First description of adenosine production by Gnomoniopsis smithogilvyi, causal agent of chestnut brown rot.

Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) is the main causal agent of chestnut brown rot on sweet chestnut worldwide. The rotting of nuts leads to alterations in the organoleptic qualities and decreased fruit production, resulting in significant economic losses. In 2021, there was an important outbreak of chestnut rot in southern Galicia (Spanish northwest). The profile of secondary metabolites from G. smithogilvyi was studied, especially to determine its capability for producing mycotoxins, as happens with other rotting fungi, due to the possible consequences on the safety of chestnut consumption. Secondary metabolites produced by isolates of G. smithogilvyi growing in potato dextrose agar (PDA) medium were identified using liquid chromatography coupled with high-resolution mass spectrometry. Three metabolites with interesting pharmacological and phyto-toxicological properties were identified based on their exact mass and fragmentation patterns, namely adenosine, oxasetin, and phytosphingosine. The capacity of G. smithogilvyi to produce adenosine in PDA cultures was assessed, finding concentrations ranging from 176 to 834 µg/kg. Similarly, the production of mycotoxins was ruled out, indicating that the consumption of chestnuts with necrotic lesions does not pose a health risk to the consumer in terms of mycotoxins.

Temporal and Spatial Variation in the Population Structure of Spanish Fusarium circinatum Infecting Pine Stands.

Fusarium circinatum is an introduced fungal pathogen extended to the northern regions of Spain that causes Pine Pitch Canker (PPC) disease. In this work, we analyzed the pathogen's genetic diversity to study changes over time and space since the first outbreak occurred in Spain. Using six polymorphic SSR markers, 15 MLGs were identified in 66 isolates, and only three haplotypes were found with frequencies higher than one. In general, genotypic diversity was low and decreased shortly over time in the northwestern regions while maintained at País Vasco, where only one haplotype (MLG32) was detected 10 years. This population also included isolates of a single mating type (MAT-2) and VCGs identified in only two groups, while isolates from NW regions were of both mating types and VCGs represented in 11 groups. The existence of haplotype MLG32 maintained on time and widely distributed suggests its good adaptation to the environment and the host. Results showed that the pathogen in País Vasco remains clearly differentiated from other northwestern populations. This fact was supported with no evidence of migration among regions. Results are explained by the asexual reproduction, but also selfing at least to a lesser extent that leads to identification of two new haplotypes.

Occurrence of mycotoxins and mycotoxigenic fungi in silage from the north of Portugal at feed-out.

Maize and grass silages are important dietary components for ruminant livestock that influence the quality of animal products for human consumption, such as milk, in many parts of the world. Infection of plants by fungi able to produce mycotoxins, either in the field or post-harvest, can result in a decrease of silage nutritional quality and, consequently, in milk quality. In this study, 45 maize and grass silage samples were collected from 25 dairy farms located in the north of Portugal. The occurrence of fungi was evaluated in samples, the most frequently isolated species being Aspergillus fumigatus, Dipodascus geotrichum, Mucor circinelloides, Penicillium paneum, and Aspergillus flavus. The mycotoxigenic profile of the fungal species was studied using the ultra-high-performance liquid chromatography coupled to mass spectrometry-ion trap-time-of-flight (UHPLC-MS-IT-TOF) detection. In addition, a new method based on a QuEChERS extraction followed by the UHPLC- tandem mass spectrometry (UHPLC-MS/MS) detection was developed for simultaneous analysis of 39 mycotoxins in silage. A high co-occurrence of Fusarium mycotoxins was found, although at low levels of contamination. Deoxynivalenol and beauvericin were found in more than 82% of maize silage samples. It can be highlighted the low occurrence of Penicillium and Aspergillus toxins in the maize and grass silages studied despite the frequent detection of species of both genera.

Synergistic abiotic and biotic stressors explain widespread decline of Pinus pinaster in a mixed forest.

