| Broad bean blister is caused by Olpidium viciae Kusano, characterizing by strumae of cell proliferation in lesion spots. Broad bean blister has only been found in Tokyo of Japan, Sichuan and some areas of its neighboring provinces in China. It is the most important disease occurred in broad bean in Northwestern Plateau of Sichuan province, especially in Aba Autonomous Prefecture and its neighboring areas. Till now, only three research papers were found in deep study of broad bean blister. The life-history and cytology of O. viciae, and the host-parasite relationship in Olpidium were studied by Shunsuke Kusano in1912. Broad bean blister was happened in Songpan county, Sichuan province what was reported first in China by Xin Zhesheng in1984. There were not reported on other aspects except studies of the broad bean blister mentioned above. Comprehensive studies of broad bean blister were carried out, some significant progresses and innovations were made especially on pathogen, histopathology, histophysiology and molecular biology.1. Symptoms of Broad Bean Blister DiseaseBroad bean blister mainly appeared on the leaf and stalk, sometimes also on petiole and carpopodium, but not pod. The early appearance of its symptom was light green nearly round spots, the surfaces of the spots appeared rough with development, proliferated gradually to form intumescent and strumae finally. The strumae was solitary or colonial, was able to fuse into irregular shape. The severe infected plants were pygmyism and leaves were dysmorphosis. Strumae and slightly rough surface were considered as characterization symptom for the broad bean blister, which are the important basis for the diagnosis. This study identified the characterization symptom for diagnosis of broad bean blister, clarified that the blister on bean pod was not the characteristic symptom, which provided scientific basis to diagnose, prevent and control this disease accurately.2. Etiology of Broad Bean Blister DiseaseThe pathogen of broad bean blister inhabited in epidermal cells of broad bean leaf, stalk and carpopodium. The fungus body was unicellular and round protoplast, holocarpic reproduction. The vegetative propagation state of O. viciae was zoosporangium, usually sphere, variation in size,12.95～62.16μm in diameter, with an average value of28.84μm. Zoospores were germinated from mature zoosporangium and liberated through spore tubes (holes). Zoospore was oval or spherical,4.1～5μm in size, with an average value of4.47μm. Each zoospore was constituted of a long posterior cilium,24～31μm long with an average value of26.56μm. Zoospore was able to function as motile isogamete and fused together to form zygote with two cilia. Resting sporangium were found in epidermal cells after zygote infected broad bean. Resting sporangium was spherical with thick wall (average value of3.81μm), has approximately uniform size with19～31μm (average value of25.51μm). Resting sporangium was mainly formed at the later period of infection and its number was able to reach more than60in a host cell. According to the morphology, the isolated microorganism was identified as O. viciae which is the disease agent of broad bean blister occurred in Sichuan province. It belongs to the genus Olpidium of the family Olpidiaceae, Chytridiales, Mastigomycotina in taxonomy.Biological studies on O. viciae indicated the mature zoosporangium in epidermal cells of disease spots was able to liberate zoospores once in the presence of rain water. The temperature range of germination of zoosporangium was from0to18℃, it will be significantly inhibited below18℃, and was unable to germinate at20℃,25℃and30℃. Light or dark did not result in distinct influence on germination of zoosporangium. K+and Na+inhibited the germination to some degree, and completely inhibited the germination when their concentration reached80or100mmol/L. pH also affected the germination of zoosporangium to some degree, and it was germinated at pH values from5to8, with optimum value between7and7.5. The swarming period of zoospores were depended largely upon the temperature, gradually shortened with temperature increase. The zoospores were able to swim for72h between0and5℃; for24,10, and3h below10,20and25℃, respectively; for only5min when temperature raised to30℃. Temperature apparently affected infection and morbidity of zoospores to plants. Infection and morbidity occurred between10and25℃, no apparent symptom observed at5℃although infection occurred, and both were not observed at30℃or above it. The optimum temperature for infection and morbidity was between10and25℃. The time of keeping high humidity for infection and morbidity of zoospores was12h or longer. Leaf age affected infection and morbidity of zoospores, and young leaves were favorable.In the presence of water, zoospores were liberated from resting sporangium that lived through the summer and the winter. The temperature range of germination was between5and30℃, and zoospores were liberated in4h at17℃. The speed of germination in resting sporangia was extremely different. However the release mode of zoospores was the same with zoosporangium.3. Occurences and Dynamic of Broad Bean Blister DiseaseOutbreak and distribution of broad bean blister were limited in ecological zone above2400m altitude suitable for spring broad bean growth, Aba Tibetan Autonomous Prefecture, Northwestern Plateau of Sichuan province. Life cycle and clinical characteristics of this disease were as follows:The resting sporangia of O. viciae were lived with sick plant residue through summer and winter together, which was the agent for early infection. Zoospores liberated from resting sporangium would infect seedlings of broad bean in next spring, and then the symptoms of disease were found early in May. Zoosporangiums were constantly reproduced after the disease occurred in field, liberated zoospores with the presence of rain or dew for secondary infection. With highly repeated secondary infection, the disease