Horizontally Transferred DNA in the Genome of the Fungus Pyricularia oryzae is Associated With Repressive Histone Modifications.

Horizontal gene transfer (HGT) is a means of exchanging genetic material asexually. The process by which horizontally transferred genes are domesticated by the host genome is of great interest but is not well understood. In this study, we determined the telomere-to-telomere genome sequence of the wheat-infecting Pyricularia oryzae strain Br48. SNP analysis indicated that the Br48 strain is a hybrid of wheat- and Brachiaria-infecting strains by a sexual or parasexual cross. Comparative genomic analysis identified several megabase-scale "insertions" in the Br48 genome, some of which were possibly gained by HGT-related events from related species, such as P. pennisetigena or P. grisea. Notably, the mega-insertions often contained genes whose phylogeny is not congruent with the species phylogeny. Moreover, some of the genes have a close homolog even in distantly related organisms, such as basidiomycetes or prokaryotes, implying the involvement of multiple HGT events. Interestingly, the levels of the silent epigenetic marks H3K9me3 and H3K27me3 in a genomic region tended to be negatively correlated with the phylogenetic concordance of genes in the same region, suggesting that horizontally transferred DNA is preferentially targeted for epigenetic silencing. Indeed, the putative HGT-derived genes were activated when MoKmt6, the gene responsible for H3K27me3 modification, was deleted. Notably, these genes also tended to be up-regulated during infection, suggesting that they are now under host control and have contributed to establishing a fungal niche. In conclusion, this study suggests that epigenetic modifications have played an important role in the domestication of HGT-derived genes in the P. oryzae genome.

Evaluation of Bacillus isolates as a biological control agents against soilborne phytopathogenic fungi.

Pesticides, used in agriculture to control plant diseases, pose risks to the environment and human health. To address this, there's a growing focus on biocontrol, using microorganisms instead of chemicals. In this study, we aimed to identify Bacillus isolates as potential biological control agents. We tested 1574 Bacillus isolates for antifungal effects against pathogens like Botrytis cinerea, Fusarium solani, and Rhizoctonia solani. Out of these, 77 isolates formed inhibition zones against all three pathogens. We then investigated their lytic enzyme activities (protease, chitinase, and chitosanase) and the production of antifungal metabolites (siderophore and hydrogen cyanide). Coagulase activity was also examined to estimate potential pathogenicity in humans and animals. After evaluating all mechanisms, 19 non-pathogenic Bacillus isolates with significant antifungal effects were chosen. Molecular identification revealed they belonged to B. subtilis (n = 19) strains. The 19 native Bacillus strains, demonstrating strong antifungal effects in vitro, have the potential to form the basis for biocontrol product development. This could address challenges in agricultural production, marking a crucial stride toward sustainable agriculture.

Antifungal capabilities of gut microbial communities of three dung beetle species (Scarabaeidae: Scarabaeinae).

Gut microbial communities are part of the regulatory array of various processes within their hosts, ranging from nutrition to pathogen control. Recent evidence shows that dung beetle's gut microbial communities release substances with antifungal activity. Because of the enormous diversity of gut microorganisms in dung beetles, there is a possibility of discovering novel compounds with antifungal properties. We tested the antifungal activity mediated by gut microbial communities of female dung beetles against nine phytopathogenic fungi strains (Colletotrichum asianum-339, C. asianum-340, C. asianum-1, C. kahawae-390, C. karstii-358, C. siamense-220, Fusarium oxysporum-ATCC338, Nectria pseudotrichia-232, Verticillium zaelandica-22). Our tests included the gut microbial communities of three species of dung beetles: Canthon cyanellus (roller beetle), Digitonthophagus gazella (burrower beetle), and Onthophagus batesi (burrower beetle), and we followed the dual confrontation protocol, i.e., we challenged each fungal strain with the microbial communities of each species of beetles in Petri dishes containing culture medium. Our results showed that gut microbial communities of the three dung beetle species exhibit antifungal activity against at least seven of the nine phytopathogenic fungal strains. The gut microbial communities of Onthophagus batesi significantly decreased the mycelial growth of the nine phytopathogenic fungi strains; the gut microbial communities of Canthon cyanellus and Digitonthophagus gazella significantly reduced the mycelial growth of seven strains. These results provide a basis for investigating novel antifungal substances within gut microbial communities of dung beetles.

