Lactic acid induced defense responses in tobacco against Phytophthora nicotianae

Induced resistance is considered an eco-friendly disease control strategy, which can enhance plant disease resistance by inducing the plant’s immune system to activate the defense response. In recent years, studies have shown that lactic acid can play a role in plant defense against biological stress; however, whether lactic acid can improve tobacco resistance to Phytophthora nicotianae, and its molecular mechanism remains unclear. In our study, the mycelial growth and sporangium production of P. nicotianae were inhibited by lactic acid in vitro in a dose-dependent manner. Application of lactic acid could reduce the disease index, and the contents of total phenol, salicylic acid (SA), jasmonic acid (JA), lignin and H2O2, catalase (CAT) and phenylalanine ammonia–lyase (PAL) activities were significantly increased. To explore this lactic acid-induced protective mechanism for tobacco disease resistance, RNA-Seq analysis was used. Lactic acid enhances tobacco disease resistance by activating Ca2+, reactive oxygen species (ROS) signal transduction, regulating antioxidant enzymes, SA, JA, abscisic acid (ABA) and indole-3-acetic acid (IAA) signaling pathways, and up-regulating flavonoid biosynthesis-related genes. This study demonstrated that lactic acid might play a role in inducing resistance to tobacco black shank disease; the mechanism by which lactic acid induces disease resistance includes direct antifungal activity and inducing the host to produce direct and primed defenses. In conclusion, this study provided a theoretical basis for lactic acid-induced resistance and a new perspective for preventing and treating tobacco black shank disease.

patterns (PAMPs), leading to PTI 11,12,15 ; (2) activation of early signaling pathways, including accumulation of calcium by influx, ROS and mitogen-activated protein kinase (MAPK) cascade reactions 16 ; (3) hormone-mediated signaling pathways that induce downstream reactions mainly dependent on the cross-communication network between SA, JA or ethylene (ET)-mediated signaling pathways 17,18 .Molecules that trigger PTI responses, such as Flg22, induce PTI reaction-related gene expression that causes ROS levels to increase, stomatal closure and callose accumulation 17,19 .However, under increasingly fierce competition between plants and pathogens, pathogens have acquired the ability to encode effectors into host cells, and inhibit PTI by secreting these effectors, thus improving pathogen virulence.These effectors may directly inhibit plant immune-related proteins or change their activity status 20,21 .In response, plants acquire a skill that can also recognize specific effectors of these attackers, called resistance (R) proteins.When cognate R proteins detect pathogen effects directly or indirectly, they trigger a second layer of the immune system: effector-triggered immunity (ETI) 22,23 .ETI usually initiates an hypersensitive response and is conceptually equivalent to classic gene-to-gene resistance 11,12 .Thus, various plant regulatory factors coordinate to form a highly regulated regulatory network.The activation of a plant's defense system enhances its defense ability against future pathogens and pests, known as induced resistance (IR), which can be achieved through the application of inducers that mimic pathogen invasion 24 .Some chemical inducers used for IR stimulation include organic acid, cyclopeptides and steroid hormone and so on, such as SA, cyclodipeptide, and brassinoids, which have been shown to induce plant disease resistance [25][26][27] .These inducers limit pathogen development by activating a variety of molecular and host-cell defense responses, including the recognition of signaling molecules such as NO, Ca 2+ , H 2 O 2 regulation, and the expression of disease-resistant genes, lignin deposition, phenol and callosity accumulation, the hypersensitive response (HR) and complex hormonal signaling (i.e., SA, JA, and ET) networks 14,25,27 .The use of these IR mechanisms to protect plants may be a more environmentally friendly control method because the metabolic investment in plants is reduced compared with constructive defense activation 14,25,27 .There have been many studies on induced plant resistance, which shed light on resistance expression signaling pathways in plant IR action, whose pathways and characteristics are more widely understood 27,28 .
Studies on tobacco resistance to black shank disease through IR are rarely reported.Previous studies have shown that thiamine can significantly up-regulate PR-1 gene expression in tobacco and induce resistance to tobacco mosaic virus (TMV) through the SA signaling pathway 29 .The defensive response of riboflavin to tobacco black shank and bacterial wilt involves the accumulation of two total phenolic compounds, scopolamine and lignin 30 .Lactic acid is a naturally occurring organic acid widely found in human animals and plants.Recent studies have shown that spraying Chlorella fusca on leaves can effectively induce resistance in Arabidopsis to Pseudomonas syringae and displays a good control effect.Moreover, it is also proven that D-lactic acid in Chlorella fusca can be used as an inducer to enhance ROS production, corpus callosum deposition and SA and JA signalrelated gene expression in Arabidopsis 31 .Importantly, the underlying mechanisms by which lactic acid induces plant resistance remain largely unknown, especially P. nicotianae infection, which has not been tested previously.In conclusion, this study aims to elucidate the defense response of lactic acid-activated tobacco to induce resistance to tobacco black shank and highlight the innovative application of lactic acid in plant disease control.
In this study, the antifungal activity of lactic acid against P. nicotianae was determined, and its potential role in inducing plant disease resistance and related molecular mechanisms were further studied.We tested the H 2 O 2 , SA, and JA contents and defense enzyme activity in tobacco leaves.In addition, we used RNA-Seq analysis of the lactic acid-induced disease resistance molecular mechanism in tobacco.Our results confirm that lactic acid can induce resistance of tobacco to P. nicotianae through SA and JA mediated plant defense pathways.This study first elucidates the mechanisms by which lactic acid triggers a defense response and highlights innovative applications of lactic acid in controlling plant diseases.

