Plant Pathogens Research Articles

Genome-wide identification and expression analysis of the U-box gene family related to biotic and abiotic stresses in Coffea canephora L.

Plant U-box genes play an important role in the regulation of plant hormone signal transduction, stress tolerance, and pathogen resistance; however, their functions in coffee (Coffea canephora L.) remain largely unexplored. In this study, we identified 47 CcPUB genes in the C. canephora L. genome, clustering them into nine groups via phylogenetic tree. The CcPUB genes were unevenly distributed across the 11 chromosomes of C. canephora L., with the majority (11) on chromosome 2 and none on chromosome 8. The cis-acting elements analysis showed that CcPUB genes were involved in abiotic and biotic stresses, phytohormone responsive, and plant growth and development. RNA-seq data revealed diverse expression patterns of CcPUB genes across leaves, stems, and fruits tissues. qRT-PCR analyses under dehydration, low temperature, SA, and Colletotrichum stresses showed significant up-regulation of CcPUB2, CcPUB24, CcPUB34, and CcPUB40 in leaves. Furthermore, subcellular localization showed CcPUB2 and CcPUB34 were located in the plasma membrane and nucleus, and CcPUB24 and CcPUB40 were located in the nucleus. This study provides valuable insights into the roles of PUB genes in stress responses and phytohormone signaling in C. canephora L., and provided basis for functional characterization of PUB genes in C. canephora L.

Phylogeographic analysis of Begomovirus coat and replication-associated proteins.

Begomoviruses are globally distributed plant pathogens that significantly limit crop production. These viruses are traditionally described according to phylogeographic distribution and categorized into two groups: begomoviruses from the Africa, Asia, Europe and Oceania (AAEO) region and begomoviruses from the Americas. Monopartite begomoviruses are more common in the AAEO region, while bipartite viruses predominate in the Americas, where the begomoviruses lack the V2/AV2 gene involved in inter-cellular movement and RNA silencing suppression found in AAEO begomoviruses. While these features are generally accepted as lineage-defining, the number of known species has doubled due to sequence-based discovery since 2010. To re-evaluate the geographic groupings after the rapid expansion of the genus, we conducted phylogenetic analyses for begomovirus species representatives of the two longest and most conserved begomovirus proteins: the coat and replication-associated proteins. Both proteins still largely support the broad AAEO and Americas begomovirus groupings, except for sweet potato-infecting begomoviruses that form an independent, well-supported clade for their coat protein regardless of the region they were isolated from. Our analyses do not support more fine-scaled phylogeographic groupings. Monopartite and bipartite genome organizations are broadly interchanged throughout the phylogenies, and the absence of the V2/AV2 gene is highly reflective of the split between Americas and AAEO begomoviruses. We observe significant evidence of recombination within the Americas and within the AAEO region but rarely between the regions. We speculate that increased globalization of agricultural trade, the invasion of polyphagous whitefly vector biotypes and recombination will blur begomovirus phylogeographic delineations in the future.

Molecular Identification and Genetic Diversity Analysis of Papaya Leaf Curl China Virus Infecting Ageratum conyzoides.

Papaya leaf curl China virus (PaLCuCNV) is a damaging plant pathogen causing substantial losses to crop. The complete genomes of three PaLCuCNV isolates from Ageratum conyzoides were obtained and combined with the 68 reference isolates in GenBank for comprehensive genetic diversity analyses using specialized computational tools. Sequence alignment revealed nucleotide sequence similarity ranging from 85.3% to 99.9% among 71 PaLCuCNV isolates. Employing phylogenetic analysis, 71 PaLCuCNV sequences were clustered into five groups, with no significant correlation observed between genetic differentiation and either host species or geographical origin. Additionally, 13 recombination events across all PaLCuCNV isolates were identified. Genetic diversity analysis indicated the ongoing expansion and evolution of PaLCuCNV populations, supported by a neutral model. Moreover, significant genetic differentiation was observed among distinct viral populations, primarily attributed to genetic drift. Overall, our findings provide valuable insights into the detection, genetic variation, and evolutionary dynamics of PaLCuCNV.

Phytochemistry and Pharmacological Activity of Malva sylvestris L: A Detailed Insight.

