Fungal Plant Research Articles

Two new endophytic fungi of Colletotrichum and Diaporthe isolated from Berchemia polyphylla var. leioclada in Guizhou Province, China

Berchemia polyphylla var. leioclada is an important traditional Chinese medicinal plant, it has been proved to produces a variety of unique metabolites, such as glycosides, quinones, lignans, terpenes and flavonoids, with different bioactivities including antioxidant, anti-tumor, anti-diarrheal, anti-inflammatory, analgesic and antibacterial activities. Studies showed that endophytic fungi may secrete compounds that are similar to those of the host. Thus, the endophytic fungi of medicinal plants are important medicinal resources and are beneficial to the research and development of natural medicines. Therefore, studying the diversity of endophytic fungi in the medicinal plant of Berchemia polyphylla var. leioclada is of great significance to discovering new drugs. However, the endophytic fungi in Berchemia polyphylla var. leioclada have been poorly studied. This study investigates the endophytic fungi in Berchemia polyphylla var. leioclada from Guizhou Province, China. Two new species Diaporthe berchemiae and Colletotrichum berchemiae are introduced based on morphological characters and multi-locus phylogenetic analysis. This is the first report of endophytic fungi in the medicinal plant Berchemia polyphylla var. leioclada, which enhances our understanding of the endophytic fungi in the medicinal plant Berchemia polyphylla var. leioclada.

Biogenic Synthesis of MgNOPs: A Revolutionary Frontier in Biomedical Advancements

At present, nanotechnology is developing at exponential rate because of its widespread applications in biomedical field. Traditionally, nanoparticles are fabricated through variety of physical and chemical approaches but because of their limited efficiency in life sciences, biogenic synthesis of nanoparticles has gained much attention. Biogenically fabricated MgONPs possess unique features that enabled their use in therapeutics and environmental biotechnology. Biogenically MgONPs can be fabricated by employing plant extracts (Manihot esculenta, Rhizophora lamarckii, Ocimum sanctum, Rosemary, Aloe vera, Orange, Artemisia abrotanum, Neem, Bauhinia purpurea), bacteria (S. coelicolor, A. johnsonii RTN), fungi (Aspergillus flavus TFR-12, Aspergillus brasiliensis TFR 2, white-button mushroom’s) and algae (S. whigti). These biogenically synthesized MgONPs are being reported as anti-fungal, antioxidant, antibacterial, anti-pyretic, anti-inflammatory and anti-cancer agents. Moreover, their photocatalytic activity is being discovered against certain organic dyes. This review focuses on biogenic synthesis of MgONPs, their applications in therapeutics and as antimicrobial agents as well as future prospects associated with their application in biomedical field.

Defoliation modifies the response of arbuscular mycorrhizal fungi to drought in temperate grassland

Arbuscular mycorrhizal (AM) fungi predominate in grasslands, where they play a key role in enhancing plant water uptake and plant tolerance to drought. However, how plant defoliation, which is common to grazed and cut grasslands, modifies plant and AM fungi responses to drought remains unknown. Here, we examined how defoliation intensity modified plant and AM fungi responses to drought.This was done using an in situ factorial field experiment at Colt Park meadows, northern England, whereby mixed grassland communities were subjected to different intensities of defoliation in combination with simulated summer drought. We measured a range of plant, AM fungal, and soil microbial and chemical responses to defoliation and drought, both individually and in combination, and extracted neutral lipid fatty acid (NLFA) as an indicator of AM fungi energy storage status.High intensity defoliation significantly reduced root colonization by AM fungi, and the negative impact of drought on AM fungi extraradical hyphal length was amplified under high intensity defoliation. Drought had no effect on total shoot biomass but reduced specific root length and increased soil NLFA concentrations, indicating an increase in AM fungi energy reserves. However, defoliation dampened this response, implying that defoliation suppresses AM fungi energy reserves under drought.Our findings suggest that plant communities can maintain above-ground productivity under drought with the assistance of AM fungi symbiosis, but that defoliation suppresses positive plant-fungi interactions under drought. Taken together, our findings provide new insights into how drought and defoliation, a key feature of grassland management, combine to negatively impact the plant-AM fungi associations in grassland.

