Mediterranean agroforestry systems (AFSs), typified by the Iberian Dehesas and Portuguese Montados, are multifunctional landscapes where Quercus species act as ecological keystones sustaining biodiversity, soil fertility, and rural livelihoods. These systems are increasingly affected by complex oak decline syndromes driven by drought, soil degradation, and climate-induced pathogen outbreaks. Conventional chemical controls are often ineffective and environmentally detrimental, underscoring the need for ecologically sound management alternatives. This review synthesizes recent advances in the application of microbial biological control agents (MBCAs) to manage diseases in Mediterranean Quercus species, including Q. ilex, Q. suber, Q. faginea, and Q. pyrenaica. We conducted a structured literature review using predefined keyword searches in Web of Science and Scopus, followed by the screening of records to identify 22 relevant peer-reviewed studies on microbial disease control in Mediterranean Quercus species. We identified 20 peer-reviewed studies that reported that MBCAs—primarily from Bacillus, Serratia, Streptomyces, Trichoderma, Simplicillium and Alternaria—exert biocontrol effects through antibiosis, mycoparasitism, competition for ecological niches, and the induction of host defense responses. Although most experiments were conducted in vitro, some demonstrated significant disease suppression in seedlings infected by Phytophthora cinnamomi, Diplodia corticola, and Biscogniauxia mediterranea. Future research should integrate field-based validation and microbiome-oriented forest management approaches to enable the operational use of microbial-based disease control strategies in AFS landscapes.

Urban forests are highly valued for the multiple benefits they provide to city dwellers. The strategic provision of ecosystem services by these forests is threatened by climate change, warming conditions being responsible for heat waves and chronic droughts that inflict stress and mortality on trees. A three-year study (2011-2013) conducted at Parco Nord Milano (PNM) (Milano, Italy) assessed the impact of thinning interventions on the dynamics of fungal pathogens in declining forest plots. Symptomatic trees of the genera Alnus, Acer, Fraxinus, Platanus, Quercus and Ulmus, exhibited in thinned subplot pronounced decline/dieback, exhibiting symptoms like microphyllia, leaf yellowing, leaf shedding, sunken cankers, shoot wilting and branch dieback. Comparative analyses between the thinned and unthinned subplots revealed a significantly higher incidence of pathogens in the thinned one. Five species of Botryosphaeriaceae, namely Botryosphaeria dothidea, Diplodia corticola, Diplodia seriata, Dothiorella omnivora and Neofusicoccum parvum, were consistently isolated from tissues of declining hosts. There is evidence that thinning altered plot-level microclimate conditions and microbial equilibrium, favoring the proliferation of latent, pathogenic Botryosphaeriaceae. In fact, during the study period, the presence of N. parvum increased tenfold and that of B. dothidea fivefold in thinned subplot. Conversely, in unthinned subplot, the same pathogenic taxa maintained stable proportions. These results demonstrate that thinning altered ecological balances increasing tree susceptibility to harmful, cosmopolitan botryosphaeriaceous fungi. Our findings challenge assumptions about thinning as a universally beneficial practice, emphasizing the need for silvicultural strategies that take into account host and pathogen ecology and the microclimatic resilience of forest stands. This study emphasizes the importance of adaptive management in urban forestry to mitigate the unintended ecological consequences of climate change.

Flavoenzymes of the 4-phenol oxidoreductase family are versatile biocatalysts that catalyze the oxidation of a wide variety of phenol derivatives to alcohols, aldehydes, ketones or alkenes. The promiscuous FAD-dependent vanillyl alcohol oxidases from Penicillium simplicissimum (PsVAO) and Diplodia corticola (DcVAO) have been described to catalyze the oxidative deamination of p-hydroxybenzylamines, giving rise to valuable flavor compounds, but starting from p-alkyl substituted phenols, the ketones are usually not accessible as these oxidases preferably stop at chiral benzylic alcohols. Here we took a closer look into the fungal VAO family with the aim to identify new members that can perform this deamination reaction and also the overoxidation of benzylic alcohols to ketones at a sufficient rate for application. Phylogenetic and amino acid cluster analysis revealed one clade that differed significantly in the constitution of the active site, while maintaining residues essential for catalysis. From this clade, five candidates were chosen for investigation, which revealed that VAO from Paecilomyces variotii (PvVAO) showed promising activities with vanillylamine and 4-(1-amino)ethylphenol, especially above pH 9.0, while also offering the ability to perform the overoxidation of p-alkyl substituted phenols toward ketones. Hence, the identified PvVAO offers two reaction routes toward benzylic ketones.

