Next-generation methods for early disease detection in crops.

Plant pathogens are commonly identified in the field by the typical disease symptoms that they can cause. The efficient early detection and identification of pathogens are essential procedures to adopt effective management practices that reduce or prevent their spread in order to mitigate the negative impacts of the disease. In this review, the traditional and innovative methods for early detection of the plant pathogens highlighting their major advantages and limitations are presented and discussed. Traditional techniques of diagnosis used for plant pathogen identification are focused typically on the DNA, RNA (when molecular methods), and proteins or peptides (when serological methods) of the pathogens. Serological methods based on mainly enzyme-linked immunosorbent assay (ELISA) are the most common method used for pathogen detection due to their high-throughput potential and low cost. This technique is not particularly reliable and sufficiently sensitive for many pathogens detection during the asymptomatic stage of infection. For non-cultivable pathogens in the laboratory, nucleic acid-based technology is the best choice for consistent pathogen detection or identification. Lateral flow systems are innovative tools that allow fast and accurate results even in field conditions, but they have sensitivity issues to be overcome. PCR assays performed on last-generation portable thermocyclers may provide rapid detection results in situ. The advent of portable instruments can speed pathogen detection, reduce commercial costs, and potentially revolutionize plant pathology. This review provides information on current methodologies and procedures for the effective detection of different plant pathogens. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A collection of Trichoderma isolates from natural environments in Sardinia reveals a complex virome that includes negative-sense fungal viruses with unprecedented genome organizations.

Trichoderma genus includes soil-inhabiting fungi that provide important ecosystem services in their interaction with plants and other fungi, as well as biocontrol of fungal plant diseases. A collection of Trichoderma isolates from Sardinia has been previously characterized, but here we selected 113 isolates, representatives of the collection, and characterized their viral components. We carried out high-throughput sequencing of ribosome-depleted total RNA following a bioinformatics pipeline that detects virus-derived RNA-directed RNA polymerases (RdRps) and other conserved viral protein sequences. This pipeline detected seventeen viral RdRps with two of them corresponding to viruses already detected in other regions of the world and the remaining fifteen representing isolates of new putative virus species. Surprisingly, eight of them are from new negative-sense RNA viruses, a first in the genus Trichoderma. Among them is a cogu-like virus, closely related to plant-infecting viruses. Regarding the positive-sense viruses, we report the presence of an 'ormycovirus' belonging to a recently characterized group of bisegmented single-stranded RNA viruses with uncertain phylogenetic assignment. Finally, for the first time, we report a bisegmented member of Mononegavirales which infects fungi. The proteins encoded by the second genomic RNA of this virus were used to re-evaluate several viruses in the Penicillimonavirus and Plasmopamonavirus genera, here shown to be bisegmented and encoding a conserved polypeptide that has structural conservation with the nucleocapsid domain of rhabdoviruses.

Cultivable mycoflora on bleached, decaying and healthy Posidonia oceanica leaves in a warm-edge Mediterranean location.

Marine fungi are widely distributed in the ocean, playing an important role in the ecosystems, but only little information is available about their occurrence and activity. Seagrass bleaching is also a neglected phenomenon that seems to be linked to warm environments, even though the causes are still to be defined. In this study, the cultivable mycoflora associated to the leaf conditions (bleached, necrotic and live) and section (from the base to the tip) in the seagrass Posidonia oceanica was investigated in a Mediterranean warm-edge location (Cyprus). A total of 17 Ascomycota species/taxon were identified and results highlighted that mycoflora composition changed significantly in relation to both the leaf condition and section. A few known pathogens of terrestrial plants were detected only on bleached leaves, but it remains unknown whether they have any direct connections with P. oceanica bleaching phenomenon.

Improved YOLOX-Tiny network for detection of tobacco brown spot disease.

Introduction: Tobacco brown spot disease caused by Alternaria fungal species is a major threat to tobacco growth and yield. Thus, accurate and rapid detection of tobacco brown spot disease is vital for disease prevention and chemical pesticide inputs. Methods: Here, we propose an improved YOLOX-Tiny network, named YOLO-Tobacco, for the detection of tobacco brown spot disease under open-field scenarios. Aiming to excavate valuable disease features and enhance the integration of different levels of features, thereby improving the ability to detect dense disease spots at different scales, we introduced hierarchical mixed-scale units (HMUs) in the neck network for information interaction and feature refinement between channels. Furthermore, in order to enhance the detection of small disease spots and the robustness of the network, we also introduced convolutional block attention modules (CBAMs) into the neck network. Results: As a result, the YOLO-Tobacco network achieved an average precision (AP) of 80.56% on the test set. The AP was 3.22%, 8.99%, and 12.03% higher than that obtained by the classic lightweight detection networks YOLOX-Tiny network, YOLOv5-S network, and YOLOv4-Tiny network, respectively. In addition, the YOLO-Tobacco network also had a fast detection speed of 69 frames per second (FPS). Discussion: Therefore, the YOLO-Tobacco network satisfies both the advantages of high detection accuracy and fast detection speed. It will likely have a positive impact on early monitoring, disease control, and quality assessment in diseased tobacco plants.

