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The necrotrophic pathogen

Herba eupatorii, one of the most important Chinese medicinal herbs, belongs to the Asteraceae family. In June 2012, a previously unknown disease, tentatively identified as powdery mildew, was observed on H. eupatorii growing in Shangqiu, in eastern Henan Province, China. Symptoms began as white mycelium partially covering upper leaf surfaces; as the disease progressed, it spread to cover entire leaf surfaces. The infected leaves became yellow and necrotic at advanced stages of infection. Specimens consisting of infected leaves were maintained at the Plant-Microbe Interaction Laboratory at Shangqiu Normal University. Microscopic observations of the morphology of the fungus revealed oval primary conidia measuring 18 to 27 × 15 to 22 μm. A long unbranched germ tube that germinated laterally from the ends of conidia was observed in some samples. Conidiophores were cylindrical, simple unbranched, and composed of a basal cell with a swollen base and three to six barrel-shaped conidia formed in chains, measuring 112 to 180 × 9 to 12 μm. Mycelial appressoria were nipple-shaped. Chasmothecia were not observed in the collected samples. To verify the identity of the fungus, the internal transcribed spacer (ITS) rDNA was amplified with ITS1 and ITS4 primers (3) and sequenced. The sequences were deposited as GenBank Accession No. JX546297. Comparison with sequences in the GenBank database revealed that the ITS sequence was 100% homologous with the sequence of Podosphaera fusca on Calendula officinalis (AB525914) (2) and Syneilesis palmata (AB040349) (1). The ITS sequence analysis verified that the causal agent was P. fusca, which is reported to be a cosmopolitan powdery mildew fungus, parasitic on numerous plant species in the Asteraceae family. Koch's postulates were completed by inoculating healthy H. eupatorii plants with a conidial suspension (prepared in distilled water) of 105 conidia/ml collected from infected plants. Five plants were sprayed until the suspension ran off the leaves, while five additional plants were sprayed with distilled water as a control. Plants were maintained in a climate cell under the following conditions: day, 24°C, 16 h; night, 20°C, 8 h; 85% humidity. After 10 days, inoculated plants developed symptoms similar to those observed in the field, whereas control plants remained healthy. Further examination showed that the inoculated plants were infected by P. fusca. To our knowledge, this is the first report of P. fusca affecting H. eupatorii in China. Because there are no fungicides labeled for use on this plant, the appearance of powdery mildew caused by P. fusca could result in substantial production loss of H. eupatorii. References: (1) T. Hirata et al. Can. J. Bot. 78:1521, 2000. (2) S. Takamatsu et al. Persoonia 24:38, 2010. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

Tall fescue (Festuca arundinacea Schreb), a predominant cool-season perennial grass, is widely used as forage and turf grasses in China. In July 2013, powdery mildew was observed on 10 F. arundinacea lawns (about 0.5 ha in total) in Urumchi, Xinjiang Province, China, with 20 to 30% of the area being infected. Signs of the disease initially appeared as irregular white mycelial colonies on the adaxial surface of infected leaves. As the disease progressed, the colonies covered the whole adaxial surface and white patches appeared on the abaxial surface of infected leaves. Conidiophores were unbranched and cylindrical with swollen bases, measuring 13.3 to 15 × 16.7 to 20 μm, and borne vertically on hyphae. Each conidiophore produced 10 to 18 conidia in a chain. The conidia were oval, one-celled, and colorless, measuring 8.1 to 9.8 × 26 to 29.7 μm. Cleistothecia were black, spherical, and 164.3 to 207.3 μm in diameter, each of which contained 9 to 26 asci. Asci were oblong or ovate, measuring 32.1 to 40 × 85.7 to 96.4 μm. Asci were petiolate, containing eight ascospores. Ascospores were round to oval, colorless, one-celled, measuring 19.1 to 22.5 × 11.7 to 13.6 μm. Based on morphological characteristics of the anamorph and the