In vitro inhibitory effect of lactic acid on P. nicotianae
In vitro inhibition tests showed that mycelial growth and sporangium production of P. nicotianae were inhibited by lactic acid (LA), and inhibition increased with an increase in concentration.LA began to inhibit P. nicotianae growth from 1 mM, with an inhibition rate of 69.13% at 10 mM, and complete inhibition of P. nicotiana filament growth at 15 mM (Fig. 1A and Fig. 1B).The inhibition rate on sporangium production also showed the same trend with all LA treatments (Fig. 1C).The results showed that LA had an obvious inhibitory effect on P. nicotianae in vitro.

Pot efficacy test of different concentrations of lactic acid on tobacco black shank disease
All lactic acid concentrations did not significantly affect tobacco maximum leaf width, 10 mM-15 mM lactic acid significantly affect tobacco plant height and maximum leaf length, 15 mM lactic acid significantly affect tobacco stem diameter.Under the 10 mM and 15 mM lactic acid treatment, each index shows a slight downward trend (Table 1).The application of low-concentration lactic acid has an excellent growth-promoting effect on tobacco plants, while high concentrations have a negative impact on tobacco plants.Different lactic acid concentration treatments change the average length of lesions size to varying degrees.The disease index of tobacco plants without lactic acid treatment was 69.8, and the disease index decreased significantly from the 5 mM-15 mM treatment (Table 2).According to the morphological results and control effects of different lactic acid concentrations on tobacco black shank, 7 mM lactic acid was selected to induce resistance in pot experiments.At 2 days post inoculation (dpi), small black lesions were observed on the tobacco stems inoculated with P. nicotianae, indicating the successful infection by P. nicotianae.At 15dpi, 7 mM lactic acid pretreatment significantly inhibited P. nicotianae growth compared with the control and reduced the area of disease spots to varying degrees (Fig. 2).Under lactic acid treatment, the average length of disease spots and disease index decreased, and the   www.nature.com/scientificreports/relative control effect reached 16.04% (Table 3).These results showed that lactic acid could effectively induce tobacco resistance to P. nicotianae.
Effects of lactic acid priming on H 2 O 2 content, protein content, total phenolic and lignin content, CAT, POD and PAL activities in tobacco leaves infected with P. nicotianae At 1 dpi, compared with the control group, lactic acid/mock inoculation (LA + Mock) treatment and lactic acid/P.nicotianae inoculation (LA + P.nic) treatment significantly increased H 2 0 2 content, and lactic acid/mock inoculation (LA + Mock) treatment significantly increased protein content (Fig. 3A-B).Total phenol content, lignin content and CAT activity were no significant difference among treatments at 1 dpi.(Fig. 3C-E).At the 5 dpi, compared with the control, lactic acid/mock inoculation (LA + Mock) treatment significantly increased H 2 0 2 content by 1.28-fold, protein content by 1.56-fold, and total phenol content by 1.47-fold, lactic acid/P.nicotianae inoculation (LA + P.nic) treatment significantly increased lignin content by 1.78-fold and CAT activity by 1.52fold (Fig. 3D and Fig. 3E).There was no significant change in POD activity and PAL activity in all treatments compared with the control (Fig. 3F and Fig. 3G).

Effects of lactic acid priming on SA, JA and lactic acid contents in tobacco leaves infected with P. nicotianae
The endogenous SA, JA and d-lactic acid contents in tobacco leaves were determined at 5 dpi.The result is shown in Fig. 4; SA and JA content showed similar content changes among different treatments.When lactic acid was applied alone (LA + Mock) or simultaneously applied with distilled water (DW) and inoculated with P. nicotianae (DW + P.nic), or simultaneously applied with lactic acid and inoculated with P. nicotianae (LA + P.nic), the JA content increased by 2.7-fold and 2.86-fold respectively (Fig. 4A and Fig. 4B).In addition, the application of