Cork oak (Quercus suber) forests are threatened by emergent fungal pathogen Diplodia corticola, which causes significant economic and ecological losses. This study evaluates the efficacy of synthetic and natural fungicides, as well as Bacillus antagonistic agents, against this phytopathogen in vitro and in vivo. Eighteen fungicidal agents were tested across three concentrations, whereas the bacterial antagonistic agents Bacillus amyloliquefaciens and a mixture of B. amyloliquefaciens + Bacillus mojavensis were tested at a fixed concentration. The assayed chemicals, including penconazole, clove oil, vanillin, and belthanol, showed 100 ± 0.0% radial growth inhibition (n = 24) and conidiation (n = 24), highlighting their potential as alternatives to benomyl and methyl thiophanate (Restricted in the European Union). In vivo assays further validated the efficacy of these agents in reducing symptom incidence and seedling mortality in cork oak seedlings. Similarly, the Bacillus-based treatments showed 47.6 ± 0.9% (n = 35) in vitro antagonistic effects and in vivo application on seedlings (n = 470) significantly reduced disease symptoms and supported physiological stability (GLMs with Tukey HSD post hoc). The study aimed to evaluate chemical, natural and biological control agents against this pathogen to identify effective management alternatives for forest nurseries and cork oak.

Almond decline and dieback have become significant challenges in newly established orchards, with symptoms including internal necrosis, canker, and external gummosis. This work aims to explore the potential fungal and bacterial causative agents through metabarcoding and traditional culture plate isolation across six almond cultivars. Our results emphasize the multifactorial nature of almond decline and dieback, with possible co-infections by opportunistic fungi and bacteria playing a central role. Classical isolation identified 47 fungal species or genera, including Diaporthe amygdali, Diplodia corticola, Phytophthora sp., and several Fusarium species. Almond metabarcoding revealed a more diverse microbial community, highlighting the prevalence of soilborne pathogens such as Neocosmospora rubicola, Dactylonectria estremocensis, and Plectosphaerella niemeijerarum. Soil metabarcoding suggested that these pathogens likely originate from nursery substrates or soils shared with other crops, such as olives and vineyards, that serve as a source of inoculum. 'Soleta' generally presented lower richness when compared to the other tested cultivars, suggesting a higher degree of biotic stress and decreased plant resilience. This study highlights the value of integrating NGS approaches to comprehensively study complex diseases and the need for further research on pathogen interactions and cultivar susceptibility for the future development of new sustainable, targeted management strategies in almond orchards.

In recent years, severe decline and mortality events have been observed in holm oak (Quercus ilex L.) ecosystems in different Italian regions, including Puglia (southern Italy). Given the landscape and ecological relevance of holm oak forests in Apulia, a study was conducted to identify the causal agents related to this complex disease syndrome. The surveys, conducted in winter 2024 in three different woodlands, revealed the widespread occurrence of mature holm oak trees showing sudden death, crown thinning, shoot and branch dieback, sunken cankers, and root rot symptoms. Isolations performed from symptomatic samples collected from both stem and small roots yielded fungal and fungal-like colonies representing two distinct families: Botryosphaeriaceae and Peronosporaceae. Analysis of morphological and DNA sequence data allowed us to identify six distinct species, including Diplodia corticola and D. quercivora (Botryosphaeriaceae), Phytophthora cinnamomi, P. multivora, P. psychrophila, and P. asparagi (Peronosporaceae). For P. asparagi and P. psychrophila, isolated for the first time from declining holm oak trees in Italy, Koch’s postulates were satisfied by inoculating 1-year-old seedlings at the collar in controlled conditions. Thirty days after inoculation, all plants showed the same symptoms observed in the field. Overall, the data obtained highlights the co-occurrence of multiple Botryosphaeriaceae and Phytophthora species on declining holm oak trees and the discovery of a new haplotype of Diplodia quercivora.

