Hylotelephium erythrostictum is a perennial succulent herb of Sedum in Crassulaceae, which likes light and has strong stress resistance. It is suitable for planting environment with low rainfall and poor soil. It can survive the winter safely in the open field, so its maintenance cost is low. In the current multi-layered greening mode of arbor, shrub and grass, Hylotelephium erythrostictum grows luxuriantly and has good paving effect, which makes up for the problems of slow growth of arbor and shrub and large gap in the lower layer (Chen, 2009; Deng and Liang, 2012; Zhang et al., 2018).

Powdery mildew is a plant specific parasitic fungus, which has a serious impact on the growth of crops, vegetables, fruit trees, flowers and forages. Yan (2019) studied the resistant varieties and chemical control effects of powdery mildew in Triticum aestivum in Qinghai Province. The results showed that the loss caused by powdery mildew in Triticum aestivum could not be effectively reduced in spring period. In recent years, Hylotelephium erythrostictum has been widely planted in Qinghai Province. However, the investigation in 2017-2019 found that the Hylotelephium erythrostictum planted in various parks in Xining was seriously infected by powdery mildew, and the plant infection rate was up to more than 60%, resulting in chlorosis and early deciduous leaves, which seriously affected its growth and ornamental value. In order to identify the pathogen, the powdery mildew was identified in this experiment, which provide a theoretical basis for the further study and the prevention and control of powdery mildew.

1 Results and Analysis

1.1 Morphological identification

The white powder layer grows both on the surface and on the back of the leaf. The powder layer is thick, covering the whole leaf, and remaining. The hypha is colorless and smooth; The ascus is single-birth, long barrel columnar, long oval, columnar. Chasmothecium is aggregated or scattered, easy to grow in the mycelial layer, dark brown, spherical or oblate, with a diameter of 87~108 μm. Parietal cells are irregular polygonal; 6~16 appendages, filiform, straight or curved, unbranched, 34~183 μm long. It is nearly equal thick from top to bottom or slightly thin at the top, with 0~5 septum, full-length brown or base brown, gradually colorless upward; There are 3~5 sacs, oval or long-oval, sessile or short-stalked, 50~70 μm×33~50 μm; There are 3~5 ascospores, long-oval or elliptical, 20~32 μm×10~12 μm (Figure 1).

Figure 1 The form of Erysiphe sedi on Hylotelephium erythrostictum Note: A: Hylotelephium erythrostictum plants infected with the powdery mildew fungus Erysiphe sedi; B: Chasmothecium with appendages; C: Ascus with ascospores

1.2 Molecular phylogenetic analysis

The extracted rDNA sequence was sequenced and the results were stored in GenBank with accession number MT476970. The new sequence was phylogenetically analyzed with sequences from 31 powdery mildew families and Byssoascus striatosporus (NCBI accession No. NR111040) was used as the external supporting sequence. The phylogenetic tree constructed based on ITS gene fragment showed that the pathogen XNSF1 were strongly clustered into one branch with Erysiphe sedi in Sedum bulbiferum collected from Korea, Sedum aizoon collected from Qinling area, Echeveria DC collected from Qingdao (NCBI accession numbers were MK511841, KR048076, MN147817, respectively) (Figure 2). Through gene sequence analysis, the powdery mildew pathogen in Hylotelephium erythrostictum observed and identified in this study was identified as Erysiphe sedi.

Figure 2 The phylogenetic tree was constructed by the adjacency method between 13 powdery millet groups and Byssoascus striatosporus as the external supporting sequence

1.3 Pathogenicity test

The pathogenicity of powdery mildew observed from Hylotelephium erythrostictum was detected after morphological microscopic examination and molecular biological information analysis. Five 2-year-old Hylotelephium erythrostictum with good growth were taken, the leaves were stabbed with needles, the powdery mildew spores collected from the Hylotelephium erythrostictum were shaken off, a small amount of distilled water was sprayed, and the leaves of Hylotelephium erythrostictum sprayed with sterile water were used as the control. The inoculated and non-inoculated plants were stored in different incubators at 22℃~25℃. 8 days later, there was a small amount of white powder layer, and the leaves were gradually chlorotic. The tissues after the disease were taken for microscopic examination and observation again. The observed powdery mildew was single-birth ascus. DNA was extracted for amplification of ITS region. The result of sequence alignment was Erysiphe sedi. The pathogenicity test was repeated for 5 times, and the results were consistent.

