Gray Mold Disease Research Articles

Early detection of Botrytis cinerea symptoms using deep learning multi-spectral image segmentation

Botrytis cinerea is an airborne plant pathogen that causes grey mould disease on horticultural crops, such as tomato, cucumber, pepper, and more. Early detection is crucial for timely and effective strategies for crop loss prevention. In this study, in planta assays were set up enabling a thorough assessment of grey mould progress on cucumber plants both artificially inoculated with two different inoculation methods, and naturally infected throughout the whole growing season of the host under controlled conditions simulating the environment typically prevailing in greenhouses. Multi-spectral imaging was used to capture a novel dataset across various spectral bands containing multiple stages of infection. Deep learning (DL) segmentation architectures, combined with Convolutional Neural Networks (CNNs) and Vision Transformers (ViTs) encoders, were employed to identify B. cinerea at various infection stages. The U-Net++ model with a MobileViT-S encoder performed best, achieving a Dice Coefficient (DC) of 0.677, an Intersection over Union (IoU) of 0.656, and a recall rate of 0.807, with a high overall accuracy of 90.1 %. In regards to early detection, the model achieves an IoU of 0.375 on 2nd day post inoculation (dpi), 0.230 on 1st dpi and 0.437 on the 6th dpi. The infection stages were compared to similar visible symptoms of physiological decline or plant responses to abiotic stress. The outcomes of this study demonstrate an important advance in the Artificial Intelligence (AI) plant health detection, for early crop disease diagnosis.

First report of Botrytis cinerea causing grey mold on Corydalis yanhusuo in Panan, China.

Yanhusuo (Corydalis yanhusuo (Y. H. Chou & Chun C. Hsu) W. T. Wang ex Z.Y. Su & C.Y. Wu), a perennial herbaceous plant of the Papaveraceae family and genus Corydalis, is also known as Yuanhu and used as medicine by its tuberous roots. It is mainly planted in Zhejiang, Jiangsu and Anhui provinces of China, with the best quality produced in Panan County of Zhejiang province. Yanhusuo has the effects of promoting blood circulation, invigorating the flow of qi and relieving pain, and is widely used in Chinese traditional medicines. In surveys carried out in summer of 2020-2023, grey mold disease was found occurred on C. yanhusuo in Panan County. This disease begins at April, and lasts to July, with incidence of 20% to 70%. The diseased plants showed a large number of gray mold layers adhere to the leaves. When the disease infects from the leaf tips, it form V-shaped lesions; when the leaves are severely infected, the entire leaves die, shrink, curl, and have a large number of gray mold layers on the surface. To identify the causal agent of this leaf disease, diseased leaves were collected from Yanhusuo field at Panan County of Zhejiang province in China since 2020, and tissues at the junction of the healthy and diseased areas were cut off, disinfected with 75% ethanol for 30 seconds, rinsed with sterile water for 3 times for 1 minute each time, air-dried under sterile conditions, and then were inoculated in PDA medium and cultured in a 25℃ incubator. After 2-3 days, picked the edge hyphae of the fungi that grew on the PDA plate and cultured them on a new PDA plate. After 5 days, picked the single spore and inoculated on a new PDA plate for continuous cultivation until pure culture strains were obtained. Thirty strains were isolated from 30 samples that collected from 3 Yanhusuo fields in Panan County. One of the thirty purified strains was named "YH8" for further identification. When cultured on PDA medium, mycelia were initially whitish and turned gray with age. The hyphae accumulate into clusters, and no sclerotia are produced during the cultivation. The conidiophores are slender, septate. The base of the conidiophore is enlarged or slightly enlarged. The conidiophore often has branches and produces a large number of conidia, which are similar to grape clusters. The conidia are monosporous, ovoid, and colorless, 6.08 μm-12.76 μm×8.42 μm-19.34 μm, with an average size of 9.55 μm×14.50 μm. To further identify the species, YH8 genomic DNA was extracted, and the internal transcribed spacer (ITS), heat shock protein (HSP60), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes were amplified with the primers ITS1/4 (White et al. 1990), HSP60-F/HSP60-R, and G3PDH-F/G3PDH-R (Staats et al. 2005), respectively. A multilocus phylogenetic tree was constructed with the ITS, HSP60, and G3PDH reference sequences, and the sequences of PCR amplicons (Genbank number: PP388281, PP376066 and PP376067) were 100% (518 bp out of 518 bp), 99% (994 bp out of 995 bp) and 100% (880 bp out of 880 bp) identical to the Botrytis cinerea strain 5-3, respectively, and the grouping of strain YH8 was supported by 99% bootstrap value. To fulfill the Koch's postulates, spore suspension (approximately 103 CFU/mL) of YH8 was sprayed onto leaves of 3-week Yanhusuo seedlings, and sterile water was sprayed as negative control, 15 seedlings for each treatment, and the experiments was repeated for times. The seedlings were incubated in a growth chamber under 28℃ and 80% humidity. Seven days after inoculation, leaves of noninoculated controls were green and healthy, while the seedlings inoculated with spore suspension of YH8 showed lesions and molds, which were same with field symptoms. The causal pathogen was then reisolated from the lesions, and the gained pathogen showed same colony and spore morphology with YH8, which suggested the confirmation of Koch's postulates. Based on the morphological characteristics and molecular identification, the strain YH8 was identified as B. cinerea. To our knowledge, this is the first report of B. cinerea causing gray mold on the Corydalis yanhusuo in China. This report will provide guide to growers and local technicians for diagnostic and controlling grey mold disease of Yanhusuo.

