The disordered C-terminal tail of fungal LPMOs from phytopathogens mediates protein dimerization and impacts plant penetration.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.

Characterization of tRNA splicing enzymes RNA ligase and tRNA 2'-phosphotransferase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trl1 enzymes are present in many human fungal pathogens and are promising targets for antifungal drug discovery because their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. Here we report that Mucorales species (deemed high-priority human pathogens by WHO) elaborate a noncanonical tRNA splicing apparatus in which a monofunctional RNA ligase enzyme is encoded separately from any end-healing enzymes. We show that Mucor circinelloides RNA ligase (MciRNL) is active in tRNA splicing in vivo in budding yeast in lieu of the Trl1 ligase domain. Biochemical and kinetic characterization of recombinant MciRNL underscores its requirement for a 2'-PO4 terminus in the end-joining reaction, whereby the 2'-PO4 enhances the rates of RNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3) by ∼125-fold and ∼6200-fold, respectively. In the canonical fungal tRNA splicing pathway, the splice junction 2'-PO4 installed by RNA ligase is removed by a dedicated NAD+-dependent RNA 2'-phosphotransferase Tpt1. Here we identify and affirm by genetic complementation in yeast the biological activity of Tpt1 orthologs from three Mucorales species. Recombinant M. circinelloides Tpt1 has vigorous NAD+-dependent RNA 2'-phosphotransferase activity in vitro.

Genome-wide analysis of heat stress-stimulated transposon mobility in the human fungal pathogen Cryptococcus deneoformans.

We recently reported transposon mutagenesis as a significant driver of spontaneous mutations in the human fungal pathogen Cryptococcus deneoformans during murine infection. Mutations caused by transposable element (TE) insertion into reporter genes were dramatically elevated at high temperatures (37° vs. 30°) in vitro, suggesting that heat stress stimulates TE mobility in the Cryptococcus genome. To explore the genome-wide impact of TE mobilization, we generated transposon accumulation lines by in vitro passage of C. deneoformans strain XL280α for multiple generations at both 30° and at the host-relevant temperature of 37°. Utilizing whole-genome sequencing, we identified native TE copies and mapped multiple de novo TE insertions in these lines. Movements of the T1 DNA transposon occurred at both temperatures with a strong bias for insertion between gene-coding regions. By contrast, the Tcn12 retrotransposon integrated primarily within genes and movement occurred exclusively at 37°. In addition, we observed a dramatic amplification in copy number of the Cnl1 (Cryptococcus neoformans LINE-1) retrotransposon in subtelomeric regions under heat-stress conditions. Comparing TE mutations to other sequence variations detected in passaged lines, the increase in genomic changes at elevated temperatures was primarily due to mobilization of the retroelements Tcn12 and Cnl1. Finally, we found multiple TE movements (T1, Tcn12, and Cnl1) in the genomes of single C. deneoformans isolates recovered from infected mice, providing evidence that mobile elements are likely to facilitate microevolution and rapid adaptation during infection.

The mRNA stability factor Khd4 defines a specific mRNA regulon for membrane trafficking in the pathogen Ustilago maydis.

Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins (RBPs). However, little is known about the spatiotemporal RNA control mechanisms during fungal pathogenicity. Here, we discover that the RBP Khd4 defines a distinct mRNA regulon to orchestrate membrane trafficking during pathogenic development of Ustilago maydis. By establishing hyperTRIBE for fungal RBPs, we generated a comprehensive transcriptome-wide map of Khd4 interactions in vivo. We identify a defined set of target mRNAs enriched for regulatory proteins involved, e.g., in GTPase signaling. Khd4 controls the stability of target mRNAs via its cognate regulatory element AUACCC present in their 3' untranslated regions. Studying individual examples reveals a unique link between Khd4 and vacuole maturation. Thus, we uncover a distinct role for an RNA stability factor defining a specific mRNA regulon for membrane trafficking during pathogenicity.

Advancements and challenges in antifungal therapeutic development.