Cork oak (Quercus suber) is an economically important species in the Mediterranean region, but its decline due to fungal pathogens such as Biscogniauxia mediterranea and Diplodia corticola poses a major threat to forest sustainability. Climate change is aggravating this problem, and few control measures are currently available. The use of naturally occurring endophytic antagonists as biocontrol agents offers a sustainable strategy to mitigate cork oak decline. In this work, cork oak seedlings subjected to normal irrigation (70% field capacity) and drought (25% field capacity) were inoculated with the endophytic fungus Simplicillium aogashimaense, followed by one of the pathogens (B. mediterranea or D. corticola). The endophyte significantly alleviated the negative impacts of both pathogens, showing a stronger protective effect under drought stress, where the combined pressures of water limitation and pathogen infection were mitigated. In addition, S. aogashimaense modulated key physiological and biochemical responses in the seedlings, including phenolic accumulation, protein expression, peroxidase activity, and oxidative stress markers. The mode of action differed between pathogens: defense responses against D. corticola involved increased hydrogen peroxide (H₂O₂) production and lipid peroxidation, while interactions with B. mediterranea were associated with reduced peroxidase activity. The consistent protective effect under both water regimes highlights the potential of S. aogashimaense in climate change scenarios characterized by increased drought frequency. This study supports the use of endophytes as environmentally sustainable tools in integrated forest management, enhancing cork oak resilience to combined stresses and contributing to a better understanding of plant responses to pathogen attack.

Fungal infections are a major threat to crop productivity and quality. Synthetic antifungals have been used ensuring high production; however, the environmental negative effects and the induction of resistant strains have been rising concerns about their use. Thus, this work aims to valorize Medicago sp. extracts, as potentially safe and effective antifungals in agriculture. The chemical analysis of an ethanolic extract showed mainly flavonoids, with flavones (23.18±0.08mg g-1) and isoflavones (13.47±0.11mg g-1) as the main constituents. Dose-dependent cytotoxicity was observed against Saccharomyces cerevisiae, with concomitant loss of plasma membrane integrity, assessed with propidium iodide, and nucleus-cytosolic translocation of the necrosis marker Nhp6A. Extract's cytotoxicity in the mutant strain yca1, affected in an apoptosis-related metacaspase, further suggested a necrotic mode of cell death. Significant in vitro antifungal activity was observed against Botrytis cinerea, Colletotrichum acutatum, Colletotrichum gloeosporioides, Colletotrichum godetiae, Colletotrichum nymphaea, and Diplodia corticola, all phytopathogenic fungi with devastating impact on relevant crops worldwide. The results indicate a remarkable antifungal activity by Medicago sp. extract with potential use in the protection of crops by inducing necrosis in fungal phytopathogens.

Background/Objectives: Cork oak forests have been declining due to fungal pathogens such as Diplodia corticola. However, the preventive fungicides against this fungus have restricted use due to the deleterious effects on human health and the environment, prompting the need for sustainable alternatives. Here, we describe the antifungal activity of an aqueous extract of Hedera helix L. leaves (HAE) against D. corticola and the possible mechanism of action. Results/Methods: The chemical analysis revealed compounds like the saponin hederacoside C, quinic acid, 5-O-caffeoylquinic acid, rutin, and glycoside derivatives of quercetin and kaempferol, all of which have been previously reported to possess antimicrobial activity. Remarkable in vitro antifungal activity was observed, reducing radial mycelial growth by 70% after 3 days of inoculation. Saccharomyces cerevisiae mutants, bck1 and mkk1/mkk2, affected the cell wall integrity signaling pathway were more resistant to HAE than the wild-type strain, suggesting that the extract targets kinases of the signaling pathway, which triggers toxicity. The viability under osmotic stress with 0.75 M NaCl was lower in the presence of HAE, suggesting the deficiency of osmotic protection by the cell wall. Conclusions: These results suggest that ivy extracts can be a source of new natural antifungal agents targeting the cell wall, opening the possibility of preventing fungal infections in cork oaks and improving the cork production sector using safer and more sustainable approaches.