Global change potentially increases forest vulnerability. Different abiotic and biotic factors may interact to cause forest decline and accelerated tree mortality. We studied a mixed Mediterranean continental forest where Pinus pinaster Ait. (maritime pine) shows widespread decline to analyse the role of different abiotic and biotic factors on health status and growth dynamics both at the individual and plot levels. We also analysed stand composition and regeneration of tree species to check whether there is a change in species dominance. Fungal pathogens were seldom present and we detected no pervasive fungi or insect infestation and no presence of pathogens like Heterobasidion or Phytophthora. Infection of hemiparasite plants like Viscum album L. (mistletoe) can reduce leaf area and its abundance is generally considered an expression of host decline. Yet, the existence among declining trees of high defoliation levels without mistletoe, but not vice versa, suggests that defoliation in response to some abiotic stressor could be a predisposing factor preceding mistletoe infection. Compared to healthy trees, declining and dead trees exhibited higher defoliation rates, smaller needles and lower recent growth with steeper negative trends. Dead and declining trees showed similar negative growth trends since the early 1990s droughts, which we interpreted as early warning signals anticipating mortality of currently declining trees in the near future. Mortality of maritime pine extending across all size classes, the lower presence of this species in the smallest size classes and its lack of regeneration suggest it is potentially losing its current dominance and being replaced by other co-occurring, more drought-tolerant species. Our results unravelled that maritime pine decline seems to be mainly driven by a combination of predisposing and inciting abiotic factors (microenvironment and drought stress) and biotic factors (mistletoe). The absence of widespread fungal pathogens suggests that they may have a minor role on pine decline acting only eventually as contributing factors. Although there could be other interrelations among factors or other biotic agents at play, our results strongly suggest that water stress plays a major role in the decline process of the dominant species on an ecosystem with strong land-use legacies.

First report of Fusarium foetens as a mycotoxin producer.

Fusarium foetens, a pathogen of Begonia plants, has been recently described as a new fungal species. This Fusarium species causes a destructive vascular wilt disease which leads to the death of the plant. Moreover, Fusarium species are known to produce a huge variety of secondary metabolites such as mycotoxins and phytotoxins. Here, we studied the toxicogenic profile of one F. foetens strain, isolated from maize, employing two methods based on the use of ultra-performance liquid chromatography coupled to mass spectrometry-ion trap-time of flight detection. The mycotoxins beauvericin and fusaric acid were detected in a pure culture of F. foetens. In addition, four fusaric acid analogs (10,11-dihidroxyfusaric acid, hydroxyfusaric acid, dehydrofusaric acid, and a hydroxylated unsaturated fusaric acid analog) were tentatively identified on the basis of their accurate mass and fragmentation patterns. Therefore, these preliminary data indicate that F. foetens isolated from maize is able to produce Fusarium mycotoxins including beauvericin and fusaric acid.

Incidencia y distribución del género Armillaria en viñedos de las cinco denominaciones de origen de vino de Galicia (noroeste de España) / Incidence and distribution of the genus Armillaria in vineyards of the five protected designation of origin wines in Galicia (Northwestern Spain)

Antecedentes: el género Armillaria, y más concretamente la especie Armillaria mellea, constituye un problema muy importante en el sector vitivinícola de Galicia, lo que ha ocasionado importantes reducciones del rendimiento de los viñedos en los últimos 15 años. Este hongo ataca al sistema radicular, causando un descenso del vigor y, finalmente, la muerte de la planta. Hasta la fecha, no se conoce ningún método químico o biológico que resulte efectivo contra el patógeno una vez que ha entrado en la planta. Objetivos: el objetivo principal del presente trabajo fue conocer la incidencia y distribución del género Armillaria mediante técnicas moleculares en las distintas zonas de cultivo de vid en Galicia, abarcando las cinco denominaciones de origen (DO) vitivinícolas gallegas (Rías Baixas, Ribeiro, Ribeira Sacra, Valdeorras y Monterrei). Métodos: se analizaron 624 muestras (483 de suelo y 141 de plantas de vid sintomáticas) mediante nested-PCR/RFLP, PCR-RFLP y análisis filogenéticos. Resultados: Armillaria mellea está ampliamente distribuida en viñedos de las cinco DO, con mayor incidencia en la DO del Ribeiro. Conclusiones: es necesario establecer medidas para reducir el avance de la podredumbre radicular causada por Armillaria mellea en las cinco DO de Galicia

Blackground: the genus Armillaria, specifically Armillaria mellea, is an important phytopathological problem in the wine sector in Galicia (NW Spain), having caused yield reductions in vineyards for the last 15 years. The fungus attacks the root system, resulting in a decrease in vigour, and eventually in the death of the plant. Up to now, there is no chemical or biological method really effective against the pathogen once it has infected the plant. Aims: the main objective of this work was to study the incidence and distribution of the genus Armillaria across the five Galician protected designation of origin (DO) wines (namely Rías Baixas, Ribeiro, Ribeira Sacra, Valdeorras and Monterrei) through the application of molecular techniques. Methods: a total of 624 samples (483 soils and 141 symptomatic vines) were analyzed by nested-PCR/RFLP, PCR-RFLP and phylogeny. Results: Armillaria mellea is widely distributed in vineyards of the five DO wines, with the highest incidence in the Ribeiro DO. Conclusions: preventive control measures against Armillaria mellea must be established in the five DO wines of Galicia, in order to reduce the advance of white root rot

[Incidence and distribution of the genus Armillaria in vineyards of the five protected designation of origin wines in Galicia (Northwestern Spain)]. / Incidencia y distribución del género Armillaria en viñedos de las cinco denominaciones de origen de vino de Galicia (noroeste de España).