Community composition of phytopathogenic fungi significantly influences ectomycorrhizal fungal communities during subtropical forest succession.

Ectomycorrhizal fungi (EMF) can form symbiotic relationships with plants, aiding in plant growth by providing access to nutrients and defense against phytopathogenic fungi. In this context, factors such as plant assemblages and soil properties can impact the interaction between EMF and phytopathogenic fungi in forest soil. However, there is little understanding of how these fungal interactions evolve as forests move through succession stages. In this study, we used high-throughput sequencing to investigate fungal communities in young, intermediate, and old subtropical forests. At the genus level, EMF communities were dominated by Sebacina, Russula, and Lactarius, while Mycena was the most abundant genus in pathogenic fungal communities. The relative abundances of EMF and phytopathogenic fungi in different stages showed no significant difference with the regulation of different factors. We discovered that interactions between phytopathogenic fungi and EMF maintained a dynamic balance under the influence of the differences in soil quality attributed to each forest successional stage. The community composition of phytopathogenic fungi is one of the strong drivers in shaping EMF communities over successions. In addition, the EMF diversity was significantly related to plant diversity, and these relationships varied among successional stages. Despite the regulation of various factors, the positive relationship between the diversity of phytopathogenic fungi and EMF remained unchanged. However, there is no significant difference in the ratio of the abundance of EMF and phytopathogenic fungi over the course of successions. These results will advance our understanding of the biodiversity-ecosystem functioning during forest succession. KEY POINTS: •Community composition of both EMF and phytopathogenic fungi changed significantly over forest succession. •Phytopathogenic fungi is a key driver in shaping EMF community. •The effect of plant Shannon's diversity on EMF communities changed during the forest aging process.

Design, bioactivity and mechanism of N'-phenyl pyridylcarbohydrazides with broad-spectrum antifungal activity.

Succinate dehydrogenase inhibitors (SDHIs) as one of the fastest-growing fungicide categories for plant protection. In this study, a series of N'-phenyl pyridylcarbohydrazides as analogues of commercial SDHIs were designed and evaluated for inhibition activity on phytopathogenic fungi to search for potential novel SDHIs. The determination of antifungal activity in vitro and in vivo led to the discovery of a series of compounds with high activity and broad-spectrum property. Especially, N'-(4-fluorophenyl)picolinohydrazide (1c) and N'-(3,4-fluorophenyl)picolinohydrazide (1ae) showed 0.041-1.851 µg/mL of EC50 values on twelve fungi, superior to positive controls carbendazim and boscalid. In vivo activity, 1c at 50 µg/mL showed 61% of control efficacy at the post-treatment 9th day for the infection of P. piricola on apples, slightly smaller than 70% of carbendazim. In terms of action mechanism, 1c showed strong inhibition activity with IC50 of 0.107 µg/mL on SDH in Alternaria brassicae, superior to positive SDHI boscalid (IC50 0.182 µg/mL). Molecular docking indicated that 1c can well bind with the ubiquinone-binding region of SDH mainly by hydrogen bond, carbon hydrogen bond, π-alkyl, amide-π stacking, F-N and F-H interactions. Furthermore, scanning and transmission electron micrographs showed that 1c was able to obviously change the structure of mycelia and cell membrane. Fluorescence staining analysis showed that 1c could increase both the intracellular reactive oxygen species level and mitochondrial membrane potential. Finally, seed germination test, seedling growth test and cytotoxicity assay showed that 1c had very low toxicity to plant growth and mammalian cells. Thus, N'-phenyl pyridylcarbohydrazides especially 1c and 1ae can be considered promising fungicide alternatives for plant protection.

Biofungicides Based on Plant Extracts: On the Road to Organic Farming.