Soil and irrigation water salinity is a growing global problem affecting farmland, due to poor agricultural practices and climate change, leading to reduced crop yields. Given the limited amount of arable land and the need to boost production, hydroponic systems offer a viable solution. Additionally, endophytic fungi have been shown to mitigate salinity effects through symbiosis with plants. This study evaluated three endophytic fungi isolated from Lavandula stoechas L. in the grasslands of Extremadura (i.e., Diplodia corticola L11, Leptobacillium leptobactrum L15, and Cladosporium cladosporioides L16) for their ability to improve hydroponic forage production under saline conditions. In vitro experiments were conducted assessing plant growth promotion and fungal growth under salinity, followed by research evaluating the impact of fungal inoculation on hydroponic wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) forages irrigated with NaCl solutions (0, 100, and 200 mM). The results showed improved fungal growth and production of plant growth-promoting substances, which could explain the improved plant germination, shoot and root length, fresh and dry weight, and yield of inoculated plants growing under salinity. Plants inoculated with L15 or L16 showed the best performance overall. L15 demonstrated broader bioactivity in vitro, potentially explaining its superior performance in both wheat and barley growth. Conversely, L16 was more effective in barley, while L11 was beneficial in wheat but detrimental in barley. This study provides a preliminary exploration of the capabilities of these fungi and their optimization for hydroponic forage production.

Grapevine cultivation in Mexico is important, especially in the states of Baja California and Coahuila, which are the main wine production regions in the country. Grapevine trunk diseases (GTDs) impact productivity and cause substantial economic losses, with Botryosphaeria dieback being one of the most destructive. This disease is caused by fungi in the Botryosphaeriaceae, including species of Botryosphaeria, Diplodia, Lasiodiplodia, and Neofusicoccum. To date, Lasiodiplodia spp. are the primary Botryosphaeriaceae fungi reported in Mexico. The present study aimed to enhance the epidemiological knowledge of Botryosphaeriaceae in Mexican vineyards. Samples from grapevine plants exhibiting disease symptoms were collected from the states of Baja California and Coahuila. Of a total of 37 Botryosphaeriaceae isolates, six species were identified: Neofusicoccum parvum, N. australe, N. vitifusiforme, Botryosphaeria dothidea, Diplodia corticola, and D. seriata. Neofusicoccum parvum isolates were the most virulent, but were less virulent than previously reported Lasiodiplodia spp. The optimum growth temperatures for N. parvum and B. dothidea were from 28 to 30°C, but 25°C for D. seriata, N. vitifusiforme, and N. australe isolates. Only D. seriata isolates recovered growth when transferred to room temperature after exposure to 37°C or 40°C. This report is the first identification of B. dothidea and N. parvum as causative agents of Botryosphaeria dieback in the vine-growing regions of Mexico.

Members of Botryosphaeria s.l. have an extensive history as cankering pathogens of stressed and declining oak trees in the eastern United States (Ferreira et al. 2021). The host range, distribution, and virulence among two closely related species, Diplodia corticola and D. gallae, remains unclear (Brazee et al. 2023). On 15 August 2023, a survey was conducted at a declining natural hardwood site in Shenandoah County, Virginia (GPS coordinates 38.922089, -78.606125). One mature Quercus coccinea tree that displayed scorched leaf margins and branch dieback was felled and a cankered branch from the crown was sampled (Fig. 1A and B). A 4-mm piece of necrotic tissue was selected from the margin of the canker, disinfected with 2.5% NaOCl, again with 70% ethanol, and air-dried before being placed on half-strength acidified PDA medium (pH 4.8) and incubated in the dark at 22 ± 2°C. After 5 days, four colonies were transferred to full-strength PDA medium and incubated in the dark at 22 ± 2°C. After 10 days, all four colonies displayed thick, gray, floccose mycelium and pigmented hyphae (Fig. 1C). Mycelia was harvested from 10-day-old colonies with a sterile pin and DNA was extracted using a Qiagen DNeasy Plant Pro Kit (Germantown, MD) according to the manufacturer's instructions. A fragment of the internal transcribed spacer (ITS) and translation elongation factor 1-α (tef1) loci were amplified using ITS4/ITS5 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999) primer sets, respectively. The PCR amplicons were purified with ExoSap-IT (Affymetrix, Santa Clara, CA) and sequenced at Eurofins (Louisville, KY).&xa0; The raw nucleotide sequences were analyzed using Geneious 11.1.5 software (Biomatters, Auckland, NZ). All four colonies had identical ITS sequences. A 523 and 276-bp fragment of the ITS and tef1 loci, respectively, from isolate R1.2 was deposited into the GenBank database (accessions OR934498 and OR961039). A dataset of 43 strains consisting of 38,658 characters was aligned using MAFFT v7.49 (Katoh et al. 2013), and a concatenated ITS + tef1 maximum likelihood phylogenetic tree (1000 bootstraps) was built with PhyML 3.0 (Guindon et al. 2010) using the GTR substitution model. Isolate R1.2 was grouped with isolates of D. gallae although the species failed to form a well-supported clade (BS = 67) due to intraspecific variation (Fig. 1D). Koch's postulates were fulfilled by inoculating five healthy, containerized Q. coccinea trees (average stem caliper 5.3 cm) with isolate R1.2, with five plants as controls. After disinfecting the bark with 70% ethanol, a 0.5 mm section of the bark was removed 13 cm above the soil line with a sterile scalpel, and a 0.5 mm agar plug taken from the edge of a 10-day-old PDA culture was placed in the wound with the mycelium facing the cambial tissue, sealed with Parafilm, and maintained at 22 ± 4°C. The same procedure was performed on the control plants using sterile PDA plugs. After five weeks the bark was removed, and all five stems treated with R1.2 had necrotic lesions with a mean linear growth ([length+width]/2) of 9.2 ± 2.72 mm from the edge of the wound, which was significantly larger (P = 0.003) than the controls (1 ± 0.66 mm; Fig. 1E - L). Necrotic stem tissue was sampled as previously described, and the isolate recovered was confirmed as D. gallae based on morphology and 100% ITS sequence homology to isolate R1.2. D. gallae was not recovered from the control plants. In the United States, D. gallae has been isolated from Q. rubra and Q. velutina twig cankers in Maine, Massachusetts, New Hampshire, New York, and Vermont (Brazee et al. 2023). This is the first report of the species in Virginia causing branch cankers on Q. coccinea.