Screening of differentially expressed genes
The relationship between the 9949 obtained differentially expressed genes (DEGs) was analyzed by clustering.
The results showed that the expressions of most DEGs in the treatment group significantly differed from those in the control group (DW24h, DW_Mo) (figure.S2).Compared with DW24h (DW24h vs LA24h), the number of LA24h-DEGs in tobacco leaves was 6996; 4157 genes were up-regulated, and 2839 genes were down-regulated.
Next, DEGs Venn diagrams of DW_Mo_vs_LA_Mo and DW_Mo_vs_LA_Pn were made, representing 187 DEGs, 2428 DEGs of mock inoculation and inoculation of P. nicotianae after spraying lactic acid.DEGs Venn diagram of DW_Pn_vs_LA_Pn and DW_Mo_vs_DW_Pn showed 233 DEGs after the infection with P. nicotianae by spraying lactic acid, 3402 DEGs after infection with P. nicotianae by spraying DW, and 111 DEGs in total in the two treatment conditions (Fig. 5B).

GO annotation and KEGG enrichment analysis of DEGs induced by lactic acid in tobacco leaves
A total of 4157 up-regulated genes and 2839 down-regulated DEGs related to DW24h vs LA24h were compared using the gene annotation (GO) database (Fig. 6A).In biological processes, DEGs were mainly involved in metabolic processes, cellular processes and biological regulation.In cell components, DEGs were mainly involved in cell membrane organelles.Among molecular functions, DEGs were mainly involved in catalytic activity and binding.

GO annotation and KEGG enrichment analysis of DEGs in tobacco resistance to P. nicotianae infection induced by lactic acid
We use 2428 DEGs shown in the Venn diagram of DEGs of DW_Mo_vs_LA_Mo and DW_Mo_vs_LA_Pn, the 344 DEGs in the DEGs Venn diagram of DW_Pn_vs_LA_Pn and DW_Mo_vs_DW_Pn were compared using the GO database (Fig. 5B).It was found that these DEGs were mainly involved in biological regulation, cellular processes, and metabolic processes in biological process (Fig. 7A and Fig. 7B).
Next, 2428 DEGs were annotated using the KEGG database to enrich pathways (Fig. 7C).The results showed that these DEGs were enriched in 116 pathways.Photosynthetic-antennae protein enrichment was the highest.
A total of 344 DEGs were annotated using the KEGG database to enrich pathways.The results showed that these DEGs were significantly enriched in five pathway (Q ≤ 0.05) (Fig. 7D), as follows: flavonoid biosynthesis, plant-pathogen interactions, flavone and flavanol biosynthesis, anthocyanin biosynthesis and MAPK signaling pathway-plant.Among them, plant-pathogen interactions and MAPK signaling pathway-plant had the most enrichment genes, and the anthocyanin biosynthesis pathway had the highest rich factor value, indicating that this pathway had the highest enrichment degree.It is also enriched in plant hormone signal transduction and sesquiterpenoid and triterpenoid biosynthesis.Then 223, 111 DEGs in the DEGs Venn diagram of DW_Pn_vs_ LA_Pn and DW_Mo_vs_DW_Pn were respectively analyzed for KEGG enrichment (Figures . S3 A and B).It was found that lactic acid-induced tobacco resistance to black shank disease was induced by flavonoid biosynthesis and anthocyanin biosynthesis, and MAPK signaling pathway-plant, plant-pathogen interactions and plant hormone signal transduction were common pathways for plants and lactic acid induced tobacco resistance to black shank disease.

nicotianae
Through KEGG enrichment analysis of the above DEGs, we found that lactic acid-induced tobacco resistance to P. nicotianae mainly involved the MAPK signaling and flavonoid biosynthesis pathways through the plant-pathogen interaction plant hormone signal transduction pathway.Signal perception of pathogens and activation of downstream defense signaling molecules, including receptor kinase (RKs), MAPK and modulin-dependent calcium sensor proteins, are critical for plant defense 32 .We conducted a heat map analysis of 16 DEGs with high MAPK signaling pathway expression levels and 14 DEGs related to plant-pathogen interactions (Fig. 8A and Fig. 8B).It was found that the DEGs related to these two pathways were significantly up regulated in the LA24h, DW_Pn, LA_Mo and LA_Pn four treatments.In DW24h vs LA24h, DW_Mo vs LA_Mo and DW_Mo vs LA_Pn, the resistance gene (PR1), mitogen-activated protein kinase (MPK3), threonine protein kinase (FLS2), abscisic acid receptor (PYL), mitogen-activated protein kinase (ERK) and catalase isoenzyme (CAT ) gene were significantly up-regulated.In DW_Po vs LA_Pn, the ERK gene was significantly up-regulated, PYL, CAT and FLS2 remained unchanged, At the same time, PR1 and MPK3 were slightly down regulated, suggesting that lactic acid triggers the plant immune response and is involved in the MAPK signaling pathway.In DW24h vs LA24h, DW_Mo vs LA_Mo and DW_Mo vs LA_Pn, cysteine protease (CTSF) and calmodulin (CALM, CML) genes were significantly upregulated, and in DW_Po vs LA_Pn, CTSF and CALM genes were significantly up-regulated, while CML was slightly down-regulated.This result suggests that lactic acid may activate calcium signal transduction, which is further enhanced when plants interact with pathogens.Heat map analysis was conducted for 28 DEGs involved in hormone signaling pathways (Fig. 8C).In DW24h vs LA24h, transcription factor (WRKY 33), PYL, auxin (IAA), auxin response factor (ARF), protein phosphatase 2 (CPP2C), PR1, and JA receptor (JAZ) gene expressions were significantly up-regulated.In DW_Po vs LA_Pn, PR1 and JAZ were down-regulated, PYL, IAA, ARF, CPP2C jasmonate-amide synthase (GH3, JAR1_4_6, DELLA) and serine/threonine protein kinase (SNRK2) were significantly up-regulated in P. nicotianae infected tobacco.Therefore, this result suggests that lactic acid can activate SA and JA signaling.