Phytopathogenic fungi are responsible for diseases in commercially important crops and cause major supply problems in the global food chain. Plants were able to protect themselves from disease before humans played an active role in protecting plants. They are known to synthesize a variety of secondary metabolites (SMs), such as terpenes, alkaloids, and phenolic compounds, which can be extracted using conventional and unconventional techniques to formulate biofungicides; plant extracts have antifungal activity and various mechanisms of action against these organisms. In addition, they are considered non-phytotoxic and potentially effective in disease control. They are a sustainable and economically viable alternative for use in agriculture, which is why biofungicides are increasingly recognized as an attractive option to solve the problems caused by synthetic fungicides. Currently, organic farming continues to grow, highlighting the importance of developing environmentally friendly alternatives for crop production. This review provides a compilation of the literature on biosynthesis, mechanisms of action of secondary metabolites against phytopathogens, extraction techniques and formulation of biofungicides, biological activity of plant extracts on phytopathogenic fungi, regulation, advantages, disadvantages and an overview of the current use of biofungicides in agriculture.

Green Synthesis and Antifungal Activities of Novel N-Aryl Carbamate Derivatives.

Carbamate is a key structural motif in the development of fungicidal compounds, which is still promising and robust in the discovery of green pesticides. Herein, we report the synthesis and evaluation of the fungicidal activity of 35 carbamate derivatives, among which 19 compounds were synthesized in our previous report. These derivatives were synthesized from aromatic amides in a single step, which was a green oxidation process for Hofmann rearrangement using oxone, KCl and NaOH. Their chemical structures were characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry. Their antifungal activity was tested against seven plant fungal pathogens. Many of the compounds exhibited good antifungal activity in vitro (inhibitory rate > 60% at 50 µg/mL). Compound 1ag exhibited excellent broad-spectrum antifungal activities with inhibition rates close to or higher than 70% at 50 µg/mL. Notably, compound 1af demonstrated the most potent inhibition against F. graminearum, with an EC50 value of 12.50 µg/mL, while compound 1z was the most promising candidate fungicide against F. oxysporum (EC50 = 16.65 µg/mL). The structure-activity relationships are also discussed in this paper. These results suggest that the N-aryl carbamate derivatives secured by our green protocol warrant further investigation as potential lead compounds for novel antifungal agents.

Design, Synthesis, Antifungal Evaluation, Structure-Activity Relationship (SAR) Study, and Molecular Docking of Novel Spirotryprostatin A Derivatives.

Phytopathogenic fungi cause plant diseases and economic losses in agriculture. To efficiently control plant pathogen infections, a total of 19 spirotryprostatin A derivatives and 26 spirooxindole derivatives were designed, synthesized, and tested for their antifungal activity against ten plant pathogens. Additionally, the intermediates of spirooxindole derivatives were investigated, including proposing a mechanism for diastereoselectivity and performing amplification experiments. The bioassay results demonstrated that spirotryprostatin A derivatives possess good and broad-spectrum antifungal activities. Compound 4d exhibited excellent antifungal activity in vitro, equal to or higher than the positive control ketoconazole, against Helminthosporium maydis, Trichothecium roseum, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium graminearum, Alternaria brassicae, Alternaria alternate, and Fusarium solan (MICs: 8-32 µg/mL). Compound 4k also displayed remarkable antifungal activity against eight other phytopathogenic fungi, including Fusarium oxysporium f. sp. niveum and Mycosphaerella melonis (MICs: 8-32 µg/mL). The preliminary structure-activity relationships (SARs) were further discussed. Moreover, molecular docking studies revealed that spirotryprostatin A derivatives anchored in the binding site of succinate dehydrogenase (SDH). Therefore, these compounds showed potential as natural compound-based chiral fungicides and hold promise as candidates for further enhancements in terms of structure and properties.

Nonribosomal peptide synthetase gene clusters and characteristics of predicted NRPS-dependent siderophore synthetases in Armillaria and other species in the Physalacriaceae.

