Study on signal transmission mechanism of arbuscular mycorrhizal hyphal network against root rot of Salvia miltiorrhiza

To explore the signal transmission mechanism of the arbuscular mycorrhizal network against root rot of Salvia miltiorrhiza. In this experiment, the arbuscular mycorrhizal hyphal network was established among Salvia miltiorrhiza plants, and a two plant three-compartment culture model was established. The root of the donor Salvia miltiorrhiza was inoculated with the pathogenic fungi Fusarium solani. The changes of hormone signals such as jasmonic acid and salicylic acid and the expression of related defense genes in the recipient Salvia miltiorrhiza plants in different periods were measured, to study the underground disease resistance signal transmission mechanism among medicinal plants. Salvia miltiorrhiza can transmit the signal of resistance to root rot through the jasmonic acid pathway; When plants suffer from disease stress, the content of JA increases significantly, and the increase of JA content will inhibit the content of SA in plants; The gene expression of PR-10 gene in the roots of Salvia miltiorrhiza with arbuscular mycorrhizal network infected by pathogenic fungi was 17.56 times higher than that inoculated only with pathogenic fungi; Changes in hormone content will also cause changes in the expression of related defense genes, such as SnRK2 is inhibited by ABA in the signal transduction pathway, while JA and ABA show antagonistic changes after inoculation of pathogenic fungi in Salvia miltiorrhiza, so JA may positively regulate the expression of SnRK2 gene. Plants can transmit signals through AM hyphal network after being stressed by the pathogen Fusarium solani. In the arbuscular mycorrhizal hyphal network, JA has important significance for the signal transmission of resistance to root rot and disease resistance of Salvia miltiorrhiza, which can make Salvia miltiorrhiza ready for stress resistance and improve the stress resistance of Salvia miltiorrhiza. This experiment is of great significance to further analyze the signal transmission mechanism of the arbuscular mycorrhizal hyphal network.


Study on signal transmission mechanism of arbuscular mycorrhizal hyphal network against root rot of Salvia miltiorrhiza
Han Song 1,2,4 , Na Li 1,2,4 , Rongchao Zhang 3 , Xiuqin Hu 1 , Wenyu Zhang 1 , Bo Zhang 1 , Xinpeng Li 1 , Zhen Wang 1* & Jie Xin 1* To explore the signal transmission mechanism of the arbuscular mycorrhizal network against root rot of Salvia miltiorrhiza.In this experiment, the arbuscular mycorrhizal hyphal network was established among Salvia miltiorrhiza plants, and a two plant three-compartment culture model was established.The root of the donor Salvia miltiorrhiza was inoculated with the pathogenic fungi Fusarium solani.The changes of hormone signals such as jasmonic acid and salicylic acid and the expression of related defense genes in the recipient Salvia miltiorrhiza plants in different periods were measured, to study the underground disease resistance signal transmission mechanism among medicinal plants.Salvia miltiorrhiza can transmit the signal of resistance to root rot through the jasmonic acid pathway; When plants suffer from disease stress, the content of JA increases significantly, and the increase of JA content will inhibit the content of SA in plants; The gene expression of PR-10 gene in the roots of Salvia miltiorrhiza with arbuscular mycorrhizal network infected by pathogenic fungi was 17.56 times higher than that inoculated only with pathogenic fungi; Changes in hormone content will also cause changes in the expression of related defense genes, such as SnRK2 is inhibited by ABA in the signal transduction pathway, while JA and ABA show antagonistic changes after inoculation of pathogenic fungi in Salvia miltiorrhiza, so JA may positively regulate the expression of SnRK2 gene.Plants can transmit signals through AM hyphal network after being stressed by the pathogen Fusarium solani.In the arbuscular mycorrhizal hyphal network, JA has important significance for the signal transmission of resistance to root rot and disease resistance of Salvia miltiorrhiza, which can make Salvia miltiorrhiza ready for stress resistance and improve the stress resistance of Salvia miltiorrhiza.This experiment is of great significance to further analyze the signal transmission mechanism of the arbuscular mycorrhizal hyphal network.Salvia miltiorrhiza bge.(Can be abbreviated as S. milliorrhiza) is a widely used traditional Chinese medicine in China.Its rhizome is used as medicine and has medicinal values such as activating blood circulation and removing blood stasis 1 , improving heart function 2,3 , and delaying aging 4 .With the research on the pharmacological effects of S. miltiorrhiza, the market demand for S. miltiorrhiza is increasing, the planting regulations of S. miltiorrhiza are expanding, and the phenomenon of root diseases and pests caused by continuous cropping is becoming more prominent.Relevant studies showed that with the gradual rise of the prevalence of root rot of S. miltiorrhiza, the yield, and quality of S. miltiorrhiza showed a significant downward trend 5,6 .Therefore, the soil-borne diseases mainly caused by the root rot of S. miltiorrhiza have seriously endangered and restricted the further development and utilization of S. miltiorrhiza resources.
Arbuscular mycorrhiza (AM) fungi are a kind of soil fungi that widely exist in nature and coexist with plants 7 .Studies have shown that inoculation of AM fungi on plants significantly improves the disease resistance of host

