Effects of Endophytic Fungus MF23 on Dendrobium nobile Lindl. in an artificial primary environment

The quality of Dendrobium nobile Lindl. is related to the endophytic fungus. It had reported that the mycorrhizal fungus MF23 helps to increase the content of dendrobine, but few studies had explained the mechanism of the phenomenon. In previous study, we verified the symbiotic mechanism of the mycorrhizal fungus MF23 with D. nobile on the agar medium. In this study, the research carried on the bark medium, nearly like the natural environment, which had special meaning because of its benefits for the widely application. As a result, it showed a significant effect especially in the later period of the cultivation, suggesting that mycorrhizal fungus MF23 had a promotion for D. nobile in the natural environment, which enabled the application of the technique in the experimental field. It also implied that post-modification enzyme genes might play an important role in stimulating biosynthesis of dendrobine.


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Dendrobium nobile Lindl. is a traditional medicinal plant which has the reputation of "fairy 28 grass". With the effect of maintaining gastric tonicity, enhancing production of body fluid, 29 relieving symptom of dryness and curing the symptom of body heat [1], it has been widely used in the clinical and Chinese medicine prescription. Dendrobine, a sesquiterpene compound, is 31 considered as the effective component of D. nobile, the Pharmacopoeia of the People's Republic 32 of China regards it as an important indicator of the quality of D. nobile. The main active 33 ingredients of D. nobile include dendrobine, polysaccharides, flavonoids, bibenzyls and so on 34 [2-4]. It had reported that D. nobile has some pharmacological activities such as antioxidant, 35 antithrombotic and anti-Alzheimer's disease [5][6].

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The growth of the Orchidaceae is closely related to the effect of mycorrhizal fungus [7] , it had 37 reported that the fungi could promote the germination of the orchid species [8]. As we all know, 38 Orchidaceae seeds, only with undifferentiated embryos, are small as dust. In this case, they must 39 rely on fungal infection to provide nutrients to germinate under natural conditions. Guo et al 40 [9-10] obtained a number of fungi from the Dendrobium, which could effectively promote the 41 germination of Dendrobium seeds. Fungi also affected the growth of the plant [11], through the 42 way of invading the root cells, and forming mycorrhizal symbioses with the root. It could supply 43 the water and nutrients such as nitrogen and phosphorus to the host plant in a further thought [12]. 44 Up to date, few studies have been published referring to the stress resistance of medicinal plant., 45 one of which had shown that it could promote the stress resistance with the effect of mycorrhizal 46 fungus. The fungus elicitor of the genus could induce the activity of lipoxygenase, peroxidase and 47 phenylalanine ammonia lyase in the protocorm of D. candidum to protect itself, which were 48 involved in the synthesis of resistant substances [13]. it was not matched with the natural environment of Dendrobium, surroundings with stones or 58 barks lacking of necessary nutrition. In this case, in order to make the results of research apply for 59 the production and application in reality, this study had performed on the chestnut bark, offering a 60 general natural environment to study the effect of MF23 on D. nobile, to provide basic materials 61 and evidence for the MF23 induced symbiotic growth promoting mechanism in natural 62 environment. The purpose of the study is to explore the effect and the mechanism of mycorrhizal 63 fungi on the D. nobile with bark medium, and lay a foundation for the possibility and mechanism 64 of using mycorrhizal fungi to promote the production of D. nobile in the field.

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The survival rate of D. nobile has a decreased trend on the total level both in the experimental 68 and experimental groups, but with the extension of the time after cultivation, the experimental 69 groups had a higher survival rate than the control groups, and maintain a relatively stable trend in 70 the period of inoculation. At 12 th week, the survival rate had a significant difference between the 71 control and the experimental group, and at 24 th week, the survival rate of experimental group was 72 only 72.9% whereas the experimental group had higher rate, more than 85.4%, which formed a 73 striking contrast with the control group ( Figure 1A). In addition, the stem diameter of D. nobile 74 presented a significant difference between the experimental and the control group since the 9 th 75 week ( Figure 1B), In view of the weight of the fresh stem, the experimental groups showed 76 significant promotion than the control groups since the 3 rd week ( Figure 1C). In general, the 77 MF23 did promote the growth of the D. nobile according to the results. In general, the dendrobine content had a significant difference between the experimental and 87 the control groups. It was not so significant in the early six weeks, but as time goes on, it showed 88 that the dendrobine had been accumulated obviously both in the control and the experimental 89 group (Figure 2), especially at the 21 st week, the obvious difference of the debdrobine content 90 occurred in the experimental group (P<0.01), reaching to 0.146%, which is almost three times 91 more than that in the control groups (0.051%). Hyphae were present in the velamen at 6 th week after inoculation ( Figure 3A), then occurred 98 in the exodermis by 9 weeks (Figure 3B), while appeared in the cortex by 15 weeks (Figure 3C) 99 and in passage cells by 21 weeks (Figure 3D). Also, it showed that the mycelium in the cortex of 100 D. nobile was densely covered at 18 th week, but rarely seen by the 21 st and 24 th week. 118 The expression pattern of key enzyme genes in the methyl-D-erythritol 4-phosphate (MEP) 119 pathway ( Figure S2) was almost the same as that in the control group in prior inoculation stage, 120 only the gene DXR was up-regulated at early period (6 weeks). At the middle period (18 weeks), 121 all the genes (DXS, DXR, MCT, CMK, MDS, HDS and HDR) were up-regulated, and the activation 122 was much greater than any other times. However, during the late stage of inoculation (24 th week), 123 the expression pattern of key enzyme genes was similar to that of MVA pathway, down-regulated 124 to a low level. 125