Increasing atmospheric CO2 differentially supports arsenite stress mitigating impact of arbuscular mycorrhizal fungi in wheat and soybean plants (Chemosphere, Volume 296, June 2022, 134044)

Increasing atmospheric CO2 differentially supports arsenite stress mitigating impact of arbuscular mycorrhizal fungi in wheat and soybean plants (Chemosphere, Volume 296, June 2022, 134044)

Plant functional traits and abundance jointly shape keystone plant species in a plant–ectomycorrhizal fungus network

AbstractKeystone species are more important than others for community dynamics and stability. Keystone species can be identified and evaluated by their centrality (i.e., a relative ranking of the topological positional importance of a species) in ecological networks. Studies of node centrality of plant–fungus bipartite networks, for example, have identified the keystone species that are important for maintaining network structure and stability. However, the underlying drivers of the importance of species in a network have rarely been examined. We assessed the centrality (degree, closeness, and betweenness) of plant and fungal species in a plant–ectomycorrhizal fungus network in a subtropical forest in southern China. Based on the phylogenies of plants and fungi and plant traits, we explored ecological factors that led to a species taking a central position or not. We found one plant species (Ternstroemia gymnanthera) and four species of ectomycorrhizal fungi (Russula citrina, Scleroderma sp., and two Cenococcum sp.) were characterized by the highest centrality of degree, closeness, and betweenness among the bipartite network nodes and thus played key roles in maintaining network structure. Centrality for fungi (not for plants) was phylogenetically constrained. Plant traits and abundance together explained 46.36%, 46.0%, and 43.7% of variation in the centrality of degree, closeness, and betweenness of plant species in the bipartite network, respectively. When plant or fungal species were sequentially removed on the order of higher to lower centrality, network was less stable than randomly removed. We suggest that abundance and traits determine the positional importance of plant species in a network. This work helps understand how plant–fungus association networks will respond to species extinction and changes in species abundance and functional traits due to habitat fragmentation and human activities.

Fungi beyond limits: The agricultural promise of extremophiles.

Global climate changes threaten food security, necessitating urgent measures to enhance agricultural productivity and expand it into areas less for agronomy. This challenge is crucial in achieving Sustainable Development Goal 2 (Zero Hunger). Plant growth-promoting microorganisms (PGPM), bacteria and fungi, emerge as a promising solution to mitigate the impact of climate extremes on agriculture. The concept of the plant holobiont, encompassing the plant host and its symbiotic microbiota, underscores the intricate relationships with a diverse microbial community. PGPM, residing in the rhizosphere, phyllosphere, and endosphere, play vital roles in nutrient solubilization, nitrogen fixation, and biocontrol of pathogens. Novel ecological functions, including epigenetic modifications and suppression of virulence genes, extend our understanding of PGPM strategies. The diverse roles of PGPM as biofertilizers, biocontrollers, biomodulators, and more contribute to sustainable agriculture and environmental resilience. Despite fungi's remarkable plant growth-promoting functions, their potential is often overshadowed compared to bacteria. Arbuscular mycorrhizal fungi (AMF) form a mutualistic symbiosis with many terrestrial plants, enhancing plant nutrition, growth, and stress resistance. Other fungi, including filamentous, yeasts, and polymorphic, from endophytic, to saprophytic, offer unique attributes such as ubiquity, morphology, and endurance in harsh environments, positioning them as exceptional plant growth-promoting fungi (PGPF). Crops frequently face abiotic stresses like salinity, drought, high UV doses and extreme temperatures. Some extremotolerant fungi, including strains from genera like Trichoderma, Penicillium, Fusarium, and others, have been studied for their beneficial interactions with plants. Presented examples of their capabilities in alleviating salinity, drought, and other stresses underscore their potential applications in agriculture. In this context, extremotolerant and extremophilic fungi populating extreme natural environments are muchless investigated. They represent both new challenges and opportunities. As the global climate evolves, understanding and harnessing the intricate mechanisms of fungal-plant interactions, especially in extreme environments, is paramount for developing effective and safe plant probiotics and using fungi as biocontrollers against phytopathogens. Thorough assessments, comprehensive methodologies, and a cautious approach are crucial for leveraging the benefits of extremophilic fungi in the changing landscape of global agriculture, ensuring food security in the face of climate challenges.