Over recent decades, the global burden of fungal disease has expanded dramatically. It is estimated that fungal disease kills approximately 1.5 million individuals annually; however, the true worldwide burden of fungal infection is thought to be higher due to existing gaps in diagnostics and clinical understanding of mycotic disease. The development of resistance to antifungals across diverse pathogenic fungal genera is an increasingly common and devastating phenomenon due to the dearth of available antifungal classes. These factors necessitate a coordinated response by researchers, clinicians, public health agencies, and the pharmaceutical industry to develop new antifungal strategies, as the burden of fungal disease continues to grow. This review provides a comprehensive overview of the new antifungal therapeutics currently in clinical trials, highlighting their spectra of activity and progress toward clinical implementation. We also profile up-and-coming intracellular proteins and pathways primed for the development of novel antifungals targeting their activity. Ultimately, we aim to emphasize the importance of increased investment into antifungal therapeutics in the current continually evolving landscape of infectious disease.

Loss of the scavenger receptor MARCO results in uncontrolled vomocytosis of fungi from macrophages.

Vomocytosis, also known as nonlytic exocytosis, is a process whereby fully phagocytosed microbes are expelled from phagocytes without discernible damage to either the phagocyte or microbe. Although this phenomenon was first described in the opportunistic fungal pathogen Cryptococcus neoformans in 2006, to date, mechanistic studies have been hampered by an inability to reliably stimulate or inhibit vomocytosis. Here we present the fortuitous discovery that macrophages lacking the scavenger receptor MAcrophage Receptor with COllagenous domain (MARCO), exhibit near-total vomocytosis of internalised cryptococci within a few hours of infection. Marco-/- macrophages also showed elevated vomocytosis of a yeast-locked C. albicans strain, suggesting this to be a broadly relevant observation. We go on to show that MARCO's role in modulating vomocytosis is independent of its role as a phagocytic receptor, suggesting that this protein may play an important and hitherto unrecognised role in modulating macrophage behaviour.

Disruption of the Aspergillus fumigatus RNA interference machinery alters the conidial transcriptome.

The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

A network of transcription factors in complex with a regulating cell cycle cyclin orchestrates fungal oxidative stress responses.

BACKGROUND: Response to oxidative stress is universal in almost all organisms and the mitochondrial membrane protein, BbOhmm, negatively affects oxidative stress responses and virulence in the insect fungal pathogen, Beauveria bassiana. Nothing further, however, is known concerning how BbOhmm and this phenomenon is regulated. RESULTS: Three oxidative stress response regulating Zn2Cys6 transcription factors (BbOsrR1, 2, and 3) were identified and verified via chromatin immunoprecipitation (ChIP)-qPCR analysis as binding to the BbOhmm promoter region, with BbOsrR2 showing the strongest binding. Targeted gene knockout of BbOsrR1 or BbOsrR3 led to decreased BbOhmm expression and consequently increased tolerances to free radical generating compounds (H2O2 and menadione), whereas the ΔBbOsrR2 strain showed increased BbOhmm expression with concomitant decreased tolerances to these compounds. RNA and ChIP sequencing analysis revealed that BbOsrR1 directly regulated a wide range of antioxidation and transcription-associated genes, negatively affecting the expression of the BbClp1 cyclin and BbOsrR2. BbClp1 was shown to localize to the cell nucleus and negatively mediate oxidative stress responses. BbOsrR2 and BbOsrR3 were shown to feed into the Fus3-MAPK pathway in addition to regulating antioxidation and detoxification genes. Binding motifs for the three transcription factors were found to partially overlap in the promoter region of BbOhmm and other target genes. Whereas BbOsrR1 appeared to function independently, co-immunoprecipitation revealed complex formation between BbClp1, BbOsrR2, and BbOsrR3, with BbClp1 partially regulating BbOsrR2 phosphorylation. CONCLUSIONS: These findings reveal a regulatory network mediated by BbOsrR1 and the formation of a BbClp1-BbOsrR2-BbOsrR3 complex that orchestrates fungal oxidative stress responses.

Protective innate immunity against Pneumocystis does not require Stat6-dependent macrophage polarization.