Trunk canker poses a major threat to the production of Chinese hickory tree (Carya cathayensis Sarg.), which is primarily determined by Botryosphaeriaceae. In our previous work, we identified Botryosphaeria dothidea as the predominant pathogen of this disease. However, it is still unclear about corresponding gene families and mechanisms associated with B. dothidea's pathogenicity on Chinese hickory tree. Here, we present a comparative analysis of high-quality genome assemblies of Botryosphaeria dothidea and other isolated pathogens, showing highly syntenic relationships between B. dothidea and its closely related species and the conservative evolution of the Botryosphaeriaceae family. Higher GC contents were found in the genomes of B. dothidea and three other isolated pathogens (Botryshaeria cortices, Botryshaeria fabicerciana, and Botryshaeria qingyuanensis) compared to Macrophomina phaseolina, Neofusicoccum parvum, Diplodia corticola, and Lasiodiplodia theobromae. An investigation of genes specific to or expanded in B. dothidea revealed that one secreted glucanase, one orsellinic acid biosynthesis enzyme, and two MFS transporters positively regulated B. dothidea's pathogenicity. We also observed an overrepresentation of viral integrase like gene and heterokaryon incompatibility proteins in the B. dothidea's genome. In addition, we observed one LRR-domain-containing protein and two Sec-domain-containing proteins (Sec_1 and Sec_7) that underwent positive selection. This study will help to understand B. dothidea's pathogenicity and potential influence on the infection of Chinese hickory, which will help in the development of disease control and ensure the security of Chinese hickory production.

