Morphological and molecular identification of new records and new host plants of powdery mildews (Erysiphaceae) from Mexico

Powdery mildew is one of the most severe diseases affecting Cucurbitaceae. We identify the members of Erysiphaceae infecting cucurbits in three producing regions in Mexico. We determined that Golovinomyces ambrosiae, Neoerysiphe sechii, and Podosphaera xanthii infected cultivated and wild cucurbits species or subspecies. Leaf samples showing symptoms and signs of powdery mildew were collected from the northwestern, central western, and eastern regions of Mexico between 2017 and 2020. Species associated with the disease were identified based on morphology and ITS rDNA sequences. All powdery mildew specimens presented only the anamorph. Podosphaera xanthii was the predominant species; it was found in 85.7 % of the samples and in 13 out of 14 species or subspecies of cucurbits. Neoerysiphe sechii was found in 15.23 % of the samples and in only four cucurbit species from the central western and eastern regions of Mexico. Golovinomyces ambrosiae was documented for the first time in a Cucurbitaceae species (Sicyos deppei) from Central West Mexico. Three new hosts were registered for Neoerysiphe sechii (Cucurbita ficifolia, Echinopepon milleflorus, and Sicyos laciniatus) and eight for Podosphaera xanthii (Cucurbita argyrosperma, C. argyrosperma subsp. sororia, C. ficifolia, C. okeechobeensis subsp. martinezii, C. radicans, Sechium edule, Sicyos laciniatus, and S. deppei).

First Report of Powdery Mildew Caused by Podosphaera xanthii on Cucurbita argyrosperma in Mexico

Cucurbita argyrosperma, commonly named as winter or cushaw squash, is highly sought for its seeds, which have important uses in culinary arts. During the autumn 2021, powdery mildew-like signs and symptoms were observed on cushaw squash in several commercial fields located in Cocula, Guerrero, Mexico. Signs were initially appeared as whitish powdery patches on both sides of leaves and then covering entire leaves and causing premature senescence. The disease incidence was estimated to be 80% in about 1000 plants in two fields. The mycelium was amphigenous, persistent, white in color, and occurred in dense patches. A voucher specimen was deposited in the Herbarium of the Colegio Superior Agropecuario del Estado de Guerrero under the accession number CSAEG22. For the morphological characterization by light microscopy, fungal structures were mounted in a drop of lactic acid on a glass slide. Microscopic examination showed nipple-shaped hyphal appressoria. Conidiophores (n = 30) were straight, 100 to 190 × 10 to 12 μm and produced 2 to 6 conidia in chains. Foot-cells were cylindrical, 41 to 78 μm long, followed by 1 to 2 shorter cells. Conidia (n = 100) were ellipsoid-ovoid to barrel-shaped, 29.5 to 39.1 × 19.4 to 22.7 μm, and contained conspicuous fibrosin bodies. Germ tubes were produced from a lateral position on conidia. Chasmothecia were not observed during the growing season. The morphological characters were consistent with those of the anamorphic state of Podosphaera xanthii (Braun and Cook 2012). For further confirmation, total DNA was extracted from conidia and mycelia following the CTAB method (Doyle and Doyle 1990), and the internal transcribed spacer (ITS) region and part of the 28S gene were amplified by PCR, and sequenced. The ITS region of rDNA was amplified using the primers ITS5/ITS4 (White et al. 1990). For amplification of the 28S rRNA partial gene, a nested PCR was performed using the primer sets PM3 (Takamatsu and Kano 2001)/TW14 (Mori et al. 2000) and NL1/TW14 (Mori et al. 2000) for the first and second reactions, respectively. Phylogenetic analyses using the Maximum Likelihood method, including ITS and 28S sequences of isolates of Podosphaera spp. were performed and confirmed the results obtained in the morphological analysis. The isolate CSAEG22 grouped in a clade with isolates of Podosphaera xanthii. The ITS and 28S sequences were deposited in GenBank under accession numbers OL423329 and OL423343, respectively. Pathogenicity was confirmed by gently dusting conidia from infected leaves onto ten leaves of healthy C. argyrosperma plants. Five non-inoculated leaves served as controls. The plants were maintained in a greenhouse at 25 to 35 ºC, and relative humidity of 60 to 70%. All inoculated leaves developed similar signs to the original observation after 10 days, whereas control leaves remained symptomless. Microscopic examination of the fungus on inoculated leaves showed that it was morphologically identical to that originally observed on diseased plants, fulfilling Koch’s postulates. Podosphaera xanthii has been previously reported on C. maxima, C. moschata, and C. pepo in Mexico (Yañez-Morales et al. 2009; Farr and Rossman 2021). To our knowledge, this is the first report of P. xanthii causing powdery mildew on C. argyrosperma in Mexico. This pathogen is a serious threat to C. argyrosperma production in Mexico and disease management strategies should be developed.

