Identification Of The Pathogen Phyllactinia Moricola Causing Mulberry Powdery Mildew In Yunnan Province, China And Study Of The Conidial Germination Patterns

Powdery mildews are a group of plant obligate parasitic fungi that widely distribute throughout the world. They can produce luxuriant mycelia and conidia, which present as white powdery and cover the surface of host, and therefore this plant disease caused by these kinds of pathogens is called powdery mildew disease. Powdery mildew fungi belong to Erysiphaceae (Ascomycota, Erysiphales). There are9838species including9176dicotyledons and662monocotyledons that have been recorded as host plants of powdery mildews, and many of those belong to economic plants. The traditional taxonomic system for powdery mildews is based on morphological characteristics, especially the morphology of appendages on ascoma and the size of ascus and ascospores. However, classification and identification for powdery mildews based on this method of morphology that maybe lead to incorrect results. Over the past decade, with the application of molecular biology technology and scanning electron microscopy, classification and identification of powdery mildews fungi become more exact and reliable. In this study, according to morphological features, phylogenetic analysis and the host, the pathogenic fungus YN1109was finally identified as Phyllactinia moricola, which will lay the foundation of pathology for devising effective control strategies in local area.The main research contents and results are as followings:1. The symptoms first appeared as tiny white patches on the abaxial surfaces of the leaves, where these colonies were subsequently greatly enlarged and coalesced. Mature ascomata were found on the abaxial surfaces of the leaves in the later stage. The disease made the leaves lose nutrients and become chlorotic and necrotic. Microscopic examination revealed that mycelium was mainly hypophyllous, hemiendophytic, white, in patches or effuse and2.9to7.1μm wide. Conidiophores with the length of65-204μm arose erectly from the external mycelia and produced a single conidium or occasionally short chains of2-3conidia. The turgid conidia were clavate,55-90×18-36μm, with scattered single warts of the outer walls. Chasmothecia were globose, black,137-270μm in diameter and contained15-24asci. The acicular appendages were arranged in the equatorial zone of the ascoma. These appendages were spherical at the swollen base and pointed at the apex,109-354μm long,0.8to1.3times as long as the chasmothecial diameter. Asci were ovate-oblong and50-94×15-49um. Generally, an ascus contained two ascospores which were ellipsoid and20.1-45.1×14.8-30.5μm. Based on partly internal mycelia and acicular appendages which are the characteristic features of the genus Phyllactinia, the pathogen belonged to the genus Phyllactinia.2. Because conidial germination and appressoria of germ tubes are vital in the infection process of the powdery mildews, in this study, we cultured the conidia of pathogen YN1109in vitro to explore the infection mechanisms of mulberry powdery mildew. The results showed that there were considerable variations in conidial germination patterns of this fungus:①. The germ tubes could be produced in terminal, subterminal, apical, subapical or lateral position of conidia;②. Conidia could form one or two germ tubes, even three germ tubes;③. The appressoria of germ tubes were mostly simple unlobed, but a few are multi-lobed, occasionally, the tip of a lobed appressorium could develop colony-forming hypha which maybe relative to infect host later.3. Genomic DNA was extracted from mycelia and conidia of the anamorph stage collected from infected leaves. The nuclear rDNA internal transcribed spacer (ITS) regions were amplified by PCR, using universal primers ITS1and ITS4. The resulting sequence of594bp was deposited in GeneBank under the accession number JX843527. A BLAST search showed that the sequence shared over96%similarity with those of Phyllacinia moricola (D84385, D84384) and Phyllactinia guttata (AB080518, AB080483, AB080540, AB080561), which were isolated from mulberry of Moraceae. We construed the phylogenetic tree used by ITS sequences available in GeneBank. The result showed the strain YN1109was also clustered with Phyllactinia guttata and Phyllactinia moricola which isolated from the same host that belonged to genus Morus. Because Phyllactinia guttata is a large complex species from different host families, we selected the ITS sequences of Phyllactinia guttata from different host families and all the ITS sequences of Phyllactinia moricola to construct the phylogenetic tree. The result showed Phyllactinia guttata and Phyllactinia moricola classed together which isolated from the family Moraceae, and distinguished other Phyllactinia guttata from different host families.4. According to morphological characteristics, phylogenetic analysis, host species and the new taxonomic method on powdery mildews, the fungus YN1109was finally identified as Phyllactinia moricola.