Characterization of the mitochondrial genomes of three powdery mildew pathogens reveals remarkable variation in size and nucleotide composition

Powdery mildews comprise a large group of economically important phytopathogenic fungi. However, limited information exists on their mitochondrial genomes. Here, we assembled and compared the mitochondrial genomes of the powdery mildew pathogens Blumeria graminis f. sp. tritici, Erysiphe pisi, and Golovinomyces cichoracearum. Included in the comparative analysis was also the mitochondrial genome of Erysiphe necator that was previously analysed. The mitochondrial genomes of the four Erysiphales exhibit a similar gene content and organization but a large variation in size, with sizes ranging from 109800 bp in B. graminis f. sp. tritici to 332165 bp in G. cichoracearum, which is the largest mitochondrial genome of a fungal pathogen reported to date. Further comparative analysis revealed an unusual bimodal GC distribution in the mitochondrial genomes of B. graminis f. sp. tritici and G. cichoracearum that was not previously observed in fungi. The cytochrome b (cob) genes of E. necator, E. pisi, and G. cichoracearum were also exceptionally rich in introns, which in turn harboured rare open reading frames encoding reverse transcriptases that were likely acquired horizontally. Golovinomyces cichoracearum had also the longest cob gene (45 kb) among 703 fungal cob genes analysed. Collectively, these results provide novel insights into the organization of mitochondrial genomes of powdery mildew pathogens and represent valuable resources for population genetics and evolutionary studies.

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.

First Report of Podosphaera xanthii Causing Powdery Mildew on Melothria indica in China

Melothria indica Lour. is a wild ornamental plant widely distributed in South China. In November 2020, powdery mildew symptoms with 100% (60 plants) incidence were observed on M. indica climbing on a fence in Zhanjiang (21.17N,110.18E), Guangdong, China. The symptoms were typical for powdery mildew with white colonies on leaf surfaces and stems. Conidiophores appeared in all symptomatic tissues. Chasmothecia were observed only during the late stage of disease. Hyphae were hyaline, branched, and septate. Conidiophores were erect, hyaline, smooth, and had a dimension of 61.5 to 185.6 µm × 8.5 to 14.5 µm (n=20) and a cylindrical, flexuous foot cell, followed by 1 to 5 (−6) shorter cells. Conidia were ellipsoid to ovoid and had a dimension of 24.5 to 38.5 μm×15.5 to 21.8 μm (n=50) with well-developed fibrosin bodies. Germ tubes were in the lateral position. Chasmothecia were gregarious or scattered, subglobose, (64.8–) 65.5 μm to 115.5 (–120.5) μm (n=20) in diameter. The appendages were few, and hyphoid. Ascus one per ascomas, clavate, or subglobose, 56.5 to 78.3 (–90) μm×52.5 to 60.5 (–72) (n=20) μm. Each ascus had eight ascospores that were broadly ellipsoid and sized 13.8 to 18.6 μm×12.5 to 16.5 μm (n=30). The morphological characteristics were consistent with the previous description of Podosphaera xanthii (Castagne) U. Braun & Shishkoff (Braun and Cook 2012). Three voucher specimens, Ms-1, Ms-2, and Ms-3, were deposited in the fungus collection at Aquatic Organisms Museum of Guangdong Ocean University, and were used for molecular analysis. Their internal transcribed spacer (ITS) regions were amplified using primers ITS1/ITS4. Amplicons were sequenced and submitted to GenBank (accession no. MW512919, MW512920, and MW512921). The sequences were identical to each other and 100% similar to two of P. xanthii (Accession No. MT472035 and MN818563). On the basis of the morphological and molecular characteristics, the fungus was identified as P. xanthii. Pathogenicity was examined through inoculation by gently pressing the naturally infected leaves onto healthy ones of three potted M. indica plants with three leaves. Healthy leaves were leaves of three further plants which served as the control. White powdery mildew colonies with an incidence of 100% were similarly observed after 7 days at 28 °C and 80% relative humidity in a greenhouse. The fungal colonies on diseased leaves were morphologically identical to the specimen, and the control plants developed no symptoms. The Koch’s postulates have completed. Golovinomyces cichoracearum is known to cause powdery mildew on M. indica in China (Liu et al. 2015). P. xanthii (synonym:P. fusca p.p.) is the cause of powdery mildew on cucurbits worldwide (Braun and Cook 2000), including M. indica (synonym:M. japonica) in Korea (Kwon et al. 2015) and Japan (Takamatsu et al. 2005), but hitherto not for China. While, the teleomorph of the fungus on cucurbits is seldom found worldwide and in China only in the north (Liu et al. 2011), chasmothecia are recorded for here southern China (21.17N,110.18E).