Discussion
In vitro inhibition experiments showed that lactic acid could directly inhibit P. nicotianae mycelial growth and sporangium production in a dose-dependent manner (Fig. 1), and P. nicotianae mycelial growth was completely inhibited by 15 mM lactic acid.Our results were the first to report the direct inhibition effect of lactic acid on P. nicotianae, at least indicating that our isolated P. nicotianae was sensitive to lactic acid.The control effect of lactic acid was 1.71% ~ 20.91% (Table 1).Pot experiment showed that lactic acid had obvious promoting effect on plant height, stem diameter, maximum leaf length and other agronomic indexes of tobacco at low dose (Table 2).We found that 7 mM lactic acid pretreatment can effectively improve the resistance of tobacco to P. nicotianae without toxic effects (Fig. 2, Table 2 and Table 3), indicating that lactic acid could meets the requirements of a plant-induced resistance inducer, and could be further developed and utilized to evaluate its effectiveness in field conditions.
When pathogens infect plants, they lead to the rapid accumulation of ROS (a phenomenon called oxidation explosion), and participate in several important processes related to immune defense and infection 33 .These ROS can directly destroy invading pathogens and participate in coordinating the hypersensitive response (HR).H 2 O 2 and O 2 -are two major and stable ROS intermediates that act as diffused selection signals to induce gene expression of proteins that are involved in defense and antioxidant processes 34,35 .In this study, the accumulation of H 2 O 2 in tobacco treated with lactic acid significantly increased (Fig. 3A). Lee et al. 31 reported that d-lactic acid in Chlorella fusca can induce resistance in Arabidopsis to Pseudomonas syringae, presenting as an ROS eruption.These results suggest that lactic acid pretreatment may be involved in the H 2 O 2 -induced resistance of tobacco by directly killing invading pathogens or stimulating subsequent defense responses.
The ROS stimulated by pathogens is highly reactive and therefore, toxic, harming plant cells.However, excessive H 2 O 2 levels can also be harmful to plants.
Thus, plants use antioxidant enzyme systems to remove excess H 2 O 2 , among which CAT and POD are the main antioxidant systems protecting cells from oxidative damage 36,37 .In our study, the change in CAT activity was consistent with H 2 O 2 concentration (Fig. 3), which may mean that CAT degrades H 2 O 2 at relatively high H 2 O 2 concentrations.
Lignin is formed upon the effective polymerization of lignin monomers through the consumption of peroxidase and H 2 O 2 , and PAL is the rate-limiting enzyme and key enzyme 38,39 .Lignin deposition can improve the degree of tissue lignification and strengthen plant cell walls 40 .In this study, POD activity did not change in inoculated plants pretreated with lactic acid, but PAL and lignin levels were significantly increased (Fig. 3), suggesting that lactic acid can induce cell wall lignification after pathogen attack.It is well known that plant secondary metabolites are involved in plant defense responses against pathogens and herbivores 41 .Our study also showed an increase in total phenolic content in tobacco plants treated with lactic acid (Fig. 3).Previous studies have shown that d-lactic acid can induce resistance to Pseudomonas syringae in Arabidopsis thaliana, presenting as ROS eruption and corpus callosum deposition, accompanied by the expression of SA and JA signaling related genes.Our results also showed that lactic acid pretreatment induced a series of defense reactions, including H 2 O 2 accumulation and significantly increased of defense enzyme activity, SA, JA and total phenolic content, activation of signaling pathways, and then induced lignin production, and increased the cell wall physical barrier to resist penetration by P. nicotianae.
SA, JA and ET are major hormones that regulate plant immunity under different biological and abiotic stresses.These three main pathways can interact with other hormonal pathways to create resistance to infection by different pathogens 22 .The role of SA and JA in plant immunity may be antagonistic 22 , 42 .However, there is evidence that salicylic acid and jasmonic acid can cooperate with each other and play synergistic roles in plant immunity 2,43.This study shows that compared with the control, LA + Mock or LA + P.nic treatment significantly increased SA and JA in tobacco leaves (Fig. 4).SA is a necessary endogenous signal molecule for plant SAR production.PAL is a key enzyme in the SA synthesis pathway, and its increased activity is one of the indicators of plant resistance.In our study, at the 1 dpi and 5 dpi, pretreatment with lactic acid increased the PAL activity (Fig. 3).Therefore, lactic acid-induced resistance may be related to both SA and JA pathways.
In the defense response of plants, various hormone signaling pathways are interconnected to produce synergistic antagonistic effects, providing a powerful regulatory network for plants 44 .The upstream pathway of the SA signaling pathway is involved in G-proteins, Ca 2+ , MAP kinase cascades, ROS and NO signaling, and the downstream pathway has been known to generate SA conjugates, ROS, MAPK signaling cascade, WRKYs transcription factors, NPR1 and PR genes.WRKY is a key gene in the biological stress response and SA, JA signaling [44][45][46] .The response of the ABA signaling pathway to various abiotic stresses is mediated by the ABA receptor (PYR/PYL) and type 2C protein phosphatase (PP2C) [44][45][46] .The interaction of the auxin signaling pathway with various defense signaling systems is more complex and is regulated by Aux/IAA inhibitors and auxin response factor (ARF) 47 .Lee et al 31 showed that lactic acid induces the expression of cysteine-rich receptor-like kinase (CRKs), WRKY transcription factor genes and SA and JA signaling related genes.The synergy of SA and JA signaling in plant immunity has previously been reported in potatoes, rice, and tobacco [48][49][50] .In our transcriptomic analysis, lactic acid increased the expression levels of CAT , CALM, CMLS, MPK3/6 and PR1 in SA signaling pathways, transcription factors WRKY25/33, GH3, JAR, JAZs and DELLA in JA signaling pathways which was consistent with the above results (Fig. 8).As mentioned above, lactic acid significantly increased SA and JA content (Fig. 4).This suggests that lactic acid initiates defense responses in SA and JA signaling pathways involving Ca 2+ and ROS signaling.In addition, lactic acid up-regulates the expression of PYL, SRK2 and PP2C genes in the ABA signaling pathway, IAA, ARF and GH3 genes in the auxin signaling pathway.This is consistent with the results reported by Bian et al. 28 that VMA against pathogens by activating Ca 2+ , ROS, SA, JA, ET, ABA and IAA in the regulatory network signaling pathway.
Our results showed that lactic acid-induced tobacco resistance to black shank disease was also related to flavonoid biosynthesis and anthocyanin biosynthesis.It has been reported that flavonoids play key roles in plant development and defense signaling pathways.It has been reported that flavonoids can bind to protein kinases of pathogens, play a key role in defense signaling pathways, and play an important role in plant growth and development 28 .Du et al. found that exogenous MeJA could significantly up-regulate genes related to flavonoid synthesis and increase the production of flavonoid compounds to reduce fungal infection 51 .The results of this study were consistent with Du et al.The up-regulation of F3H, E5.5.1.6and FLS related to flavonoid biosynthesis under lactic acid treatment (LA24h, LA_Mo) was significantly higher than that in the control group, and the upregulation degree was higher after pathogen attack (Fig. 8).These results indicated that lactic acid could induce the expression of genes related to flavonoid synthesis and further stimulate the synthesis of some flavonoid related substances to resist P. nicotianae during pathogen infection.
Induced resistance includes two mechanisms: direct induction of the defense response and priming of the defense response 52,53 .Direct defense response is locally or systematically induced by inducers stimuli before plants were subjected to any stress.The priming defense response is a process in which plants can be activated only after being stimulated by inducers and attacked by pathogens [54][55][56] .Previous studies have shown that inducers can www.nature.com/scientificreports/activate direct defense responses at high concentrations and trigger the priming defense response mechanisms at low concentrations [54][55][56] .Boubakri et al. showed that thiamine effectively protects grapevines against Plasmopara viticola through a dual action mode of direct antifungal activity and inducing the host to produce direct and primed defense mechanisms 57 .Our conclusion was consistent with the study of Boubakri et al., lactic acid could directly inhibit P. nicotianae, and induce the accumulation of a large amount of H 2 O 2 and the expression of related defense genes in tobacco regardless of pathogen infection.These defense genes were up-regulated or down-regulated in different degrees during subsequent infection by P. nicotianae.These results indicated that lactic acid acted as an activator, inducing tobacco to produce a direct defense response, and priming the defense response against P. nicotianae through activating related signaling pathways.