Malva sylvestris L., is commonly referred to as Mallow and is found in Europe, Asia and Africa. This has been traditionally used for inflammation, gastrointestinal disturbances, skin disorders, menstrual pains, and urological disorders. This review covers phytoconstituents and Pharmacological activities of M. sylvestris. The plant contains a large number of phytochemical constituents having diverse pharmacological activities. The plant contains many phenolic compounds responsible for its strong antioxidant activity. Coumarins from Mallow have a potential anticancer activity. Malva sylvestris also contains essential as well as non-essential elements and minerals. Many researchers have provided evidence that Malva sylvestris is a good candidate for use as a medicinal herb and has good nutritional value. The leaves, in particular, offer properties like anticancer, skin whitening, and anti-aging. Furthermore, the aqueous extract was recently shown to have an anti-ulcerogenic effect. Malva sylvestris has a high potential for use in cosmetics such as skin whitening and anti-aging treatments. Methanolic extracts of Malva sylvestris leaves, and flowers showed strong antibacterial activity against a common plant pathogen bacterium. The plant also contains Malvone A, which is responsible for antibacterial action. The plant also possesses anti-inflammatory, analgesic, wound healing properties and various other activities.

Shift in potential pathogenic bacteria during permafrost degradation on the Qinghai-Tibet Plateau

Permafrost acts as a potential pathogen reservoir. With accelerating climate change and intensifying permafrost degradation, the release of these pathogens poses significant threats to ecosystems and public health. However, the changes in pathogenic communities during permafrost degradation remain unclear. This study utilized quantitative PCR and Illumina high-throughput sequencing to analyze the composition and quantities of potential pathogenic bacteria in four types of permafrost soil on the northeast edge of the Qinghai-Tibet Plateau (QTP): sub-stable permafrost (SSP), transition permafrost (TP), unstable permafrost (UP), and extremely unstable permafrost (EUP). The results showed that during permafrost degradation, the quantity of potential pathogenic bacteria decreased from 7.8 × 106 to 3.1 × 106 copies/g. Both the Richness and Shannon indices initially declined from SSP, to TP, UP, and then began to rise when permafrost degraded to EUP. A total of 216 potential pathogenic bacterial species were identified, including 166 animal pathogens, 28 zoonotic pathogens, and 22 plant pathogens. The pathogenic community intergroup differences (ANOSIM), unique taxa, and dominant pathogen analysis indicated the significant changes in pathogenic communities during permafrost degradation. The potential pathogenic community was significantly influenced by non-pathogenic bacterial communities (Procrustes analysis), with soil moisture being the primary environmental factor, followed by TDS, soil organic carbon, and total nitrogen. SourceTracker2 analysis indicated that the majority of potential pathogenic bacteria in the soil originated from external sources, only a small portion coming from the permafrost itself. These findings suggest that a large number of pathogens were released into the environment while also preserving amount from external sources. It elucidates that each stage of permafrost degradation presents unique biosecurity risks. This study highlights the release and redistribution of pathogenic bacteria associated with the potential public health risks. It provides the crucial insights into the ecological dynamics of permafrost degradation, emphasizing the need for ongoing monitoring and proactive management strategies.

Application of loop-mediated isothermal amplification (LAMP) in plant pathogen detection.

Plant diseases impact the production of all kinds of crops, resulting in significant economic losses worldwide. Timely and accurate detection of plant pathogens is crucial for surveillance and management of plant diseases. In recent years, loop-mediated isothermal amplification (LAMP) has become a popular method for pathogen detection and disease diagnosis due to the advantages of its simple instrument requirement and constant reaction temperature. In this review, we provide an overview of current research on LAMP, including the reaction system, design of primers, selection of target regions, visualization of amplicons, and application of LAMP on the detection of all major groups of plant pathogens. We also discuss plant pathogens for which LAMP is yet to be developed, potential improvements of plant disease diagnosis, and disadvantages that need to be considered.

Spectral Intelligence: AI-Driven Hyperspectral Imaging for Agricultural and Ecosystem Applications

Ensuring global food security amid mounting challenges, such as population growth, disease infestations, resource limitations, and climate change, is a pressing concern. Anticipated increases in food demand add further complexity to this critical issue. Plant pathogens, responsible for substantial crop losses (up to 41%) in major crops like wheat, rice, maize, soybean, and potato, exacerbate the situation. Timely disease detection is crucial, yet current practices often identify diseases at advanced stages, leading to severe infestations. To address this, remote sensing and Hyperspectral imaging (HSI) have emerged as robust and nondestructive techniques, exhibiting promising results in early disease identification. Integrating machine learning algorithms with image data sets enables precise spatial–temporal disease identification, facilitating timely detection, predictive modeling, and effective disease management without compromising fitness or climate adaptability. By harnessing these cutting-edge technologies and data-driven decision-making, growers can optimize input costs while achieving enhanced yields, making significant strides toward global food security in the face of climate change risks. This review will discuss some of the foundational concepts of remote sensing, several platforms used for remote sensing data collection, successful application of the approach, and its future perspective.