Validation and expression analysis of genes involved in post-modification.
126 The expression of genes involved in post-modification process were more consistent with the 127 change of the dendrobine content ( Figure S3). At early weeks (6 weeks), genes CYP71D10, 128 At4g10440 and ATX4 were up-regulated, and genes had no significant change during the middle 129 period. All the genes exhibited higher expression levels at 18 weeks after inoculation. Their levels 130 of expression were higher than the control groups, reaching to the significant difference at 21 131 weeks, and almost all the genes except CYP94C1, At4g10440 and ATX4 exhibited higher 132 expression levels after inoculation than any other stages, which was consistent with the 133 maximizing of dendrobine content. In conclusion, the enzyme genes involved in post-modification 134 had closer relationship with the content of dendrobine. phenomenon, one is that the process of MF23 influx into the cortex from passage cells occurred 172 precisely at the time we did not investigate. Otherwise, passage cells probably not the only 173 channel for MF23 to enter cortical cells. MF23 could pass into the cortex through non-channel 174 cells directly. We could find that the mycelium in the cortex of D. nobile densely covered at 18 th 175 week, but rarely seen at the 21 st and 24 th week, and it had reported that Orchidaceae might digest 176 endophytic fungal to obtain nutrients for growth [24]. Therefore, we may regard it as the period 177 for the large-scale digestion of fungal hyphae to achieve energy and nutrients. In addition, it also 178 reported that there were differences in the distribution of hyphae in different parts of the root, Pan 179 et al [28] found that most of the Orchidaceae had no mycorrhizal infection at the root tip, in 180 general, it may also be related to the sampling site. while the key enzyme genes in later stage were at a relatively low expression level, suggesting that 197 the key enzyme genes of MVA pathway might be affected by dendrobine negative feedback 198 regulation, to make sure that the content of dendrobine in plant could reach a relative balance.

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Through the analysis of the results in the MEP pathway, we noticed that MEP pathway may

Biological materials and culture conditions 230
The chestnut bark was washed, soaked for 24 hours, cut into pieces, and 70g of which was 231 placed into each medium bottle, 80 ml deionized water was added, then autoclaved at 121℃ for 232 180 minutes. Tissue culture seedlings of D. nobile with 3-5cm in height were cultured on bark 233 medium, an artificial primary environment, which was similar to its primordial surroundings. 234 Mycelial plugs from 20-day-old MF23 grew on the potato dextrose agar medium were placed in 235 the root of the D. nobile, covered with bark. Then, the culture bottles were kept in a conventional 236 greenhouse with 12 hours light and an illumination intensity of 2000LX, the temperature was 237 controlled at 24 to 26℃, the plants without mycelial plugs were regarded as a control.
238 Plant samples were collected at different period: 1, 3, 6, 9, 12, 15, 18, 21, 24 weeks ( Figure  239 S4) the control group and the experimental group. 12 repetitions were carried on at each point of 240 time, and the samples were selected randomly. All samples were divided into two portions; ones were used for morphological and chemical research, and the others were frozen in liquid nitrogen 242 and stored for the RNA extraction.      The key enzyme genes involved in the formation of dendrobine including the mevalonate 282 (MVA) pathway (Table S1), the methyl-D-erythritol 4-phosphate (MEP) pathway (Table S2) and 283 the post-modification pathway (

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The results of content determination and microscopic observation showed that the fungus 299 MF23 had a significant effect on the D. nobile grew on the bark medium. Its survival rate has been 300 significantly increased in the later period during the inoculation. In addition, it found that the 301 infection of MF23 was gradually progressed inwards, from root to exodermis and then to cortex 302 through the microscopic observation. The expression levels of post-modification enzymes genes 303 like cytochrome P450, aminotransferase and N-methyltransferase were studied, and it showed that 304 these enzymes genes had a significant relevance with the content of dendrobine, especially the 305 DAT, an aminotransferase enzyme gene, which was positively correlated with dendrobine 306 biosynthesis. It implied that post-modification enzyme genes might play an important role in 307 stimulating biosynthesis of dendrobine with the effect of MF23. The research laid an important 308 foundation for MF23-induced increase in dendrobine content of D. nobile, and also provided a scientific basis for regulating the biosynthesis of dendrobine by molecular means. In conclusion, it 310 reveals the proper mechanism of the mycorrhizal symbiosis mechanism in the wild, and lays a 311 foundation for the possibility of using mycorrhizal fungi to promote the production of D. nobile in 312 the field.

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What is the most important is that the research we have carried out strongly suggested that 314 MF23 was beneficial to the growth of D. nobile on the bark medium that close to the natural state, 315 indicating that the endophytic fungus-MF23 is suitable for field production applications.

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Simultaneously, further exploration such as the role and molecular mechanism of MF23 in 317 improving the quality of D. nobile on the basis of experimental fields should be carried out in the 318 future. Focusing on the basis of use of transgenic, RNAi and other technologies to verify the 319 function of candidate genes. The study of protein will be performed to intuitively and accurately 320 illuminate the molecular mechanism of how MF23 increase the effective component content of D. 321 nobile. In short, the study carried out on the bark medium not only has important meaning in the 322 application of the filed, but also lays the foundation on further researches.

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Availability of data and materials

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The datasets used and/or analyzed during the current study are available from the corresponding 330 author on reasonable request.

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The authors declare that they have no competing interests.