Conclusion
In this study, combining our results, a complex regulatory network was used to describe the molecular role of lactic acid in enhancing tobacco resistance to black shank disease (Fig. 9).We found that when P. nicotianae attacked tobacco plants, they could trigger PAMP-triggered immunity (PTI) through the plant hormone signaling pathway, MAPK and plant-pathogen interactions.Lactic acid stimulates tobacco to produce a direct defense response and primes the defense response after subsequent pathogen attack.During the primed defense responses, lactic acid stimulates an increase in intracellular Ca 2+ and activates a series of calcium-binding proteins that inhibits ROS-generating enzymes.At the same time, stimulus-specific downstream signal transduction was activated.Lactic acid induces the synthesis of some hormones (SA, JA, ABA, TAA) and the expression of genes related to signal transduction, and coordinates the expression of other hormones in the defense network; lactic acid induces the expression of transcription factors (WRKY) and related to flavonoid synthesis genes (FLS, F3H) to resist disease stress.To remove ROS, lactic acid can also induce changes in the activity of some antioxidant enzymes (POD, PAL), as well as the accumulation of representative non-enzymatic antioxidants, such as phenolic compounds, flavonoids and lignin.
In conclusion, the results of this study indicate that lactic acid can probably induce resistance to black shank disease in tobacco plants under greenhouse control through a dual mode of action of direct antifungal activity and induction of host defense mechanisms.Lactic acid can improve tobacco disease resistance by regulating the antioxidant system, plant hormones, transcription factors, flavonoid metabolism, signal molecules and other mechanisms.In this study, lactic acid was found to play an essential role in improving tobacco disease resistance, which laid a theoretical foundation for effective prevention and treatment of tobacco black shank disease.