Fungal secondary metabolites are often pathogenicity or virulence factors synthesized by genes contained in secondary metabolite gene clusters (SMGCs). Nonribosomal polypeptide synthetase (NRPS) clusters are SMGCs which produce peptides such as siderophores, the high affinity ferric iron chelating compounds required for iron uptake under aerobic conditions. Armillaria spp. are mostly facultative necrotrophs of woody plants. NRPS-dependent siderophore synthetase (NDSS) clusters of Armillaria spp. and selected Physalacriaceae were investigated using a comparative genomics approach. Siderophore biosynthesis by strains of selected Armillaria spp. was evaluated using CAS and split-CAS assays. At least one NRPS cluster and other clusters were detected in the genomes studied. No correlation was observed between the number and types of SMGCs and reported pathogenicity of the species studied. The genomes contained one NDSS cluster each. All NDSSs were multi-modular with the domain architecture (ATC)3(TC)2. NDSS clusters of the Armillaria spp. showed a high degree of microsynteny. In the genomes of Desarmillaria spp. and Guyanagaster necrorhizus, NDSS clusters were more syntenic with NDSS clusters of Armillaria spp. than to those of the other Physalacriaceae species studied. Three A-domain orthologous groups were identified in the NDSSs, and atypical Stachelhaus codes were predicted for the A3 orthologous group. In vitro biosynthesis of mainly hydroxamate and some catecholate siderophores was observed. Hence, Armillaria spp. generally contain one highly conserved, NDSS cluster although some interspecific variations in the products of these clusters is expected. Results from this study lays the groundwork for future studies to elucidate the molecular biology of fungal phyto-pathogenicity.

Myxobacterial Outer Membrane ß-1,6-Glucanase Induced the Cell Death of Fusarium oxysporum by Destroying the Cell Wall Integrity.

The ß-1,6-glucan is the key linker between mannoproteins in the outermost part of the cell wall and ß-1,3-glucan/chitin polysaccharide to maintain the rigid structure of the cell wall. The ß-1,6-glucanase GluM, which was purified from the fermentation supernatant of Corallococcus sp. EGB, was able to inhibit the germination of Fusarium oxysporum f. sp. cucumerinum conidia at a minimum concentration of 2.0 U/mL (0.08 µg/mL). The survival rates of GluM-treated conidia and monohyphae were 10.4% and 30.7%, respectively, which were significantly lower than that of ß-1,3-glucanase treatment (Zymolyase, 20.0 U/mL; equate to 1.0 mg/mL) (72.9% and 73.9%). In contrast to ß-1,3-glucanase treatment, the high-osmolarity glycerol (HOG) pathway of F. oxysporum f. sp. cucumerinum cells was activated after GluM treatment, and the intracellular glycerol content was increased by 2.6-fold. Moreover, the accumulation of reactive oxygen species (ROS) in F. oxysporum f. sp. cucumerinum cells after GluM treatment induced apoptosis, but it was not associated with the increased intracellular glycerol content. Together, the results indicate that ß-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents. IMPORTANCE Phytopathogenic fungi are the most devastating plant pathogens in agriculture, causing enormous economic losses to global crop production. Biocontrol agents have been promoted as replacements to synthetic chemical pesticides for sustainable agriculture development. Cell wall-degrading enzymes (CWDEs), including chitinases and ß-1,3-glucanases, have been considered as important armaments to damage the cell wall. Here, we found that F. oxysporum f. sp. cucumerinum is more sensitive to ß-1,6-glucanase GluM treatment (0.08 µg/mL) than ß-1,3-glucanase Zymolyase (1.0 mg/mL). The HOG pathway was activated in F. oxysporum f. sp. cucumerinum cells after GluM treatment, and the intracellular glycerol content was significantly increased. Moreover, the decomposition of F. oxysporum f. sp. cucumerinum cell wall by GluM induced the burst of intracellular ROS and apoptosis, which eventually leads to cell death. Therefore, we suggest that the ß-1,6-glucan of the fungal cell wall may be a better antifungal target compared to the ß-1,3-glucan.