Cellulose Mitochondria Growth of Fungi

Fungus were a member of eukaryotic organism included microorganisms such as yeast and maize. There were molecular methods of phylogenetic analysis. Fungi can grow on soil. These can contain membrane-bound cytoplasmic organelles such as mitochondria and ribosomes of 80S class. Fungi can be receptors of photosynthesis and phosphorylation. This was because it is tubular, elongated and filamentous structures. The results showed fungi on plants make these capable of growing in different habitats, salt concentrations and UV environment. It can be concluded the fungi were solid substrates with single cells. This had high surface area to volume ratios. They can permeate the plant to form a structure known as appressorium at 8MPa loads. Microscopy of the surface revealed compression of fossils dissolved in the cellulose of the plant (Fungus, 2022).

Rhizosphere microbial community enrichment processes in healthy and diseased plants: implications of soil properties on biomarkers.

Plant health states may influence the distribution of rhizosphere microorganisms, which regulate plant growth and development. In this study, the response of rhizosphere bacteria and fungi of healthy and diseased plants compared to bulk microbes was analyzed using high-throughput sequencing. Plant adaptation strategies of plants under potato virus Y (PVY) infection have been studied from a microbial perspective. The diversity and community structure of bacteria and fungi varied between bulk and rhizosphere soils, but not between healthy and diseased rhizosphere soils. A LEfSe analysis revealed the significant differences between different treatments on bacterial and fungal community compositions and identified Roseiflexaceae, Sphingomonas, and Sphingobium as the bacterial biomarkers of bulk (BCK), healthy rhizosphere (BHS), and diseased rhizosphere (BIS) soils, respectively; Rhodotorula and Ascomycota_unidentified_1_1 were identified as the fungal biomarkers of bulk (FCK) and healthy rhizosphere (FHS) soils. Bacterial networks were found to be more complex and compact than fungal networks and revealed the roles of biomarkers as network keystone taxa. PVY infection further increased the connectedness among microbial taxa to improve rhizosphere microbial community stability and resistance to environmental stress. Additionally, water content (WC) played an apparent influence on bacterial community structure and diversity, and pH showed significant effects on fungal community diversity. WC and pH greatly affected the biomarkers of bacterial rhizosphere communities, whereas the biomarkers of bulk bacterial communities were significantly affected by soil nutrients, especially for Sphingobium. Overall, the rhizosphere microbial community enrichment processes were different between healthy and diseased plants by changing the community compositions and identifying different biomarkers. These findings provide insight into the assemblage of rhizosphere microbial communities and soil physicochemical properties, which contributes to a deeper understanding of the establishment of an artificial core root microbiota to facilitate plant growth and bolstering resistance mechanisms. This knowledge contributes to a deeper understanding of the establishment of an artificial core root microbiota, thereby facilitating plant growth and bolstering resistance mechanisms.

Discovery of Potential Rosin-Based Triazole Antifungal Candidates to Control Valsa mali for Sustainable Crop Protection.

To investigate the potential application value of dehydroabietic acid, 27 novel dehydroabietyl-1,2,4-triazole-5-thioether-based derivatives were designed and characterized by IR, 1H NMR, 13C NMR, and LC-MS. Their antifungal activities were evaluated against five plant fungi, namely, Valsa mali, Colletotrichum orbiculare, Fusarium graminearum, Sclerotinia sclerotiorum, and Gaeumannomyces graminis; the results showed that compound 5h-1 (Co. 5h-1) exhibited a considerable inhibitory effect against V. mali. Moreover, in vivo experiments indicated that Co. 5h-1 had a certain protective effect on apple branches. The preliminary structure-activity relationship analysis suggested that the electron-withdrawing group on the benzyl group was significantly better than that of other substituent derivatives. Through electron microscopy analysis, it was found that Co. 5h-1 hindered the growth of mycelia, damaged their cell structure, and caused the large accumulation of reactive oxygen species (ROS). Preliminary research on the mode of action indicated that Co. 5h-1 could affect the activity of CAT by increasing the α-helix (0.790%), decreasing the β-sheet (0.170%), which led to the accumulation of ROS. In addition, Co. 5h-1 also affected the activity of CYP51, hindered the biosynthesis of ergosterol, and increased cell membrane permeability. Overall, this above research proposed that Co. 5h-1 can be a novel leading structure for development of a fungicide agent.