Plant material and inoculum preparation
These experiments were conducted at Yunnan Agricultural University from May to August 2021.The experimental material was tobacco; thecultivar was Honghua Dajinyuan (HD).The seeds (donated by the Yunnan China Tobacco Industry Co., Ltd.) were sown in seedling trays, and the seedlings were transferred to pots 30 cm in diameter, after 6 weeks of growing and culturing in a greenhouse (25 ± 3 ℃).All experiments were administered within three weeks after seedling transplantation.www.nature.com/scientificreports/ In previous experiments, we isolated and maintained the P. nicotianae strain, and the P. nicotianae strain was sequenced using a polymerase chain reaction and compared with sequences in the National Center for Biotechnology Information databases 58 .The strain was routinely recultured on potato dextrose agar (PDA: 200 g of potato, 20 g of glucose and 15 g of agar in 1000 mL water) at 27 ℃ in the dark for 15 days 59 .

Inhibition of different concentrations of lactic acid on P. nicotianae in vitro
The effect of lactic acid on P. nicotianae mycelial growth was evaluated on PDA plates according to a method reported previously 60,61 .The lactic acid solution was filtered through a 0.45 μm microfiltration membrane, and PDA containing different concentrations of lactic acid (0, 1, 2, 5, 7, 10, and 15 mM) were prepared.To ensure uniform dissolution of high lactic acid concentration in the medium, the sterilized PDA medium was maintained at about 50 ℃ and shaken gently.Next, the hyphal disks (7 mm diameter) of P. nicotianae cultured for 15 days were inoculated on PDA plates containing different lactic acid concentrations, and no lactic acid was added to the control culture.After incubation at 27 °C in the dark for 15 days, the colony diameter was measured to calculate the inhibition rates.Inhibition rate (%) = [(the diameter of control colony − treated colony)/the diameter of control colony] × 100%.
According to reported method 62 , the inhibition effect of lactic acid on the sporangia of P. nicotianae was determined.A raw lactic acid solution was mixed with 0.1% KNO 3 to prepare the induced sporulation solution containing different lactic acid concentrations.The concentrations of induced sporulation fluid were 1, 2, 5, 7, 10, and 15 mM, respectively.The hyphal disks of P. nicotianae cultured for 15 days were placed in a petri dish with induction solution, and no lactic acid was added to the control culture.These were cultured at 27 °C in the dark for 48 h.The agar medium, with a thickness of about 1mm, was cut from the lower part of the fungal hyphae disc in parallel and placed on a slide.The number of sporangia was observed and recorded under a 10 × 20 light microscope.Three biological replicates were performed for each experiment.

Pot experiment of different lactic acid concentrations on tobacco black shank disease
Following a method reported previously 63 , pot experiments were used to test the effect of different lactic acid concentrations on tobacco black shank disease.Tobacco plants were pretreated with different concentrations of lactic acid and inoculated with pathogen to determine the effect of lactic acid on tobacco black shank disease and tobacco growth and development.When tobacco plants grew to 5-6 leaves, the leaf surface and stem base wered sprayed lactic acid of different concentrations, and P. nicotianae was inoculated using the mycelium block basal-trauma inoculation method 64 .First, the tobacco plant petiole base was scratched with a blade, then the hyphal disks (7 mm diameter) of P. nicotianae were placed on the wound surface and moisturized with sterile cotton, inoculating sterile PDA medium as a blank control.
After 10 days, the length of lesion size on the stem was measured, the disease grade was recorded, and the control effect was calculated.It was recorded as 0 d on the first application day, and the increase in each morphological index was statistically analyzed only 18 d after only application.