Experimental design
In the experiment, a two plant three chamber culture model was used.The bottom of a container was divided into three-chambers.The donor S. miltiorrhiza and the recipient S. miltiorrhiza were planted, and the donor S. miltiorrhiza was cultured in a single plant two-chamber culture.Four treatment groups A, B, C, and D, were established based on whether the S. miltiorrhiza plants were symbiotic with AM fungi, whether the AM hyphal network was established between the donor and the recipient, and whether the donor S. miltiorrhiza was applied with the pathogen Fusarium solani.
Group A: the donor and recipient of S. miltiorrhiza were inoculated with AM fungi, and the hyphal network was connected to impose pathogenic fungi on the donor S. miltiorrhiza; Group B: the donor and recipient of S. miltiorrhiza were completely isolated without a hyphal network connection, and pathogenic fungi were applied to the donor S. miltiorrhiza; Group C: the donor and recipient of S. miltiorrhiza were inoculated with AM fungi, with hyphal network connection, but no pathogenic fungi were applied to the donor S. miltiorrhiza; Group D: the donor and recipient of S. miltiorrhiza were not inoculated with AM fungi, there was no hyphal network between them, and pathogenic fungi were applied to the donor S. miltiorrhiza.
In this experimental model, the left side is the donor (G) S. miltiorrhiza, and the right side is the recipient (ST) S. miltiorrhiza.The root system of the donor S. miltiorrhiza is divided into two chambers, written as G1 and G2 respectively, and the recipient S. miltiorrhiza is written as ST.To exclude the influence of leaf volatiles, the aboveground part of S. miltiorrhiza was covered and sealed by self-sealing bags during the experiment.

AM hyphal network construction
In the test, the donor S. miltiorrhiza was cultured in a single plant with two chambers.The root system of a donor S. miltiorrhiza was divided into two parts, which were placed in two culture chambers, separated by plastic partitions.See Fig. 1.Both sides of the culture chamber were inoculated with AM fungi, 45 d later, the pathogen Fusarium solani was applied to the left root of the donor S. miltiorrhiza, and the changes of hormone content in the root system of S. miltiorrhiza on both sides over time were detected.
To test whether the arbuscular mycorrhizal hyphal network can mediate the signal transmission of resistance to root rot among S. miltiorrhiza plants, four treatment groups were set up, as shown in Fig. 2. In group A, 35 μM screen separation for AM fungal hyphae to pass through; In group B, the two strains of S. miltiorrhiza were completely separated by a waterproof partition.The donor and recipient of S. miltiorrhiza were in an independent space.Although both of them were symbiotic with AM fungi, there was no hyphal network connection; In group C, the donor and recipient of S. miltiorrhiza were separated by a mesh, and a hyphal network could be formed between the two plants; In group D, the two strains of S. miltiorrhiza were separated by a screen and were not inoculated with AM fungi, so there was no hyphal network connection.

Planting, management and experimental treatment
The bottom of the planting box was drilled and washed with running water, dried, and disinfected with 75% alcohol.Pour the sterilized and cool dry mixed culture medium into the incubator, and plant healthy S. miltiorrhiza seedlings with uniform size.Three replicates in each group were randomly placed.After planting for 1 d, water it thoroughly.After the soil is dry, water 1 L every 5 d.Use concentrated seaweed nutrient solution at the ratio of 1:200, and supplement it every 15 d.After 4 months of growth of S. miltiorrhiza, 50 g of AM fungi were inoculated into the three chambers of S. miltiorrhiza, and the same amount of sterilized fungi was inoculated into the control.After 40 d of inoculation, the infection rate of AM fungal roots was detected by the trypan blue staining method.On 45 d, the root of S. miltiorrhiza was inoculated with 20 mL of Fusarium Pythium spore suspension (1.0 × 10 7 CFU), and the control was replaced with an equal amount of dd H 2 O 23 .
The sampling time of materials was set as five time periods.The pathogenic fungi Fusarium solani was applied to S. miltiorrhiza and set at 0 h.The root system of S. miltiorrhiza was sampled at 24 h, 72 h, 120 h, 144 h, and 192 h 24 .

