Indole alkaloids isolated from the Nicotiana tabacum-derived Aspergillus fumigatus 0338 as potential inhibitors for tobacco powdery mildew and their mode of actions.

To explore active natural products against tobacco powdery mildew caused by Golovinomyces cichoracearum, an extract from the fermentation of endophytic Aspergillus fumigatus 0338 was investigated. The mechanisms of action for active compounds were also studied in detail. As a result, 14 indole alkaloid derivatives were isolated, with seven being newly discovered (1-7) and the remaining seven previously described (8-14). Notably, compounds 1-3 are rare linearly fused 6/6/5 tricyclic prenylated indole alkaloids, with asperversiamide J being the only known natural product of this kind. The isopentenyl substitutions at the 5-position in compounds 4 and 5 are also rare, with only compounds 1-(5-prenyl-1H-indol-3-yl)-propan-2-one (8) and 1-(6-methoxy-5-prenyl-1H-indol3-yl)-propan-2-one currently available. In addition, compounds 6 and 7 are new framework indole alkaloid derivatives bearing a 6-methyl-1,7-dihydro-2H-azepin-2-one ring. The purified compounds were evaluated for their activity against G. cichoracearum, and the results revealed that compounds 7 and 9 demonstrated obvious anti-G. cichoracearum activities with an inhibition rate of 82.6% and 85.2%, respectively, at a concentration of 250 µg/mL, these rates were better than that of the positive control agent, carbendazim (78.6%). The protective and curative effects of compounds 7 and 9 were also better than that of positive control, at the same concentration. Moreover, the mechanistic study showed that treatment with compound 9 significantly increased the structural tightness of tobacco leaves and directly affect the conidiospores of G. cichoracearum, thereby enhancing resistance. Compounds 7 and 9 could also induce systemic acquired resistance (SAR), directly regulating the expression of defense enzymes, defense genes, and plant semaphorins, which may further contribute to increased plant resistance. Based on the activity experiments and molecular dockings, the indole core structure may be the foundation of these compounds' anti-G. cichoracearum activity. Among them, the indole derivative parent structures of compounds 6, 7, and 9 exhibit strong effects. Moreover, the methoxy substitution in compound 7 can enhance their activity. By isolating and structurally identifying the above indole alkaloids, new candidates for anti-powdery mildew chemical screening were discovered, which could enhance the utilization of N. tabacum-derived fungi in pesticide development.

Golovinomyces cichoracearum effector-associated nuclear localization of RPW8.2 amplifies its expression to boost immunity in Arabidopsis.

Arabidopsis RESISTANCE TO POWDERY MILDEW 8.2 (RPW8.2) is specifically induced by the powdery mildew (PM) fungus (Golovinomyces cichoracearum) in the infected epidermal cells to activate immunity. However, the mechanism of RPW8.2-induction is not well understood. Here, we identify a G. cichoracearum effector that interacts with RPW8.2, named Gc-RPW8.2 interacting protein 1 (GcR8IP1), by a yeast two-hybrid screen of an Arabidopsis cDNA library. GcR8IP1 is physically associated with RPW8.2 with its REALLY INTERESTING NEW GENE finger domain that is essential and sufficient for the association. GcR8IP1 was secreted and translocated into the nucleus of host cell infected with PM. Association of GcR8IP1 with RPW8.2 led to an increase in RPW8.2 in the nucleus. In turn, the nucleus-localized RPW8.2 promoted the activity of the RPW8.2 promoter, resulting in transcriptional self-amplification of RPW8.2 to boost immunity at infection sites. Additionally, ectopic expression or host-induced gene silencing of GcR8IP1 supported its role as a virulence factor in PM. Altogether, our results reveal a mechanism of RPW8.2-dependent defense strengthening via altered partitioning of RPW8.2 and transcriptional self-amplification triggered by a PM fungal effector, which exemplifies an atypical form of effector-triggered immunity.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on Solanum carolinense in Korea.