Induction of tobacco resistance against P. nicotianae using 7 mM lactic acid
Lactic acid treatment: According to the morphological results and control effects in the pot experiment testing different lactic acid concentrations on tobacco black shank, 7 mM LA was selected for the following experiment.LA was sprayed evenly on tobacco leaves, and distilled water DW was used as a control.LA was sprayed every 2 days, three times in total.Leaves were detached from the same layer of each plant for assays at 24 h after the third treatment.The total induction time was 7 days.Experimental design and inoculation of P. nicotianae: 2 days after the third spray of DW or LA on the leaves, P. nicotianae was inoculated.The test design was as follows: (a) Control: Sprayed with DW/mock inoculation (DW + Mock); (b) Sprayed with DW/inoculated with P. nicotianae (DW + P.nic); (c) Sprayed with lactic acid/mock inoculation (LA + Mock); (d) Sprayed with lactic acid/inoculated with P. nicotianae (LA + P.nic).
At 15 dpi, the disease state was investigated, and the disease grade was recorded in each treatment.The classification standards and investigation methods of diseases were carried out according to GB/T23222-2008 65 ; Classification and Investigation Methods of Tobacco Diseases and Pests.Ten tobacco plants were treated using each treatment, and this experiment was replicated three times.Leaves were taken at 1 and 5 dpi, respectively, and immediately frozen in liquid nitrogen, and stored at − 80 °C until used for enzyme analysis and transcriptomic analyses.
H 2 O 2 content, protein content, total phenol content, lignin content, lactic acid content, and enzyme activity determination H 2 O 2 content, protein content, total phenol content, lignin content, lactic acid content and the activities of CAT, PAL, and POD were determined using an H 2 O 2 content assay kit (Suzhou Grace Biotechnology Co., Ltd., Suzhou, China), a protein assay kit (Suzhou Grace Biotechnology Co., Ltd., Suzhou, China), a total phenol assay kit (Suzhou Grace Biotechnology Co., Ltd., Suzhou, China), a lignin assay kit (Suzhou Grace Biotechnology Co., Ltd., Suzhou, China), a lactic acid assay kit (Suzhou Grace Biotechnology Co., Ltd., Suzhou, China) and the CAT, PAL and POD activity assay kit (Suzhou Greys Biological Technology Co., Ltd, Suzhou, China), respectively, H 2 O 2 content, protein content, total phenol content, lignin content, lactic acid content and enzyme activity were determined precisely according to the instructions in these kits.

SA and JA measurements
The SA and JA contents in the tobacco leaves of seedlings were measured using high-performance liquid chromatography (HPLC).SA content was determined as described in previous studies with some slight modifications 66,67 .
JA content was determined based on the method of Liu et al. 68 .The same part of tobacco leaves under different treatments was sampled, each treatment was repeated three times, and the sterilized scissors were used for picking.The external standard method is used for the quantitative analysis of SA and JA.
Determination of SA content: weigh 0.1 g of sample, grind, add 1 mL of pre-cooled 70% methanol and soak overnight at 4 °C.Centrifuge 8000 g for 10 min, take the supernatant, and extract the residue with 0.5 mL extraction solution for 2 h.After centrifugation, remove the supernatant, combine the supernatant twice, and then filter through 0.45 µm membrane for machine detection.Liquid chromatography conditions: Shimadzu LC-20AT high performance Liquid chromatograph, C18 reversed phase column (250 mm * 4.6 mm, 5 µm), mobile phase A: methanol, B: 0.1% acetic acid water, A: B 35:65, injection volume 10 μL, flow rate 0.8 mL/min, column temperature 35℃, sample removal time: 40 min, UV detector wavelength was 302 nm.The retention time is 8.33 min.
JA content was measured 69 , 0.1 g sample was accurately weighed, 1 mL extraction solution was added and ground into pulp, 1 mL 2 M HCl was added to adjust pH to 2.5-3.0, and then transferred into EP tube after shock and mixing, ultrasonic extraction for 30 min, centrifugation for 8000 g, supernatant was removed, 0.3 mL extraction solution was added into the residue, and the supernatant was combined twice.Dry the nitrogen blower in ice bath, add 0.3 mL mobile phase to redissolve, filter the membrane, and test on the machine.Liquid chromatography conditions: Wufeng LC-100 high performance liquid chromatograph, C18 reversed-phase column (250 mm * 4.6 mm, 5 µm), mobile phase A: acetonitrile, B: 1% formic acid in water, A:B 45:55, injection volume of 10 μL, flow rate of 1 mL/min, column temperature of 35 ℃, the walk-around time of :40 min, and the wavelength of the ultraviolet detector was 210 nm.The retention time is 24.75 min.