Since the beginning of the twentieth century oak decline has been documented in central and eastern hardwood forests of the United States as a stress-mediated disease (Oak et al. 2016). Opportunistic canker pathogens, including Diplodia corticola, D. quercivora, D. sapinea, and Botryosphaeria dothidea have been associated with crown dieback of declining oak trees in several mid-Atlantic states (Ferreira et al. 2021). On 02 August 2022, a survey was conducted at two natural hardwood sites in Fredrick and Shenandoah Counties, Virginia that exhibited symptoms of decline (Fig. 1A). At both sites, mature Quercus montana trees were observed with bole and branch cankers, bleeding and sooty lesions, and discolored sapwood. Pycnidia were present on the margin of seven branch cankers from three trees that were felled, with hyaline, elliptical to oblong conidia 19.0 - 26.8 × 8.5 - 11.2 µm (n = 40) in size (Fig. 1C and D). Six cultures were derived from single spores that were placed on PDA medium and incubated for 10 days in the dark at 22 ± 2°C. Additionally, a 4-mm piece of necrotic tissue was selected from the margin of each of the seven cankers, disinfected with 2.5% NaOCl, again with 70% ethanol, and air-dried before being placed on half-strength acidified PDA medium (pH 4.8) and incubated in the dark at 22 ± 2°C. After 5 days, seven colonies from each canker assayed were transferred to full-strength PDA plates and incubated for 10 days in the dark at 22 ± 2°C. Colonies derived from spores and the necrotic wood were morphologically identical, with white, aerial, floccose mycelium that turned dark gray to olivaceous after five days (Fig. 1B). DNA was individually extracted from four, 10-day-old cultures (two from spores and two from wood). Mycelia was harvested with a sterile pin and extracted using a Qiagen DNeasy Plant Pro Kit (Germantown, MD) according to the manufacturer's instructions. A segment of the internal transcribed spacer (ITS), large subunit rRNA (LSU), and translation elongation factor 1-α (tef1) loci were amplified using ITS4/ITS5 (White et al. 1990), LR5/LROR (Vilgalys and Hester 1990), and EF1-728F/EF1-986R (Carbone and Kohn 1999) primer sets, respectively. The PCR amplicons were purified with ExoSap-IT (Affymetrix, Santa Clara, CA) and sequenced at Eurofins (Louisville, KY). The nucleotide sequences were analyzed using Geneious 11.1.5 software (Biomatters, Auckland, NZ). The resulting ITS sequences from the four isolates were identical. A 544-bp, 1131-bp, and 273-bp segment of the ITS, LSU, and tef1 loci from isolate GS22-DSB-17 was deposited into the GenBank database (accessions OQ597712, OQ597714, and OR754429, respectively). A Genbank BLAST analysis revealed that the ITS and tef1 fragments shared 510/516 (99%) and 271/273 (99%) nucleotides with the D. quercivora ex-type BL8 (JX894205/JX894229). Koch's postulates were fulfilled by inoculating five healthy, containerized Q. montana trees (average stem caliper 6.5 cm) with D. qercivora isolate GS22-DSB-17, while five plants were used as controls. After disinfecting the bark with 70% ethanol, a 0.5 mm section of the bark was removed 13 cm above the soil line with a sterile scalpel, and a 0.5 mm agar plug taken from the edge of a 10-day-old PDA culture was placed in the wound with the mycelium facing the cambial tissue, sealed with Parafilm, and maintained at 22 ± 6°C. The same procedure was performed on the control plants using sterile PDA plugs. After six weeks the bark was carefully removed, and all five stems treated with D. quercivora had necrotic lesions with a mean canker linear growth ([length+width]/2) of 15.6 mm from the edge of the wound, which was significantly larger (P = 0.001) than the controls (2.3 mm; Fig. 1E-M). Necrotic stem tissue was sampled as previously described, and the isolate recovered was confirmed as D. quercivora based on morphology and 100% ITS sequence homology to isolate GS22-DSB-17. D. quercivora was not recovered from the control plants. In the United States, D. quercivora has been isolated from declining white oak trees in Maryland, Massachusetts, West Virginia, and Florida (Dreaden et al. 2014; Ferreira et al. 2021; Haines et al. 2019). More surveys are needed to understand the host range and distribution of D. quercivora in the United States, as well as the environmental and site factors that impact oak health and predispose trees to infection from opportunistic cankering pathogens.

In recent years, nutmeg (Myristica fragans Houtt.) has attracted considerable attention in the field of phytochemistry due to its diverse array of bioactive compounds. However, the potential application of nutmeg as a biorational for crop protection has been insufficiently explored. This study investigated the constituents of a nutmeg hydroethanolic extract via gas chromatography-mass spectrometry and vibrational spectroscopy. The research explored the extract's activity against phytopathogenic fungi and oomycetes, elucidating its mechanism of action. The phytochemical profile revealed fatty acids (including tetradecanoic acid, 9-octadecenoic acid, n-hexadecanoic acid, dodecanoic acid, and octadecanoic acid), methoxyeugenol, and elemicin as the main constituents. Previously unreported phytochemicals included veratone, gelsevirine, and montanine. Significant radial growth inhibition of mycelia was observed against Botrytis cinerea, Colletotrichum acutatum, Diplodia corticola, Phytophthora cinnamomi, and especially against Fusarium culmorum. Mode of action investigation, involving Saccharomyces cerevisiae labeled positively with propidium iodide, and a mutant strain affected in ERG6, encoding sterol C-24 methyltransferase, suggested that the extract induces a necrotic type of death and targets ergosterol biosynthesis. The evidence presented underscores the potential of nutmeg as a source of new antimicrobial agents, showing particular promise against F. culmorum.