teleomorph, the fungus was identified as Blumeria graminis (DC.) Speer. Additionally, the internal transcribed spacer (ITS) of 563 bp was amplified from DNA of conidia using ITS1 and ITS4 primers (4). The ITS sequence was deposited in GenBank (Accession No. KF545644). The ITS sequence showed 100% homogeneity with those of B. graminis on Poa pratensis in Swizerland (AB273540) and on P. bulbosa in Iran (AB273551) (1), which further confirmed the identification. Ten 3-week-old healthy plants were inoculated by spraying a spore suspension (1 × 105 conidia ml−1) made from conidia brushed from infected plants, and 10 plants sprayed with sterile distilled water were served as controls. All the plants were placed in the same growth chamber at 20°C, 80% humidity, and 16-h photoperiod. Twenty days after inoculation, typical signs and symptoms of powdery mildew were observed on all the inoculated plants, whereas no symptoms were observed on the controls. Microscopic and ITS analysis showed that the fungus on the inoculated plants is identical to that on diseased field plants. B. graminis on F. arundinacea has been observed in a few European countries (1), Israel (3), and the United States (2). To our knowledge, this is the first report of powdery mildew caused by B. graminis on F. arundinacea in China, which will increase the difficulty to prevent powdery mildew on grasses including cereals. References: (1) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena-Stuttgart-New York, 1995. (2) F. M. Dugan and G. Newcombe. Pacific Northwest Fungi. 2:1-5, 2007. (3) S. O. Voytyuk et al. Biodiversity of the Powdery Mildew Fungi (Erysiphales, Ascomycota) of Israel Vol. 7. Biodiversity of Cyanoprocaryotes, Algae and Fungi of Israel. Koeltz Scientific Books, 2009. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Swiss chard (Beta vulgaris L. subsp. cicla) is a widely planted vegetable in China. From May to June 2013, an outbreak of powdery mildew on Swiss chard cultivar Fangzheng was observed in the commercial fields in Zhoukou city of Henan Province, located in central China. More than 80% of the plants exhibited symptoms of the disease. At the beginning of infection, circular, white, dust-like colonies of powdery mildew occurred mainly on adaxial surfaces of leaves. As the disease progressed, white mycelia covered the foliar parts of plant. No cleistothecia were found on or in collected samples. Upon microscopic evaluation, conidiophores were unbranched with the length of 63 to 126 and width of 7 to 10 μm (n = 50), produced conidia singly, and composed of a cylindrical foot cell followed by one to three short cells. Conidia were colorless, hyaline, ovoid, measured 29 to 40 × 12 to 18 μm (n = 100), lacked fibrosin bodies, and produced germ tubes on the ends of the conidia. The fungus was identified as Erysiphe betae according to the morphological features (1). To verify the identity, the internal transcribed spacer (ITS) region was amplified with the universal primers ITS1 and ITS4 (2) and sequenced. The ITS sequence obtained was assigned as Accession No. KF268348 in GenBank, which showed 100% homogeneity with two ITS sequences of E. betae isolates from UK (DQ164432 and DQ164436). Koch's postulates were conducted by inoculating 15 healthy 5-week-old plants (cv. Fangzheng) with detached infected leaves, which grew in a growth chamber under 22/16°C (day/night), 50% relative humidity, 120 μmol/m2/s light and a 16-h photoperiod. Fifteen non-inoculated plants grew in another growth chamber with the same conditions as control. Symptoms consistent with the infected field plants were observed on the inoculated plants, while no symptoms were found on the control plants. Microscopic observation revealed that the pathogen growing on the inoculated plants was consistent with the morphology of the original fungus. To our knowledge, this is the first report of E. betae infection on Swiss chard in China (3). References: (1) S. Francis. Mol. Plant Pathol. 3:119, 2002. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990. (3) R. Y. Zheng et al. Page 63 in: Flora Fungorum Sinicorum, Vol. 1, Erysiphales. Science Press, Beijing, 1987.