Solanum carolinense L. (Solanaceae) is a perennial herbaceous plant native to the southeastern United States. The plant has been invasive in Europe, Australia, and Asia. It was accidentally introduced to Korea in the 1960s (Oh et al. 2002) and is now widely naturalized mostly in the southern area of the Korean peninsula. Since 2002, it has been designated as one of 'harmful non-indigenous plants' by the Korean Ministry of Environment due to its adverse effects on native plants. In September 2014, several S. carolinense plants were found infested with a powdery mildew disease in Busan, which was the first for Korea. Later in 2020-2022, hundreds of plants were continuously observed with an approximately 50% disease incidence in Jeonju, Korea. Symptoms first appeared as circular to irregular white patches, which subsequently coalesced to develop into abundant hyphal growth on both sides of the leaves. Representative voucher specimens were deposited in the Korea University herbarium (KUS-F28240 and F32541). Hyphal appressoria were nipple-shaped. Conidiophores were cylindrical, 110 to 190 × 10 to 12 µm, and produced 2 to 5 immature conidia in chains with a sinuate outline. Foot-cells of conidiophores were straight, cylindrical, and 58 to 90 µm long, followed by 2 to 3 cells. Conidia were ellipsoid to barrel-shaped, 29 to 36 × 15 to 20 µm (l/w 1.6 to 2.0), and devoid of distinct fibrosin bodies. Germ tubes were at the perihilar position of the conidia. Sexual stage was not developed. These morphological characteristics are consistent with those of Golovinomyces ambrosiae (Schwein.) U. Braun & R.T.A. (Braun and Cook 2012). Sequences of the internal transcribed spacer (ITS) region and large subunit (LSU) gene of rDNA were determined using primer pairs ITS1/PM6 and PM3/TW14, respectively (Bradshaw and Tobin 2020). A comparison of the resulting sequences using the BLASTn algorithm showed 100% identity with reference sequences of G. ambrosiae (MT355556, AB769425) and G. spadiceus (MN365027) for ITS and LSU in NCBI. Obtained sequences were deposited in GenBank (Accession Nos: OP585651-OP585654). A Maximum parsimony tree was constructed based on ITS+LSU dataset consisting of 23 sequences. Our sequences were clustered with sequences of G. ambrosiae, G. latisporus and G. cichoracearum, and supported with 100% BS value. A pathogenicity test was performed by gently dusting conidia onto leaves of five healthy potted plants. Five non-inoculated plants served as controls. Powdery mildew colonies developed on all inoculated plants after 5 days, whereas the control plants remained symptomless. The fungus present on the inoculated leaves was morphologically identical to that observed on the originally diseased leaves, which supports Koch's postulate. Previously, Erysiphe cichoracearum (syn. of G. cichoracearum) was recorded on this plant in theUSA (Farr and Rossman 2022). Since G. cichoracearum has been divided into several distinct species (Takamatsu et al. 2013, Qiu et al. 2020), the current taxonomic position of the North American isolate is unknown. In Korea, G. ambrosiae was reported on several asteraceous hosts such as Bellis perennis, Brachyscome multifida, Helianthus annuus, H. salicifolius, and Verbena bonariensis (Farr and Rossman 2022). To our knowledge, this is the first report of powdery mildew on this plant outside the USA. According to our field observations, powdery mildew infestation had little effect on plant growth and vigor.   References: Braun, U., and Cook, R. T. A. 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, Netherlands. Bradshaw, M., and Tobin, P.C. 2020. Phytopathology 110:1248. Farr, D. F., and Rossman, A. Y. Fungal Databases, Syst. Mycol. Microbiol. Lab., Online publication. ARS, USDA. Retrieved October 5, 2022. Oh, S. M., et al. 2002. Kor. J. Weed Sci. 22:280. Qiu, P-L., et al. 2020. BMC Microbiology 20:51. Takamatsu S., et al. 2013. Mycologia 105:1135.

First Report of Powdery Mildew Caused by Golovinomyces ambrosiae on New York Aster (Symphyotrichum novi-belgii) in Korea.