spread quickly in the field and reached peak outbreak in flowering and pod formation stages, after that formed resting sporangium in following pod stage and then stopped gradually. This disease was able to spread in short distance by wind and rain. Continuous cropping and application of animal manure in which sick plant residue existed could cause this disease seriously. No disease-resistant breed is available in all the44breeds used for planting and disease-resistant identification. Pathogen in the sick plant residue from the same year did not infect autumn broad bean in basin zone according to preliminary epidemiological analysis.4. Histopathology of Broad Bean Blister DiseaseTo understand the histopathology of broad bean blister caused by O. viciae, the anatomy of epidermal tissues and cross sections of leaf and stem of broad bean, and the growth of the fungus in host cells were observed with light microscope. The results showed that the pathogen infected and parasitized in the epidermal cells of leaf and stem. The whole thallus of the pathogen was converted into a zoosporangium or a resting sporangium. The host responded quickly to the invasion. Once the fungal body in the individual infected cells began to develop, the epidermal cells adjacent infected cell and differentiated cells (including mesophyll cells of leaf and cortex cells of stem) beneath the immediate area of invasion of the pathogen were stimulated to meristematic activity and converted into meristematic cells. The cells of normal basic tissues were replaced by the cells of largely undifferentiated parenchyma with capacity of division. These cells continued to dividing by periclinal or radial division resulting in hyperplasia at local infected site. This histopathological change of hyperplasia was the mechanism of inducing symptom of broad bean blister. The result also indicated the interaction of the pathogen and broad bean at cytological and histological level.5. Pathological physiology of Broad Bean Blister DiseaseBroad bean blister was characterized by strumae as major symptom in infected spots, caused brown stain and necrosis of infected cells. The abnormal pathological change indicated phytohormones changed in host tissues, the activity of antioxidant enzymes in disease tissues changed evidenced by brown stain and necrosis of infected tissues, and then hurt cell to death. In order to deep understand the histophysiology of broad bean blister, concentrations of phytohormones, activity of antioxidant enzyme and concentrations of malonaldehyde in diseased tissues were tested in this study. Results showed that concentrations of ZT and KT increased greatly, together with slight increase in IAA and GA3in early stage. They stimulated cells that were divided and proliferated in diseased tissue, which beginning make the spot verrucose in the early stage. At the same time, increased ABA hurt seedling of broad bean plants, caused them to dwarfing and dysmorphia, delayed their growth stage; damaged old plants and made their leaf pro-senescence and easy to abscission as results. Concentrations of GA3increased dramatically in the late-mid-period of infection, higher than that in health tissue by3times, slight increases were observed for both ZT and KT, as a result, cells what were divided and proliferated in early stage were swelled quickly, which making spot verrucose seriously. ABA increased to some degree in these stages, caused infected cells chlorisis and brown stain, Ultimately Death and leaf senescence. However, concentrations of IAA decreased in the late-mid-period, which was reflected the physiological response of suscept. In the early stage, the analysis of antioxidant enzymes activity in diseased tissues indicated that there are no significant differences of both SOD and POD in diseased tissue compared with health tissue as control; however both of them significantly increased in diseased tissues in late-mid-stages. Because active oxygen removing system was disrupted and active oxygen accumulated in the cells of spot. So, the increased activity of SOD and POD was the biochemical characteristic by infection. Activity of CAT in diseased tissues what in early or in late-mid-stage was lower than that in health tissues. It is well known CAT has high affinity to H2O2, so the decreased CAT led to accumulation of H2O2and then damaged the infected tissues deeply. The concentrations of MDA was higher significantly in diseased tissues in late-mid-stage indicated damages to cellular membrane were intensified, and the inreversible damages disrupted cell and ulcerated tissues finally. These studies uncovered the histophysiological mechanism of symptom caused by broad bean blister, and also discovered the change characteristics of major antioxidant enzymes in diseased tissues in the whole process of broad bean blister.6. Molecular biology of O. viciaeThe Internal Transcribed Spacer (ITS) was amplified with universal primers ITS1/ITS4using total DNA isolated from infected tissues, further identified by sequencing and deposited at NCBI GenBank under accession number HQ677595.1, which added new information of molecular biology for O. viciae. It was most closely related to Olpidium brassicae and Olpidium bornovanus=O radical, elucidated that taxonomic relationships of O. viciae and other Olpidiums at molecular biology level. The ITS sequences were amplified, compared and analyzed for polymorphism using broad bean blister samples collected from different regions, and the homology was reached above99%, which indicates the ITS sequence of O. viciae is highly conservative. Using designed primers of P1/P2, ITS of O. viciae was amplified specifically from diseased tissues with sensitivity of10pg rDNA after optimizing the amplification conditions, and it had practical value for application in early detection and pathogenic identification of broad bean blister when the characteristic symptom did not appear. |
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