Cucumber PGIP2 is involved in resistance to gray mold disease

Polygalacturonase inhibitor protein (PGIP) restricts fungal growth and colonization and functions in plant immunity. Gray mold in cucumber is a common fungal disease caused by Botrytis cinerea, and is widespread and difficult to control in cucumber (Cucumis sativus L.) production. In this study, Cucumis sativus polygalacturonase-inhibiting protein 2 (CsPGIP2) was found to be upregulated in response to gray mold in cucumber. CsPGIP2 was detected in the endoplasmic reticulum, cell membrane, and cell wall after transient transformation of protoplasts and tobacco. A possible interaction between Botrytis cinerea polygalacturonase 3 (BcPG3) and CsPGIP2 was supported by protein interaction prediction and BiFC analysis. Transgenic Arabidopsis plants expressing CsPGIP2 were constructed and exhibited smaller areas of gray mold infection compared to wild type (WT) plants after simultaneous inoculation. Evans blue dye (EBD) confirmed greater damage for WT plants, with more intense dyeing than for the transgenic Arabidopsis. Interestingly, compared to WT, transgenic Arabidopsis exhibited higher superoxide dismutase (AtSOD1) expression, antioxidant enzyme activities, lignin content, net photosynthetic rate (Pn), and photochemical activity. Our results suggest that CsPGIP2 stimulates a variety of plant defense mechanisms to enhance transgenic Arabidopsis resistance against gray mold disease.

Agronomic evaluation and adaptability of Pinot gris to an environment with high grey mould disease pressure: a comparative analysis on 17 clones

The grapevine (Vitis vinifera L.) cultivar Pinot gris is an international variety that has spread world wide and is oneof the most important in Italy, especially in the northern regions where almost half of the world's total is grown. Over a three-year period (2019–2021), a comparative study was conducted on 17 clones to evaluate their agronomic aptitudes, susceptibility of grapes to Botrytis cinerea, fertility of buds, vigour, and oenological characteristics. We aimed to provide local viticulture with agronomic, sanitary status, and oenological indications that help to choose the genotype that best fits and enhances the clone/environment synergy. This is important given climate change, one of the most significant factors affecting viticulture. Hence, productive, qualitative, and health surveys were conducted on grapes during harvest. Vigour and vegetative/productive balance were also evaluated regarding the weight of the pruned wood. The Ravaz index and fertility of buds were studied when the inflorescences were separated and clearly visible (between DOY 131 and DOY 141 each year). The confirmation of the bunch was also evaluated (shape and number of wings) according to the Organisation Internationale de la Vigne et du Vin (OIV) descriptors. Through the use of digital photogrammetry, some descriptors of the cluster that most correlate with the severity of Botrytis cinerea were identified. In 2020 and 2021, microvinifications were carried out in white and with maceration of skins, respectively, following tested protocols. Sensory analysis of the wines was performed with a trained panel of experts. The study revealed interesting differences between clones concerning grapes' qualitative, productive, and sanitary aspects. Some clones stood out for their sugar accumulation, yield and resistance to Botrytis cinerea, as well as for the oenological olfactory profile. Statistical analysis highlighted that the severity of Botrytis cinerea and bunch rot was not correlated with the vigour of the plant but rather with the hardness of skin, content of soluble solids, soluble solids/total acidity ratio, pH, and total acidity. From the sensory analysis, several clones had a higher score for some descriptors in both study years and were more appreciated than others.