Selective deployment of virulence effectors correlates with host specificity in a fungal plant pathogen.

The hemibiotrophic fungal plant pathogen Colletotrichum orbiculare is predicted to secrete hundreds of effector proteins when the pathogen infects cucurbit crops, such as cucumber and melon, and tobacco (Nicotiana benthamiana), a distantly related Solanaceae species. Here, we report the identification of sets of C. orbiculare effector genes that are differentially required for fungal virulence to two phylogenetically distant host species. Through targeted gene knockout screening of C. orbiculare 'core' effector candidates defined based on in planta gene expression, we identified: four host-specific virulence effectors (named effector proteins for cucurbit infection, or EPCs) that are required for full virulence of C. orbiculare to cucurbit hosts, but not to the Solanaceae host N. benthamiana; and five host-nonspecific virulence effectors, which collectively contribute to fungal virulence to both hosts. During host infection, only a small subset of genes, including the host-specific EPC effector genes, showed preferential expression on one of the hosts, while gene expression profiles of the majority of other genes, including the five host-nonspecific effector genes, were common to both hosts. This work suggests that C. orbiculare adopts a host-specific effector deployment strategy, in addition to general host-blind virulence mechanisms, for adaptation to cucurbit hosts.

Incompatibility between proliferation and plant invasion is mediated by a regulator of appressorium formation in the corn smut fungus Ustilago maydis.

Plant pathogenic fungi often developed specialized infection structures to breach the outer surface of a host plant. These structures, called appressoria, lead the invasion of the plant by the fungal hyphae. Studies in different phytopathogenic fungi showed that appressorium formation seems to be subordinated to the cell cycle. This subordination ensures the loading in the invading hypha of the correct genetic information to proceed with plant infection. However, how the cell cycle transmits its condition to the genetic program controlling appressorium formation and promoting the plant's invasion is unknown. Our results have uncovered how this process occurs for the appressorium of Ustilago maydis, the agent responsible for corn smut disease. Here, we described that the complex Clb2-cyclin-dependent kinase (Cdk)1, one of the master regulators of G2/M cell cycle progression in U. maydis, interacts and controls the subcellular localization of Biz1, a transcriptional factor required for the activation of the appressorium formation. Besides, Biz1 can arrest the cell cycle by down-regulation of the gene encoding a second b-cyclin Clb1 also required for the G2/M transition. These results revealed a negative feedback loop between appressorium formation and cell cycle progression in U. maydis, which serves as a "toggle switch" to control the fungal decision between infecting the plant or proliferating out of the plant.

First report of Fusarium oxysporum Species Complex causing root and rhizome rot of Canna edulis in Colombia.