Pneumocystis species are respiratory fungal pathogens that cause life-threatening opportunistic infections in immunocompromised hosts. Pneumocystis typically evade pulmonary innate immunity but are efficiently eradicated by a functional adaptive immune response. FVB/NJ mice are unique in that they display protective alveolar macrophage-dependent innate immunity against Pneumocystis, and remain resistant to infection even in the absence of CD4+ T lymphocyte function. FVB/NJ alveolar macrophages (AMs) were found to display an M2-biased phenotype at baseline, which was potentiated after stimulation with Pneumocystis, suggesting that macrophage polarization may dictate the outcome of the Pneumocystis-macrophage interaction. To determine whether Stat6, a key global regulator of M2 polarization, was required for FVB/NJ innate immunity, FVB Stat6-/- mice were generated. FVB Stat6-deficient AMs were markedly impaired in their ability to polarize to an M2 phenotype when stimulated with Th2 cytokines. However, FVB Stat6-/- mice remained highly resistant to infection, indicating that Stat6 signaling is dispensable for innate FVB/NJ resistance. Despite the loss of Stat6 signaling, primary AMs from FVB Stat6-/- mice maintained baseline expression of M2 markers, and also strongly upregulated M2-associated genes following direct stimulation with Pneumocystis. Additional FVB/NJ knockout strains were generated, but only FVB MerTK-/- mice showed a marginally increased susceptibility to Pneumocystis infection. Together, these findings demonstrate that effective FVB/NJ innate immunity against Pneumocystis does not require Stat6 signaling and suggest that alternative pathways regulate M2 bias and macrophage-mediated innate resistance in FVB/NJ mice.

The Zymoseptoria tritici Avirulence Factor AvrStb6 Accumulates in Hyphae Close to Stomata and Triggers a Wheat Defense Response Hindering Fungal Penetration.

Zymoseptoria tritici, the causal agent of Septoria tritici blotch, is one of Europe's most damaging wheat pathogens, causing significant economic losses. Genetic resistance is a common strategy to control the disease, Stb6 being a resistance gene used for more than 100 years in Europe. This study investigates the molecular mechanisms underlying Stb6-mediated resistance. Utilizing confocal microscopy imaging, we determined that Z. tritici epiphytic hyphae mainly accumulate the corresponding avirulence factor AvrStb6 in close proximity to stomata. Consequently, the progression of AvrStb6-expressing avirulent strains is hampered during penetration. The fungal growth inhibition co-occurs with a transcriptional reprogramming in wheat characterized by an induction of immune responses, genes involved in stomatal regulation, and cell wall-related genes. Overall, we shed light on the gene-for-gene resistance mechanisms in the wheat-Z. tritici pathosystem at the cytological and transcriptomic level, and our results highlight that stomatal penetration is a critical process for pathogenicity and resistance. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Paranannizziopsis spp. Infection in Wild Vipers, Europe.

We describe the detection of Paranannizziopsis sp. fungus in a wild population of vipers in Europe. Fungal infections were severe, and 1 animal likely died from infection. Surveillance efforts are needed to better understand the threat of this pathogen to snake conservation.

A Burkholderia cenocepacia-like environmental isolate strongly inhibits the plant fungal pathogen Zymoseptoria tritici.

Fungal phytopathogens cause significant reductions in agricultural yields annually, and overusing chemical fungicides for their control leads to environmental pollution and the emergence of resistant pathogens. Exploring natural isolates with strong antagonistic effects against pathogens can improve our understanding of their ecology and develop new treatments for the future. We isolated and characterized a novel bacterial strain associated with the species Burkholderia cenocepacia, termed APO9, which strongly inhibits Zymoseptoria tritici, a commercially important pathogenic fungus causing Septoria tritici blotch in wheat. Additionally, this strain exhibits inhibitory activity against four other phytopathogens. We found that physical contact plays a crucial role for APO9's antagonistic capacity. Genome sequencing of APO9 and biosynthetic gene cluster (BGC) analysis identified nine classes of BGCs and three types of secretion systems (types II, III, and IV), which may be involved in the inhibition of Z. tritici and other pathogens. To identify genes driving APO9's inhibitory activity, we screened a library containing 1,602 transposon mutants and identified five genes whose inactivation reduced inhibition efficiency. One such gene encodes for a diaminopimelate decarboxylase located in a terpenoid biosynthesis gene cluster. Phylogenetic analysis revealed that while some of these genes are also found across the Burkholderia genus, as well as in other Betaproteobacteria, the combination of these genes is unique to the Burkholderia cepacia complex. These findings suggest that the inhibitory capacity of APO9 is complex and not limited to a single mechanism, and may play a role in the interaction between various Burkholderia species and various phytopathogens within diverse plant ecosystems. IMPORTANCE: The detrimental effects of fungal pathogens on crop yields are substantial. The overuse of chemical fungicides contributes not only to environmental pollution but also to the emergence of resistant pathogens. Investigating natural isolates with strong antagonistic effects against pathogens can improve our understanding of their ecology and develop new treatments for the future. We discovered and examined a unique bacterial strain that demonstrates significant inhibitory activity against several phytopathogens. Our research demonstrates that this strain has a wide spectrum of inhibitory actions against plant pathogens, functioning through a complex mechanism. This plays a vital role in the interactions between plant microbiota and phytopathogens.