Evaluation of Agronomic Characteristics, Disease Incidence, Yield Performance, and Aflatoxin Accumulation among Six Peanut Varieties (Arachis hypogea L.) Grown in Kenya.

Diseases contribute to attainment of less than 50% of the local groundnut potential yield in Kenya. This study aimed to evaluate the agronomic characteristics (flowering and germination), disease incidence, yield performance (biomass, harvest index, 100-pod, 100-seed, and total pod weight), and aflatoxin accumulation in six peanut varieties. A field experiment was conducted using four newly improved peanut varieties: CG9, CG7, CG12, and ICGV-SM 90704 (Nsinjiro), and two locally used varieties: Homabay local (control) and 12991, and in a randomized complete block design with three replications. The disease identification followed the International Crop Research Institute for the Semi-Arid Tropics (ICRISAT) rating scale and further isolation of fungal contaminants was conducted by a direct plating technique using potato dextrose agar. The aflatoxin levels in the peanuts were determined after harvesting using the ultrahigh performance liquid chromatography and fluorescence detection (UHPLC-FLD) technique. ICGV-SM 90704 showed the least average disease incidence of 1.31 ± 1.75%, (P < 0.05); the lowest total aflatoxin levels (1.82 ± 1.41 µg kg-1) with a range 0.00-0.85 µg kg-1 for total aflatoxins and a range 0.00-1.24 µg kg-1 for Aflatoxin B1. The locally used varieties (12991 and the control) revealed the highest disease incidence (5.41 ± 8.31% and 7.41 ± 1.88%), respectively. ICGV-SM 90704 was the best performing among all the six varieties with an average total pod weight (9.22 ± 1.19 kg), 100-pod weight (262.93 ± 10.8 g), and biomass of (27.21 ± 5.05 kg) per row. The 12991 variety and the control showed the least total pod weight (1.60 ± 0.28 and 1.50 ± 1.11 kg, respectively) (P = 0.0001). The newly improved varieties showed lower disease rates, low levels of aflatoxins, and higher yields than the locally used varieties.

Different diagnostic approaches for the characterization of the fungal community and Fusarium species complex composition of Italian durum wheat grain and correlation with secondary metabolite accumulation.

BACKGROUND: The evolution of the fungal communities associated with durum wheat was assessed using different diagnostic approaches. Durum wheat grain samples were collected in three different Italian cultivation macro-areas (north, center and south). Fungal isolation was realized by potato dextrose agar (PDA) and by deep-freezing blotter (DFB). Identification of Fusarium isolates obtained from PDA was achieved by partial tef1α sequencing (PDA + tef1α), while those obtained from DFB were identified from their morphological characteristics (DFB + mc). The fungal biomass of eight Fusarium species was quantified in grains by quantitative polymerase chain reaction (qPCR). Fungal secondary metabolites were analyzed in grains by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Correlations between Fusarium detection techniques (PDA + tef1α; DFB + mc and qPCR) and mycotoxins in grains were assessed. RESULTS: Alternaria and Fusarium showed the highest incidence among the fungal genera developed from grains. Within the Fusarium community, PDA + tef1α highlighted that F. avenaceum and F. graminearum were the most represented members, while, DFB + mc detected a high presence of F. proliferatum. Alternaria and Fusarium mycotoxins, principally enniatins, were particularly present in the grain harvested in central Italy. Deoxynivalenol was mainly detected in northern-central Italy. CONCLUSIONS: The adoption of the different diagnostic techniques of Fusarium detection highlighted that, for some species, qPCR was the best method of predicting their mycotoxin contamination in grains. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Perfume Guns: Potential of Yeast Volatile Organic Compounds in the Biological Control of Mycotoxin-Producing Fungi.