Sonchus asper is an exotic annual herb belonging to the Asteraceae family. Crude extracts of S. asper leaves have antiphlogistic and antibacterial properties. In May 2011, severe infection of powdery mildew was observed on S. asper in Henan Province, a central area of China. Symptoms began as small, nearly circular white spots that later enlarged and coalesced. As the disease progressed, white mycelia covered the entire plant, including the inflorescence. The voucher specimens are kept at the Key Laboratory of Plant-Microbe Interaction, Shangqiu Normal University. Conidiophores were erect, 108 to 220 × 10 to 12 μm, and composed of a cylindrical foot cell followed by two to three short cells. Nipple-shaped appressoria were formed on ectophytic mycelia. Conidia were hyaline and cylindrical, without distinct fibrosin bodies, and ranging from 29 to 42 × 19 to 24 μm. Long, unbranched germ tubes were formed from the ends of the conidia. These morphological characteristics suggested that the powdery mildew fungus is the anamorph of Golovinomyces cichoracearum (1,2). Chasmothecia were not observed in the collected samples. To confirm the identification, the internal transcribed spacer (ITS) region of the rDNA was amplified with ITS1 and ITS4 primers (4) and sequenced. The ITS sequence obtained was assigned Accession No. JQ010848 in GenBank, and had 99% similarity to two ITS sequences from different G. cichoracearum accessions (HM449077 and AY739111). The ITS sequence analysis further verified that the causal agent of the powdery mildew on S. asper was G. cichoracearum. Pathogenicity was confirmed by inoculating five healthy plants of S. asper with a conidial suspension (105 conidia/ml) from the infected plants, with five noninoculated plants serving as control. Inoculated plants developed the original powdery mildew symptoms after 10 days, whereas control plants remained healthy. G. cichoracearum (synonym Erysiphe cichoracearum) is a cosmopolitan powdery mildew fungus, parasitic on numerous plant species in the Asteraceae family. It was reported to be infecting S. asper in Bulgaria (3). To our knowledge, this is the first report of G. cichoracearum infections on S. asper in China. S. asper is one of the most important Chinese herbal medicines. The sudden appearance of powdery mildew caused by G. cichoracearum on this plant could result in substantial production loss of S. asper unless fungicide applications are employed. References: (1) U. Braun. Beih. Nova Hedwigia 89:1, 1987. (2) R. T. A. Cook and U. Braun. Mycol. Res. 113:616, 2009. (3) G. Negrean and C. M. Denchev. Mycologia Balcanica, 2004, 1:63. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

Euonymus japonicus Thunb. is a popular, woody, ornamental plant in China. From June to September 2010, severe outbreaks of powdery mildew occurred on all investigated E. japonicus plants used for hedges in Zhoukou and Shangqiu cities of Henan province in central China, which is the second largest province in terms of ornamental plant cultivation. A large amount of fungicide was used to prevent the fungal disease, which caused serious environmental pollution and was estimated to result in a 40 to 60% increase in preservation cost of E. japonicus hedges. Gray-white colonies of powdery mildew fungi occurred mainly on the leaves of E. japonicus, on average approximately 50% of the leaves of an individual plant were infected by the fungi. On severely infected leaves, mycelia were amphigenous, thick, forming irregular white patches, and effused to cover the whole surface. Subsequently, these leaves became chlorotic, curled, and withered. The fungus was identified as Erysiphe euonymi-japonici (Vienn.-Bourg.) U. Braun & S. Takam on the basis of the following data of microscopic morphology with scanning electron microscopy and molecular phylogenetic analysis of the internal transcribed spacer (ITS) region. The fungus had unbranched conidiophores with an average length of 57.3 μm and width of 8.8 μm, which was composed of a cylindrical foot cell, 15 to 40 × 6 to 10 μm and one to three shorter cells or cells of about the same length. Conidia were borne singly, cylindrical, or ellipsoid-cylindrical, with an average length of 27.7 μm and width of 10.4 μm. Chasmothecia were not observed in the collected samples during the whole outbreak period. PCR amplification and sequencing of the ITS region was amplified using the universal primers ITS1 and ITS4 (4). The obtained ITS sequence was assigned Accession No. HQ012432 in GenBank, which had a 99 and 98% nt similarity with the ITS sequences (ITS region including the 5.8S rDNA) of two Erysiphe euonymi-japonici isolates from Japan and Argentina in GenBank (Accession Nos. AB250228 and AB250229 (3), respectively). To our knowledge, Erysiphe euonymi-japonici has not been reported previously from central China, although synonymous species, Oidium euonymi-japonici and Microsphaera euonymi-japonici, were reported in southwestern (Sichuan Province) (1) and eastern (Shandong Province) (2) regions. Herbarium specimens are available at the Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University, Zhoukou, China. References: (1) H. Feng. J. Sichuan For. Sci. Technol. 13:57, 1992. (2) S. Z. Li et al. J. Shandong For. Sci. Technol. 46:40, 1995. (3) S. Limkaisang et al. Mycoscience 47:327, 2006. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.