Resilience to summer bushfire in the threatened orchid, Caladenia tessellata, in terms of pollination success, herbivory, and mycorrhizal associations

Abstract In some biogeographic regions, many threatened plant species occur in habitats that periodically experience bushfire. However, we currently have relatively little information on how important plant–animal and plant–fungus interactions are affected by these fires. For the threatened sexually deceptive orchid Caladenia tessellata we test whether pollination rate, frequency of florivory, and the species of mycorrhizae the plant associates with differ between burnt and unburnt sites. Interestingly, pollination rates were unaffected by fire, demonstrating that populations of the thynnine wasp pollinator can persist post-fire. However, there was a significant negative relationship between number of flowers in a population and pollination success, which is likely a by-product of a deceptive pollination strategy. Despite the presence of vertebrate herbivores, florivory rates were low in both burnt and unburnt sites. Caladenia tessellata associated primarily with the mycorrhiza Serendiptia australiana, regardless of fire history. While our results suggest resilience to a one-off summer fire for the ecological interactions that we measured, it would be interesting to investigate the effects of fire frequency and time of year. High reproductive rates in small populations of C. tessellata suggest these populations may be viable and that retaining them is a high priority for conservation.

Tiny but mighty: metal nanoparticles as effective antimicrobial agents for plant pathogen control.

Metal nanoparticles (MNPs) have gained significant attention in recent years for their potential use as effective antimicrobial agents for controlling plant pathogens. This review article summarizes the recent advances in the role of MNPs in the control of plant pathogens, focusing on their mechanisms of action, applications, and limitations. MNPs can act as a broad-spectrum antimicrobial agent against various plant pathogens, including bacteria, fungi, and viruses. Different types of MNPs, such as silver, copper, zinc, iron, and gold, have been studied for their antimicrobial properties. The unique physicochemical properties of MNPs, such as their small size, large surface area, and high reactivity, allow them to interact with plant pathogens at the molecular level, leading to disruption of the cell membrane, inhibition of cellular respiration, and generation of reactive oxygen species. The use of MNPs in plant pathogen control has several advantages, including their low toxicity, selectivity, and biodegradability. However, their effectiveness can be influenced by several factors, including the type of MNP, concentration, and mode of application. This review highlights the current state of knowledge on the use of MNPs in plant pathogen control and discusses the future prospects and challenges in the field. Overall, the review provides insight into the potential of MNPs as a promising alternative to conventional chemical agents for controlling plant pathogens.

Profiling of deubiquitinases that control virulence in the pathogenic plant fungus Fusarium graminearum.

Eukaryotes have evolved sophisticated post-translational modifications to regulate protein function and numerous biological processes, including ubiquitination controlled by the coordinated action of ubiquitin-conjugating enzymes and deubiquitinating enzymes (Dubs). However, the function of deubiquitination in pathogenic fungi is largely unknown. Here, the distribution of Dubs in the fungal kingdom was surveyed and their functions were systematically characterized using the phytopathogen Fusarium graminearum as the model species, which causes devastating diseases of all cereal species world-wide. Our findings demonstrate that Dubs are critical for fungal development and virulence, especially the ubiquitin-specific protease 15 (Ubp15). Global ubiquitome analysis and subsequent experiments identified three important substrates of Ubp15, including the autophagy-related protein Atg8, the mitogen-activated protein kinase Gpmk1, and the mycotoxin deoxynivalenol (DON) biosynthetic protein Tri4. Ubp15 regulates the deubiquitination of the Atg8, thereby impacting its subcellular localization and the autophagy process. Moreover, Ubp15 also modulates the deubiquitination of Gpmk1 and Tri4. This modulation subsequently influences their protein stabilities and further affects the formation of penetration structures and the biosynthetic process of DON, respectively. Collectively, our findings reveal a previously unknown regulatory pathway of a deubiquitinating enzyme for fungal virulence and highlight the potential of Ubp15 as a target for combating fungal diseases.