RNA-Seq and data analysis
According to the reported method 70 .RNA-Seq and data analysis were carried out.The treatments used for transcriptome sequencing were as follows: 7 mM lactic acid treatment for 24 h (LA24h) or DW treatment for 24 h (DW24h), after spraying with 7 mM lactic acid, and inoculated with P. nicotianae for 24 h (LA_Pn) or mock inoculation for 24 h (LA_Mo), after spraying with DW, and inoculated with P. nicotianae for 24 h (DW_Pn) or mock inoculation for 24 h (DW_Mo).Leaves from the same part of three plants were collected and immediately frozen using liquid nitrogen and stored at − 80 °C for transcriptome sequencing.
Total RNA was extracted using TRIzol reagent (Invitrogen), and each sample was purified with Plant RNA Purification Reagent (Invitrogen) according to the manufacturer's instructions.The samples were analyzed by 2100 Agilent Technologies for RNA size quantification and quality control.Each transcriptome library consists of 1 μg total RNA.According to the manufacturer's instructions, the TruSeqTM RNA sample preparation kit (Illumina, Inc., San Diego, CA) was used to generate a sequencing library.The library preparation and highthroughput RNA sequencing were completed using HiSeq 4000 equipment (Illumina) operated by Major Genome Center, Shanghai, China.
RawData were obtained by sequencing on an Illumina high-throughput sequencing platform, and CleanData were obtained by removing linker sequences and low-quality reads.CleanData and tobacco reference genome (https:// www.ncbi.nlm.nih.gov/ genome/ 425?Genome_ assem bly_ id= 274804) to obtain MappedData.RSEM software was used to analyze the differential expression of transcripts.Then, selected DEGs were annotated using the GO function and enriched using the KEGG pathway.DEGs were screened using DESeq2, and p < 0.01 and a multiple change (FC) ≥ 2 was selected as the threshold for significant differentially expressed genes (SDEGs).Based on the GO and KEGG databases, the functional enrichment analysis of all SDEGs was carried out using GoTools (github.com/TangHaibao/GoTools) and Kobas software (kobas.cbi.pku.edu.cn/home.do).In Mapman software, absolute log2 times change data are used, and a heat map is constructed using the gene cluster method.

Statistical analysis
The repetitions and replications for each assay are repeated in triplicate.Data were input into Microsoft Excel 2016 software, and Origin 2018 was used to analyse the data.These graphs were generated using Origin 2018.Origin 2018, with values expressed as means ± SE.Significant differences between means were analyzed at p < 0.05 using a one-way analysis of variance (ANOVA).

Ethics approval and consent to participate
The use of plant parts in the present study complies with international, national and/or institutional guidelines for plants.

Figure 1 .
Figure 1.Inhibition of lactic acid on P. nicotianae in vitro.(A) P. nicotianae cultures in Petri dishes illustrating the inhibition of mycelial radial growth with increasing lactic acid concentrations.The mycelial colonies were 15 days old.(B) Inhibition of P. nicotianae mycelial growth using different lactic acid concentrations.(C) Inhibition of P. nicotianae sporangium production at different lactic acid concentrations.Data (means ± SE, n = 4) followed by different letters above the bars indicate significant differences (p < 0.05).

Figure 2 .
Figure 2. The symptoms of tobacco resistant to P. nicotianae treated with exogenous 7 mM lactic acid pretreatment.(A) Distilled water was applied as a control, large black necrotic areas in tobacco stems, (B) Application of lactic acid reduced the area of disease spots.

Figure 6 .
Figure 6.RNA-seq data analysis of DEGs induced by 7 mM lactic acid in Tobacco leaves.(A) GO functional annotation analysis, (B, C) KEGG functional enrichment analysis of up and down-regulated DW24h vs LA24h genes.

Figure 7 .
Figure 7. RNA-seq data analysis of DEGs in tobacco resistant to P. nicotianae infestans induced by 7 mM lactic acid for 24 h.(A) GO function annotation analysis of 2428 DEGs, (B) GO function annotation analysis of 344 DEGs, (C)KEGG functional enrichment analysis of 2428 DEGs.(D)KEGG functional enrichment analysis of 344 DEGs.

Figure 9 .
Figure 9. Schematic diagram of molecular mechanism of lactic acid induced tobacco resistance to P. nicotianae.

Table 1 .
Effects of different concentrations of lactic acid on growth indexes of tobacco.Data (mean ± SD, n = 15)

Table 2 .
Effects of different concentrations of lactic acid on tobacco black shank disease.Data (mean ± SD, n = 6) followed by the different letters among treatments indicate significant differences at p < 0.05.

Table 3 .
Effect of 7 mM lactic acid on tobacco black shank.