Detection of AM fungal infection rate
The root system of S. miltiorrhiza Bunge was stained by using the method of staining with triphenyl blue.The root system was added to the 5% KOH solution.After the 90 °C water bath, the waste liquid was discarded to clean the root system, 2% HCl solution was added, and the root system was placed at room temperature for 5 min.the waste liquid was discarded to clean the root system, 0.05% triphenyl blue was added, and the 90 °C The left S. miltiorrhiza is the donor S. miltiorrhiza, and the root is divided into two parts using a waterproof partition.The left part is marked as G1, and the right part is marked as G2; The other S. miltiorrhiza is the recipient S. miltiorrhiza, separated from the donor S. miltiorrhiza by a sieve or waterproof partition, denoted as ST.AMF was applied to the soil of groups A, B and C flowerpots, while group D S. miltiorrhiza did not apply AMF.At the same time, Fusarium solani FS was applied to A-G1, B-G1, and D-G1.Seal the above ground part with a self sealing bag.water bath was 30 min; Discarded the waste liquid to clean the root system, added the decolorizing solution, decolorized at room temperature, and stood overnight 25 .Finally, the mycorrhizal infection rate was calculated using the conventional method of root segment frequency.

Hormone content determination
Preparation of test article: take out the sample stored at -80 °C and grind it to dry powder in liquid nitrogen; Add 7 mL of isopropanol water hydrochloric acid mixed extraction solution to the glass test tube (2-propanol/H20/ concentrated HCl(2:1:0.002,vol/vol/vol));Add 8 µL of 1 µg/mL internal standard solution and shake at 4 °C for 30 min; Add 8 mL of dichloromethane, shake at 4 °C for 30 min, centrifuge at 13,000 r/min for 5 min, and take the lower organic phase; Protected from light, the organic phase was blown dry with nitrogen and redissolved with methanol (0.1% formic acid); Centrifuge at 4 °C for 10 min (13,000 g), take the supernatant and filter through a 0.22 µM filter membrane, and detect it by HPLC-MS/MS.
Standard solution preparation.The standard solutions of SA, JA, and ABA with gradients of 0.1 ng/mL, 0.2 ng/mL, 0.5 ng/mL, 2 ng/mL, 5 ng/mL, 20 ng/mL, 50 ng/mL, and 200 ng/mL were prepared with methanol (0.1% formic acid) as solvent, and the internal standard solution with final concentration of 20 ng/mL was added.
The data acquisition system mainly includes high-performance liquid chromatography (HPLC) (Agilent 1290) and tandem mass spectrometry (MS/MS) (Applied Biosystems 6500 quadrupole trap).Mass spectrometry data were processed using the software analyst, the specific parameters are shown in Table 1.
Drawn a standard curve for hormones used in the internal standard method experiment and carried out methodological validation.The hormone standard curve and methodological validation data are shown in Table 2.

Relative gene expression assay
Use the Sangon Biotech Spin Column Plant Total RNA Purification Kit (NO.: B518661) to extract total RNA from the root system, Store at -80 °C.Perform integrity, quality, and concentration tests on RNA, and synthesize cDNA using the Dongyang Spin Reverse Transcription Reaction Kit for RNA that meets quality requirements.Based on transcriptome data from previous experiments, specific primers were designed for the PR-10 gene and SnRK2 gene sequences using Primer Premier 5 software, and the Actin gene was selected as the internal reference gene 26 .The primer sequences are shown in Table 3.The RT-PCR reaction system is 20 μL.Amplification program settings: 95 °C for 5 min, 95 °C for 10 s, 55 °C for 20 s, 72 °C for 20 s, 40 cycles, instrument default for melting curve stage.

Ethical approval
We confirm that all methods are carried out in accordance with the relevant guidelines in the methods section.Meanwhile, the use of medicinal plants in this experiment complies with relevant institutions, national and international guidelines and legislation.

Result and analysis
Infection rate of AM fungi in the root system of S. miltiorrhiza

Detection of Trimethylbenzene blue staining
After sampling the root system, a staining experiment was conducted using triphenyl blue to observe whether AM fungi had successfully infected the root system of S. miltiorrhiza.Treatment groups A, B, and C were treated with AM fungi, while group D was not inoculated with AM fungi.Observe the root systems of the four groups under a microscope by staining them with a triphenyl blue reagent.As shown in Fig. 3, spores and endophytic hyphae can be seen in Fig. 3a

Calculate the AM fungal infection rate of the S. miltiorrhiza root system
As shown in Table 4, after 40 d of AM fungi inoculation, the root system of four groups of S. miltiorrhiza donors and recipients were sampled, and the infection rate of AM fungi was calculated by observing whether the AM fungi successfully infected the root system of S. miltiorrhiza through the triphenyl blue staining experiment.