Transient Expression of Nicotiana tabacum Silicon-Induced Histidine-Rich Defensins in Nicotiana benthamiana Limits Necrotic Lesion Development Caused by Phytopathogenic Fungi.

Silicon (Si) supplementation permits plants to better deter infection. Supplementing hydroponically-propagated Nicotiana tabacum with 1 mM potassium silicate (K2SiO3) reduced necrotic lesion development on detached leaves by both Botrytis cinerea and Sclerotinia sclerotiorum. Previously, a family of Si-induced genes was identified in N. tabacum. These genes were members of the Solanaceous Histidine-Rich Defensin (HRD) superfamily and were termed NtHRD1s (the first identified family of Nicotiana tabacum Histidine-Rich Defensins). Defensins were originally identified to participate in innate immunity. Thus, the NtHRD1s were tested for antimicrobial effects on plant pathogens. Transient expression of NtHRD1 genes within Nicotiana benthamiana leaves restricted the development of necrotic lesions caused by B. cinerea and S. sclerotiorum. Thus, the NtHRD1s may be an additional Si-responsive factor conferring beneficial effects on plants.

Profiling the biocontrol agents, nitrogen-fixing bacteria, and indole acetic acid-producing bacteria in anthill soil

Due to the adverse ecological impacts of prolonged and excessive use of agrochemicals, many researchers have called for the adoption of eco-friendly materials in farming. Consequently, the aim of this study is to profile the biocontrol agents, nitrogen-fixing, and indole-acetic-acid-producing bacteria present in anthill soil. Anthill soils and adjacent soils were collected, and their physicochemical properties were analysed using standard analytical methods. Viable bacteria were isolated and screened for plant growth-promoting (PGP) activity using standard biochemical, morphological, and bacteriological methods. The PGP capacity of the isolates was evaluated using standard protocol for nitrogen fixation and indole-acetic-acid production, while antagonistic effect against plant pathogens was evaluated using the disk diffusion assay. Results revealed that Pseudomonas aeruginosa, Klebsiella pneumoniae, and Bacillus sp. isolated from the anthill soil demonstrated PGP characteristics including IAA production and nitrogen fixation. Bacillus sp. exhibited zones of clearance with values of 20.00 ± 1.50 mm, 20.00 ± 1.75 mm, 17.00 ± 1.33 mm, and 17.00 ± 1.33 mm against Aspergillus niger, Trichoderma sp., Penicillium sp., and Fusarium sp., respectively. This study demonstrated that anthill soils contain beneficial microbes with the potential to stimulate plant growth and suppress soil-borne plant pathogens.

The evolution of plant responses underlying specialized metabolism in host-pathogen interactions.

In the course of plant evolution from aquatic to terrestrial environments, land plants (embryophytes) acquired a diverse array of specialized metabolites, including phenylpropanoids, flavonoids and cuticle components, enabling adaptation to various environmental stresses. While embryophytes and their closest algal relatives share candidate enzymes responsible for producing some of these compounds, the complete genetic network for their biosynthesis emerged in embryophytes. In this review, we analysed genomic data from chlorophytes, charophytes and embryophytes to identify genes related to phenylpropanoid, flavonoid and cuticle biosynthesis. By integrating published research, transcriptomic data and metabolite studies, we provide a comprehensive overview on how these specialized metabolic pathways have contributed to plant defence responses to pathogens in non-vascular bryophytes and vascular plants throughout evolution. The evidence suggests that these biosynthetic pathways have provided land plants with a repertoire of conserved and lineage-specific compounds, which have shaped immunity against invading pathogens. The discovery of additional enzymes and metabolites involved in bryophyte responses to pathogen infection will provide evolutionary insights into these versatile pathways and their impact on environmental terrestrial challenges.This article is part of the theme issue 'The evolution of plant metabolism'.