2 Discussion

Powdery mildew is an important plant specific parasitic fungus in the world, which cannot be artificially cultured on the culture medium. It can infect a variety of angiosperms and is very popular (Liu, 2010, Science Press, pp. 1-322). According to statistics, it can reduce the yield of crops, such as the yield of Cucumis sativus decreased by 75%, the yield of Amygdalus persica decreased by 80%, the yield of Vitis vinifera decreased by 33%~90% and so on. (Yarwood, 1973; 1978). Powdery mildew is a serious hazard to strawberries and melons grown in greenhouses, and the yield can generally decrease by 50% (Li and Ma, 2003). According to Liu (2007), the host plant of Erysiphe sedi is Ser. Aizoonta. According to the classification and flora of powdery mildew in Inner Mongolia, Erysiphe sedi parasitized on Hylotelephium pallescens and Sedum aizoon; Braun et al. (2012) reported that Erysiphe sedi parasitized on Aeonium arboreum and Chiastophyllum oppositifolium; Bai et al. (2012) recorded that the host plant of powdery mildew in Qinling area is Sedum aizoon; Götz et al. (2019) studied Pseudoidium kalanchoe based on the phylogenetic analysis of ITS and 28S rDNA regions, and classified Pseudoidium kalanchoe under Erysiphe sedi.

In this experiment, the parasitic powdery mildew on Hylotelephium erythrostictum was examined by microscope, and the ascocarps, appendages, asci and ascospores were observed. At the same time, the molecular biology identification was carried out. The phylogenetic tree constructed based on ITS gene fragment showed that the pathogen XNSF1 and Erysiphe sedi (MK511841) collected from Korea were clustered into one branch. Therefore, through morphological observation and molecular systematical analysis, the powdery mildew parasitic on Hylotelephium erythrostictum was identified as Erysiphe sedi, which was the same as the pathogen parasitic on Ser. Aizoonta, Sedum aizoon and Aeonium arboreum.

3 Materials and Methods

3.1 Material collection and preservation

Using the method of collecting samples by reconnaissance, the powdery mildew samples of Hylotelephium erythrostictum were collected in Xining People’s Park (36°38’20.22’’N, 101°45’39.39’’E) and Cultural Park (36°38’37.32’’N, 101°44’44.48’’E), the collection information was recorded, and the samples were dried and preserved for microscopic observation; At the same time, the fresh leaves with typical powdery mildew symptoms and thin powder layer were selected and cut into about 0.4 cm×0.4 cm square. And then they were stored in a centrifuge tube containing 4% glutaraldehyde fixing solution for scanning electron microscope observation.

3.2 Morphological research methods

The morphological characteristics mainly referred to Braun et al. (2012). The sexual and asexual forms of powdery mildew were also morphologically classified with reference to Kirk and Cannon (2008) and the Annals of Powdery Mildew in China (Zheng and Yu, 1987, Science Press, pp. 552).

Under the light microscope, the mature chasmothecium or mycelium was picked up with a needle to make temporary water loading. Under the microscope (Olympus–CX31), the morphological characteristics of chasmothecium, ascus and ascospore, as well as the number and length of appendages were observed, their sizes were measured and the photos were taken.

Through the relevant steps of sample preparation and drying, the scanning electron microscope was used to observe the characteristics of ascus and surface decoration for classification.

3.3 Research methods of molecular biology

3.3.1 Extraction of powdery mildew DNA

From 2017 to 2019, Hylotelephium erythrostictum plants infected with powdery mildew were collected in Xining People’s Park, Nanshan Park and Beishan Park. The mycelium and propagules of powdery mildew were scraped off with a sterilized knife in the ultra clean platform, and the total DNA extraction kit of fungi (Sangon Biotech Co., Ltd.) was used to extract DNA.

3.3.2 PCR amplification

The rDNA-ITS segment of pathogenic bacteria was amplified by fungal universal primers ITS1 and ITS4 (Table 1). Table 2 for PCR amplification system. The PCR procedure was as follows: pre-denaturation at 95℃ for 4~5 min; Denaturation at 94℃ for 1 min; Annealing at 52℃ for 1 min, extension at 72℃ for 1 min, and for 32 cycles; And then extension again for 8~10 min at 72℃ (Hirata and Takamatsu, 1996; Takamatsu and Kano, 2001).

Table 1 rDND-ITS PCR primer sequencesrDND-ITS PCR primer sequences

Table 2 PCR amplification system

3.3.3 Sequence analysis

1% agarose gel electrophoresis was used, and the gel imaging analysis system was used to take pictures. The PCR product was sent to the Tianqi company for sequencing. The sequencing results were compared by GenBank, and the phylogenetic tree was constructed based on neighbor-joining (NJ) by using MEGA-6 software.

3.4 Pathogenicity detection of pathogenic bacteria

According to Koch postulates, the leaf surface of five healthy 2-year-old Hylotelephium erythrostictum plants were pierced by acupuncture. The Hylotelephium erythrostictum plants infected with powdery mildew was pressed on the pierced leaves of Hylotelephium erythrostictum, and then the plants inoculated with sterile water were used as CK. The inoculated plants were cultured in a 22℃~25℃ incubator, and the experiment was repeated for 5 times.

Authors’ contributions

BLC was the designer and director of this study. ZJF was the executor of this study, carrying out disease investigation, sample collection, pathogen isolation and identification, and finally writing the manuscript. Both authors read and approved the final manuscript.

Acknowledgments

Thank Professor Li Qiangfeng, College of Agricultural and Animal Husbandry, Qinghai University for his revision and correction.

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