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Horticultural Plant Disease Chapter 3 Plant Pathogens section 2 Plant Pathogenic Prokaryote Prokaryote: A kind of lower organism, no nucleus, no nuclear membrane, and the genetic substances disperse in the cytoplasm forming a circular or elliptoid Nucleolus region. Including bacterium, actinomycete(放 线菌纲), cyanobacterium(蓝细菌), mycoplasma(菌原体)etc. 1. Brief summery of plant pathogenic bacterium The number and harm of bacterium disease is inferior to fungus disease and virus disease, and bacterium is situated on third pathogen. Bacterium pertains to unicellular organism of prokaryota , has cell wall, no nucleus. General conformation of bacterium are globular, bacilliform, helical, and bacteria are held together as pairs, long chains or clusters; most of the plant pathogenic bacteria are bacilliform, a few of the bacteria are globular. Most bacteria have flagella(鞭毛);some bacteria have polar flagella attached to end of the cell; others have many flagella distributed over the cell surface. Few of pathogenic bacteria produce spore(芽孢,endospore, a resting body of bacterium in specific phase of life cycle). Bacteria are non-obligate parasites, and can live on substrate; bacteria with stronger parasitism can infect the green leaf of plant, weaker parasitism bacteria only infect fruit or storage organ of plant. Thermal death point of pathogenic bacteria is in 48-53℃ for 10 minutes; killing the spore of bacteria need in 120 ℃ for 10-20 minutes. Bacteria multiply by dividing, that is binary fission( Each bacteria grows until it reaches a certain size, then the cell becomes pinched in the middle and separates into two equal parts or daughter cells.). In suitable condition, bacteria can divide every 20 minutes. Plant pathogenic bacteria can grow well in 26-30℃, bacteria colony appear after 28-48 hours. Bacteria variance often occur, and the virulence is easy to be weaken by cultivation. Inoculation can make bacteria to acquire the virulence. 1.1 Shape and structure 1.1.1 Shape and size General conformation of bacterium are globular, bacilliform, helical, and have great difference in size. Diameter of globular bacteria is 0.5-1.3µm; size of bacilliform is 0.5-0.8µm×1-5µm, helical bacteria are bigger with the size of 13-14µm×1-3µm. Most of bacteria are single, few of the bacteria are held together as pairs, long chains or clusters. Most of the plant pathogenic bacteria are bacilliform, the sizes are 0.5-0.8µm×1-3µm ; a few of the bacteria are globular. 1.1.2 General characters of bacterial spore(芽孢) Spore is a kind of endospore producing in bacillus(芽孢杆菌属). Spore is not the reproduction organ because one bacterium only forms one spore and a spore germinates to form one bacterium. Spore has strong resistance to abnormal environment. Thermal death point of pathogenic bacteria is 48-53℃ , killing bacteria spore need in 120 ℃ for 10-20 minutes. 1.1.3 Gram’s staining reaction Gram’s method involves the use of crystal violet(结晶 紫) and iodine (碘液)to determine if bacteria will absorb these chemicals and turn dark (gram positive), most plant pathogenic bacteria do not retain the crystal-violet stain and are called gram negative. Gram’s staining reaction relate to other characters. E.g. Gram positive bacteria can tolerate concentrated saline solution, and can not be decomposed by protease, but are sensitive to basic(碱性的) dyes and detergent(去垢剂) ( e.g. soap). Having different reaction to antibiotics, penicillin(青霉 素) only can act on Gram positive bacteria, while streptomycin(链霉素) can affect gram positive and negative bacteria. Needing different nutrient substances, in general, gram positive bacteria need complex nutrient substances. Cell wall of Gram positive bacteria is not slick( 光滑) by electron microscope. It is obvious that gram’s staining reaction reflected essential differences in constitution and structure. So gram’s staining reaction is an important trait in bacteria classification. 1.2 Reproduction, inheritance and variance of prokaryote Prokaryote multiply by dividing. Bacteria multiply rapidly, in suitable condition, E.coli can divide each 20 minutes. Temperature also can influence growth and reproduction of bacteria when other conditions are in order. The suitable growth temperature is 2630 ℃ for plant pathogenic prokaryote; a few ones grow well in high or low temperature, for example, P. solanacearum(茄青枯菌) grow well in 35 ℃, while Clavibacter michiganensis subsp. Sepedonicus(马铃 薯环腐病菌) grow well in 20-23 ℃. Genetic material of prokaryote is DNA which mainly lies in nucleus region , but there is unattached genetic material in bacterium cell, i.e. plasmid. Nucleoplasm(核质) and plasmid compose the genetic information library of prokaryote---genomics. Prokaryotes often appear variation, for instance, the virulence of plant pathogenic bacteria is easy to be weaken by cultivation, and the variation may be owing to the selection to living condition. The similar phenomenon appear in different host plant, In different host plant which have diverse resistance, the virulence of plant pathogenic bacteria can be weakened or enhanced. 