Symphyotrichum novi-belgii (L.) G.L. Nesom (syn. Aster novi-belgii L.), known as New York aster, is a perennial herb used in gardens and as a potted plant. The plant is native to North America but has been developed into various horticultural varieties. In Korea, it is one of the most common plants used for autumn bloom. In September 2011, New York asters (variety unknown) showing typical signs of powdery mildew were observed in a public garden in Seoul, Korea. Since then, the disease on New York asters has been continuously found in parks and flower markets in different regions of Korea. Voucher specimens (n=3) were deposited in the Korea University Herbarium (KUS-F 30752, 31865, and 32103). On leaves, circular to irregular white patches appeared which subsequently showed abundant hyphal growth on both sides of the leaves and on young stems and inflorescences, reducing the aesthetic value and vigor of the plants affected. Hyphae were septate, branched, and 4 to 8 µm wide. Appressoria on the mycelium were nipple-shaped. Conidiophores measured 110 to 200 × 9 to 11.5 µm, were simple, and produced 2 to 5 immature conidia in chains with a sinuate outline, followed by 2 to 3 cells. Foot-cells of conidiophores were straight, cylindric, and 55 to 125 µm long. Conidia were hyaline, ellipsoid to barrel-shaped, measured 22 to 52 × 15 to 20 µm (length/width ratio = 1.5-2.5), lacked distinct fibrosin bodies, and produced germ tubes on the subterminal position, with reticulate wrinkling of the outer walls. No chasmothecia were observed. The structures described above were typical of the Oidium subgenus Euoidium anamorph of the genus Golovinomyces, and the fungus measurements were consistent with those of G. ambrosiae (Schwein.) U. Braun & R.T.A. Cook (Braun and Cook 2012, Qiu et al. 2020). To confirm the identity of the causal fungus, the internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA were amplified with primers PM10/ITS4 for ITS and PM3/TW14 for LSU (Mori et al. 2000, Bradshaw and Tobin 2020). The resulting sequences were deposited in GenBank (Accession No. OP028065-7 for ITS and OP028053-5 for LSU). A GenBank BLAST search of these sequences revealed 100% identity with sequences of G. ambrosiae on many asteraceous plants, including S. novi-belgii from China (MK452575-9 for ITS and MK452648-52 for LSU). Pathogenicity was confirmed through inoculation by gently pressing diseased leaves onto leaves of five healthy potted New York aster plants. Five non-inoculated plants served as controls. Plants were maintained in an incubator at 24°C. Inoculated plants developed signs and symptoms after three weeks, whereas the control plants remained symptomless. The fungus present on the inoculated plants was morphologically identical to that observed initially on diseased plants, fulfilling Koch's postulates. The powdery mildew infections of S. novi-belgii associated with G. ambrosiae have been widely known in Europe and North America but only recently in China (Qiu et al. 2020, Farr and Rossman 2022). In Japan, Podosphaera fuliginea was known to be associated with powdery mildew of S. novi-belgii (Farr and Rossman 2022). To our knowledge, this is the first report of powdery mildew caused by G. ambrosiae on S. novi-belgii in Korea. The powdery mildew on this ornamental plant can be considered a severe threat.

Involvement of S-nitrosothiols modulation by S-nitrosoglutathione reductase in defence responses of lettuce and wild Lactuca spp. to biotrophic mildews.

MAIN CONCLUSION: Resistant Lactuca spp. genotypes can efficiently modulate levels of S-nitrosothiols as reactive nitrogen species derived from nitric oxide in their defence mechanism against invading biotrophic pathogens including lettuce downy mildew. S-Nitrosylation belongs to principal signalling pathways of nitric oxide in plant development and stress responses. Protein S-nitrosylation is regulated by S-nitrosoglutathione reductase (GSNOR) as a key catabolic enzyme of S-nitrosoglutathione (GSNO), the major intracellular S-nitrosothiol. GSNOR expression, level and activity were studied in leaves of selected genotypes of lettuce (Lactuca sativa) and wild Lactuca spp. during interactions with biotrophic mildews, Bremia lactucae (lettuce downy mildew), Golovinomyces cichoracearum (lettuce powdery mildew) and non-pathogen Pseudoidium neolycopersici (tomato powdery mildew) during 168 h post inoculation (hpi). GSNOR expression was increased in all genotypes both in the early phase at 6 hpi and later phase at 72 hpi, with a high increase observed in L. sativa UCDM2 responses to all three pathogens. GSNOR protein also showed two-phase increase, with highest changes in L. virosa-B. lactucae and L. sativa cv. UCDM2-G. cichoracearum pathosystems, whereas P. neolycopersici induced GSNOR protein at 72 hpi in all genotypes. Similarly, a general pattern of modulated GSNOR activities in response to biotrophic mildews involves a two-phase increase at 6 and 72 hpi. Lettuce downy mildew infection caused GSNOR activity slightly increased only in resistant L. saligna and L. virosa genotypes; however, all genotypes showed increased GSNOR activity both at 6 and 72 hpi by lettuce powdery mildew. We observed GSNOR-mediated decrease of S-nitrosothiols as a general feature of Lactuca spp. response to mildew infection, which was also confirmed by immunohistochemical detection of GSNOR and GSNO in infected plant tissues. Our results demonstrate that GSNOR is differentially modulated in interactions of susceptible and resistant Lactuca spp. genotypes with fungal mildews and uncover the role of S-nitrosylation in molecular mechanisms of plant responses to biotrophic pathogens.