Development of a Multiplex Polymerase Chain Reaction Assay for Detecting Five Previously Unreported Papaya Viruses for Quarantine Purposes in Korea

There are concerns about the introduction and spread of plant pests and pathogens with globalization and climate change. As commercial control agents have not been developed for plant viruses, it is important to prevent virus spread. In this study, we developed a multiplex polymerase chain reaction (PCR) detection method to rapidly diagnose and control three DNA (papaya golden mosaic virus, Lindernia anagallis yellow vein virus, and melon chlorotic leaf curl virus) and two RNA (papaya leaf distortion mosaic virus and lettuce chlorosis virus) viruses that infect papaya. Specific primer sets were designed for the virus coat protein. Performing PCR, clear bands were observed with no non-specific reaction. Our multiplex PCR method can simultaneously detect small amounts of DNA/RNA to diagnose five viruses infecting papaya and prevent the spread of the virus.

Antibacterial Properties of Plant Extracts against the Pathogen of Rosaceae Plants Erwinia amylovora

Erwinia amylovora is a devastating pathogen of plants of the family Rosaceae causing blight on the leaves, shoots, and flowers. Orchard trees suffer the most, however, other plants, like roses, strawberries, raspberries, and hawthorn, can also be affected. Due to the extreme use of chemicals for plant protection, alternatives like plant extracts have been studied recently. Some of the research shows a good potential to control plant diseases, however, only a symbolic number of plant species have been tested, and also, on a restricted number of pathogens. The information on the activity of plant extracts against E. amylovora is scarce. In this study, we prepared plant extracts from fifteen plant species and tested them against the plant pathogenic bacteria E. amylovora. The products were prepared by Soxhlet extraction with methanol and the following concentration according to an established procedure. The finished products were diluted and tested for antibacterial activity in different concentrations in vitro against seven reference strains of E. am¬ylovora, isolated from different hosts, such as pear, apple, quince, aronia, hawthorn, strawberry, and musk strawberry. Extracts from the roots of Geranium macrorrhizum and fruits of Chaenomeles sp. genotypes showed promising potentialities for further experiments on the control of E. amylovora.

OBTAINING TRANSGENIC POTATO PLANTS FOR COUNTERING PHYTOPHTHORA INFESTANS VIA “HIGS” METHOD IMPLEMENTATION

Oomycetes are a group of organisms that include some of the most dangerous plant pathogens capable of causing epiphytoties that lead to reduced crop yields and famine in some countries. The only available method of combating them – repeated application of fungicides in fields and careful selection of seed material – is ineffective for most of these pathogens. This paper proposes to use the HIGS (host-induced gene silencing) approach so that genetically modified plants, when infected with late blight, would cause the pathogen to silence its effector protein genes, which would prevent the pathogen from growing and reproducing in plant. To implement the HIGS method against the pathogen Phytophthora infestans, we developed two genetically engineered constructs, one of which uses the P. infestans RXLR-effector gene AVR3a-b as the RNA interference target, and the other uses a region of the PITG_03155 effector gene, which is conserved for both P. infestans and P. cactorum. After Agrobacterium-mediated genetic transformation of potato plants, we obtained four transgenic plants carrying a construct based on the AVR3a-b gene (two each of the “Milena” and “Gala” varieties) and one of “Milena” variety carrying a construct based on the PITG_03155 gene. These plants will be micro clonally propagated and tested for the presence of interfering RNA to the effector genes of P. infestans, after which the plant lines are planned to be planted under natural pathogenic background conditions and a comparative analysis with control plants for disease incidence and resistance will be conducted.

Phytophthora zoospores display klinokinetic behaviour in response to a chemoattractant.

Microswimmers are single-celled bodies powered by flagella. Typical examples are zoospores, dispersal agents of oomycete plant pathogens that are used to track down hosts and infect. Being motile, zoospores presumably identify infection sites using chemical cues such as sugars, alcohols and amino acids. With high-speed cameras we traced swimming trajectories of Phytophthora zoospores over time and quantified key trajectory parameters to investigate chemotactic responses. Zoospores adapt their native run-and-tumble swimming patterns in response to the amino acid glutamic acid by increasing the rate at which they turn. Simulations predict that tuneable tumble frequencies are sufficient to explain zoospore aggregation, implying positive klinokinesis. Zoospores thus exploit a retention strategy to remain at the plant surface once arriving there. Interference of G-protein mediated signalling affects swimming behaviour. Zoospores of a Phytophthora infestans G⍺-deficient mutant show higher tumbling frequencies but still respond and adapt to glutamic acid, suggesting chemoreception to be intact.

