover, an in vivo germination test showed no major reduction in seed germination when the substances were applied as seed treatment. A rapid molecular screening method has been developed, through real-time PCR, for the specific quantification of Cmm in the presence of a vegetable matrix to test in vivo the antimicrobial efficacy of oregano and cinnamon oil on seed treatment without resorting to whole plant essays, which are time- and space-consuming.