Inactivation of siderophore iron-chelating moieties by the fungal wheat root symbiont Pyrenophora biseptata.

We investigated the ability of four plant and soil-associated fungi to modify or degrade siderophore structures leading to reduced siderophore iron-affinity in iron-limited and iron-replete cultures. Pyrenophora biseptata, a melanized fungus from wheat roots, was effective in inactivating siderophore iron-chelating moieties. In the supernatant solution, the tris-hydroxamate siderophore desferrioxamine B (DFOB) underwent a stepwise reduction of the three hydroxamate groups in DFOB to amides leading to a progressive loss in iron affinity. A mechanism is suggested based on the formation of transient ferrous iron followed by reduction of the siderophore hydroxamate groups during fungal high-affinity reductive iron uptake. P. biseptata also produced its own tris-hydroxamate siderophores (neocoprogen I and II, coprogen and dimerum acid) in iron-limited media and we observed loss of hydroxamate chelating groups during incubation in a manner analogous to DFOB. A redox-based reaction was also involved with the tris-catecholate siderophore protochelin in which oxidation of the catechol groups to quinones was observed. The new siderophore inactivating activity of the wheat symbiont P. biseptata is potentially widespread among fungi with implications for the availability of iron to plants and the surrounding microbiome in siderophore-rich environments.

Morphology and multi-gene phylogeny reveal a new Brunneofusispora species from coffee in Yunnan Province, China

Coffee is one of the most important cash crops native to north-central Africa, and the second most popular beverage ingredient worldwide. Coffee-associated fungal pathogens have been well studied, but few studies have reported saprobes of coffee. In China, Yunnan Province is the main region of coffee plantations and production. To investigate saprobic fungi of coffee plants, dead branches of Coffea with fungal fruiting bodies were collected in Baoshan City, Yunnan Province, China. Multi-gene phylogenetic analyses of SSU, ITS, LSU, RPB2, and TEF1-α sequence data indicated that our collections formed an independent branch within Brunneofusispora and sister to B. sinensis. The new species B. baoshanensis, characterized by uniloculate ascomata with an erumpent long neck, and inequilateral, ends acute, 1-septate, constricted at the septum ascospores with mucilaginous sheath, was identified based on morphology and multi-gene phylogeny. This is the first report of Brunneofusispora species isolated from Coffea in China. A complete description, micrographs, and a phylogenetic tree showing the placement of the new species are provided.

Aabrm1-mediated melanin synthesis is essential to growth and development, stress adaption, and pathogenicity in Alternaria alternata.

Scytalone dehydratase (brm1) is one of the key enzymes in 1, 8-dihydroxynaphthalene (DHN) melanin synthesis, which mediates melanin biosythesis and regulates cell biological process of plant fungi, but its function in Alternaria alternata, the causal agent of pear black spot, is unclear. Brm1 in A. alternata was cloned, identified, and named as Aabrm1. An Aabrm1-deletion mutant was generated and revealed that the deletion of Aabrm1 leads to a significant decrease in melanin production and forms orange colony smooth spores. In addition, the deletion of Aabrm1 gene impaired infection structure information and penetration. The external stress resistance of ΔAabrm1 was significantly weakened, and, in particular, it is very sensitive to oxidative stress, and the contents of H2O2 and O2.- in ΔAabrm1 were significantly increased. Virulence of ΔAabrm1 was reduced in non-wound-inoculated pear leaves but not changed in wound-inoculated pear fruit. These results indicated that Aabrm1-mediated melanin synthesis plays an important role in the pathogenicity of A. alternata.