The identification of a key gene highlights macrocyclic ring’s role in trichothecene toxicity

Trichothecenes are toxins produced by certain species from several fungal genera, including Aspergillus, Fusarium, Isaria, Paramyrothecium, Stachybotrys, Trichoderma, and Trichothecium. These toxins are of interest because they contribute to the toxigenicity, plant pathogenicity, and/or biological control activities of some fungi. All trichothecenes have the same core (12,13-epoxytrichothec-9-ene or EPT) structure but can differ from one another by the presence or absence of a macrocyclic ring formed from polyketide and isoprenoid substituents esterified to carbon atoms 4 and 15 of EPT, respectively. Genes required for formation and some modifications of EPT have been elucidated, but almost nothing is known about genes specific to the formation of the macrocyclic ring. Therefore, we used genomic, transcriptomic, metabolomic, and gene deletion analyses to identify genes that are required specifically for the formation of the macrocyclic ring. These analyses identified one gene, TRI24, that is predicted to encode an acyltransferase and that is required for macrocyclic ring formation during biosynthesis of macrocyclic trichothecenes by the fungus Paramyrothecium roridum. In addition, a TRI24 deletion mutant of P. roridum caused less severe disease symptoms on common bean and had less antifungal activity than its wild-type progenitor strain. We propose that the reduced aggressiveness and antifungal activity of the mutant resulted from its inability to produce trichothecenes with a macrocyclic ring. To our knowledge, this is the first report of a gene required specifically for the formation of the macrocyclic ring of trichothecenes and that loss of the macrocyclic ring of trichothecenes can alter the biological activities of a fungus.Key points• TRI24 gene is found in all known macrocyclic trichothecene-producing fungi.• A tri24-deletion mutant exhibits a reduction in antifungal and plant disease activities.• TRI24 is the first described gene specific to macrocyclic trichothecene biosynthesis.Graphical abstract

Isolation, diagnosis, and antibiotic resistance of the plant pathogen Erwinia tracheiphila

This study was conducted with the aim of identifying the cause of bacterial wilt disease in cucurbits, which is considered one of the most threatening diseases for melon and cucumber production because it causes great losses that can reach up to 75% of the annual production, and the study proved that Erwinia tracheiphila is the main cause of this disease after isolating (50) isolates of it from melons and cucumbers that showed symptoms of infection and performing several chemical tests to diagnose it. (50) isolates were isolated from melons and cucumbers that showed symptoms of infection, and several chemical tests were conducted to diagnose it, and the effect of (6) different types of antibiotics (rifampin, cefotazima, piperacillin, ceftriaxone, tobramycin and ciprofloxacin) on it was studied by the sensitivity test method. The antibiotic ciprofloxacin gave the highest inhibition value, as indicated by the diameter of the inhibition zone around the disc, which reached (35) mm, followed by tobramycin (20) mm and piperacillin (15) mm, while other antibiotics were ineffective due to resistance. This result indicates that this bacterium is resistant to several drugs, but at the same time it can be treated with antibiotics that are sensitive to them, which was indicated in this study for the purpose of controlling its infection of plants, especially cucurbits. The results of this study are therefore very important in terms of promoting the overcoming of its natural resistance to many antibiotics, which limits the possibility of controlling it when resistant strains appear in a particular country.

Citrus exosome-modified exogenous dsRNA delivery reduces plant pathogen resistance and mycotoxin production

Plant-derived exosome-like nanoparticles (PENs) are crucial for intercellular communication. However, PEN-based transport of pathogenic fungal genes remains unclear. This study isolated and purified PENs from lane late navel orange citrus juice by following the sucrose gradient ultracentrifugation technique. Citrus PENs were round and oval-shaped with an average size of 154.5 ± 1.9 nm. Electroporation-based exogenous dsRNA to PENs loading efficiency remained at 6.0 %. Laser confocal microscopy was employed to investigate citrus PEN uptake by fungal spores. dsCrcB loaded PENs inhibited the CrcB gene expression in spores to alleviate Penicillium italicum resistance against prochloraz fungicide, which promoted resistant strains' mortality by 10-fold. Moreover, dsFUM21-loaded PENs suppressed the FUM21 gene expression in spores, which significantly reduced FB1 production in Fusarium proliferatum. These findings suggest that citrus PENs could potentially serve as nano-carriers to counter fungicide resistance and mycotoxin production in pathogenic plant fungi.

Manipulation of juvenile hormone signaling by the fire blight pathogen Erwinia amylovora mediates fecundity enhancement of pear psylla.