Heterologous Expression of the Grapevine JAZ7 Gene in Arabidopsis Confers Enhanced Resistance to Powdery Mildew but Not to Botrytis cinerea.

Jasmonate ZIM-domain (JAZ) family proteins comprise a class of transcriptional repressors that silence jasmonate-inducible genes. Although a considerable amount of research has been carried out on this gene family, there is still very little information available on the role of specific JAZ gene members in multiple pathogen resistance, especially in non-model species. In this study, we investigated the potential resistance function of the VqJAZ7 gene from a disease-resistant wild grapevine, Vitis quinquangularis cv. "Shang-24", through heterologous expression in Arabidopsis thaliana. VqJAZ7-expressing transgenic Arabidopsis were challenged with three pathogens: the biotrophic fungus Golovinomyces cichoracearum, necrotrophic fungus Botrytis cinerea, and semi-biotrophic bacteria Pseudomonas syringae pv. tomato DC3000. We found that plants expressing VqJAZ7 showed greatly reduced disease symptoms for G. cichoracearum, but not for B. cinerea or P. syringae. In response to G cichoracearum infection, VqJAZ7-expressing transgenic lines exhibited markedly higher levels of cell death, superoxide anions (O2¯, and H2O2 accumulation, relative to nontransgenic control plants. Moreover, we also tested the relative expression of defense-related genes to comprehend the possible induced pathways. Taken together, our results suggest that VqJAZ7 in grapevine participates in molecular pathways of resistance to G. cichoracearum, but not to B. cinerea or P. syringae.

Taxonomy, Phylogeny, and Host Expansion of Powdery Mildew Fungi Parasitic to Veronica Species.

Powdery mildew is a prevalent fungal disease that affects various Veronica spp. (Plantaginaceae), caused by species in the genera Podosphaera and Golovinomyces (Erysiphales). There have been recent taxonomic changes in these groups and ongoing disease occurrence on a range of Veronica species previously unrecognized as hosts; however, the pathogen found in Korea remains unidentified. This study aimed to identify the causative agent of powdery mildew on Veronica spp. in Korea through molecular phylogenetic analysis of internal transcribed spacer (ITS) and large subunit (LSU) sequences of rDNA and morphological examination. Our study identified Golovinomyces bolayi as the sole pathogen affecting all nine Veronica species in Korea and confirmed that G. cichoracearum s. str., which was previously identified as the pathogen, was not the causal agent. These results expand the known host range of G. bolayi, including the addition of five Veronica species in Korea: V. hederifolia, V. kiusiana var. glabrifolia, V. longifolia, V. polita, and V. serpyllifolia. Notably, V. kiusiana has been added as a new global host of powdery mildew. These findings substantially enhance our understanding of the taxonomy of powdery mildew pathogens on Veronica species and provide valuable insights into their evolutionary host expansion.

Effect of zinc imbalance and salicylic acid co-supply on Arabidopsis response to fungal pathogens with different lifestyles.

In higher plants, Zn nutritional imbalance can affect growth, physiology and response to stress, with effect variable depending on host-pathogen interaction. Mechanisms through which Zn operates are not yet well known. The hormone salicylic acid (SA) can affect plant ion uptake, transport and defence responses. Thus, in this study the impact of Zn imbalance and SA co-supply on severity of infection with the necrotrophic fungal pathogen B. cinerea or the biotroph G. cichoracearum was assessed in A. thaliana Col-0. Spectrophotometric assays for pigments and malondialdehyde (MDA) content as a marker of lipid peroxidation, plant defensin 1.2 gene expression by semi-quantitative PCR, callose visualization by fluorescence microscopy and diseases evaluation by macro- and microscopic observations were carried out. Zinc plant concentration varied with the supplied dose. In comparison with the control, Zn-deficit or Zn-excess led to reduced chlorophyll content and PDF 1.2 transcripts induction. In Zn-deficient plants, where MDA increased, also the susceptibility to B. cinerea increased, whereas MDA decreased in G. cichoracearum. Zinc excess increased susceptibility to both pathogens. Co-administration of SA positively affected MDA level, callose deposition, PDF 1.2 transcripts and plant response to the two pathogens. The increased susceptibility to B. cinerea in both Zn-deficient and Zn-excess plants could be related to lack of induction of PDF 1.2 transcripts; oxidative stress could explain higher susceptibility to the necrotroph and lower susceptibility to the biotroph in Zn-deficient plants. This research shows that an appropriate evaluation of Zn supply according to the prevalent stress factor is desirable for plants.