The Diversity and Growth-Promoting Potential of the Endophytic Fungi of Neuwiedia singapureana (Orchidaceae) in China

Neuwiedia singapureana is a rare and endangered plant of the Apostasioideae subfamily. The Apostasioideae subfamily has a unique evolutionary status, as it is considered to be the most primitive group forming the base of the Orchidaceae evolutionary tree. Therefore, N. singapureana has high scientific research and conservation value. The endophytic fungal communities associated with orchids are rich and diverse, but few studies have investigated the endophytic fungi of Neuwiedia orchid plants. In the present study, the aim was to examine the endophytic fungal community structures associated with wild N. singapureana rhizomes and normal roots in the ground and with bare prop roots in the air at two sampling sites in China. High-throughput sequencing of nuclear ribosomal DNA fragments of the internal transcribed spacer regions was conducted, and cultivable methods were adopted. A total of 2161 endophytic fungal operational taxonomic units (OTUs) were obtained at a 97% sequence similarity threshold. The endophytic fungal diversity differed among the samples but not significantly. There were many more non-mycorrhizal endophytic fungal than orchid mycorrhizal (OM) fungal species detected in the N. singapureana orchid, about 98.33% OTUs of non-mycorrhizal fungi contrasting with 1.67% OTUs of potential orchid mycorrhizal fungi, among which Ceratobasidiaceae, Russulaceae, and Thelephoraceae were the dominant orchid mycorrhizal fungi. One culturable OM fungal Epulorhiza sp. isolated from the rhizome was capable of significantly promoting the seed germination and seedling growth of Dendrobium officinale and Epidendrum secundum orchids, respectively, with different efficiencies. These endophytic fungal strains with growth-promoting functions will provide materials for orchid conservation and for the study of the mechanisms underlying orchid symbiotic associations.

Analysis of Plant Virus-Induced Immunity by Using Viral-Derived Double-Stranded RNA in Arabidopsis thaliana.

Pattern-triggered immunity is the first line of defense against infection by pathogens such as bacteria and fungi in plants, and this mechanism remains poorly defined in plant viruses. Double-stranded RNA (dsRNA) is an intermediate in the replication of plant RNA viruses, and is considered to be a conserved structure of plant viruses similar to pathogen-associated molecular pattern. Whether dsRNA is the elicitor that activates plant immunity in response to virus infection remains obscure. In this method, we use the cDNA of turnip mosaic virus genome as the template to in vitro synthesis of viral dsRNA and examine whether viral dsRNA could activate plant immunity in Arabidopsis thaliana, including MAPK kinase cascade and reactive oxygen burst. In order to provide some references for researchers studying dsRNA in terms of research methodology and experimental methods, we use western blot to measure MAPK kinase cascade and luminol-based assay to measure ROS burst in Arabidopsis thaliana treated by viral dsRNA.

Phenolylazoindole scaffold for facilely synthesized and bis-functional photoswitches combining controllable fluorescence and antifungal properties using theoretical methods.

Functionalization is a major challenge for the application of photoswitches. With the aim to develop novel bis-functional azo photoswitches with stationary photophysical properties, a series of phenolylazoindole derivatives were designed, synthesized, and characterized via NMR spectroscopy studies and high-resolution mass spectrometry (HRMS). Herein, UV/Vis and 1H NMR spectra revealed that the photostationary state (PSS) proportions for PSScis and PSStrans were 76-80% and 68-81%, respectively. Furthermore, the thermal half-lives (t1/2) of compounds A2-A4 and B2 ranged from 0.9 to 5.3 h, affected by the diverse substituents at the R1 and R2 positions. The results indicated that azo photoswitches based on the phenolylazoindole scaffold had stationary photophysical properties and wouldn't be excessively affected by modifying the functional groups. Compounds A4 and B2, which were modified with an aryl group, also exhibited fluorescence emission properties (the quantum yields of A4 and B2 were 2.32% and 13.34%) through the modification of the flexible conjugated structure (benzene) at the R2 position. Significantly, compound C1 was obtained via modification with a pharmacophore in order to acquire antifungal activities against three plant fungi, Rhizoctonia solani (R. solani), Botrytis cinerea (B. cinerea), and Fusarium graminearum (F. graminearum). Strikingly, the inhibitory activity of the cis-isomer of compound C1towards R. solani (53.3%) was significantly better than that of the trans-isomer (34.2%) at 50 μg mL-1. In order to further reveal the antifungal mechanism, molecular docking simulations demonstrated that compound C1 effectively integrates into the cavity of succinate dehydrogenase (SDH); the optically controlled cis-isomer showed a lower binding energy with SDH than that of the trans-isomer. This research confirmed that phenolylazoindole photoswitches can be appropriately applied as molecular regulatory devices and functional photoswitch molecules via bis-functionalization.