First Report of Golovinomyces cichoracearum Causing Powdery Mildew on Zinnia elegans in China

Zinnia elegans, common zinnia, is an annual plant with highly ornamental values. It is widely planted in many nurseries, city parks, universities and home gardens in China. From August to October 2020, powdery mildew-like signs and symptoms were observed on leaves of Z. elegans growing on the campus of Henan Normal University, Henan Province, China. White powdery colonies in circular- or irregularly shaped-lesions were abundant on both surfaces of leaves and covered up to 95 % of the leaf area. Any infected leaves were chlorotic, deformed or senescence. More than 70 % of the monitored Z. elegans plants showed these signs and symptoms. Conidiophores (n = 20) were 100 to 200 × 9 to 13 μm and composed of foot cells, followed by straight cells and conidia. Mycelial appressoria were single and nipple-shaped. The oval-shaped conidia (n = 30) were 22 to 36 × 12 to 18 μm, with a length/width ratio of 1.4 to 2.7, and produced germ tubes from the polar ends of the spore. No chasmothecia were found. Based on these morphological characteristics, the pathogen was initially identified morphologically as Golovinomyces cichoracearum (Braun and Cook 2012). Structures of the pathogen were scraped from infected leaves and total genomic DNA was isolated using the method previously described by Zhu et al. (2019). The internal transcribed spacer (ITS) region of rDNA was amplified by PCR using the primers ITS1/ITS4 (White et al. 1990) and the amplicon was sequenced by Invitrogen (Shanghai, China). The sequence for the fungus was deposited into GenBank under Accession No. MW029904 and was 99.83 % identical (595/596 bp) to G. cichoracearum on Symphyotrichum novi-belgii (HM769725)(Mørk et al. 2011). To perform pathogenicity analysis, leaf surfaces of five healthy plants were fixed in a settling tower and then inoculated by blowing fungal conidia from mildew-infested leaves using pressurized air. Five non-inoculated plants served as a control. The inoculated and non-inoculated plants were separately maintained in two growth chambers (humidity, 60 %; light/dark, 16 h/8 h; temperature, 18 ℃). Eleven- to twelve-days post-inoculation, powdery mildew signs were conspicuous on inoculated plants, while control plants remained healthy. Similar results were obtained by conducting two repeated pathogenicity assays. Thus, based on the morphological characteristics and molecular analysis, the pathogen was identified and confirmed as G. cichoracearum. This pathogen has been reported on Z. elegans in India, Israel, Jordan, Korea, Nepal, Sri Lanka, Switzerland, and Turkey (Farr and Rossman 2020). To our best knowledge, this is the first report of G. cichoracearum on Z. elegans in China. The sudden outbreak of powdery mildew caused by G. cichoracearum on Z. elegans may adversely impact the plant health and ornamental value in China. Therefore, the confirmation of G. cichoracearum infecting Z. elegans expands the understanding of this pathogen and provides the fundamental knowledge for future powdery mildew control.

Arabidopsis EDR1 Protein Kinase Regulates the Association of EDS1 and PAD4 to Inhibit Cell Death

ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) are sequence-related lipase-like proteins that function as a complex to regulate defense responses in Arabidopsis by both salicylic acid–dependent and independent pathways. Here, we describe a gain-of-function mutation in PAD4 (S135F) that enhances resistance and cell death in response to infection by the powdery mildew pathogen Golovinomyces cichoracearum. The mutant PAD4 protein accumulates to wild-type levels in Arabidopsis cells, thus these phenotypes are unlikely to be due to PAD4 over accumulation. The phenotypes are similar to loss-of-function mutations in the protein kinase EDR1 (Enhanced Disease Resistance1), and previous work has shown that loss of PAD4 or EDS1 suppresses edr1-mediated phenotypes, placing these proteins downstream of EDR1. Here, we show that EDR1 directly associates with EDS1 and PAD4 and inhibits their interaction in yeast and plant cells. We propose a model whereby EDR1 negatively regulates defense responses by interfering with the heteromeric association of EDS1 and PAD4. Our data indicate that the S135F mutation likely alters an EDS1-independent function of PAD4, potentially shedding light on a yet-unknown PAD4 signaling function.