Changes in hormone content in the root system of S. miltiorrhiza after AMF treatment alone
In the research model of signal transduction in S. miltiorrhiza, after applying Fusarium solani to B-G1, Fusarium solani can be transmitted to B-G2 through the roots.However, due to the presence of waterproof barriers between B-ST and B-G2, will hinder the transmission of Fusarium solani between B-ST and B-G2.Therefore, a mycelial network cannot be formed between B-ST and B-G2.In group D, although Fusarium solani can be transmitted from D-G1 to D-G2 through the root of S. miltiorrhiza, and the screen between D-G2 and D-ST can carry out signal transmission, due to the absence of AMF fungi in the soil, a mycelium network cannot be formed between D-G and D-ST.
As can be seen in Fig. 4, B-ST is symbiotic due to the application of AM fungi.Without receiving external stimuli, the contents of JA, SA, and abscisic acid (ABA) of S. miltiorrhiza itself were significantly higher than those of D-ST without AM fungi.The JA, SA, and ABA contents of B-ST are 2.98 times, 1.84 times, and 2.50 times of D-ST respectively.
Under the stimulation of no pathogenic fungi, the hormone content of S. miltiorrhiza with AM fungi was much higher than that of S. miltiorrhiza without AM fungi, which indicated that a certain amount of endogenous hormones would accumulate after the participation of S. miltiorrhiza in the symbiosis of AM fungi.JA 27 , SA 28 , and ABA 29 as important signaling molecules in the process of plant disease resistance, are closely related to various physiological and biochemical reactions of plants.The accumulation of JA and SA can effectively improve the disease resistance of plants 30 .The accumulation of ABA can also improve the ability of plants to resist stress.ABA signaling also plays an important role in plant disease resistance.Therefore, the symbiosis of AM fungi and S. miltiorrhiza can effectively improve the resistance of S. miltiorrhiza and the tolerance to diseases.C-GT and C-ST applied AM fungi, but were not inoculated with Fusarium solani, so the change of hormone content is consistent with that of B-ST, which can be used to exclude the influence of AM fungi and S. miltiorrhiza after forming mycorrhizas.