Arabidopsis Toxicos en Levadura 12 (ATL12): A Gene Involved in Chitin-Induced, Hormone-Related and NADPH Oxidase-Mediated Defense Responses.

Plants, as sessile organisms, have evolved complex systems to respond to changes in environmental conditions. Chitin is a Pathogen-Associated-Molecular Pattern (PAMP) that exists in the fungal cell walls, and can be recognized by plants and induce plant pattern-triggered immunity (PTI). Our previous studies showed that Arabidopsis Toxicos en Levadura 12 (ATL12) is highly induced in response to fungal infection and chitin treatment. We used the model organism Arabidopsis thaliana to characterize ATL12 and explore its role in fungal defense. Histochemical staining showed that pATL12-GUS was continually expressed in roots, leaves, stems, and flowers. Subcellular co-localization of the ATL12-GFP fusion protein with the plasma membrane-mcherry marker showed that ATL12 localizes to the plasma membrane. Mutants of atl12 are more susceptible to Golovinomyces cichoracearum infection, while overexpression of ATL12 increased plant resistance to the fungus. ATL12 is highly induced by chitin after two hours of treatment and ATL12 may act downstream of MAPK cascades. Additionally, 3,3'-diaminobenzidine (DAB) staining indicated that atl12 mutants generate less reactive oxygen species compared to wild-type Col-0 plants and RT-PCR indicated that ATL12-regulated ROS production may be linked to the expression of respiratory burst oxidase homolog protein D/F (AtRBOHD/F). Furthermore, we present evidence that ATL12 expression is upregulated after treatment with both salicylic acid and jasmonic acid. Taken together, these results suggest a role for ATL12 in crosstalk between hormonal, chitin-induced, and NADPH oxidase-mediated defense responses in Arabidopsis.

Expression of antimicrobial peptides thanatin(S) in transgenic Arabidopsis enhanced resistance to phytopathogenic fungi and bacteria.

Thanatin(S) is an analog of thanatin, an insect antimicrobial peptide possessing strong and broad spectrum of antimicrobial activity. In order to investigate if the thanatin could be used in engineering transgenic plants for increased resistance against phytopathogens, the synthetic thanatin(S) was introduced into Arabidopsis thaliana plants. To increase the expression level of thanatin(S) in plants, the coding sequence was optimized by plant-preference codon. To avoid cellular protease degradation, signal peptide of rice Cht1 was fused to N terminal of thanatin(S) for secreting the expressed thanatin(S) into intercellular spaces. To evaluate the application value of thanatin(S) in plant disease control, the synthesized coding sequence of Cht1 signal peptide (Cht1SP)-thanatin(S) was ligated to plant gateway destination binary vectors pGWB11 (with FLAG tag). Meanwhile, in order to observe the subcellular localization of Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP, the sequences of Cht1SP-thanatin(S) and thanatin(S) were respectively linked to pGWB5 (with GFP tag). The constructs were transformed into Arabidopsis ecotype Col-0 and mutant pad4-1 via Agrobacterium-mediated transformation. The transformants with Cht1SP-thanatin(S)-FLAG fusion gene were analyzed by genomic PCR, real-time PCR, and western blots and the transgenic Arabidopsis plants introduced respectively Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP were observed by confocal microscopy. Transgenic plants expressing Cht1SP-thanatin(S)-FLAG fusion protein showed antifungal activity against Botrytis cinerea and powdery mildew, as well as antibacterial activity against Pseudomonas syringae pv. tomato. And the results from confocal observation showed that the GFP signal from Cht1SP-thanatin(S)-GFP transgenic Arabidopsis plants occurred mainly in intercellular space, while that from thanatin(S)-GFP transgenic plants was mainly detected in the cytoplasm and that from empty vector transgenic plants was distributed uniformly throughout the cell, demonstrating that Cht1 signal peptide functioned. In addition, thanatin(S) and thanatin(S)-FLAG chemically synthesized have both in vitro antimicrobial activities against P. syringae pv. tomato and B. cinerea. So, thanatin(S) is an ideal candidate AMPs for the construction of transgenic crops endowed with a broad-spectrum resistance to phytopathogens and the strategy is feasible to link a signal peptide to the target gene.