BLACKGROUND: The genus Armillaria, specifically Armillaria mellea, is an important phytopathological problem in the wine sector in Galicia (NW Spain), having caused yield reductions in vineyards for the last 15 years. The fungus attacks the root system, resulting in a decrease in vigour, and eventually in the death of the plant. Up to now, there is no chemical or biological method really effective against the pathogen once it has infected the plant. AIMS: The main objective of this work was to study the incidence and distribution of the genus Armillaria across the five Galician protected designation of origin (DO) wines (namely Rías Baixas, Ribeiro, Ribeira Sacra, Valdeorras and Monterrei) through the application of molecular techniques. METHODS: A total of 624 samples (483 soils and 141 symptomatic vines) were analyzed by nested-PCR/RFLP, PCR-RFLP and phylogeny. RESULTS: Armillaria mellea is widely distributed in vineyards of the five DO wines, with the highest incidence in the Ribeiro DO. CONCLUSIONS: Preventive control measures against Armillaria mellea must be established in the five DO wines of Galicia, in order to reduce the advance of white root rot.

Critical environmental and genotypic factors for Fusarium verticillioides infection, fungal growth and fumonisin contamination in maize grown in northwestern Spain.

In northwestern Spain, where weather is rainy and mild throughout the year, Fusarium verticillioides is the most prevalent fungus in kernels and a significant risk of fumonisin contamination has been exposed. In this study, detailed information about environmental and maize genotypic factors affecting F. verticillioides infection, fungal growth and fumonisin content in maize kernels was obtained in order to establish control points to reduce fumonisin contamination. Evaluations were conducted in a total of 36 environments and factorial regression analyses were performed to determine the contribution of each factor to variability among environments, genotypes, and genotype × environment interactions for F. verticillioides infection, fungal growth and fumonisin content. Flowering and kernel drying were the most critical periods throughout the growing season for F. verticillioides infection and fumonisin contamination. Around flowering, wetter and cooler conditions limited F. verticillioides infection and growth, and high temperatures increased fumonisin contents. During kernel drying, increased damaged kernels favored fungal growth, and higher ear damage by corn borers and hard rainfall favored fumonisin accumulation. Later planting dates and especially earlier harvest dates reduced the risk of fumonisin contamination, possibly due to reduced incidence of insects and accumulation of rainfall during the kernel drying period. The use of maize varieties resistant to Sitotroga cerealella, with good husk coverage and non-excessive pericarp thickness could also be useful to reduce fumonisin contamination of maize kernels.

In vitro selection of an effective fungicide against Armillaria mellea and control of white root rot of grapevine in the field.

Armillaria mellea (Vahl ex Fr) Kummer is an aggressive pathogen which causes white root rot in a wide range of hosts. Most chemicals tested so far against Armillaria, both in vitro and in the field, have not been effective in reducing fungal growth and/or preventing plant decline and mortality. In the present work the effects of four DMI (sterol demethylation inhibitor) fungicides, cyproconazole, hexaconazole, propiconazole and tetraconazole, and another six downwardly mobile systemic chemicals, azoxystrobin, cubiet (copper bis(ethoxy-dihydroxy-diethylamino)sulfate), fosetyl-Al, potassium phosphite, sodium tetrathiocarbonate (STTC) and 2-(thiocyanomethylthio)benzothiazole (TCMTB), on the mycelial growth of A. mellea were compared and evaluated; the product yielding the best results in in vitro experiments was selected to determine its efficacy in preventing decline and mortality of grapevines in the field. Best results on in vitro fungal growth reduction were obtained with the four azoles tested, in particular with cyproconazole and hexaconazole, achieving 67-72% mycelial growth inhibition at the lowest dose. Results obtained in the field showed that a dose of 50 mg AI litre(-1) of cyproconazole once or twice a year was efficient in controlling the disease even in vines seriously affected by the pathogen. However, further research is required to study minimum effective doses, residual effects and the convenience of the application of annual dressings in damaged vineyards, so as to gradually reduce the pathogen inoculum potential in soil and control the disease while reducing chemical residues in the plant and preventing development of fungal resistance.