Changes of hormone content in the roots of S. miltiorrhiza treated with AMF and Fusarium solani
Changes of JA content in root system of S. miltiorrhiza The difference between A-G1, B-G1, and C-G1 is that A-G1 and B-G1 apply pathogenic fungi, while C-G1 does not.From the expression level of JA in Fig. 5, it can be seen that the JA content of C-G1 without the application of pathogenic fungi is stable, while the JA content of A-G1 and B-G1 shows a trend of first increasing and then decreasing, indicating that the application of pathogenic fungi can indeed increase the JA content.Compared with D-G1, A-G1, B-G1 both applied pathogenic fungi, with the difference being that D-G1 did not apply AMF fungi.From Fig. 5, it can be seen that the variation trend of JA content in A-G1, B-G1, and D-G1 is consistent.However, it is evident that at the same time, the JA content in A-G1 and B-G1 is approximately twice that of D-G1 without the application of AMF.
After AM fungi were applied to group A, a hyphal network could be formed between A-G2 and A-ST for signal transmission.The JA content in donor G1 increases from 24 to 72 h, and the JA content of S. miltiorrhiza roots after applying pathogenic fungi showed a trend of first increasing and then decreasing, and the JA content of A-G1 was always higher than that of D-G1 without AM fungi.In group A, the JA content of G1 increased significantly at 72 h and then decreased slowly.However, through the analysis of G2, it was found that the JA content continued to increase from 24 to 120 h, and then decreased at 144 h, in the group with Fusarium solani, the change of hormone content in G2 lagged behind that in G1.It can be found that hormones play an important role in signal transmission.Meanwhile, this also indicates that S. miltiorrhiza can rapidly induce its own JA content to increase after infection with pathogenic fungi.The JA content of D-G1 infected by pathogenic fungi was higher than that of B-ST of S. miltiorrhiza inoculated with AM fungi.This is because when S. miltiorrhiza is invaded by pathogenic fungi, JA content as an important signal molecule in the process of plant disease resistance will significantly increase, thus playing a role in disease resistance in the root system 31 .The JA content of S. miltiorrhiza has been at a high level after being inoculated with AM fungi in advance.When the plant is under disease stress, it can rapidly increase JA content to enhance the resistance to root rot.
As shown in Fig. 6, when A-G1 was subjected to disease stress, the JA content of the A-G2 S. miltiorrhiza root showed a trend of first increasing and then decreasing.At 120 h, the JA content of A-G2 reached the highest point, but at the same time, the JA content in A-G2 is always lower than that in A-G1.Compared with the JA content of D-G2 roots, the JA content of A-G2 roots was always higher than that of D-G2 without AM fungi inoculation, but  www.nature.com/scientificreports/ the difference was not as obvious as that of A-G1, and the JA content of D-G2 reached the highest point at 144 h.This may be because the stress occurs in the G1 root system, but not directly in the G2 root system.When AM fungi are applied to S. miltiorrhiza, AM fungi and S. miltiorrhiza are symbiotic.Both B-ST and D-ST did not apply Fusarium solani fungi, but B-ST applied AMF fungi, while D-ST did not.From the comparison of the hormone levels in Fig. 4, it can be seen that the three hormone levels in B-ST with AMF fungi were higher than those in D-ST.Therefore, Under the condition of not applying pathogenic fungi, JA accumulates in a certain amount in S. miltiorrhiza.When S. miltiorrhiza is under disease stress, the disease resistance signal can be transmitted to neighboring plants through the hyphal network, so that the JA content of neighboring plants increases rapidly, so that plants can prepare for disease defense in advance and cope with stress.
As shown in Figs.7 and 8, after the pathogen infection treatment of S. miltiorrhiza donor G1, the receptor JA content in groups B, C, and D had been stably expressed without obvious change.Although A-ST was not directly threatened by the pathogen, it showed a trend of first stable change and then increasing., which indicated that S. miltiorrhiza was induced to activate JA in vivo after suffering from the pathogen Fusarium solani, and the JA content increased to resist stress.When there is a hyphal network connection between the donor and the recipient S. miltiorrhiza, JA can stimulate the recipient healthy S. miltiorrhiza to receive defense signals, so that JA in the recipient's S. miltiorrhiza is activated and expressed, and the content gradually increases in response to stress.By comparing the JA content of B-ST with four samples of C-G1, C-G2, and C-ST, which were only treated with AMF fungi and not with Fusarium solani fungi, as shown in Fig. 8, the JA content remained unchanged, while B-G2 showed an increase from 24 to 120 h and a decrease thereafter.Moreover, the JA content of B-G1 reaches its peak at 72 h, while B-G2 only reaches its peak at 120 h, indicating that the transmission of disease resistance signals from B-G1 to B-G2 takes time.When one side of the root system is attacked by pathogenic fungi, the disease resistance signal will be transmitted from one side of the root system to the other side of the healthy root system, and then through the hyphal network, the disease resistance signal will be transmitted to the adjacent nonstressed plants.After receiving the disease resistance signal, the plants will make a defense mechanism in advance.In group D, no AM fungi were applied to S. miltiorrhiza, so there was no hyphal network connection between the recipient and the donor, and the JA content in D-ST showed a low-level change.
Changes in salicylic acid content in the root system of S. miltiorrhiza Group A was treated with AM fungi, while group D was not treated with AM fungi.The root system of S. miltiorrhiza of A-G1 and D-G1 were inoculated with Fusarium solani.The changing trend of SA in the roots of the donors exposed to pathogenic fungi stresses is similar to that of JA.As shown in Figs. 9 and 10, the change of SA content in the roots of both groups of S. miltiorrhiza donors in groups A and D shows a trend of first increasing and then decreasing, and the SA content in the roots of A-G1 is higher than that of A-G2, and that of D-G1 is higher than that of D-G2; SA content in roots of A-G1 and A-G2 with AM fungi was higher than that of D-G1 and D-G2 without AM fungi.
However, different from the changes in JA, the content of SA in S. miltiorrhiza after the application of pathogenic fungi was lower than that of S. miltiorrhiza without pathogenic fungi, which may be due to the antagonistic effect of JA and SA.When plants suffer from disease stress, the content of JA increases significantly, and the increase of JA content will inhibit the content of SA in plants 32 .Therefore, after the application of pathogenic fungi, the content of SA in S. miltiorrhiza is lower than that without pathogenic fungi.
There is a hyphal network between the recipient and the donor of S. miltiorrhiza in group A, so A-ST can receive the disease resistance signal transmitted by the donor S. miltiorrhiza.Before the disease resistance signal is transmitted to the recipient, the SA content of A-ST is not much different from that of B-ST inoculated with AM fungi alone.Due to the increase of JA content of the recipient S. miltiorrhiza, the SA content in the recipient S. miltiorrhiza shows an upward trend, but due to the antagonistic effect of JA, the content is lower than that of S. miltiorrhiza inoculated with AM fungi alone.