Two-speed genomes of Epichloe fungal pathogens show contrasting signatures of selection between species and across populations.

Antagonistic selection between pathogens and their hosts can drive rapid evolutionary change and leave distinct molecular footprints of past and ongoing selection in the genomes of the interacting species. Despite an increasing availability of tools able to identify signatures of selection, the genetic mechanisms underlying coevolutionary interactions and the specific genes involved are still poorly understood, especially in heterogeneous natural environments. We searched the genomes of two species of Epichloe plant pathogen for evidence of recent selection. The Epichloe genus includes highly host-specific species that can sterilize their grass hosts. We performed selection scans using genome-wide SNP data from seven natural populations of two co-occurring Epichloe sibling species specialized on different hosts. We found evidence of recent (and ongoing) selective sweeps across the genome in both species. However, selective sweeps were more abundant in the species with a larger effective population size. Sweep regions often overlapped with highly polymorphic AT-rich regions supporting the role of these genome compartments in adaptive evolution. Although most loci under selection were specific to individual populations, we could also identify several candidate genes targeted by selection in sweep regions shared among populations. The genes encoded small secreted proteins typical of fungal effectors and cell wall-degrading enzymes. By investigating the genomic signatures of selection across multiple populations and species, this study contributes to our understanding of complex adaptive processes in natural plant pathogen systems.

Regional diversity and leaf microbiome interactions of the fungal maize pathogen Exserohilum turcicum in Switzerland: A metagenomic analysis.

The spread and adaptation of fungal plant pathogens in agroecosystems are facilitated by environmental homogeneity. Metagenomic sequencing of infected tissues allowed us to monitor eco-evolutionary dynamics and interactions between host, pathogen and plant microbiome. Exserohilum turcicum, the causal agent of northern corn leaf blight (NCLB) in maize, is distributed in multiple clonal lineages throughout Europe. To characterize regional pathogen diversity, we conducted metagenomic DNA sequencing on 241 infected leaf samples from the highly susceptible Swiss maize landrace Rheintaler Ribelmais, collected over 3 years (2016-2018) from an average of 14 agricultural farms within the Swiss Rhine Valley. All major European clonal lineages of E. turcicum were identified. Lineages differ by their mating types which indicates potential for sexual recombination and rapid evolution of new pathogen strains, although we found no evidence of recent recombination. The associated eukaryotic and prokaryotic leaf microbiome exhibited variation in taxonomic diversity between years and locations and is likely influenced by local weather conditions. A network analysis revealed distinct clusters of eukaryotic and prokaryotic taxa that correlates with the frequency of E. turcicum sequencing reads, suggesting causal interactions. Notably, the yeast genus Metschnikowia exhibited a strongly negative association with E. turcicum, supporting its known potential as biological control agent against fungal pathogens. Our findings show that metagenomic sequencing is a useful tool for analysing the role of environmental factors and potential pathogen-microbiome interactions in shaping pathogen dynamics and evolution, suggesting their potential for effective pathogen management strategies.

Transcriptional landscape of pathogen-responsive lncRNAs in tomato unveils the role of hydrolase encoding genes in response to Botrytis cinerea invasion.