Achira, Canna edulis Ker, a plant native to South America, is the source of a starch used for food and industrial purposes. Since 2016, Colombian growers of the main cropping regions, Cundinamarca (CU), Nariño (NA), and Huila (HU) are experiencing yield losses due to rhizome rots. Surveys of the affected areas evidenced wilting and collapsed plants, with oxidized rhizomes and affected root masses. Disease incidence per field was around 10%, but diseased plants were found in all 44 visited farms. To study this problem, wilting plants were collected, and symptomatic tissues, pseudo-stems, roots, and rhizomes, were cut and disinfested in 1.5% hypochlorite, rinsed in sterile water, and plated onto PDA amended with 0.01% tetracycline. A total of 121 isolates were recovered; of these, 77 Fusarium-like isolates stood out, given their recovery frequency (64.7%) and cross-region distribution. To morphologically study the isolates, carnation leaf agar cultures of NA01, NA16, NA48, CU08-1 and HU02, were made. Isolates had hyaline, mostly aseptated microconidia, oval in shape, developing in false heads with short monophialides. Macroconidia were hyaline and falcate, straight to slightly curved, 2 to 4 septate, with apical cells curved and basal cells foot shaped. For NA01 the average size and width of the microconidia was 4.3 x 3.2 µm (n=80), while macroconidia averaged 18.9 × 5.7 µm (n =80); NA16 was slightly bigger (6.5 x 3 and 22.9 x 5.5 um respectively). This morphology resembles Fusarium oxysporum (Fox) (Leslie et al. 2006). Identity confirmation was obtained by Sanger sequencing of the rRNA internal transcribed spacer (ITS) and the translation elongation factor 1α (TEF1α) loci using protocols of White et al. 1994, and O'Donnell et al. 1998. Blast comparisons against NCBI databases, showed a very high identity (>99.5%) to MN528565.1 (ITS), and KU985430.1 (TEF 1α), both, F. oxysporum sequences. The identity of NA01 and CU08 was further confirmed by sequencing the DNA-directed RNA polymerase II (RPB1) locus (O'Donnell et al. 2015), observing more than 99% identity to CP052885.1 (RPB1) a F. oxysporum strain. BLAST check against the Fusarium MLSD database confirmed the identity. The obtained sequences were deposited in NCBI as MN963788, MN963793, MN963801, MN963782, MN963786 (ITS); OK143597, OK141601, OK143596 MW594202, OK169575 (TEF1α); and ON297670 and MZ670431 RPB1). To confirm causality, pathogenicity assays were conducted using NA01, NA48 and CU08. To this end, 25, 35 day-olds sprouted rhizomes, from each of the "purple", "green" and "white" varieties, were inoculated by drench with 30 ml of conidium suspension (1x106 conidia/ml) (Schmale 2003). Control rhizomes (25 per variety) were treated with sterile distilled water. Greenhouse conditions were 25 °C, 40% RH, and photoperiod 12h. Disease symptoms were detected 10 days after inoculation and evolved to resemble those from the field. While symptom and severity of infection varied with the isolate and host combination used, pathogen re-isolation and identification was successful fulfilling Koch´s postulates. Control plants remained healthy. The data shows that F. oxysporum species complex is the causal agent of this achira root and rhizome rot. To our knowledge, this is the first report of this problem in Colombia and clarifies local reports of Fusarium sp. causing disease in this crop (Caicedo et al. 2003). The disease affects the food security of local communities and strategies for control are being developed.

Synthesis and Evaluation on the Fungicidal Activity of S-Alkyl Substituted Thioglycolurils.

A series of S-alkyl substituted thioglycolurils was prepared through the alkylation of corresponding thioglycolurils with halogenoalkanes and tested for their fungicidal activity against six phytopathogenic fungi from different taxonomic classes: Venturia inaequalis, Rhizoctonia solani, Fusarium oxysporum, Fusarium moniliforme, Bipolaris sorokiniana, and Sclerotinia sclerotiorum, and two pathogenic yeasts: Candida albicans and Cryptococcus neoformans var. grubii. A number of S-alkyl substituted thioglycolurils exhibited high activity against Venturia inaequalis and Rhizoctonia solani (85-100% mycelium growth inhibition), and moderate activity against other phytopathogens. S-Ethyl substituted thioglycolurils possessed a high activity against Candida albicans. Additionally, the hemolytic and cytotoxic properties of promising derivatives were determined using human red blood cells and human embryonic kidney cells, respectively. Two S-ethyl derivatives possessed both low cytotoxicity against normal human cells and high fungicidal activity against Candida albicans.

Impact of bacterial volatiles on phytopathogenic fungi: an in vitro study on microbial competition and interaction.