Induced expression of PR-10 gene in the roots of donor and recipient S. miltiorrhiza
According to the detection of fluorescence quantitative PCR technology, as shown in Figs.11 and 12, at 144 h, the PR-10 gene had the highest expression level in the A-G1 root system of S. miltiorrhiza, much higher than that in the root system of S. miltiorrhiza treated only with pathogenic fungi without AM fungi.Only S. miltiorrhiza inoculated with Fusarium solani could induce the expression of the PR-10 gene at 72 h, and the gene expression reached the highest at 120 h.For the roots of S. miltiorrhiza inoculated with Fusarium solani, the amount of gene changes showed a trend of first increasing and then decreasing.The expression of the PR-10 gene changed in different groups.The highest expression of A-G1 was 2.16 times that of D-G1; The expression level of PR-10 gene in the root system of C-G1 S. miltiorrhiza, which was only treated with AMF fungi, was similar to that of D-ST, which was not treated with both, and the expression level of PR-10 gene in C-G1 increased only after 24 h.Due to the absence of pathogenic fungal infection in C-G1, the defense mechanism for disease resistance was not induced, and the gene expression level of PR-10 showed a low level of change.
In the healthy root system on the other side of the S. miltiorrhiza donor, for S. miltiorrhiza inoculated with AM fungi and Fusarium solani, the PR-10 gene showed a trend of first increasing and then decreasing.The PR-10 gene was gradually induced to express in 24 h-120 h, and the gene expression reached the highest point in 120 h.For S. miltiorrhiza inoculated with Fusarium solani only, the expression of the PR-10 gene also showed a trend of increasing first and then decreasing and reached the highest expression at 120 h, but the expression of A-G2 S. miltiorrhiza root system inoculated with double inoculation was 2.28 times of its expression.The G1 roots of S. miltiorrhiza inoculated only with AM fungi were not infected by Fusarium solani, and there was no transmission of disease resistance signal.The expression of PR-10 gene changed steadily and was at a low level.
In the root system of recipient S. miltiorrhiza, there were roots connected by hyphal network with donor S. miltiorrhiza.After the pathogen Fusarium solani was inoculated into donor S. miltiorrhiza G1, the expression of PR-10 gene showed a trend of first increasing and then decreasing, and reached the highest level at 120 h.However, the root G1 was only inoculated with S. miltiorrhiza of Fusarium solani, because no AM fungi were applied, and there was no hyphal network connection between donor S. miltiorrhiza and recipient S. miltiorrhiza, The above data show that the expression of PR-10 gene can only be induced after inoculation of S. miltiorrhiza with the pathogenic fungi Fusarium solani.In S. miltiorrhiza without inoculation of the pathogenic fungi Fusarium solani, the PR-10 gene expression of S. miltiorrhiza inoculated with AM fungi and without AM fungi has always been low.Under the condition that the recipient S. miltiorrhiza in group A was not directly infected by the pathogen fungi, the PR-10 gene showed a high expression, which was different from that of the other three groups.This showed that the hyphal network between the recipient and the donor could transmit the signal of resistance to root rot.after the infection of Fusarium solani, the defense response of S. miltiorrhiza was rapidly activated, and the relevant disease resistance signal was transmitted to the recipient S. miltiorrhiza through the hyphal network, making it start the relevant defense response in advance, In response to the upcoming coercion.
The symbiosis between arbuscular mycorrhizal fungi and plants forms a mycorrhizal hyphal network, which is a reciprocal effect for plants and fungi.The formation of mycorrhizas not only increases the absorption of mineral elements in the soil, but also improves the disease resistance of plants 33 .This study proved that the defense response of S. miltiorrhiza inoculated with AM fungi was rapidly activated when the plant was subjected to disease stress.Compared with S. miltiorrhiza not inoculated with AM fungi, the increased expression level of defense gene PR-10 can enhance the disease resistance of S. miltiorrhiza.