The emergence of new plant diseases is an increasingly important concern. Climate change is likely to be among the factors causing most of the emerging diseases endangering forest and tree heritage around the world. Such diseases may be caused by latent pathogens or microorganisms cryptically associated with plants. The shift from a non-pathogenic to a pathogenic stage may depend on physiological alterations of the host, environmental changes, and/or stress factors. In some woods of the Salento Peninsula (Apulia Region, Italy), sudden declines of holm oak plants (Quercus ilex L.) have been observed since 2016. The morphological and molecular characterization of representative fungal isolates associated with cankers and necrosis in declining plants indicated that these isolates belong to the Botryosphaeriaceae family, and the most frequent species were Diplodia corticola and Diplodia quercivora, followed by Neofusicoccum vitifusiforme. In artificially inoculated young holm oak plants, both D. corticola and D. quercivora species produced intense and severe subcortical and leaf margin necrosis. N. vitifusiforme, although less aggressive, induced the same symptoms. Our research, in addition to confirming the involvement of D. corticola in olm oak decline, represents the first report of D. quercivora as a new pathogen of Q. ilex in Italy. Furthermore, to the best of our knowledge, we also found N. vitifusiforme as a new pathogen of Q. ilex.

Diplodia corticola is a fungal pathogen contributing to oak (Quercus spp.) decline in the Mediterranean and US (Félix et al., 2017; Ferreira et al., 2021). In 2021, this pathogen was detected in Tennessee (TN) causing branch dieback in Q. alba (Onufrak et al., 2022). In September 2021, a matured pin oak (Q. palustris) with wilted leaves and elongated branch cankers was observed in the State Botanical Garden of Tennessee-Knoxville (TN, US). Small sections of the phloem were sampled from canker margins of a symptomatic branch using a sterile scalpel, surface sterilized, and plated onto potato dextrose agar amended with antibiotics (PDA++) (Gazis et al. 2018). Three days later, a fungal isolate resembling D. corticola was cultured on ½ PDA. Diplodia corticola is characterized on half-strength PDA by fast growth, irregular margins, and dense white mycelium that turns dark, grayish as the mycelium matures (Úrbez-Torres et al., 2010; Alves et al., 2004). Total genomic DNA was extracted from this isolate following Gazis et al. (2018), and the internal transcribed spacer (ITS), large ribosomal subunit (LSU), and transcription elongation factor 1-α (ef1-α) were amplified (Ferreira et al. 2021). Resulting PCR products were sequenced and assembled into consensus sequences using Unipro UGENE v. 44.0 (Okonechnikov et al., 2012). Each consensus sequence identity was determined using BLAST on the NCBI nucleotide database, restricted to type material. The ITS (accession OQ189888), ef-1α (accession OQ201608), and LSU (accession OQ189887) sequences had a 99.6% (accession KF766156.1), 98.6% (accession XM_020275852.1), and 100% (accession KF766323.1) identity match with D. corticola type culture CBS112549, respectively. To complete Koch's postulates and assess potential pathogenicity on economically and ecologically relevant oaks, 10 pin (Q. palustris; caliper 15.6 ± 2.0 mm), 10 overcup (Q. lyrata; caliper 15.1 ± 2.4 mm), and 10 sawtooth (Q. acutissima; 16.1 ± 2.1 mm) oaks were acclimated in the greenhouse for 1 week prior to the experiment. Five trees of each species were then randomly inoculated at 30 cm above the soil line with a 3 mm diameter plug of D. corticola (grown for 10 days on PDA; Sitz et al. 2017). To serve as a control, the remaining 5 trees for each species received a 3 mm diameter PDA plug. Fifteen days post-inoculation, seepage was observed in D. corticola-inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks. No seepage from wound sites was noted in control trees. Cankers were exposed, photographed, and then measured using ImageJ (Rasband, 2012). Using a sterile scalpel, four wood chips were excised from canker margins and plated onto PDA++. We recovered D. corticola from symptomatic inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks and confirmed species identity by extracting DNA and amplifying the ITS, ef-1α, and LSU regions as described above (Gazis et al., 2018; Ferreira et al., 2021). The resulting consensus sequences matched the D. corticola type culture (CBS112549) ITS (99.0%-99.8% identity), ef-1α (91.0%-99.1% identity), and LSU (96.9%-100% identity) barcoding regions. Cankers were significantly larger in D. corticola-inoculated pin (4.7 ± 1.5 cm2; P = 0.003), overcup (6.8 ± 2.9 cm2; P = 0.009), and sawtooth (5.1 ± 1.3 cm2; P = 0.001) oaks in comparison to the control trees from these groups. Based on current reports, this is the first record of D. corticola causing dieback in pin oak (Q. palustris) in TN.