1.3 Physiology and biochemistry of bacteria Requiring simple nutrition, and can grow in ordinary substrate. The suitable growth temperature is 26-30 ℃ for plant pathogenic bacteria, and can be killed in 50 ℃ for 10 minutes; In moderate and weak alkaline condition, bacteria can grow well; most of the pathogenic bacteria are aerobic, and few ones are facultative anaerobe(厌氧生物). Enzyme produced by bacteria can bring different biochemical reaction to diverse substrate or chemicals, this can be used to identify different bacteria, e.g. biochemical reaction test. Biochemical reaction test including following ones: ① glucide fermentation(糖类发酵) ② hydrolyzing amylum(淀粉) ③ liquefying gelatin(明胶) ④ utilization of lactose(乳糖) and protein on litmus-milk (石蕊牛乳)substrate ⑤ testing metabolic product in peptone(蛋白胨) nutrient solution, e.g. hydrogen sulphide(硫化氢), ammonia(胺) and indole(吲哚) ⑥ reducing nitrate(还原硝酸盐) ⑦ decomposing grease(分解脂肪) 1.4 Cultivation characters of bacteria Forming different colony with various shapes and colors on solid substrate. In general situation, the colors mostly are white and yellow , and the shape mainly is circle, now and then brown or irregular . 1.5 Main groups The main groups of plant pathogenic prokaryotes are bacteria and mycoplasma, the two clusters are ascribe to Gracilicutes(薄壁菌门)、Firmicutes(厚 壁菌门)and Tenericutes(软壁菌门). 1.2 Classification of plant pathogenic bacteria 1.2.1 Classification basis Morphological characters Nourishing and living style Cultivation properties Physiological and biochemical properties Pathogenicity Symptom traits Antigen conformation Genetic character 1.2.2 Main groups of plant pathogenic bacteria 1.2.2.1 Agrobacterium(土壤杆菌属) Soil inhabiting fungus, short bacilliform, single bacterium or holding together as pairs. Having 1-6 flagella distributed over the cell surface. Aerobic, metabolism by breathing. Gram staining reaction negative, no spore. The colony is circular, bulging and smooth; offwhite to white, ropy(粘稠的) and pigment can not be produced Most of the bacteria have plasmid which separate from bacteria chromosome. Plasmid control the pathogenicity and drug resistance of bacteria. 1.2.2.2 Burkholderia(布克氏菌属) Separating from Psedomonas Forming smooth, moist, ridgy and offwhite colony on combined substrate. Host range is wide, can harm more than 30 families, 100 species. Typical symptom is acute wilt, vascular bundle of diseased stem turn into brown. Spilling white bacteria ooze(菌脓) by extruding. Soil inhabiting bacteria, can spread by soil, water, contaminated stock and diseased plant. infecting host plant from wound. High temperature and humidity are benefited to occurring disease. 1.2.2.3 Erwinia(欧文氏杆菌属) short bacilliform, more than one flagella around the bacteria cell surface. Offwhite colony, gram staining reaction negative .causing rot, e.g. Chinese cabbage soft rot. E. Carotovora have wide host range, and can infect more than 20 families including crucifers(十字花科), gramineae(禾本科), solanaceae( 茄科) etc. infecting host plant from wound or by insect vector activity. In general, the symptom appear during storage stage. 1.2.2.4 Pseudomonas(假单胞杆菌属) Short bacilliform or slightly flexual(弯 曲). Having more than one flagella attached to one end of bacteria cell. The colony is circular, bulging , smooth, offwhite. Producing fluorescence on agar substrate, some species can produce brown pigment infusing into substrate. Gram staining reaction negative. Aerobic, metabolism by breathing, no spore. Causing leaf spot, rot and wilt. E.g. Cucumber angular leaf spot(黄瓜细菌性角斑 病), eggplant bacterial wilt(茄科青枯病), Morus blight(桑疫病). 1.2.2.5 Xanthomonas(黄单胞菌属) Short bacilliform, having one flagellum attached to one end of bacteria cell. Gram negative. Aerobic, metabolism by breathing. The colony is circular, bulging , yellow. Producing non-watersoluble yellow pigment. Model species is Xanthomonas Campestris(野油菜黄单胞菌) Causing cabbage black rot, rice leaf blight, etc. 1.2.2.6 Clavibacter(棒形杆菌属) Short bacilliform to irregular bacilliform, no flagellum, no endospore. Gram positive. Aerobic, metabolism by breathing. The colony is circualr, smooth, bulging, opaque(不透明), offwhite on agar substrate C.Michiganensis subsp. Sepedonicum (马铃薯环腐亚种) cause potato ring rot. 3. Plant mycoplasma 3.1 Common characters Spherical, no cell wall, having cell membrane Spiroplasma(螺原体) can be cultivated on substrate, forming omelette-like(煎蛋形) colony Sensitive to tetracycline(四环素). Because of no cell wall, mycoplasma is not sensitive to penicillin and streptomycin Causing chlorosis, dwarf, clustering, leaflet. depending on insect vector to spread. E.g. Leafhopper(叶蝉) and planthopper(飞虱). 3.2 Main group of plant mycoplasma 3.2.1 Spiroplasma(螺原体) Helical, producing helical offshoot(分 支). Main host are dicotyledon(双子叶植 物) and hexapod(昆虫). Causing Citrus stubborn (柑橘僵化病), maize corn stunt (玉米矮化病). Leafhopper can diffuse Spiroplasma Spiroplasma can winter in Johnsongrass(假高粱) or leafhopper. 3.2.2 Phytoplasma(植原体属) Orbicular or elliptic. The size of phytoplasma is 80-1000nm. to date, it can not be cultivated on substrate. Spreading by hishimonoides sellatifrons Ishihara(桑菱纹叶蝉). Causing Morus virus disease(桑萎缩病), Jujube witches’ broom(枣疯病), paulownia witches’ broom(泡桐丛枝病), Pear decline(梨 衰退病)and sweet potato witches’ broom(甘 薯丛枝病).