Kiwifruit resistance to gray mold is enhanced by yeast-induced modulation of the endophytic microbiome

The influence of endophytic microbial community on plant growth and disease resistance is of considerable importance. Prior research indicates that pre-treatment of kiwifruit with the biocontrol yeast Debaryomyces hansenii suppresses gray mold disease induced by Botrytis cinerea. However, the specific underlying mechanisms remain unclear. In this study, Metagenomic sequencing was utilized to analyze the composition of the endophytic microbiome of kiwifruit under three distinct conditions: the healthy state, kiwifruit inoculated with B. cinerea, and kiwifruit treated with D. hansenii prior to inoculation with B. cinerea. Results revealed a dominance of Proteobacteria in all treatment groups, accompanied by a notable increase in the relative abundance of Actinobacteria and Firmicutes. Ascomycota emerged as the major dominant group within the fungal community. Treatment with D. hansenii induced significant alterations in microbial community diversity, specifically enhancing the relative abundance of yeast and exerting an inhibitory effect on B. cinerea. The introduction of D. hansenii also enriched genes associated with energy metabolism and signal transduction, positively influencing the overall structure and function of the microbial community. Our findings highlight the potential of D. hansenii to modulate microbial dynamics, inhibit pathogenic organisms, and positively influence functional attributes of the microbial community.

The mechanistic insights of essential oil of Mentha piperita to control Botrytis cinerea and the prospection of lipid nanoparticles to its application

Botrytis cinerea is the phytopathogenic fungus responsible for the gray mold disease that affects crops worldwide. Essential oils (EOs) have emerged as a sustainable tool to reduce the adverse impact of synthetic fungicides. Nevertheless, the scarce information about the physiological mechanism action and the limitations to applying EOs has restricted its use. This study focused on elucidating the physiological action mechanisms and prospection of lipid nanoparticles to apply EO of Mentha piperita. The results showed that the EO of M. piperita at 500, 700, and 900 μL L−1 inhibited the mycelial growth at 100 %. The inhibition of spore germination of B. cinerea reached 31.43 % at 900 μL L−1. The EO of M. piperita decreased the dry weight and increased pH, electrical conductivity, and cellular material absorbing OD260 nm of cultures of B. cinerea. The fluorescence technique revealed that EO reduced hyphae width, mitochondrial activity, and viability, and increased ROS production. The formulation of EO of M. piperita loaded- solid lipid nanoparticles (SLN) at 500, 700, and 900 μL L−1 had particle size ∼ 200 nm, polydispersity index < 0.2, and stability. Also, the thermogravimetric analysis indicated that the EO of M. piperita-loaded SLN has great thermal stability at 50 °C. EO of M. piperita-loaded SLN reduced the mycelial growth of B. cinerea by 70 %, while SLN formulation (without EO) reached 42 % inhibition. These results supported that EO of M. piperita-loaded SLN is a sustainable tool for reducing the disease produced by B. cinerea.

Growth Inhibition of Botrytis cinerea Fungus on Strawberry (Fragaria sp.) Using Kaffir Lime (Citrus hystrix) Leaf Essential Oil Emulsion

Post-harvest damage is caused by biological factors such as the attack of Botrytis cinerea pathogenic fungi. causes gray mold rot disease to be a source of problems in strawberry production. This study aims to determine the potential of kaffir lime leaf essential oil emulsion as an antifungal against Botrytis cinerea. The fungus was isolated from strawberry fruit samples that showed symptoms of gray mold disease. Fungal isolation was carried out through the direct planting method and antifungal inhibitory ability test by food poisoned method with potato extract agar media. This experiment used a completely randomized design consisting of 6 treatments with 5 repetitions. The results showed that kaffir lime leaf essential oil emulsion contains secondary metabolites that can inhibit 100% of the growth of Botrytis cinerea pathogenic fungal colonies. Essential oil concentrations of 0.10%, 0.15%, and 0.20% had a significant effect on the growth of Botrytis cinerea pathogenic fungi.