Diplodia corticola is one of the most aggressive fungal pathogens of Quercus species involved in the decline of Mediterranean oak forests. In this study, three strains of D. corticola associated with holm (Quercus ilex) and cork (Quercus suber) oak trees exhibiting dieback symptoms and cankers in Algeria were selected to investigate the production of secondary metabolites. Metabolomic analyses revealed the production of several known compounds, such as sphaeropsidins, diplopyrones and diplofuranones. Moreover, the comparative investigation of secondary metabolites produced by the analyzed strains with different degrees of virulence revealed possible implications of these compounds in the fungal virulence. In particular, sphaeropsidins seem to be the main phytotoxic compounds of D. corticola involved in the infections of Quercus species, with a possible synergistic influence of the less representative compounds in the fungal virulence.

In 2016, an almond (Prunus dulcis) decline syndrome (ADS) emerged in intensive almond plantations in the Andalusia region (southern Spain), showing branch dieback, gummosis, and general tree decline. The aim of this work was to elucidate the etiology of this disease complex. For this purpose, surveys were conducted across the Andalusia region, and a wide collection of fungi was recovered from wood samples showing gum and internal discoloration. Representative isolates were selected and identified by sequencing ITS, TEF1, TUB, ACT, LSU, and/or RPB2 genes. The following fungal species were identified to be associated with the disease: Botryosphaeria dothidea, Diplodia corticola, Di. seriata, Dothiorella iberica, Lasiodiplodia viticola, Macrophomina phaseolina, Neofusicoccum mediterraneum, N. parvum, N. vitifusiforme, Diaporthe neotheicola, Dia. rhusicola, Dia. ambigua, Eutypa lata, E. tetragona, Eutypella citricola, Eu. microtheca, Fusarium oxysporum s.l., Pleurostoma richardsiae, Phaeoacremonium iranianum, Pm. krajdenii, Pm. parasiticum, and Cytospora sp. All isolates were tested for pathogenicity by inoculating detached or attached almond shoots. Di. corticola and N. parvum were the most aggressive species, showing the largest lesions and most gummosis in attached shoots. The results suggest that the species belonging to Botryosphaeriaceae play a key role in disease development, while the remaining identified species may act as secondary pathogens or endophytes. However, further research to determine the interaction between all these fungal species and other biotic and abiotic factors in the ADS progress is needed.

Abstract The 2015–2018 outbreak of spongy moth (Lymantria dispar) in southern New England initiated a severe oak decline and mortality event. While defoliation was a primary driver, increased secondary pest and pathogen activity contributed to decline and death. Following this large defoliation event, Diplodia was frequently recovered from northern red oaks (Quercus rubra) with serious outbreaks of twig cankering. Given the many recent reports of Diplodia corticola on oak in eastern North America, it was presumed to be the causal agent. To confirm, a limited survey was conducted from five states in the region (Maine, Massachusetts, New Hampshire, New York, and Vermont). Based on ITS, tef1 and tub2 sequences generated from 28 isolates collected primarily from northern red oak, Diplodia gallae, two potentially novel Diplodia species and Diplodia sapinea were identified. Surprisingly, D. corticola was not found in this study. ITS sequences alone failed to discriminate among isolates of D. gallae and D. corticola, creating uncertainty over previous reports of D. corticola in eastern North America. Only a combined ITS + tef1 dataset successfully distinguished D. gallae and D. corticola along with two other closely related species that also occur on oak (Diplodia quercicola and Diplodia quercivora). Additional cankering and endophytic fungi (Coryneum, Dendrostoma, Gnomoniopsis, Pestalotiopsis and Tubakia) were also found on symptomatic oaks in the region. Identification of Diplodia isolates from non‐Quercus hosts also detected Diplodia neojuniperi on Juniperus chinensis and Microbiota decussata, which has not been reported previously in North America.