Triplex real-time qPCR for the simultaneous detection of Botryosphaeriaceae species in woody crops and environmental samples.

Species of Botryosphaeriaceae fungi are relevant pathogens of almond causing trunk cankers, extensive gumming, necrosis of internal tissues and plant dieback and dead, threatening almond productivity. A novel triplex quantitative real-time PCR (qPCR) assay was designed for the simultaneous detection and quantification of Neofusicoccum parvum, Botryosphaeria dothidea and the Botryosphaeriaceae family. The method was validated in symptomatic and asymptomatic almond, avocado, blueberry and grapevine plants and in environmental samples, such as cropping soil and rainwater and in artificially inoculated trapped spores, demonstrating the same performance on several matrices. The limit of detection of the triplex qPCR was 10 fg of genomic DNA for the three fungal targets, with high correlation coefficients (R2) and amplification efficiencies between 90 and 120%. Although the triplex qPCR demonstrated to be more sensitive and accurate than the traditional plate culturing and further sequencing method, a substantial agreement (kappa index = 0.8052 ± 0.0512) was found between the two detection methods. The highly sensitive qPCR assay allows for accurate diagnosis of symptomatic plants and early detection of Botryosphaeriaceae fungi in asymptomatic plants (rootstocks and grafting scions from almond nurseries). Furthermore, the triplex qPCR successfully detected Botryosphaeriaceae fungi in environmental samples, such as cropping soils and rainwater. It was also capable of detecting as few as 10 conidia in artificially inoculated tapes. Therefore, the triplex qPCR is a valuable tool for accurate diagnosis, aiding in the implementation of suitable control measures. It enables preventive detection in asymptomatic samples, helping to avoid the introduction and spread of these pathogens in production fields. Moreover, it assists in identifying inoculum sources and quantifying inoculum levels in crop environments, contributing to a precise phytosanitary application schedule, thereby reducing production costs and preserving the environment.

Synthesis and biological evaluation of burnettiene A derivatives enabling discovery of novel fungicide candidates.

An antifungal polyene-decalin polyketide natural product, burnettiene A (1) has been re-discovered from the culture broth of Lecanicillium primulinum (current name: Flavocillium primulinum) FKI-6715 strain utilizing our original multidrug-sensitive yeast system. This polyene-decalin polyketide natural product was originally isolated from Aspergillus burnettii. The antifungal activity of 1 against Candida albicans has been reported. However, only one fungal species for the antifungal activity of 1 has been revealed, and details of the antifungal activity against other pathogenic fungus remain unknown. After extensive screening for antifungal activity, we found that 1 exhibits broad antifungal activity against pathogenic plant fungi, including Colletotrichum gloeosporioides, Botrytis cinerea, Pyricularia oryzae, Leptosphaeria maculans, and Rhizoctonia solani. Furthermore, we synthesized 12 derivatives from 1 and evaluated their antifungal activity to reveal the detailed structure-activity relationship. The methyl ester derivative showed antifungal activity against Saccharomyces cerevisiae 12geneΔ0HSR-iERG6 100-fold more potent than that of 1. Our research indicates that 1 would be a promising natural product as a new fungicidal candidate and the methyl ester derivative especially has great potential.