First Report of Golovinomyces orontii Causing Hydrangea macrophylla Powdery Mildew in Qinghai-Tibet Plateau, China

Hydrangea macrophylla (Thunb.) Ser. (Hydrangeaceae) is the most popular hydrangea species grown in home gardens and landscapes in China. Plants of H. macrophylla with symptoms of powdery mildew were found in a commercial wholesale nursery in Huzhu, Haidong (36°49'11.87" N, 101°57′03.36″E, alt. 2490 m), in May 2020, with disease incidence reaching 80%. Symptoms included yellowing and necrosis of leaves. Upon microscopic observation, masses of conidia and mycelium were observed covering the symptomatic tissues. Fungal isolates displayed nipple-shaped hyphal appressoria, often poorly developed, conidiophores erect, arising laterally or from the upper surface of hyphal mother cells, and positioned almost centrally or towards one end of the cells, up to about 160 μm long (n = 30), with foot cells straight or flexuous, 32 to 86 × 8 to 13 μm (n = 50), followed by one to three shorter cells about 11 to 24 × 10 to 15 μm (n = 50), forming catenescent conidia in usually predominantly chains, conidia doliiform to limoniform, hyaline, 24 to 35 × 13 to 25μm (n = 50). Conidial germination was of the Euoidium type. Chasmothecia were not observed. To confirm fungal classification, single spores were isolated and cultured on detached leaf bioassay following the protocol described in Farinas et al. (2019). Total DNA was extracted directly from single-spore cultures using a Chelex extraction method (Walsh et al. 1991). The rDNA internal transcribed spacer (ITS) regions were amplified by polymerase chain reaction (PCR) utilizing the universal primer pairs ITS1/ITS4 (White et al. 1990). The sequences (726-727 bp) were deposited in GenBank (accessions no. MT568633, MT757924 and MT757925). The ITS sequences showed 99.9-100% identity with a sequence of Golovinomyces orontii reported on Papaver rhoeas (AB769466) in Switzerland. Based on the ITS rDNA phylogenetic tree, the sequences retrieved from the specimen clustered within a strongly supported clade with G. orontii (AB769466), confirming the identity of the pathogen (Takamatsu 2013). Cladistic trees were constructed using the neighbor-joining method with the Kimura 2-parameter substitution model in MEGA 6.0. Branch robustness was assessed via bootstrap analysis with 1,000 replicates. To confirm pathogenicity, eight H. macrophylla plants were sprayed until run-off with a suspension containing 1 × 105 conidia/ml. Four plants were used for fulfilling Koch’s postulates and four plants were used as mock-inoculated controls sprayed only with sterile distilled water. Inoculated and non-inoculated plants were covered with plastic bags separately and maintained overnight in a greenhouse at 25 ± 2°C and 50 to 60% relative humidity. Typical powdery mildew colonies developed on inoculated plants 10 to 15 days after inoculation, which were morphologically identical to those originally observed on the diseased plats, whereas the control plants remained symptomless. To our knowledge, this is the first report of powdery mildew caused by G. orontii on H. macrophylla in Qinghai-Tibet Plateau, China (Braun and Cook 2012).

De novo indol-3-ylmethyl glucosinolate biosynthesis, and not long-distance transport, contributes to defence of Arabidopsis against powdery mildew

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolates are constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolates are activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis indicate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.