Induced expression of ABA signal transduction-related gene SnRK2 in the roots of donor and recipient S. miltiorrhiza
There was also a significant difference in ABA content between B-ST inoculated with AM fungi and D-ST not inoculated with AM fungi.According to the comparison of ABA content in Figs. 4 and 13, we can get that the ABA content of S. miltiorrhiza inoculated with AM fungi is 2.50 times that of uninoculated.Meanwhile, the content of ABA varied between recipients and donors in the four treatment groups, which was not the same as JA and SA.
Therefore, when no pathogenic fungi were applied to S. miltiorrhiza, the ABA content of B-ST inoculated with AMF fungi is higher than that of D-ST not inoculated with AMF fungi, indicating that AM fungi can promote the accumulation of ABA content in S. miltiorrhiza.The ABA content of C-G1, which was only applied with AM fungi and not inoculated with pathogenic fungi, had not changed significantly, but after the application of pathogenic fungi to S. miltiorrhiza, the ABA content of S. miltiorrhiza had interesting changes.
As shown in Fig. 14, in treatment group A, the ABA content of A-G1 increased first and then decreased, but the change was not obvious, and its content was greater than that of the other groups.Although D-G1 was not inoculated with AM fungi, it may be that Fusarium solani was applied at the G1 end, so the content was also high.The opposite phenomenon occurred in the G2 root system of S. miltiorrhiza 34 .The ABA content of the D-G2 root system was always at the highest level, which may be because S. miltiorrhiza without AM fungi was not resistant to disease stress, but when it was stressed by pathogenic fungi, S. miltiorrhiza might enhance its disease resistance by controlling its own stomatal movement and water content changes.The study showed that the closure of stomata and the reduction of ion permeability are conducive to plants' resistance to the invasion of fungi, and when plants are stressed by bacteria, fungi, and viruses, the endogenous ABA level of plants will increase 35 .The low ABA content in A-G2 may be due to the application of AM fungi by S. miltiorrhiza, which improved the resistance of S. miltiorrhiza through symbiosis when no stress occurred, so the ABA content in the root system of S. miltiorrhiza G2 did not significantly increase during this period.The data of recipient S. miltiorrhiza showed that the ABA content of A-ST roots was lower than that of B-ST.Increasing the content of ABA and jasmonic acid can improve plant biological activity 36 and enhance plant resistance to insect pests 37 , but many evidences also indicate that ABA and jasmonic acid signaling pathway may antagonize each other.In the study of resistance to cabbage clubroot disease, it was found 38 .When exogenous hormone MeJA was applied to cabbage, the JA content of the cabbage itself increased, while the ABA content decreased.So the ABA content of donors in group A was lower than that of donors in group B, probably because the JA content of donors in group A was significantly increased, which inhibited the synthesis and accumulation of ABA.
As the most important stress resistance hormone of plants, ABA has the functions of promoting plant dormancy, reducing plant water loss, inhibiting plant growth and development 39 , inducing the expression of stressrelated genes 40 , etc., which can promote the adaptation of plants to the stress environment.Nowadays, studies have shown that the ABA signaling system also interacts with many hormone pathways during transcription [41][42][43] .Our research group completed the sequencing of the transcriptome of S. miltiorrhiza symbiosis with mycorrhiza in the previous experiment and screened the resistance gene SnRK2.As a gene related to the self-resistance response of S. miltiorrhiza, SnRK2 type protein kinase can phosphorylate arebs / ABFs and transcription factor ABI5, and then induce the expression of the target gene through arebs in the promoter, promoting ABA response 44 .
As shown in Figs. 15, 16, SnRK2 gene changes were also different in the four S. miltiorrhiza treatment groups with different treatment methods.It can be seen that for the treatment group D without AM fungi, the expression of the SnRK2 gene is up-regulated relative to the other three groups with AM fungi, which is consistent with our results obtained in transcriptome sequencing.If there is no communication between the S. miltiorrhiza donor and the recipient, the gene expression of group A recipients should be consistent with that of group B recipients.If the change of gene expression is caused by mycorrhizal infection, the receptor S. miltiorrhiza in group A and group C should have the same change, but relative to the receptors in group B and group D, the expression of A-ST is higher than that of b-st and lower than that of D-ST, which indicates that there is one or more transmission of disease resistance signals between the donor S. miltiorrhiza and the recipient S. miltiorrhiza connected by the hyphal network, which makes the SnRK2 gene of S. miltiorrhiza make corresponding changes.
The data results of the detection of plant endogenous hormones JA, SA, and ABA showed that after inoculation with pathogenic fungi, JA showed antagonistic changes with SA and ABA, the increase of JA content inhibited the expression of SA and ABA hormones, SnRK2 was inhibited by ABA in the signal transduction pathway, so we speculated that JA might positively regulate the expression of SnRK2 gene.When the root of S. miltiorrhiza is subjected to stress, the JA content increases, and the SnRK2 gene is positively regulated by it, thereby inhibiting ABA signal transduction in the plant body, reducing the inhibition of root growth, promoting the decomposition of proteins, sugars, and other substances in the plant body, and improving the plant's resistance.

