Interaction Between Endophytic Fungi And Its Microbial Biology And Its Effects On Host Drought Resistance

In natural condition, there are various endophytes and rhizospheric/ phyllospheric microbes companied with Atractylodes lancea, which made it as a good plant materials to study the microbe-microbe interaction in phytosphere. By the method of "tissue culture propagation-acclimatation-transplantation to soil", we erected the symbiosis of known strain and plant, which facilitate the studies of interaction between endophytes in vivo and the interaction between endophytes and rhizospheric/phyllospheric microbes. However this research system had some problems:due to the un-adaptation to water potential, tissue culture plantlets had a low survival rate. And due to the undeveloped root system and delicate characteristics, some of young plantlets which were transplanted in soils will die. It made a obstacle for our research. To solve this problem, we inoculated the endophytes which came from previous research, expecting to confer the drought resistance to the host and enhance the adaptability of At. lancea.Firstly, we investigated interactions between endophytic fungi infecting the same host; Gilmaniella sp. AL12 and Cunninghamella sp. AL4 are endophytes of At. lancea (Asteraceae). We studied the effect on the host’s essential oils of the single inoculation of one fungus and the mixed inoculation of both. We also observed the effects of these inoculations on endophytic fungal colonization and distribution. Single inoculation included two groups:single inoculation of AL12 (AL12 group), and single inoculation of AL4 (AL4 group). Mixed inoculation included three groups:AL4 inoculation first, followed by AL12 (AL4/AL12), simultaneous inoculation of AL4 and AL12 (AL4_VS_AL12), and AL12 inoculation first, followed by AL4 (AL12/AL4). The control (CK) did not include any fungi. AL4 was observed to prefer to colonize rhizomes, and AL12 to colonize blades of plants. The isolation rate of each fungus in the mixed inoculation group was lower than in the single inoculation group; this may have been due to niche competition. When we inoculated AL12 first and then AL4, the isolation rate of AL4 in stems, leaves, and roots decreased. Similarly, when we inoculated AL4 first and then AL12, the isolation rate of AL12 decreased in the stems and leaves. However, in the roots it appeared that the invasion of AL4 changed the volatile oil levels and that this helped the colonization of AL12; the colonization rate of AL12 following single inoculation was slightly lower than in the AL12/AL4 group and the AL4_VS_AL12 group. This may illustrate that these endophytes have a long history of mutual colonization of roots, and thus have synergies with each other. We used tissue staining to observe inoculated plant tissue. No hyphae were observed intruding into plant cells, and the hyphae of the two fungi were not observed intertwining; meaning that there was no physical interaction between them. However, we found hyphae colonizing the interspaces of leaf tissues and between root cortical cells. These hyphae were single bend fold and unbranched. These mycelia exhibited adaptive morphology compared with those in in vitro pure culture. Transmission electron microscopy (TEM) allowed us to more clearly observe endophytic colonization between root cortical cells. Gas chromatography showed that both single and mixed inoculation significantly increased levels of atractylone and atractylodin in the essential oils of At. lancea. In particular, the relative percentage of atractylodin in groups AL12/AL4 and AL4/AL12 was twice as much as in the control group, and the relative percentage of atractylone in AL4_VS_AL12 was more than three times that of the control. The gas chromatograph of the host’s essential oil was more complex following mixed inoculation than it was in the control.Then, tissue-cultured plantlets of At. lancea were inoculated with the endophytes AL4 (Cunninghamella sp.) and AL12 (Gilmaniella sp.), and subsequently transplanted into soil for one year growth period. We investigated rhizospheric and phyllospheric microbial communities by culture-based and culture-independent methods. We used energy spectrum analysis, HPLC, and other assay methods to quantify basic elements in the leaves, and soluble sugars, free amino acids, and organic acids in the rhizosphere. The result showed endophytes significantly enhanced the diversity and size of rhizospheric bacterial populations and fungal populations. The effect of endophytes AL4 is more significant. Different endophytes had different effect on the phyllospheric microbes. The most biggest size and diversity of phyllospheric bacterial population is on AL4 group, while the phyllospheric fungal population is on AL12 group. The dominant bacterial genera were Microbacterium, Kocuria, and Sphingomon in endophyte-inoculated groups and Acinetobacter in the endophyte-free group. Fusarium and Penicillum were dominant fungal genera, but endophytes inhibited them in the phyllosphere. The dominant fungal genera in AL4 group and AL12 group were Acremonium and Curvularia, respectively. The types and concentrations of basic elements in the phyllosphere and the nutrients in the rhizosphere differed among treatment groups. These differences affected microbial communities. This research firstly used At. lancea as the plant material, by tissue culture propagation, single endophyte inoculation, refinement of tissue culture plantlet and transplantation to soil for one year growth, to give the systematic research on the effects of the symbiosis between type II fungal endophytes and host plant on rhizosphere and phyllosphere microbial communities. Additionally, we also further explore the possible reason for the phytospheric microbial communities changes from plant metabolites affected by endophyte, which give the references on the succession of phytospheric micrbes. Together with the interactions of the different endophytic fungi in vivo, our paper systematically research on the micobe-microbe interactions in phytosphere, in which the endophytes are the main participant.Finally, we screened one fungal endophyte AL16, which is dominant and special within At. lance a in Mao Shan region. It was able to enhanced the survival rate in the process of acclimatation. It is identified as Acremonium strictum based on the molecular phylogeny and morphological characteristics including an interesting and unique sporulation structure. After inoculation and acclimatation this fungus was allowed to colonize At. lancea plantlets. Plantlets which were inoculated endophytes or not were subjected to a range of drought treatments (control group-regularly water, mild drought-PEG6000 10% and severe drought-PEG6000 30%). We research on endophytes’effects on drought-resistance of host plant. Results show in mild drought AL16 conferred drought tolerance to plantlets by enhancing leaf soluble sugar, protein, proline content and antioxidant enzyme activity, decreasing degree of plasmalemma oxidation, facilitating transfer of abscisic acid from roots to leaves, and increasing root shoot ratio. When exposed to control group and severe drought the beneficial effect of the endophyte disappeared, or was detrimental. This research explored the special endophytes which enhanced the host resistance to drought stress, and then by the establishment of plant symbiosis system alleviate the negative effect of abiotic factor. It helped us have a better understanding and make full use of plant-microbes interaction research system.