LncRNAs have gained increasing attention owing to their important regulatory roles on growth and stress responses of plants. However, the mechanisms underlying the functions of lncRNAs in fruit-pathogen interaction are still largely unknown. In this study, a total of 273 lncRNAs responding to Botrytis cinerea infection were identified in tomato fruit, among which a higher percentage of antisense lncRNAs were targeted to the genes enriched in hydrolase activity. To ascertain the roles of these lncRNAs, seven hydrolase-related transcripts were transiently knocked-down by virus-induced gene silencing. Silencing of lncRNACXE20 reduced the expression level of a carboxylesterase gene, further enhancing the resistance of tomato to B. cinerea. In contrast, silencing of lncRNACHI, lncRNAMMP, lncRNASBT1.9 and lncRNAPME1.9 impaired the resistance to B. cinerea, respectively. Further RT-qPCR assay and enzymatic activity detection displayed that the attenuated resistance of lncRNAMMP and lncRNASBT1.9-silenced plants was associated with the inhibition on the expression of JA-related genes, while the decreased resistance of lncRNACHI-silenced plants resulted in reduced chitinase activity. Collectively, these results may provide references for deciphering the mechanisms underlying specific lncRNAs to interfere with B. cinerea infection by regulating the expression of defence-related genes or affecting hydrolase activity.

Terpenoids are involved in the expression of systemic-induced resistance in Austrian pine.

Terpenoids are defense metabolites that are induced upon infection or wounding. However, their role in systemic-induced resistance (SIR) is not known. Here, we explored the role of terpenoids in this phenomenon at a very early stage in the interaction between Austrian pine and the tip blight and canker pathogen Diplodia pinea. We induced Austrian pine saplings by either wounding or inoculating the lower stems with D. pinea. The seedlings were then challenged after 12 h, 72 h, or 10 days with D. pinea on the stem 15 cm above the induction. Lesion lengths and terpenoids were quantified at both induction and challenge locations. Key terpenoids were assayed for antifungal activity in in vitro bioassays. SIR increased with time and was correlated with the inducibility of several compounds. α-Pinene and a cluster of ß-pinene, limonene, benzaldehyde, dodecanol, and n-dodecyl acrylate were positively correlated with SIR and were fungistatic in vitro, while other compounds were negatively correlated with SIR and appeared to serve as a carbon source for D. pinea. This study shows that, overall, terpenoids are involved in SIR in this system, but their role is nuanced, depending on the type of induction and time of incubation. We hypothesize that some, such as α-pinene, could serve in SIR signaling.

First Report of Phyllosticta capitalensis Causing Postharvest Brown Spot Disease on Guava Fruit in Thailand.

Guava (Psidium guajava L.) is a popular fruit crop that is widely cultivated in Thailand. In November 2023, brown spot disease on guava was observed during postharvest storage at 22 to 31°C and 70 to 75% relative humidity over a period of 3 to 7 days in Fang District, Chiang Mai Province, Thailand. The disease incidence was ~20% of 100 fruits per pallet box. The disease severity on each fruit ranged from 40 to 70% of the surface area affected by lesions. The symptoms appeared as circular to irregular brown to dark brown spots, ranging from 5 to 30 mm in diameter. Fungi were isolated from lesions using a single conidial isolation method (Choi et al. 1999). Two fungal isolates (SDBR-CMU497 and SDBR-CMU498) with similar morphology were obtained. Colonies on potato dextrose agar (PDA) and malt extract agar (MEA) were 65 to 67 and 29 to 38 mm in diameter, respectively after incubation for 1 week at 25°C. Colonies on PDA and MEA were flat, slightly undulate, greenish gray in the center, greyish green at the margin; reverse black. Both isolates produced asexual structures. Pycnidia were black, granular, and grouped. Conidiogenous cells were hyaline, subcylindrical to cylindrical, 8.5 to 17.5 × 3 to 5.5 µm. Conidia were single-celled, hyaline, obovoid to ellipsoid, 5.2 to 9.4 × 3.6 to 7.5 µm (n = 50), smooth-walled, with a single apical appendage. Morphologically, both isolates resembled Phyllosticta capitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, large subunit (nrLSU), translation elongation factor 1-alpha (tef1-α), actin (act), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using primer pairs ITS5/ITS4, LROR/LRO5, EF1-728F/EF2, ACT-512F/ACT-783R, and GPD1-LM/GPD2-LM, respectively (White et al. 1990; Zhang et al. 2022). Sequences were deposited in GenBank (ITS: PP946770, PP946771; nrLSU: PP948677, PP948678; tef1-α: PP948012, PP948013; act: PP948014, PP948015; GAPDH: PP948016, PP948017). Maximum likelihood phylogenetic analyses of the concatenated five genes identified both isolates as P. capitalensis. Thus, both morphology and molecular data confirmed the fungus as P. capitalensis. To confirm pathogenicity, healthy commercial guava fruits cultivar Kim Ju were surface disinfected by 0.1% NaClO for 3 min, rinsed three times with sterile distilled water, and wounded (Cruz-Lagunas et al. 2023). Conidia were collected from 2-week-old cultures on PDA and suspended in sterile distilled water. Fifteen microliters of a 1 × 106 conidia/ml suspension were dropped onto the wounded fruits. Mock inoculations were used as a control with sterile distilled water. Ten replications were conducted for each treatment and repeated twice. The inoculated fruits were stored in individual sterile plastic boxes at 25°C with 80 to 90% relative humidity. After 7 days, all inoculated fruits exhibited brown to dark brown lesions, while control fruits were asymptomatic. Phyllosticta capitalensis was consistently reisolated from the inoculated tissues on PDA to complete Koch's postulates. Prior to this study, P. capitalensis was known to cause brown or black spot disease on guava fruits cultivated in fields in China (Liao et al. 2020), Egypt (Arafat 2018), and Mexico (Cruz-Lagunas et al. 2023). To our knowledge, this is the first report of P. capitalensis causing postharvest brown spot disease on guava fruit in Thailand. The results will inform epidemiological investigations and future approaches to managing this disease.