In nature, plant pathogens often rely on insect vectors for transmission. Through long-term evolution, plant pathogens and insect vectors have established a mutually beneficial symbiotic relationship. Fire blight, caused by the Gram-negative bacterium Erwinia amylovora (Eam), poses a significant global threat to apple and pear production due to its rapid dissemination among host plants of the Rosaceae family. Despite evidence of E. amylovora transmission by various insects, the association between this pathogen and the pear psylla Cacopsylla chinensis, a common vector insect in pear orchards, remains unclear. Sampling investigations and qRT-PCR results revealed that C. chinensis, from 11 pear orchards severely affected by fire blight disease in Xinjiang of China, harbored varying levels of this pathogen. Eam-positive females exhibited significantly higher fecundity compared to Eam-negative individuals, displaying accelerated ovarian development and a notable increase in egg production. Further RNAi results revealed that juvenile hormone (JH) receptor methoprene-tolerant (CcMet) and a crucial downstream gene Krüppel-homologue 1 (CcKr-h1) mediated the fecundity improvement of C. chinensis induced by Eam. Additionally, miR-2b, which targets CcKr-h1, was identified as being involved in Eam-induced fecundity enhancement in C. chinensis. This study unveils, for the first time, that Eam colonize and amplify the fecundity of C. chinensis females. Host miR-2b targets CcKr-h1 of the JH signaling pathway to regulate the heightened fecundity of C. chinensis induced by Eam. These findings not only broaden our understanding of the interaction between plant pathogens and insect vectors, but also provide novel strategies for managing fire blight and pear psylla. © 2024 Society of Chemical Industry.

Conservation of molecular responses upon viral infection in the non-vascular plant Marchantia polymorpha

After plants transitioned from water to land around 450 million years ago, they faced novel pathogenic microbes. Their colonization of diverse habitats was driven by anatomical innovations like roots, stomata, and vascular tissue, which became central to plant-microbe interactions. However, the impact of these innovations on plant immunity and pathogen infection strategies remains poorly understood. Here, we explore plant-virus interactions in the bryophyte Marchantia polymorpha to gain insights into the evolution of these relationships. Virome analysis reveals that Marchantia is predominantly associated with RNA viruses. Comparative studies with tobacco mosaic virus (TMV) show that Marchantia shares core defense responses with vascular plants but also exhibits unique features, such as a sustained wound response preventing viral spread. Additionally, general defense responses in Marchantia are equivalent to those restricted to vascular tissues in Nicotiana, suggesting that evolutionary acquisition of developmental innovations results in re-routing of defense responses in vascular plants.

Multi-spectral image transformer descriptor classification combined with molecular tools for early detection of tomato grey mould

Accurate early detection of plant diseases is a critical milestone in the development of self-navigating robots for precision agriculture and still remains one of the most significant challenges. Plant pathogens, like Botrytis cinerea causing grey mould disease, pose significant threats to agriculture and food safety. This study focuses on the early detection of B. cinerea in tomato crops using Artificial Intelligence. Specifically, the Deep Learning (DL) YOLOv8 architecture was employed to apply single class segmentation on plant captures to extract the tomato leaves locational information. Each plant capture includes five images from hyperspectral wavelengths (at 460, 540, 640, 775 and 875 nm) and one RGB images. The leaf segmentation achieved 81.7 % Mean Average Precision (mAP) at Intersection over Union (IoU) threshold 0.5 (mAP50). Then the for each leaf segment, and array of descriptors is extracted through various Transformer models. Finally, the descriptors are classified through a KNN or an LSTM solution and the results of all the descriptors for each leaf from each of the Transformer models, for each of the six images of the capture, are ensembled to yield the class of each leaf. The best ensemble results were produced by only accumulating results from some of the Transformer and specifically the MaxViT-L, the ViT-B (P:16 × 16 – C), the VOLO (D:5) and the XCIT-L (L:24 – P:16 × 16), by only using the results provided by the images at the hyperspectral wavelengths at 540, 640 nm and the RGB image, and by applying the LSTM solution. The process achieved a 79.41 % classification accuracy and 71.12 % F1-Score for all classes. Additionally, early, rapid and accurate detection of grey mould at the early stages of the infection or at latent infections when symptoms are not visible was assessed by comparative qPCR-based methods (qRT-PCR) for fungal biomass estimation in plant tissues and the rest of this study's methodology. The findings of this study represent a significant advancement in crop disease detection and management, and highlight the potential for integrating these methods into on-site digital systems (robots, mobile apps, etc.) under real-world settings. The results, demonstrate the effectiveness of combining segmentation and transformer-based classification models for early and accurate detection of grey mould disease.