Transcriptional Analysis Revealing the Improvement of ε-Poly-L-lysine Production from Intracellular ROS Elevation after Botrytis cinerea Induction.

Gray mold, caused by Botrytis cinerea, poses significant threats to various crops, while it can be remarkably inhibited by ε-poly-L-lysine (ε-PL). A previous study found that B. cinerea extracts could stimulate the ε-PL biosynthesis of Streptomyces albulus, while it is unclear whether the impact of the B. cinerea signal on ε-PL biosynthesis is direct or indirect. This study evaluated the role of elevated reactive oxygen species (ROS) in efficient ε-PL biosynthesis after B. cinerea induction, and its underlying mechanism was disclosed with a transcriptome analysis. The microbial call from B. cinerea could arouse ROS elevation in cells, which fall in a proper level that positively influenced the ε-PL biosynthesis. A systematic transcriptional analysis revealed that this proper dose of intracellular ROS could induce a global transcriptional promotion on key pathways in ε-PL biosynthesis, including the embden-meyerhof-parnas pathway, the pentose phosphate pathway, the tricarboxylic acid cycle, the diaminopimelic acid pathway, ε-PL accumulation, cell respiration, and energy synthesis, in which sigma factor HrdD and the transcriptional regulators of TcrA, TetR, FurA, and MerR might be involved. In addition, the intracellular ROS elevation also resulted in a global modification of secondary metabolite biosynthesis, highlighting the secondary signaling role of intracellular ROS in ε-PL production. This work disclosed the transcriptional mechanism of efficient ε-PL production that resulted from an intracellular ROS elevation after B. cinerea elicitors' induction, which was of great significance in industrial ε-PL production as well as the biocontrol of gray mold disease.

Neem (Azadirachta indica) seed extract formulation for managing anthracnose and gray mold diseases in strawberry

Anthracnose caused by Colletotrichum nymphaeae and gray mold caused by Botrytis cinerea are the most epidemic fungal diseases causing significantly losses in strawberry plantings. The objective of the study is to determine the antifungal activity of neem (Azadirachta indica A. Juss.) seed extracts and preparing new formulated antifungal agent. Plant extracts including alcoholic, aqueous and oil were extracted from neem seeds and used in concentrations of 1000, 2000, 4000, 8000 and 10,000 µl/l to control the vegetative mycelial growth of pathogens. The results showed that alcoholic extract had highest antifungal effect and used for the formulation. The concentration of 10,000 microliters of alcoholic extract showed 91.6 % inhibition of gray mold disease, while it inhibited 80.66 % in anthracnose disease and a significant difference was observed between them. For encapsulation of alcoholic neem extract with plant biopolymers with new changes, the separation method of water-miscible solvent through precipitation with salt was used. The results showed that the best effect of the formulation was observed at a dose of four per thousand. Inhibition at this concentration as the best option in terms of controlling gray mold and anthracnose disease was 86.3 % and 79.38 %, respectively, and they were placed in the same statistical group (d). This formulation is designed to determine the minimum concentration threshold and reduce the consumption of alcoholic extract. Based on the results, the use of formulated alcoholic extract of neem seed as an alternative to chemical fungicides can help control anthracnose and gray mold in strawberries.

Identification of Laccase Genes in Grapevine and Their Roles in Response to Botrytis cinerea

Laccases are the key enzymes responsible for plant lignin biosynthesis and responses to environment stress. However, the roles of LAC genes in plant disease resistance are still largely unknown, especially in grapevine, one of the most important horticultural crops in the world. Its quality and yield are very vulnerable to gray mold disease caused by Botrytis cinerea. In total, 30 VvLAC genes were identified and found to be unevenly distributed on seven chromosomes; they were classified into seven groups based on phylogenetic analysis according to the criteria applied in Arabidopsis thaliana. Collinearity and synteny analyses identified some orthologous gene pairs in Vitis vinifera and a few paralogous gene pairs among grape and peach. The VvLAC gene family has diverse gene structures and a highly conserved motif composition. The prominent presence of the MYB cis-elements in each VvLAC promoter highlighted MYB transcriptional factors as the main regulators of VvLAC genes. Furthermore, twenty-five VvLAC genes with functional redundancy are probably implicated in grape lignin biosynthesis. The expression patterns of the LAC genes in grape leaves of Chinese wild V. amurensis ‘Shuangyou’ (SY), a germplasm highly resistant to B. cinerea, were investigated through transcriptomic data and qRT-PCR verification. Combined with the phylogenetic analysis, with AtLACs participating in lignin metabolism, and the cis-element analysis, VaLAC14, VaLAC19, VaLAC24 and VaLAC30 were identified as key candidate genes for lignin biosynthesis in the grape response to B. cinerea. This study supplies a comprehensive understanding of the classification, evolution, structure and responses of the grape LAC genes against B. cinerea. It also provides valuable genetic resources for functional characterization towards enhancing grapevine disease resistance.

