The Study on the Pathogenic Mechanism of Mulberry Root Rot and Selection of Antagonistic Bacteria

Mulberry (Morus alba L.) root rot could cause damage rotting of the root,withering of the leaves and the death of whole plant, and has caused serious damagein mulberry planting, this disease that can be found in Yunnan and Guangxi Provincehas caused seriously affected economic loss in sericulture in China, but there was lackof correlation study about the pathogenic agent and prevention measures. Thepathogenic strain was isolated from a disease mulberry root, which was collectedfrom a mulberry field in Guangxi Province. In this study we did some research aboutisolation and identification pathogenic fungus, pathogenicity test, pathogenicmechanism and biological control. A preliminary analysis was determined that thepathogen belong to Fusarium solani. Using TEM, the hyphae were found and blockedat cortical cell walls; the hyphae were attached and clogged at almost all plasma.Clogging can broke the cell integrity that reduce water passage and cause waterdeficiency in the plants. Bacillus strains were isolated from the rhizosphere soilaround mulberry root. The isolated strain was tested its antagonistic effects and thesurvey of the ultra-structure of the strain were analyzed. The result of the co-cultureon plate showed that the antagonistic strain could colonization the surface of thehyphae by using SEM; this phenomenon revealed that it had an antagonistic effect topathogen. The main conclusions were obtained as follows:(1) The isolated pathoigen grew quickly and produced abundance of the mycelium.The cottony mycelium acquired a white color on the culture dish. The microscopicappeared that non-septate ovoid micro-conidia formed in false heads in short chains,chlamydospores occurred both singly and in pairs and were thick-walled with a bluntand rounded apical cell. For PCR amplification, the ITS primers amplified a singlefragment with a size of552bp. A blast analysis of the sequence indicated that theamplified gene fragment shared over99%similarity with the ITS gene sequencesfrom Fusarium solani. Evolutionary tree construction were performed that the treeshowed that it has a high similarity with Fusarium solani. Sp. Radicicola. Thereforethe strain was denominated as FS-1.(2) Select healthy mulberry seedlings at different growth stages and test theirpathogenicity. Lesion formation in the root tissues was found in seedlings afterinoculation in a short time. The pathological tissues of the main root expanded the healthy tissue, the development of lesions on all tap roots that rotting of the wholeroot and withering of the leaves followed by sudden death. The ultra-structures of thecortex parenchyma cells in the primary root tissue were observed using TEM. Theimages showed that some hyphae that were growing within the cortex cell wall andproduced thick net-like mycelia over the plant plasma membrane. Prominent wallappositions were observed in the host cells. These papillae were visible within thenecrotised cortical cell. These structures decreased the cells permeability anddamaged moisture and nutrients transport. These observations provided support forthe assumption that the mechanisms in mulberry caused by Fusarium solani aresimilar with other Fusarium species.(3) One strain named JK-7which had antagonistic effects to the pathogen of FS-1wasisolated by face-to-face-culturing. The strain was observed that the morphology of thecolony was translucent, and its surface was smooth and waxy, the edge of colony wasirregular on LB. The strain was Gram-positive bacteria and the shape of the single cellwas rod-shaped. The shape of the spore was oval and located in the middle or subendof the strain. The JK-7strain’s16S rDNA gene sequence was amplified by PCR usinga pair of universal primers, and its fragment size was1477bp. The phylogenetic treebased on16S rDNA revealed that JK-7had the closed genetic relationship with theBacillus cereus. The result of the co-culture on plate showed that the pathogen grewquickly on PDA, but it could be found that there had inhibition zone on the edge ofthe JK-7bacterial colony. The ultra-structure of the antagonistic mycelium on theedge observed that the JK-7strain could colonization the surface of the hyphae, andcaused mycelia growth deformity and induced mycelia broken by using scanningelectron microscope; this phenomenon revealed that it had an antagonistic effect topathogen.This study is the first report that the Fusarium solani was pathogen to mulberrytrees in China，we also did the research about the study on the pathogenic mechanismand selection of effective antagonistic bacteria, there had important practicalsignificance in the sustainable development of sericulture economy and greenprevention and control of mulberry disease.