Microorganisms in the rhizosphere are abundant and exist in very high taxonomic diversity. The major players are bacteria and fungi, and bacteria have evolved many strategies to prevail over fungi, among them harmful enzyme activities and noxious secondary metabolites. Interactions between plant growth promoting rhizobacteria and phytopathogenic fungi are potentially valuable since the plant would benefit from fungal growth repression. In this respect, the role of volatile bacterial metabolites in fungistasis has been demonstrated, but the mechanisms of action are less understood. We used three phytopathogenic fungal species (Sclerotinia sclerotiorum, Rhizoctonia solani, and Juxtiphoma eupyrena) as well as one non-phytopathogenic species (Neurospora crassa) and the plant growth promoting rhizobacterium Serratia plymuthica 4Rx13 in co-cultivation assays to investigate the influence of bacterial volatile metabolites on fungi on a cellular level. As a response to the treatment, we found elevated lipid peroxidation, which indirectly reflected the loss of fungal cell membrane integrity. An increase in superoxide dismutase, catalase, and laccase activities indicated oxidative stress. Acclimation to these adverse growth conditions completely restored fungal growth. One of the bioactive bacterial volatile compounds seemed to be ammonia, which was a component of the bacterial volatile mixture. Applied as a single compound in biogenic concentrations ammonia also caused an increase in lipid peroxidation and enzyme activities, but the extent and pattern did not fully match the effect of the entire bacterial volatile mixture.

Antifungal activity screening of fractions from Annona cherimola Mill. leaf extract against Fusarium oxysporum.

The antifungal effect of ethanolic extract fractions of Annona cherimola leaves against the mycelial growth of Fusarium oxysporum was studied. The ethanolic crude extract was solvent partitioned and the ethyl acetate phase was fractionated by column or preparative thin-layer chromatography. All fractions were developed on TLC and analyzed for acetogenins (ACG) with Kedde reagent. The antifungal effect assays were carried out in vitro by the diffusion method on PDA plates. The ethanolic extract of A. cherimola leaves was highly active against F. oxysporum growth; subfractions obtained from the antifungal screening had a significant effect (p < 0.05) on the F. oxysporum growth parameters. The screening showed that as the purification steps progressed, the inhibition of mycelial growth increased. Six bioactive ACG (Annomolon-B, 34-epi annomolon B, almunequin, cherimoline 1, cherimoline 2, and isocherimoline 1) were identified by LC-QTOF-MS/MS. These findings suggested that bioactive ACG from A. cherimola leaves could be an alternative resource of a promising botanical fungicide to control plant diseases caused by F. oxysporum.

Conserved fungal effector suppresses PAMP-triggered immunity by targeting plant immune kinases.

Plant pathogens have optimized their own effector sets to adapt to their hosts. However, certain effectors, regarded as core effectors, are conserved among various pathogens, and may therefore play an important and common role in pathogen virulence. We report here that the widely distributed fungal effector NIS1 targets host immune components that transmit signaling from pattern recognition receptors (PRRs) in plants. NIS1 from two Colletotrichum spp. suppressed the hypersensitive response and oxidative burst, both of which are induced by pathogen-derived molecules, in Nicotiana benthamianaMagnaporthe oryzae NIS1 also suppressed the two defense responses, although this pathogen likely acquired the NIS1 gene via horizontal transfer from Basidiomycota. Interestingly, the root endophyte Colletotrichum tofieldiae also possesses a NIS1 homolog that can suppress the oxidative burst in N. benthamiana We show that NIS1 of multiple pathogens commonly interacts with the PRR-associated kinases BAK1 and BIK1, thereby inhibiting their kinase activities and the BIK1-NADPH oxidase interaction. Furthermore, mutations in the NIS1-targeting proteins, i.e., BAK1 and BIK1, in Arabidopsis thaliana also resulted in reduced immunity to Colletotrichum fungi. Finally, M. oryzae lacking NIS1 displayed significantly reduced virulence on rice and barley, its hosts. Our study therefore reveals that a broad range of filamentous fungi maintain and utilize the core effector NIS1 to establish infection in their host plants and perhaps also beneficial interactions, by targeting conserved and central PRR-associated kinases that are also known to be targeted by bacterial effectors.

Isolation and Identification of Secondary Metabolites Produced by Phytopathogenic Fungus Corynespora cassiicola from Hevea brasiliensis.