The potent effect of selenium nanoparticles: insight into the antifungal activity and preservation of postharvest strawberries from gray mold diseases.

Most microorganisms that cause food decay and the lower the shelf life of foods are fungi. Nanotechnologies can combat various diseases and deal with the application of nanomaterial to target cells or tissues. In this study selenium nanoparticles (Se-NPs) were synthesized using ascorbic acid and characterized by ultraviolet-visible spectroscopy, transmission electron microscopy (TEM), X-ray diffraction and zeta potential. The different concentrations of As/Se-NPs were tested against various fungi, including Alternaria linicola, Alternaria padwickii, Botrytis cinerea, Bipolaris sp., Cephalosporium acremonium, Fusarium moniliform and Fusarium semitectum. This study tested the influence of coated As/Se-NPs on healthy strawberry fruits and those infected with Botrytis cinerea during 16 days of storage, with regard to shelf life, decay percentage, weight loss, total titratable acidity percentage, total soluble solids content (TSS) and anthocyanin content. Energy-dispersive X-ray analysis showed only two elements: selenium and oxygen. TEM images showed that the nanoparticles ranged in size between 26 to 39 nm and were rhombohedral in shape. Se-NPs showed antifungal activity against all tested fungi, the most effective being against Botrytis cinerea, Cephalosporium acremonium and Fusarium semitectum. During storage periods of strawberries fruits coated with As/Se-NPs, the shelf life was increased, and the number of decaying fruits was less than in control (uncoated) and coated infected fruits. The decline in weight loss was lower in coated fruits than in control fruits. These findings demonstrated that As/Se-NPs could effectively maintain the postharvest quality of strawberries, even when the fruit was infected with B. cinerea. © 2024 Society of Chemical Industry.

Efficacy of different fungicides against Ascochyta rabiei and Botrytis cinerea associated with chickpea

Ascochyta rabiei and Botrytis cinerea have been reported to cause blight and gray mold diseases in chickpea in different parts of the world and cause a significant economic loss to crop. In the present investigation, an in vitro evaluation of fungicides was conducted to find out the sensitivity and fungicide concentration in inhibiting the growth of Ascochyta rabiei and Botrytis cinerea. In vitro evaluation of fungicides revealed that, among five fungicides, bavistin (carbendazim 50 WP) a systemic fungicide was highly effective in inhibiting the mycelial growth with 100% inhibition at all the concentrations tested followed by baycor (bitertenol WP (25% w/w), with 98.88% inhibition at 500 ppm concentration and captan with 80% mycelia growth inhibition in A.rabiei. The fungicides such as chlorothalonil, copper oxychloride, and bitertanol at all concentrations were ineffective against B. cinerea. The fungicides that were effective under laboratory conditions could be further evaluated under field conditions to validate their efficacy and they could be incorporated as a component in integrated disease management in chickpea.

Modes of action and potential as a peptide-based biofungicide of a plant defensin MtDef4.

Due to rapidly emerging resistance to single-site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens. Its MoA involves plasma membrane disruption and binding to intracellular targets. However, specific biochemical processes inhibited by this defensin and causing cell death have not been determined. Here, we show that MtDef4 exhibited potent antifungal activity against Botrytis cinerea. It induced severe plasma membrane and organelle irregularities in the germlings of this pathogen. It bound to fungal ribosomes and inhibited protein translation invitro. A MtDef4 variant lacking antifungal activity exhibited greatly reduced protein translation inhibitory activity. A cation-tolerant MtDef4 variant was generated that bound to β-glucan of the fungal cell wall with higher affinity than MtDef4. It also conferred a greater reduction in the grey mould disease symptoms than MtDef4 when applied exogenously on Nicotiana benthamiana plants, tomato fruits and rose petals. Our findings revealed inhibition of protein synthesis as a likely target of MtDef4 and the potential of its cation-tolerant variant as a peptide-based fungicide.