Silicon modulates multi-layered defense against powdery mildew in Arabidopsis

Silicon (Si) has been widely employed in agriculture to enhance resistance against pathogens in many crop plants. However, the underlying molecular mechanisms of Si-mediated resistance remain elusive. In this study, the Arabidopsis-powdery mildew pathosystem was employed to investigate possible defense mechanisms engaged for Si-mediated resistance. Because Arabidopsis lacks efficient Si transporters and thus is a low Si-accumulator, two heterologous Si influx transporters (from barley and muskmelon) were individually expressed in wild-type Arabidopsis Col-0 and a panel of mutants defective in different immune signaling pathways. Results from infection tests showed that while very low leaf Si content slightly induced salicylic acid (SA)-dependent resistance, high Si promoted PAD4-dependent but EDS1- and SA-independent resistance against the adapted powdery mildew isolate Golovinomyces cichoracearum UCSC1. Intriguingly, our results also showed that high Si could largely reboot non-host resistance in an immune-compromised eds1/pad4/sid2 triple mutant background against a non-adapted powdery mildew isolate G. cichoracearum UMSG1. Taken together, our results suggest that assimilated Si modulates distinct, multi-layered defense mechanisms to enhance plant resistance against adapted and no-adapted powdery mildew pathogens, possibly via synergistic interaction with defense-induced callose

Arabidopsis ENHANCED DISEASE RESISTANCE1 Protein Kinase Regulates the Association of ENHANCED DISEASE SUSCEPTIBILITY1 and PHYTOALEXIN DEFICIENT4 to Inhibit Cell Death

ABSTRACTENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) are sequence-related lipase-like proteins that function as a complex to regulate defense responses in Arabidopsis by both salicylic acid-dependent and independent pathways. Here we describe a gain-of-function mutation in PAD4 (S135F) that enhances resistance and cell death in response to infection by the powdery mildew pathogen Golovinomyces cichoracearum. The mutant PAD4 protein accumulates to wild-type levels in Arabidopsis cells, thus these phenotypes are unlikely to be due to PAD4 over accumulation. The phenotypes are similar to loss of function mutations in the protein kinase Enhanced Disease Resistance1 (EDR1), and previous work has shown that loss of PAD4 or EDS1 suppresses edr1-mediated phenotypes, placing these proteins downstream of EDR1. Here we show that EDR1 directly associates with EDS1 and PAD4 and inhibits their interaction in yeast and plant cells. We propose a model whereby EDR1 negatively regulates defense responses by interfering with the heteromeric association of EDS1 and PAD4. Our data indicate that the S135F mutation likely alters an EDS1-independent function of PAD4, potentially shedding light on a yet unknown PAD4 signaling function.

Parthenocarpic Cucumber Cultivar Evaluation in High-tunnel Production

Fresh-consumed parthenocarpic cucumbers (Cucumis sativus) are a popular and high-value crop sold in local food markets. The parthenocarpic plant characteristics and climbing growth habit make cucumbers an ideal crop for high-tunnel production. Major types of parthenocarpic cucumbers include Beit alpha and mini, Dutch greenhouse, American slicer, and Japanese. Information regarding yield performance, plant growth, and disease resistance of the four types grown in high-tunnel conditions is limited. In this study, 16 parthenocarpic cucumber cultivars from the four major types were evaluated in high tunnels at three locations in Indiana and Illinois during Spring 2018. Plants were pruned to a single stem that was supported on a string. At all locations, the cultivars that had the most total yields were Beit alpha and mini, although their total yields were not always significantly higher than that of all the others. However, Beit alpha and mini cucumbers had high percentages of unmarketable fruit, mainly because of insect feeding damage and mechanical injuries on the skins that led to scarred fruit. Dutch greenhouse cultivars had relatively lower marketable yields at two of the three locations where there was a high percentage of misshaped fruit. ‘Tasty Green’ Japanese cucumber consistently had the lowest yields at all three locations. This cultivar also produced the most side shoot growth and, therefore, more pruning waste. The Japanese types ‘Tasty Jade’ and ‘Taurus’ had yields comparable to those of other cultivars, and they were more tolerant to two-spotted spider mites (Tetranychus urticae). However, ‘Tasty Jade’ was the cultivar most susceptible to powdery mildew (Podosphaera xanthii and Golovinomyces cichoracearum). ‘Corinto’ American slicer cucumber had relatively high yields at two of the three locations. This cultivar also had the highest percentage of marketable fruit. Information provided in the study is readily useful for growers using high tunnels when selecting parthenocarpic cucumber cultivars. It is also valuable for seed companies wishing to breed new cultivars adaptive for high-tunnel production.