First Report of Black Leaf Spot Caused by Epicoccum latusicollum on Chinese Yam in China.

Chinese yam (Dioscorea polystachya Turczaninow cv. Tiegun), which belongs to the family Dioscoreaceae, is widely cultivated throughout China due to its high economic and medicinal value. In June 2023, black leaf spots on Chinese yam (cv. Purple 1) were observed in Nanchang city (28.45° N, 115.49° E) of Jiangxi province, southeastern China. The incidence of the disease ranged between 70 and 85% of plants, and up to 30% of the leaves per plant were affected in the field over a 2-week period of study. Infected foliage displayed brown necrotic lesions, elliptical or irregular, with yellow halo at the edge of the lesion (0.5 to 3 cm diam.). To identify the causal agent, 32 symptomatic leaves of eight symptomatic plants were collected. Small pieces from the margin of necrotic leaf tissue (about 3 x 3 mm) were surface sterilized in 75% ethanol for 30 s followed in 0.1% HgCl2 for 1 min, and washed three times with ddH2O. Then, the pieces were transferred onto potato dextrose agar (PDA) plates and incubated at 26°C for 3 days with a 12-h light-dark cycle. From the 32 isolates, 21 exhibited similar morphology after hyphal tipping resulting in an isolation frequency of 65.6%. Colonies on PDA were initially white aerial hyphae but became grayish with age, and a reddish orange pigment on the underside. After 16 days of incubation, pycnidia were observed, which were dark, spherical or flat spherical, and 64.1 to 172.5 µm (n = 25) in diameter. Conidia were ellipsoidal, aseptate, hyaline, and 4.1 to 5.6 × 1.8 to 2.7 µm (n = 80). In addition, a blackish green discoloration was produced on malt extract agar (MEA) using the NaOH spot test. The isolates were tentatively identified as Epicoccum spp. based on morphological characteristics (Chen et al. 2017). Isolate AYZ-1 was randomly selected for identification and pathogenicity testing. Genomic DNA of the isolate (AYZ-1) was extracted and amplified by polymerase chain reaction (PCR) using ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990), Btub2Fd/Btub4Rd for the ß-tubulin (TUB) region (Woudenberg et al. 2009), LROR/LR7 for the large ribosomal RNA gene (LSU) region (Rehner and Samuels 1994), and RPB2-5F2/fRPB2-7cR for RNA polymerase II second largest subunit (RPB2) region (Liu et al. 1999), respectively. The concatenated sequences (GenBank Accession No. OR574165, OR567827, OR574166, OR567828, respectively) shared 99.8 to 100% identity with Epicoccum latusicollum (OP788080, MN329871, OR428532, and OL422485, respectively). A neighbor-joining phylogenetic tree was generated based on the concatenated sequences in MEGA7, placed isolate (AYZ-1) within E. latusicollum. To fulfill Koch's postulates, healthy leaflets from three one-year-old Chinese yam (cv. Purple 1) were used as inoculation materials, using isolate AYZ-1. Two sites of each leaf were wounded with a sterile needle and covered with a piece of cotton drenched with 200 µL spore suspension (106 conidia/mL) on the left sides, while sterilized water served as the control on the right sides of leaves. All inoculated leaves were covered with clear polyethylene bags for 24 h. Plants were grown outdoors at a daily average temperature of 26°C with relative humidity over 45%. After 7 days of incubation, the leaves showed the same symptoms as the original diseased leaves. The E. latusicollum isolate was re-isolated from diseased leaves and confirmed by morphology and sequencing analysis, fulfilling Koch's postulates. E. latusicollum has been previously reported to cause black root on yam in China's south-western province of Sichuan (Han et al. 2019). Meanwhile, leaf spot have been reported on many plants by this genus, such as tobacco (Guo et al. 2020) and banana (Liu et al. 2023). According to our knowledge, this is the first report of E. latusicollum causing black leaf spot on Chinese yam in China. This finding will provide an important reference for understanding the biology of E. latusicollum and the distribution of the disease, but more research is needed to determine if management is warranted.