Deep Learning Approaches for Sunflower Disease Classification: A Study of Convolutional Neural Networks with Squeeze and Excitation Attention Blocks

Diseases in agricultural plants are one of the most important problems of agricultural production. These diseases cause decreases in production and this poses a serious problem for food safety. One of the agricultural products is sunflower. Helianthus annuus, generally known as sunflower, is an agricultural plant with high economic value grown due to its drought-resistant and oil seeds. In this study, it is aimed to classify the diseases seen in sunflower leaves and flowers by applying deep learning models. First of all, it was classified with ResNet101 and ResNext101, which are pre-trained CNN models, and then it was classified by adding squeeze and excitation blocks to these networks and the results were compared. In the study, a data set containing gray mold, downy mildew, and leaf scars diseases affecting the sunflower crop was used. In our study, original Resnet101, SE-Resnet101, ResNext101, and SE-ResNext101 deep-learning models were used to classify sunflower diseases. For the original images, the classification accuracy of 91.48% with Resnet101, 92.55% with SE-Resnet101, 92.55% with ResNext101, and 94.68% with SE-ResNext101 was achieved. The same models were also suitable for augmented images and classification accuracies of Resnet101 99.20%, SE-Resnet101 99.47%, ResNext101 98.94%, and SE-ResNext101 99.84% were achieved. The study revealed a comparative analysis of deep learning models for the classification of some diseases in the Sunflower plant. In the analysis, it was seen that SE blocks increased the classification performance for this dataset. Application of these models to real-world agricultural scenarios holds promise for early disease detection and response and may help reduce potential crop losses.

Biofilm containing the Thymus serpyllum essential oil for rice and cherry tomato conservation.

Fungal pathogens cause major yield losses in agriculture and reduce food quality and production worldwide. To evaluate new safer alternatives to chemicals for disease management and preserve the shelf life of food, this research was conducted to: determine the chemical composition of the essential oils (EOs) of Thymus serpyllum and Thymus piperella chemotypes 1 and 2; investigate the antifungal potential of EOs in vitro against: Alternaria alternata, Bipolaris spicifera, Curvularia hawaiiensis, Fusarium oxysporum f. sp. lycopersici, Penicillium italicum, Botryotinia fuckeliana; evaluate a natural T. serpyllum extract biofilm to conserve rice grain and cherry tomatoes. EOs were analyzed by GC-MS+GC-FID. EOs' antifungal activity was evaluated by dissolving Thymus extracts in PDA. Petri dishes were inoculated with disks of each fungus and incubated at 25°C for 7 days. The T. serpyllum EO displayed the best Mycelial Growth Inhibition. The antifungal effect of the T. serpyllum EO biofilm was evaluated on rice caryopsis. Disinfected grains were dipped in a conidial suspension of each fungus and sprayed with EO (300 and 600 μg/mL) prepared in Tween 20. Grains were stored. The percentage of infected grains was recorded for 30 days. The T. serpyllum EO effect on cherry tomato conservation was evaluated in vivo. Wounded fruit were immersed in the T. serpyllum EO (300 and 400 μg/mL) and inoculated with Fusarium oxysporum f. sp. lycopersici. Fruit were evaluated for 7 and 14 days. Chemical profiles thymol/carvacrol for T. serpyllum, carvacrol for T. piperella Tp1 and thymol for T. piperella Tp2 were defined. The three evaluated EOs reduced all the studied phytopathogens' fungal growth. The T. serpyllum biofilm was effective with rice storage and against Fusarium oxysporum f. sp. lycopersici for extending the shelf life of tomatoes in warehouses and storing postharvest cherry tomatoes. We suggest applying these EOs as biofilms for safe food conservation to replace synthetic products.

Plant Growth Promotion and Plant Disease Suppression Induced by Bacillus amyloliquefaciens Strain GD4a.