Pathogenic fungi in the genera Alternaria, Aspergillus, Botrytis, Fusarium, Geotrichum, Gloeosporium, Monilinia, Mucor, Penicillium, and Rhizopus are the most common cause of pre- and postharvest diseases of fruit, vegetable, root and grain commodities. Some species are also able to produce mycotoxins, secondary metabolites having toxic effects on human and non-human animals upon ingestion of contaminated food and feed. Synthetic fungicides still represent the most common tool to control these pathogens. However, long-term application of fungicides has led to unacceptable pollution and may favour the selection of fungicide-resistant mutants. Microbial biocontrol agents may reduce the incidence of toxigenic fungi through a wide array of mechanisms, including competition for the ecological niche, antibiosis, mycoparasitism, and the induction of resistance in the host plant tissues. In recent years, the emission of volatile organic compounds (VOCs) has been proposed as a key mechanism of biocontrol. Their bioactivity and the absence of residues make the use of microbial VOCs a sustainable and effective alternative to synthetic fungicides in the management of postharvest pathogens, particularly in airtight environments. In this review, we will focus on the possibility of applying yeast VOCs in the biocontrol of mycotoxigenic fungi affecting stored food and feed.

Bioprospecting Phenols as Inhibitors of Trichothecene-Producing Fusarium: Sustainable Approaches to the Management of Wheat Pathogens.

Fusarium spp. are ubiquitous fungi able to cause Fusarium head blight and Fusarium foot and root rot on wheat. Among relevant pathogenic species, Fusarium graminearum and Fusarium culmorum cause significant yield and quality loss and result in contamination of the grain with mycotoxins, mainly type B trichothecenes, which are a major health concern for humans and animals. Phenolic compounds of natural origin are being increasingly explored as fungicides on those pathogens. This review summarizes recent research activities related to the antifungal and anti-mycotoxigenic activity of natural phenolic compounds against Fusarium, including studies into the mechanisms of action of major exogenous phenolic inhibitors, their structure-activity interaction, and the combined effect of these compounds with other natural products or with conventional fungicides in mycotoxin modulation. The role of high-throughput analysis tools to decipher key signaling molecules able to modulate the production of mycotoxins and the development of sustainable formulations enhancing potential inhibitors' efficacy are also discussed.

Molecular Docking and Comparative Inhibitory Efficacy of Naturally Occurring Compounds on Vegetative Growth and Deoxynivalenol Biosynthesis in Fusarium culmorum.

The fungal pathogen Fusarium culmorum causes Fusarium head blight in cereals, resulting in yield loss and contamination of the grain by type B trichothecene mycotoxins such as deoxynivalenol (DON), and its acetylated derivatives. Synthesis of trichothecenes is driven by a trichodiene synthase (TRI5) that converts farnesyl pyrophosphate (FPP) to trichodiene. In this work, 15 naturally occurring compounds that belong to the structural phenol and hydroxylated biphenyl classes were tested in vitro and in planta (durum wheat) to determine their inhibitory activity towards TRI5. In vitro analysis highlighted the fungicidal effect of these compounds when applied at 0.25 mM. Greenhouse assays showed a strong inhibitory activity of octyl gallate 5, honokiol 13 and the combination propyl gallate 4 + thymol 7 on trichothecene biosynthesis. Docking analyses were run on the 3D model of F. culmorum TRI5 containing the inorganic pyrophosphate (PPi) or FPP. Significant ligand affinities with TRI-PPi and TRI-FPP were observed for the same sites for almost all compounds, with 1 and 2 as privileged sites. Octyl gallate 5 and honokiol 13 interacted almost exclusively with sites 1 and 2, by concurrently activating strong H-bonds with common sets of amino acids. These results open new perspectives for the targeted search of naturally occurring compounds that may find practical application in the eco-friendly control of FHB in wheat.

Prenylated Trans-Cinnamic Esters and Ethers against Clinical Fusarium spp.: Repositioning of Natural Compounds in Antimicrobial Discovery.

Onychomycosis is a common nail infection mainly caused by species belonging to the F. oxysporum, F. solani, and F. fujikuroi species complexes. The aim of this study was to evaluate the in vitro susceptibility of six representative strains of clinically relevant Fusarium spp. toward a set of natural-occurring hydroxycinnamic acids and their derivatives with the purpose to develop naturally occurring products in order to cope with emerging resistance phenomena. By introducing a prenylated chain at one of the hydroxy groups of trans-cinnamic acids 1-3, ten prenylated derivatives (coded 4-13) were preliminarily investigated in solid Fusarium minimal medium (FMM). Minimal inhibitory concentration (MIC) and lethal dose 50 (LD50) values were then determined in liquid FMM for the most active selected antifungal p-coumaric acid 3,3'-dimethyl allyl ester 13, in comparison with the conventional fungicides terbinafine (TRB) and amphotericin B (AmB), through the quantification of the fungal growth. Significant growth inhibition was observed for prenylated derivatives 4-13, evidencing ester 13 as the most active. This compound presented MIC and LD50 values (62-250 µM and 7.8-125 µM, respectively) comparable to those determined for TRB and AmB in the majority of the tested pathogenic strains. The position and size of the prenylated chain and the presence of a free phenol OH group appear crucial for the antifungal activity. This work represents the first report on the activity of prenylated cinnamic esters and ethers against clinical Fusarium spp. and opens new avenues in the development of alternative antifungal compounds based on a drug repositioning strategy.