The Auto-Regulation of ATL2 E3 Ubiquitin Ligase Plays an Important Role in the Immune Response against Alternaria brassicicola in Arabidopsis thaliana.

The ubiquitin/26S proteasome system is a crucial regulatory mechanism that governs various cellular processes in plants, including signal transduction, transcriptional regulation, and responses to biotic and abiotic stressors. Our study shows that the RING-H2-type E3 ubiquitin ligase, Arabidopsis Tóxicos en Levadura 2 (ATL2), is involved in response to fungal pathogen infection. Under normal growth conditions, the expression of the ATL2 gene is low, but it is rapidly and significantly induced by exogenous chitin. Additionally, ATL2 protein stability is markedly increased via chitin treatment, and its degradation is prolonged when 26S proteasomal function is inhibited. We found that an atl2 null mutant exhibited higher susceptibility to Alternaria brassicicola, while plants overexpressing ATL2 displayed increased resistance. We also observed that the hyphae of A. brassicicola were strongly stained with trypan blue staining, and the expression of A. brassicicola Cutinase A (AbCutA) was dramatically increased in atl2. In contrast, the hyphae were weakly stained, and AbCutA expression was significantly reduced in ATL2-overexpressing plants. Using bioinformatics, live-cell confocal imaging, and cell fractionation analysis, we revealed that ATL2 is localized to the plasma membrane. Further, it is demonstrated that the ATL2 protein possesses E3 ubiquitin ligase activity and found that cysteine 138 residue is critical for its function. Moreover, ATL2 is necessary to successfully defend against the A. brassicicola fungal pathogen. Altogether, our data suggest that ATL2 is a plasma membrane-integrated protein with RING-H2-type E3 ubiquitin ligase activity and is essential for the defense response against fungal pathogens in Arabidopsis.

Use of natural-based commercial products as an alternative for providing bioprotection against verticillium wilt of olive.

BACKGROUND: As a result of the ineffectiveness of existing control methods against Verticillium dahliae, the causal agent of verticillium wilt of olive (Olea europaea; VWO), it is necessary to search for sustainable and environmentally friendly alternatives, such as bioprotection by products based on plant extracts and other naturally synthesized compounds. Therefore, present study aimed to evaluate the effects of seven natural-based commercial products on the inhibition of mycelial growth, the germination of V. dahliae conidia and microsclerotia, and disease progression in olive plants (cv. Picual). Aluminium lignosulfonate and a copper phosphonate salt (copper phosphite) were included for comparative purposes. RESULTS: The seaweed and willow extracts and copper phosphite inhibited V. dahliae mycelial growth by more than 50% at the high doses tested. Most of the products inhibited conidial germination by up to 90% compared to the control at the high doses tested. However, none of the products showed efficacy above 50% in inhibiting microsclerotia germination. The willow extract was the most effective at reducing disease severity and progression in olive plants, with no significant differences compared to the non-inoculated negative control. CONCLUSION: The results of the present study suggest that the use of natural-based products (i.e. seaweed and willow extracts) is a potential sustainable alternative in an integrated VWO control strategy. © 2024 Society of Chemical Industry.