Botrytis cinerea, the causative agent of gray mold disease (GMD), invades plants to obtain nutrients and disseminates through airborne conidia in nature. Bacillus amyloliquefaciens strain GD4a, a beneficial bacterium isolated from switchgrass, shows great potential in managing GMD in plants. However, the precise mechanism by which GD4a confers benefits to plants remains elusive. In this study, an A. thaliana-B. cinerea-B. amyloliquefaciens multiple-scale interaction model was used to explore how beneficial bacteria play essential roles in plant growth promotion, plant pathogen suppression, and plant immunity boosting. Arabidopsis Col-0 wild-type plants served as the testing ground to assess GD4a's efficacy. Additionally, bacterial enzyme activity and targeted metabolite tests were conducted to validate GD4a's potential for enhancing plant growth and suppressing plant pathogens and diseases. GD4a was subjected to co-incubation with various bacterial, fungal, and oomycete pathogens to evaluate its antagonistic effectiveness in vitro. In vivo pathogen inoculation assays were also carried out to investigate GD4a's role in regulating host plant immunity. Bacterial extracellular exudate (BEE) was extracted, purified, and subjected to untargeted metabolomics analysis. Benzocaine (BEN) from the untargeted metabolomics analysis was selected for further study of its function and related mechanisms in enhancing plant immunity through plant mutant analysis and qRT-PCR analysis. Finally, a comprehensive model was formulated to summarize the potential benefits of applying GD4a in agricultural systems. Our study demonstrates the efficacy of GD4a, isolated from switchgrass, in enhancing plant growth, suppressing plant pathogens and diseases, and bolstering host plant immunity. Importantly, GD4a produces a functional bacterial extracellular exudate (BEE) that significantly disrupts the pathogenicity of B. cinerea by inhibiting fungal conidium germination and hypha formation. Additionally, our study identifies benzocaine (BEN) as a novel small molecule that triggers basal defense, ISR, and SAR responses in Arabidopsis plants. Bacillus amyloliquefaciens strain GD4a can effectively promote plant growth, suppress plant disease, and boost plant immunity through functional BEE production and diverse gene expression.

Ethylene-MPK8-ERF.C1-PR module confers resistance against Botrytis cinerea in tomato fruit without compromising ripening.

The plant hormone ethylene plays a critical role in fruit defense against Botrytis cinerea attack, but the underlying mechanisms remain poorly understood. Here, we showed that ethylene response factor SlERF.C1 acts as a key regulator to trigger the ethylene-mediated defense against B. cinerea in tomato fruits without compromising ripening. Knockout of SlERF.C1 increased fruit susceptibility to B. cinerea with no effect on ripening process, while overexpression enhanced resistance. RNA-Seq, transactivation assays, EMSA and ChIP-qPCR results indicated that SlERF.C1 activated the transcription of PR genes by binding to their promoters. Moreover, SlERF.C1 interacted with the mitogen-activated protein kinase SlMPK8 which allowed SlMPK8 to phosphorylate SlERF.C1 at the Ser174 residue and increases its transcriptional activity. Knocking out of SlMPK8 increased fruit susceptibility to B. cinerea, whereas overexpression enhanced resistance without affecting ripening. Furthermore, genetic crosses between SlMPK8-KO and SlERF.C1-OE lines reduced the resistance to B. cinerea attack in SlERF.C1-OE fruits. In addition, B. cinerea infection induced ethylene production which in turn triggered SlMPK8 transcription and enhanced the phosphorylation of SlERF.C1. Overall, our findings reveal the regulatory mechanism of the 'Ethylene-MPK8-ERF.C1-PR' module in resistance against B. cinerea and provide new insight into the manipulation of gray mold disease in fruits.

Antifungal activities of vineyard-habitat wild yeast for grape gray-mold disease and its effects on spontaneous winemaking.