The secondary metabolites of the phytopathogenic fungus Corynespora cassiicola CC01 from Hevea brasiliensis were investigated. As a result, two new compounds, 5-acetyl-7-hydroxy-6- methoxybenzofuran-2(3H)-one (1) and (S)-2-(2,3-dihydrofuro [3,2-c]pyridin-2-yl)propan-2-ol (2), together with seven known compounds, 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (3), 3,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (4), curvulin acid (5), 2-methyl-5-carboxymethyl- 7-hydroxychromone (6), tyrosol (7), p-hydroxybenzoic acid (8) and cerevisterol (9), were isolated from the fermentation extract by comprehensive silica gel, reverse phase silica gel, Sephadex-LH20 column chromatography and high-performance liquid chromatography (HPLC). The structures of these compounds were identified by using high-resolution electrospray mass spectrometry (HRESIMS), nuclear magnetic resonance spectroscopy (NMR), optical rotation, ultraviolet and infrared spectroscopy techniques and a comparison of NMR data with those reported in the literature. Compounds 1 and 2 were new compounds, and compounds 3-9 were discovered from this phytopathogenic fungus for the first time. Compounds 1-9 were tested for phytotoxicity against the fresh tender leaf of Hevea brasiliensis, and the results show that none of them were phytotoxic. Additionally, these compounds were subjected to an antimicrobial assay against three bacteria (E. coli, methicillin-resistant Staphylococcus aureus and Micrococcus luteus), but they showed no activity.

Antioxidant, Antibacterial, and Antifungal Activities of the Ethanolic Extract Obtained from Berberis vulgaris Roots and Leaves.

This work assessed the phenolic and flavonoid components and their antioxidant, antifungal, and antibacterial effects in the ethanolic extract of barberry leaf and roots. The antibactericidal activity of root and leaf extracts against pathogenic bacteria was tested using agar diffusion and microdilution broth production for the lowest inhibitory concentration (MIC). Berberis vulgaris root and leaf extracts inhibited Staphylococcus aureus ATCC9973, Escherichia coli HB101, Staphylococcus enteritis, and Escherichia coli Cip812. The disc assay technique was used to assess the bactericidal activity of the extracts versus both pathogenic Gram-positive and Gram-negative strains. Hydro alcoholic extract was more effective against bacterial than fungal strains. The results showed that Berberis vulgaris leaf and roots extract had similar antifungal activities. Berberis vulgaris root extract inhibited the mycelial growth of Penicillium verrucosum, Fusarium proliferatum, Aspergillus ochraceous, Aspergillus niger, and Aspergillus flavus. Berberis vulgaris root extract has excellent antioxidant, antibacterial, and antifungal effects. Berberis vulgaris exhibited antimicrobial activity in vitro, and MIC showed that Berberis vulgaris parts efficiently affected pathogens in vitro. In conclusion, both Berberis vulgaris roots and leaves have considerable antibacterial activity and can be used as a source of antibacterial, antioxidant, and bioactive compounds to benefit human health.

Metabolite Profiling and Bioassay-Guided Fractionation of Zataria multiflora Boiss. Hydroethanolic Leaf Extracts for Identification of Broad-Spectrum Pre and Postharvest Antifungal Agents.

Hydroethanolic leaf extracts of 14 Iranian Zataria multiflora Boiss. populations were screened for their antifungal activity against five plant pathogenic fungi and metabolically profiled using a non-targeted workflow based on UHPLC/ESI-QTOFMS. Detailed tandem mass-spectrometric analyses of one of the most active hydroethanolic leaf extracts led to the annotation of 68 non-volatile semi-polar secondary metabolites, including 33 flavonoids, 9 hydroxycinnamic acid derivatives, 14 terpenoids, and 12 other metabolites. Rank correlation analyses using the abundances of the annotated metabolites in crude leaf extracts and their antifungal activity revealed four O-methylated flavones, two flavanones, two dihydroflavonols, five thymohydroquinone glycoconjugates, and five putative phenolic diterpenoids as putative antifungal metabolites. After bioassay-guided fractionation, a number of mono-, di- and tri-O-methylated flavones, as well as three of unidentified phenolic diterpenoids, were found in the most active subfractions. These metabolites are promising candidates for the development of new natural fungicides for the protection of agro-food crops.