Conclusion and discussion
In nature, abiotic stress and biotic stress are the main threats to plant growth and development.At present, most studies are aimed at abiotic stresses, such as drought stress 45 .Plants need water for growth.For their growth and development, plants have formed a mechanism to resist drought stress after long-term evolution 46 .We have little knowledge about the signaling mechanisms by which plants respond to abiotic stress, but in this experiment, we found that plants respond much faster to pathogenic fungi when containing mycorrhizal hyphal networks than we imagine.
This research shows that plants and plant roots can form a huge mycorrhizal hyphal network through the hyphal bridge between the soil.When the plants on one side are given drought stress, the plants can sense the stress signal in advance, and then prepare for drought and drought resistance in advance, which is conducive to the plants' resistance to the adverse environment.Biological stress is mainly caused by various diseases, such as leaf spots 47,48 , blight 49 , etc. Fruitful research achievements have been made in model plants and important such as Arabidopsis, tobacco, rice, soybean, corn, etc., but the research on disease resistance signal transmission of medicinal plants is still relatively backward.This experiment to some extent compensates for this deficiency.
After plants and AM fungi form symbionts, the cell structure in plant roots changes and is rich in abundant Arbuscular structures 50 .It is the existence of Arbuscular structures that makes the communication between plants and fungi more favorable.Through hyphal connection, it can realize distant communication between plants.The mycorrhizal structure enables plants to transmit signals to adjacent roots faster when they encounter stress 51 .
This experiment confirmed that the inoculation of AM fungi can increase the content of JA, SA, and ABA in S. miltiorrhiza.JA and SA are important signal molecules in plants.The increase in hormone content can enhance the ability of plants to resist external stress.In the analysis of PR-10 gene expression, it was found that S. miltiorrhiza, which formed a mycelial network between AM fungi and plant roots, could promote healthy roots to transmit disease resistance signals to recipient S. miltiorrhiza through mycelial bridges when infected by pathogenic fungi.The SnRK2 gene is down-regulated in mycorrhizal symbiotic plants.When the pathogenic fungi threaten the donor plants, it will also cause a series of corresponding changes in the receptor plant SnRK2 gene: ABA accumulation inhibits the expression of SnRK2, and SnRK2 is in the ABA transduction pathway.When SnRK2 is inhibited, it weakens the ABA signal transduction in the root, resulting in ABA reducing the inhibition of root growth and other biological processes, which may be the response feedback of ABA hormone signal to AM fungi and pathogenic fungi.S. miltiorrhiza can transmit the signal of resistance to root rot through the jasmonic acid pathway; When plants suffer from disease stress, the content of JA increases significantly, and the increase of JA content will inhibit the content of SA in plants; The gene expression of PR-10 gene in the roots of S. miltiorrhiza with arbuscular mycorrhizal network infected by pathogenic fungi was 17.56 times higher than that inoculated only with pathogenic fungi; Changes in hormone content will also cause changes in the expression of related defense genes, such as SnRK2 is inhibited by ABA in the signal transduction pathway, while JA and ABA show antagonistic changes after inoculation of pathogenic fungi in S. miltiorrhiza, so JA may positively regulate the expression of SnRK2 gene.
With the development of ecological agriculture, more and more local planting enterprises begin to pay attention to environmental protection and the good quality of products.In future agriculture, the use of AM fungi for biological prevention and control has a good industry prospect.Therefore, modern scientific and technological means should be used to promote the greening of agriculture and develop green agriculture in the future.

Figure 2 .
Figure 2. Model for signaling studies of Salvia miltiorrhiza.Figure shows a signal transduction research model of S. miltiorrhiza, divided into four groups.Each basin of S. miltiorrhiza consists of two S. miltiorrhiza plants.The left S. miltiorrhiza is the donor S. miltiorrhiza, and the root is divided into two parts using a waterproof partition.The left part is marked as G1, and the right part is marked as G2; The other S. miltiorrhiza is the recipient S. miltiorrhiza, separated from the donor S. miltiorrhiza by a sieve or waterproof partition, denoted as ST.AMF was applied to the soil of groups A, B and C flowerpots, while group D S. miltiorrhiza did not apply AMF.At the same time, Fusarium solani FS was applied to A-G1, B-G1, and D-G1.Seal the above ground part with a self sealing bag.

Figure 3 .
Figure 3. Salvia miltiorrhiza is involved in the symbiotic mycorrhizal staining map in AM fungi.Note: (a-d) show the root system of S. miltiorrhiza with AMF applied; (e) shows the root system of S. miltiorrhiza without AMF applied (10 × ,10 ×).

Figure 8 .
Figure 8. Changes in root JA content of Salvia miltiorrhiza in each group.

Figure 10 .
Figure 10.Comparison of root SA content between donor and recipient roots in A group and D group.

Figure 12 .
Figure 12.Changes in root PR-10 gene expression in each group.

Figure 14 .
Figure 14.Changes in root ABA content in each group.

Figure 15 .
Figure 15.Changes in SnRK2 gene expression between miltiorrhiza donors and recipients.

Table 2 .
Hormone name, Linear range, Calibration curve, Correlation coefficient and weight of the phytohormones.

Table 3 .
Primers used for genes in real-time PCR.