Seed-borne Bacteria Of Lucerne(Medicago Sativa)and Their Pathogenicity

Lucerne(Medicago sativa) is the main cultivated forage legume in China and high-quality seeds guarantee its high yield. In this study,53seed samples from the United States, Canada, and Gansu province of China were collected as research materials. The objectives of this dissertation were (i) to understand the diversity of lucerne seed-borne bacteria and their functions,(ii) to investigate the patterns and pathogen of bacterial diseases of lucerne, and (iii) to probe the environmental adaptability of pathogenic microbes and to identify some specific pathogen and their hosts, as well as the infection way of the pathogen, so that we can provide basic information and theoretical evidence for controlling bacterial diseases of lucerne.1. Diversity of lucerne seed-borne bacteria. From the tested lucerne seeds,382bacterial strains were isolated and among which19strains were the predominant bacterial populations. These19bacterial strains were identified to be9genera and18species using the methods of phenotype characteristics, Biolog and16S rDNA In these trains,10species of five genera were Gram negative bacteria, and eight species of four genera were Gram positive bacteria. Eleven species of bacteria were reported worldwide for the first time and they were Pseudomonas orientalis2Ap1, Bacillus simplex ARI, Bacillus atrophaeus BF, Erwinia persicina1Cp2, Paenibacillus polymyxa3Cp1, Kocuria rosea fF6, Ochrobactrum intermedium Gp1, Bacillus pumilus1gF2, Arthrobacter crystallopoietes HT7, Ochrobactrum rhizosphaerae HT13, and Enterobacter cloacae YZ-DS1. Seven species of bacteria first reported in China were Pantoea agglomerans1Ap1, Enterobacter agglomerans1Bp1, Pseudomonas fluorescens Biovar Ⅰ3Bp3, Bacillus cereus Fp, Enterobacter sakazakii1gF3, Pseudomonas fluorescens Biovar Ⅳ2Hp1, and Bacillus subtilis HT17.2. Pathogenicity of lucerne seed-borne bacteria. Algonquin lucerne seeds were soaked in a dominant bacterial suspension and several varieties such as E. persicina1Cp2, P. agglomerans1Ap1, E. agglomerans1Bp1, E. cloacae YZ-DS1, O. intermedium GPl and O. rhizosphaerae HT13were found to be pathogenic to lucerne by hindering the plant growth and affecting the yield. In fact, only three bacterial diseases of lucerne were found in previous germination trials and they were bacterial sprout wilt caused by E. persicina, bacterial sprout decay caused by E. cloacae, and bacterial seed necrosis caused by P. agglomerans. In the study, two beneficial bacteria, P. polymyxa3Cp1and B. subtil is HT17, were identified.3. Biological characteristics of Erwinia persicina. The growth temperature for E. persicina was5～40℃and the optimal temperature was28℃. The growth rate of E. persicina was reduced as water content decreased in the culture media, but E. persicina could tolerate low water content. The best light treatment condition for E. persicina growth was the12h light/dark photoperiod. E. persicina showed the lowest growth rate when incubated in complete darkness and a medium growth rate under full light conditions. Although E. persicina could tolerate pH values between4and10, the highest growth rate was observed at pH7. Salt concentration at0.5%NaCl was beneficial to the growth of E. persicina. The bacteria could tolerate3%NaCl; however, they could not grow in4%NaCl. The results showed that maltose, glucose, sucrose, glycerol and fucose could be used as carbon sources, soy peptone as the nitrogen source, and ammonium acetate and sulfanilamide as the adverse carbon and nitrogen source, respectively, in selective media for E. persicina4. Transmission of Erwinia persicina. It was verified that E. persicina could infect mature lucerne and the incidence rate was100%. The transmission might be achieved through water source, soil and seeds. Using the above inoculation method, we found that E. persicina could invade the tested lucerne through natural openings, which could result in the necrosis of stems, leaves, flowers and bean pods as well as the water-soaked lesion of seeds. Once E. persicina enter lucerne, they could move with the help of flagella to rhizosphere, stems, leaves, flowers and subsequently to seeds for preservation. Once the environment is suitable for the growth of E. persicina, they would infect new plants. 5. Hosts of Erwinia persicina. It was proved that E. persicina could infect23species in15genera of legumes and all organs in each plant including stems, leaves, flowers, bean pods and seeds. Among11leguminous crops and12leguminous forages used in this experiment, it was known that lucerne, pea, kidney bean and soybean could be infected by E. persicina, while the other species were newly identified hosts, which include grain legumes such as Vigna angularis, V. unguiculata, V. radiate, Vicia faba, Dolichos lablab, Lens culinaris, Arachis hypogaea, and Phaseolus lunatus, as well as forage legumes such as Onobrychis viciaefolia, Astragalus adsurgens, Trifolium repens, T. pretense, Vicia sativa, V. sepium, V. unijuga, Lespedeza bicolor, Lotus cornioulatus, Melilotus albus, and M. officinalis.