Nutrient Status of Cucumber Plants Affects Powdery Mildew (Podosphaera xanthii)

We examined the effects of applications of N, P, K, Mg, and Ca through an irrigation solution and spraying K, Ca, and Mg salts on cucumber powdery mildew (CPM, Podosphaera xanthii) in potted plants and under commercial-like conditions. Spraying CaCl2 and MgCl2, or KCl and K2SO4, decreased CPM. There were significant negative correlations between the anion-related molar concentrations of the salts and disease severity. Among the sprayed treatments, NaCl provided significantly less CPM control when applied at a low (0.05 M) concentration, as compared with CaCl2 and MgCl2. When sprayed applications of Mg and K salts were analyzed separately from the untreated control, the Cl− salts were found to be more effective than the SO4−2 salts. High N and Mg concentrations in the irrigation water delivered to young, fruit-less cucumber plants reduced CPM, whereas more CPM was observed when the irrigation solution contained a medium amount of P and a high amount of K. In contrast, mature, fruit-bearing plants had less severe CPM at higher N, lower P, and higher K levels. Spraying mature plants with monopotassium phosphate, polyhalite (K2Ca2Mg(SO4)4·2H2O), and the salts mentioned above over an entire growing season suppressed CPM. CPM severity was also reduced by spray applications of Ca, Mg, and KSO4−2 and Cl− salts. Spray applications provided better CPM control than fertigation treatments. Induced resistance is probably involved in the effects of nutrients on CPM.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Sigesbeckia orientalis in China

Sigesbeckia orientalis L., (St Paul’s wort) is an annually grown natural herb of Asteraceae with a long therapeutic history for a wide range of inflammation-related diseases in China (Zhong et al. 2019). In June 2020, typical symptoms of powdery mildew were observed on 30% of wild S. orientalis plants grown along the roadsides and gardens in Minjiang University, Fuzhou, China. Circular to irregular white powdery fungal colonies were observed on both surfaces of the leaves and young stems, causing necrosis and premature senescence. Fungal hyphae were epigenous, flexuous to straight, branched, and septate. Appressoria on the hyphae were nipple-shaped or nearly absent. Conidiophores were straight, 30 to 210× 8 to 12 μm, and produced 3 to 7 immature conidia in chains with a crenate outline. Foot-cells were cylindrical, 45 to 75 ×10 to 12 μm, followed by 1 to 2 shorter cells. Conidia were hyaline, ellipsoid-ovoid to barrel-shaped, 25 to 38 × 18 to 23 μm with distinct fibrosin bodies. Germ tubes were produced from a lateral position on the conidia. Chasmothecia were not observed on the infected leaves. Based on anamorph characteristics, fungus was identified as Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Braun and Cook 2012). For molecular identification, total genomic DNA was extracted (Mukhtar et al. 2018) from fungal colonies on infected leaves of five collections separately. For each DNA sample, the part of LSU and ITS regions were amplified using primers LSU1/LSU2 and ITS1/ITS4 (Scholin et al. 1994; White et al. 1990), respectively. A BLAST search revealed 100 % sequences similarity with P. xanthii sequences reported on Ageratum conyzoides (KY274485), Eclipta prostrata (MT260063), Euphorbia hirta (KY388505), Sonchus asper (MN134013), and Verbena bonariensis (AB462804). Representative sequences (ITS: MZ613309; LSU: MZ614707) of an isolate were deposited in GenBank. The phylogenetic analysis also grouped the obtain sequences into P. xanthii clade. Pathogenicity was confirmed by gently pressing the infected leaves onto young leaves of five healthy one-month-old S. orientalis plants, while three non-inoculated plants were used as controls. All plants were maintained in a greenhouse at 25 ± 2°C. After, seven days, white powdery colonies were observed on inoculated plants, whereas controls remained mildew-free. On inoculated leaves, the fungus was morphologically and molecularly identical to the fungus on the original specimens. P. xanthii has been reported as a significant damaging pathogen on a wide range of plants in China (Farr and Rossman 2021). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on S. orientalis in China as well as worldwide. S. orientalis is one of the most important commercial Chinese medicinal herbs and the occurrence of powdery mildew is a threat to its production, quality, and marketability. References: Braun, U., and Cook, R. T. A. 2012. The Taxonomic Manual of the Erysiphales (Powdery Mildews). CBS Biodiversity Series 11: CBS. Utrecht, The Netherlands. Farr, D. F., and Rossman, A. Y. 2021. Fungal Databases. Syst. Mycol. Microbiol. Lab., USDA ARS, 9 October 2021. Mukhtar, I., et al. 2018. Sydowia.70:155. Scholin, C. A., et al. 1994. J. Phycol. 30:999. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Zhong, Z., et al., 2019. Chin. Med. (U. K.) 14, 1–12. 10.1186/s13020-019-0260-y

Gene Mining for Conserved, Non-Annotated Proteins of Podosphaera xanthii Identifies Novel Target Candidates for Controlling Powdery Mildews by Spray-Induced Gene Silencing

The powdery mildew fungus Podosphaera xanthii is one of the most important limiting factors for cucurbit production worldwide. Despite the significant efforts made by breeding and chemical companies, effective control of this pathogen remains elusive to growers. In this work, we examined the suitability of RNAi technology called spray-induced gene silencing (SIGS) for controlling cucurbit powdery mildew. Using leaf disc and cotyledon infiltration assays, we tested the efficacy of dsRNA applications to induce gene silencing in P. xanthii. Furthermore, to identify new target candidate genes, we analyzed sixty conserved and non-annotated proteins (CNAPs) deduced from the P. xanthii transcriptome in silico. Six proteins presumably involved in essential functions, specifically respiration (CNAP8878, CNAP9066, CNAP10905 and CNAP30520), glycosylation (CNAP1048) and efflux transport (CNAP948), were identified. Functional analysis of these CNAP coding genes by dsRNA-induced gene silencing resulted in strong silencing phenotypes with large reductions in fungal growth and disease symptoms. Due to their important contributions to fungal development, the CNAP1048, CNAP10905 and CNAP30520 genes were selected as targets to conduct SIGS assays under plant growth chamber conditions. The spray application of these dsRNAs induced high levels of disease control, supporting that SIGS could be a sustainable approach to combat powdery mildew diseases.