Naturally Occurring Phenols Modulate Vegetative Growth and Deoxynivalenol Biosynthesis in Fusarium graminearum.

To assess the in vitro activity of five naturally occurring phenolic compounds (ferulic acid, apocynin, magnolol, honokiol, and thymol) on mycelial growth and type B trichothecene mycotoxin accumulation by Fusarium graminearum, three complementary approaches were adopted. First, a high-throughput photometric continuous reading array allowed a parallel quantification of F. graminearum hyphal growth and reporter TRI5 gene expression directly on solid medium. Second, RT-qPCR confirmed the regulation of TRI5 expression by the tested compounds. Third, liquid chromatography-tandem mass spectrometry analysis allowed quantification of deoxynivalenol (DON) and its acetylated forms released upon treatment with the phenolic compounds. Altogether, the results confirmed the activity of thymol and an equimolar mixture of thymol-magnolol at 0.5 mM, respectively, in inhibiting DON production without affecting vegetative growth. The medium pH buffering capacity after 72-96 h of incubation is proposed as a further element to highlight compounds displaying trichothecene inhibitory capacity with no significant fungicidal effect.

Honokiol, magnolol and its monoacetyl derivative show strong anti-fungal effect on Fusarium isolates of clinical relevance.

The antifungal activity of magnolol and honokiol, two naturally occurring hydroxylated biphenyls, and of their synthetic derivatives was evaluated on a collection of representative isolates of Fusarium oxysporum, F. solani and F. verticillioides of clinical and ecological concern. The tested compounds were proposed as a 'natural' alternative to conventional fungicides, even though a larger range of concentrations (5-400 µg/ml) was applied. The activity of magnolol and honokiol was compared with that of terbinafine (0.1-10 µg/ml), and fluconazole (1-50 µg/ml), two fungicides widely used in treating fungal infections on humans. Magnolol showed similar fungicidal activity compared to fluconazole, whereas honokiol was more effective in inhibiting mycelium growth compared to this fungicide on all tested clinical Fusarium spp. isolates. Compared to terbinafine, honokiol showed similar antifungal activity when tested on clinical F. solani isolates, whereas magnolol was less effective at all selected concentrations (5-400 µg/ml). The different position of the phenol-OH group, as well as its protection, explain different in vitro activities between magnolol, honokiol, and their derivatives. Furthermore, magnolol showed mycelium dry weight reduction at a concentration of 0.5 mM when tested on a set of agricultural isolates of Fusaria, leading to complete inhibition of some of them. Magnolol and honokiol are proposed as efficient and safe candidates for treating clinically relevant Fusaria.

FcRav2, a gene with a ROGDI domain involved in Fusarium head blight and crown rot on durum wheat caused by Fusarium culmorum.

Fusarium culmorum is a soil-borne fungal pathogen which causes foot and root rot and Fusarium head blight on small-grain cereals, in particular wheat and barley. It causes significant yield and quality losses and results in the contamination of kernels with type B trichothecene mycotoxins. Our knowledge of the pathogenicity factors of this fungus is still limited. A transposon tagging approach based on the mimp1/impala double-component system has allowed us to select a mutant altered in multiple metabolic and morphological processes, trichothecene production and virulence. The flanking regions of mimp1 were used to seek homologies in the F. culmorum genome, and revealed that mimp1 had reinserted within the last exon of a gene encoding a hypothetical protein of 318 amino acids which contains a ROGDI-like leucine zipper domain, supposedly playing a protein-protein interaction or regulatory role. By functional complementation and bioinformatic analysis, we characterized the gene as the yeast Rav2 homologue, confirming the high level of divergence in multicellular fungi. Deletion of FcRav2 or its orthologous gene in F. graminearum highlighted its ability to influence a number of functions, including virulence, trichothecene type B biosynthesis, resistance to azoles and resistance to osmotic and oxidative stress. Our results indicate that the FcRav2 protein (and possibly the RAVE complex as a whole) may become a suitable target for new antifungal drug development or the plant-mediated resistance response in filamentous fungi of agricultural interest.