Microorganisms, including native yeasts, are abundant in vineyard fields. Herein, we studied the possibility of using vineyard-derived wild yeast as a microbial pesticide against Botrytis cinerea, a pathogen that causes grape gray mold disease, to boost the initial alcohol production of spontaneously fermented wine. We identified the Saccharomyces cerevisiae strain KONDO170908, which showed the most effective antifungal activity in an ex vivo yeast dripping experiment on grape berries. This strain was utilized in an in vivo spray test on grape bunches in vineyard fields and was proven to significantly suppress gray mold disease on the grape berries in test plot #16 when the yeast was sprayed during both the flowering and ripening periods (morbidity 11.2% against 15.3% of the control plot, χ2 test, p < 0.0001). However, in test plot #17, spraying the yeast during only the ripening period had no effect (morbidity 16.3%). The grapes from each test plot were also submitted for spontaneous wine fermentation. Alcoholic fermentation of the grapes from test plot #16 provided the most active bubbling of CO2 gas and the highest ethanol production and colony counts over seven days of fermentation. Unique changes in the different strains of S. cerevisiae among the plots were observed throughout the early fermentation stage. Thus, yeast spraying during the flowering period might trigger modification of the entire microbiota and could ultimately contribute to promoting alcohol production in the spontaneously fermented wine, although it decreased the grape yield by 20%.

Development of a longevity prediction model for cut roses using hyperspectral imaging and a convolutional neural network.

Hyperspectral imaging (HSI) and deep learning techniques have been widely applied to predict postharvest quality and shelf life in multiple horticultural crops such as vegetables, mushrooms, and fruits; however, few studies show the application of these techniques to evaluate the quality issues of cut flowers. Therefore, in this study, we developed a non-contact and rapid detection technique for the emergence of gray mold disease (GMD) and the potential longevity of cut roses using deep learning techniques based on HSI data. Cut flowers of two rose cultivars ('All For Love' and 'White Beauty') underwent either dry transport (thus impaired cut flower hydration), ethylene exposure, or Botrytis cinerea inoculation, in order to identify the characteristic light wavelengths that are closely correlated with plant physiological states based on HSI. The flower bud of cut roses was selected for HSI measurement and the development of a vase life prediction model utilizing YOLOv5. The HSI results revealed that spectral reflectance between 470 to 680 nm was strongly correlated with gray mold disease (GMD), whereas those between 700 to 900 nm were strongly correlated with flower wilting or vase life. To develop a YOLOv5 prediction model that can be used to anticipate flower longevity, the vase life of cut roses was classed into two categories as over 5 d (+5D) and under 5 d (-5D), based on scoring a grading standard on the flower quality. A total of 3000 images from HSI were forwarded to the YOLOv5 model for training and prediction of GMD and vase life of cut flowers. Validation of the prediction model using independent data confirmed its high predictive accuracy in evaluating the vase life of both 'All For Love' (r2 = 0.86) and 'White Beauty' (r2 = 0.83) cut flowers. The YOLOv5 model also accurately detected and classified GMD in the cut rose flowers based on the image data. Our results demonstrate that the combination of HSI and deep learning is a reliable method for detecting early GMD infection and evaluating the longevity of cut roses.

Long amplicon nanopore sequencing of Botrytis cinerea and other fungal species present in infected grapevine leaf samples.

Botrytis cinerea is a well-known plant pathogen responsible for grey mould disease infecting more than 500 plant species. It is listed as the second most important plant pathogen scientifically and economically. Its impact is particularly severe in grapes since it affects both the yield of grape berries and the quality of wines. While various methods for detecting B. cinerea have been investigated, the application of Oxford Nanopore Technology (ONT) for complete ribosomal operon sequencing, which has proven effective in human and animal fungal research and diagnostics, has not yet been explored in grapevine (Vitis vinifera) disease research. In this study, we sequenced complete ribosomal operons (∼5.5 kb amplicons), which encompass the 18S, ITS1, 5.8S, ITS2, and 28S regions, from both pure cultures of B. cinerea and infected grapevine leaf samples. Minimap2, a sequence alignment tool integrated into the EPI2ME software, served as a taxonomy classifier, utilizing the custom reference database FRODO. The results demonstrate that B. cinerea was detectable when this pathogen was not the dominant fungal species in leaf samples. Additionally, the method facilitates host DNA-free sequencing and might have a good potential to distinguish other pathogenic and non-pathogenic fungal species hosted within grapevine's infected leaves, such as Alternaria alternata, Saccharomyces cerevisiae, Saccharomyces boulardii, Mucor racemosus, and Ascochyta rabie. The sequences were uploaded to the NCBI database. Long amplicon sequencing method has the capacity to be broadened to other susceptible crops and pathogens, as a valuable tool for early grey rot detection and mycobiome research. Future large-scale studies are needed to overcome challenges, such as comprehensive reference databases for complete fungal ribosomal operons for grape mycobiome studies.