| The endophytic Herbaspirillum sp.WTOOC and WTOOF,isolated from the branches of tea plant in the park by our laboratory.Previous study found that two endophytic Herbaspirillum strains were able to grow under the laboratorial conditions,and had the capability of producing indole-3-acetic acid(IAA),ammonia and siderophore.Thus,two trains were thought to have the potential of promoting plant growth.In order to explore the potential application of the endophytic Herbaspirillum sp.WTOOC and WTOOF,we focused on bacterial infection,colonization,plant-growth-promoting effects and safety,and developed techniques for tea-field application.The results obtained from this study are as follows:1.Both Herbaspirillum sp.WTOOC and WTOOF strains do not contain any plasmid.Plasmid DNA was extracted respectively from two strains,and then run on agarose gel by electrophoresis.The result showed both bacterial strains did not hold a plasmid DNA.2.Both Herbaspirillum sp.WTOOC and WTOOF strains display phenol/polyphenol oxidase activity.Oxidase activity assay by non-denatured PAGE demonstrated both strains contained phenol/polyphenol oxidase.3.Construction of an novel strain with more ampicillin resistance.Since Herbaspirillum sp.WTOOC and WTOOF displayed less resistance(?10 ?g/ml)against spectinomycin and ampicillinhave.Such a low antibiotic resistance was not suitable for further studying bacterial invasion and colonization.To enhance the ampicillin resistance of Herbaspirillum sp.WTOOC,a pUC18 plasmid was introduced into the bacterial cells via electroporation.After screened on the LB plate containing 100 mg/ml ampicillin,the Herbaspirillum sp.WTOOC containing pUC18,named as ZWI00C with more ampicillin resistance(>100 mg/ml)was finally obtained.4.Two endophytic bacterial strains get into plants via traumatic infection and colonize only in tea plants.Three different methods,irrigation,sprinkling and traumatic infection were used to test how Herbaspirillum sp.WTOOC invaded Brassica campestrisy B.rape,Oryza sative,Triticum aestivum,Chlorophytum comosum and Camellia sinensis.All plants were cultivated at a green house.After treated with tree methods,the samples were collected at different times.Surface disinfectants of different parts of each plant were performed initially,these sterilized roots,stems or leaves were grinded with a mortar,and then those grinded liquids were inoculated onto LB plates containing 10?g/ml spectinomycin and 100 ?g/ml ampicillin.The number of bacterial colonies was counted.Whether irrigation or sprinkling was applied,Herbaspirillum sp.WTOOC was not detectable in roots,stems or leaves of all tested plants.In traumatic infection,the bacterium was only found in stems of plants,suggesting that the tea endophytic Herbaspirillum entered into plants via traumatic infection.Further study found that the bacterium was not detectable inside the stems of rice,wheat and spider plant after 10-day postinnoculation,wheras the bacterium was also undetectable after 20-day postinnoculation inside the stems of B.campestris and B.rapa.Different from rice,wheat,spider plant and Chinese cabbages,the bacterial cells was detectable inside the stems of tea plant after a month.Moreover,bacterial cells were found to migrate upward and downward inside the stem of tea plant.Our result demonstrated that Herbaspirillum sp.WTOOC colonizes only in tea plants but not in gramineae rice,wheat,Chinese cabbage and spider plant.5.Two endophytic bacterial strains do not cause any plant-disease symptom.When two endophytic Herbaspirillum strains got into plants by traumatic infection,the growth of all tested plants was continuously observed.The results showed that the experimental groups and the control groups for rice,wheat,Chinese cabbage and spider plant did not show any noticeable difference,and also did not display any plant disease symptom.As compared with the control group,tea-plant seedling grew more vigorous after inoculation by endophytic Herbaspirillum.Our results showed that the endophytic Herbaspirillum strains did not have any inhibition for plant growth and pathogenic feature.6.The growth-promoting effects of two bacterial strains on the growth of crops are not noticeable.Chinese cabbage,rice and wheat were tested.The seeds of rice,wheat,and two Chinese cabbages were respectively soaked in bacterial suspension,and then their growth was observed.Compared to the control groups,all tested plants did not show any marked difference,suggesting that Herbaspirillum sp.WTOOC and WTOOF may not have an obvious promoting effect on the growth of rice,wheat,rape and Chinese cabbages.7.Herbaspirillum sp.WTOOC and WTOOF have the capability of stimulating lateral root formation and bud growth in tea-cuttage propagation.Tea cuttings including tender green-cutting and hard red-cutting were separately soaked in the bacterial cultures of Herbaspirillum sp.WTOOC and WTOOF for 1 to 3 hours.Data were collected at the 160th and 280th day posttreatment.Newborn shoot length and root number were significantly different between experimental groups and control groups(P<0.05).The tea seedling survival rate was 100%.The bacterium was only found in those tea cuttings treated with the bacterial culture.In newborn shoots,leaves and roots,the bacterium was undetectable.8.Two endophytic Herbaspirillum strans have the ability to stimulate the growth of lateral buds in the field experiments.Tea twigs at the top of tea bush were pruned with a trimmer,and then inoculated the bacterium with different dilutions to the incision on the upper part of tea twigs.At the 60th day post treatment,length and weight of lateral buds were measured,and the results were shown that length of newborn lateral shoots and average weight of 200 lateral shoots in all experimental groups were significantly different between experimental groups and control groups.All of studies in this project demonstrated that Herbaspirillum sp.WTOOC and WTOOF was a tea-specific endophyte and entered into plants via traumatic infectionwith.They had the ability to enhance the survival rate of tea cutting,stimulate the lateral root formation and bud growth of tea cuttings,and raise the output of tea.Our study has paved the way for developing two endophytic Herbaspirillum strains as a novel bioaccelerator. |
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