Resistance Of Panax Notoginseng To Black Spot Disease Induced By 2,3-Butanediol From Leachates Of Pinus Armandii Needles

Panax notoginseng is a precious herbal in China,but the sustainable development of the industry has been constrained by problems of continuous cropping obstacles,excessive use of chemical fertilizers and pesticides,high yield and low quality.In view of these problems,our research group has carried out innovative research on the organic planting of P.notoginseng under the forest in accordance with the principle of efficacy first.This innovative model uses the advantages of "biodiversity" and "habitat coupling" under the forest to help P.notoginseng resist diseases and promote growth through the allelochemicals in pine volatiles,leachates,secretions and decomposition products.The research group has studied the volatiles of pine trees in the early stage.This paper takes the leachates from Pinus armandii needles as the research object to study the effect and mechanism of the leachates on the resistance of P.notoginseng.Firstly,this paper evaluated the effects of leachates from pine needles on plant growth,pathogen inhibition and resistance induction of P.notoginseng.Then the substance composition of leachates from pine needles was identified,and 2,3-butanediol was chosen for further study at the effect and mechanism of directly and indirectly inducing the disease resistance of P.notoginseng via affecting rhizosphere microorganisms.The specific research results are as follows:1.The leacheates from P.armandii needles has the effects of promoting growth and inducing resistance to P.notoginseng.The bioassay results showed that the leachates could promote the germination of P.notoginseng seeds and the growth of seedlings,and reduce the severity of black spot disease on leaves,but the leachates had no significant inhibitory effect on the growth of Alternaria panax.This shows that the leachates could promote the growth of P.notoginseng and induce the disease resistance of P.notoginseng.GC-MS was used to analyze the volatile and water-soluble components of the leachates from P.armandii needles.35 compounds were identified,of which 2,3-butanediol existed in both components with high relative content.The results of bioassay showed that 2,3-butanediol had allelopathic effects on P.notoginseng similar to leachates from P.armandii needles.At 11 μm,it could promote the seed germination and seedling growth of P.notoginseng,induce the resistance of P.notoginseng to A.panax,and change the rhizosphere microbial community structure of P.notoginseng,but had no direct antibacterial activity against A.panax.2.Pre-spraying of 2,3-butanediol on leaves could help P.notoginseng resist black spot disease by inducing systemic resistance(ISR)and activating systemic acquired resistance(SAR).Transcriptome sequencing was used to analyze the changes of P.notoginseng gene expression in four treatments,CK,A.panax infection(i),2,3-butanediol induction(E),and A.panax infection after 2,3-butanediol induction(Ei).The results showed that the induction of 2,3-butanediol with or without A.panax infection(E and Ei)mainly regulated the transcription factor activity and mitogen activated protein kinases(MAPKs)signaling pathways of P.notoginseng to activate SAR and ISR.But after 2,3-butanediol induction,A.panax infection could more strongly upregulate the expression of genes related to resistance.Among them,SAR is activated mainly through two modes.One is to start the downstream MAPK cascade initiation mode to trigger pattern-triggered immunity(PTI)through pathogen-or microbial associated molecular pattern(P / MAMP).This pattern triggers the immune response by upregulating the coding genes of FLS2-BAK1 complex,MKK5,MPK3 and WRKY22.Another way is to start the MAPK cascade of downstream reactive oxygen species outbreak through the upregulation of reactive oxygen species signaling factors,and then trigger effector triggered immunity(ETI).This pattern triggers the immune response by up-regulating the coding genes of Rboh D,OXI1,MKK5,MPK3 and WRKY22.In these two processes,a large number of transcription factors of WRKY family are activated.At the same time,the upregulation of FLS2-BAK1 complex,MKS1 and WRKY33 coding genes also activates the synthesis of camalexin.Induction of 2,3-butanediol with or without A.panax infection(E and Ei)could activate ISR in different ways.During the activation process,a large number of ERF and MYB family transcription factors were regulated.Further analysis demonstrated that induction of 2,3-butanediol without A.panax infection(E)activates jasmonic acid(JA)/ ethylene(ET)-mediated ISR,which is activated by the expression of genes encoding MPK3,EBF2 and ERF1.Induction of 2,3-butanediol with A.panax infection(Ei)could activate JA / abscisic acid(ABA)-mediated ISR,which was activated by the expression of the genes encoding MYC2.The q RT-PCR was used to further verify the expression of the above key genes.3.2,3-butanediol helps P.notoginseng resist the infection of A.panax by recruiting beneficial microorganisms in the rhizosphere.The structural changes of P.notoginseng rhizospheric microorganisms in the above four treatments(CK,i,E and Ei)were analyzed by using high-throughput sequencing of microbiome.The results showed that the induction of 2,3-butanediol(E)could significantly upregulate the beneficial fungi Trichoderma and Chaetomium and significantly downregulate the harmful bacteria Sphingobium in P.notoginseng rhizosphere soil.Comparison of A.panax infection(i)and A.panax infection after 2,3-butanediol induction(Ei)suggested that pre-spraying of 2,3-butanediol could ameliorate the negative plant-soil feedbacks(NPSFs)caused by A.panax infection.Specifically,the significantly upregulated pathogenic fungi(Fusarium,Ilyonectria and Gibberella)and harmful bacteria(Sphingobium and Sphingopyxis)in A.panax infection(i),were significantly downregulated in A.panax infection after 2,3-butanediol induction(Ei).The significantly downregulated beneficial microorganisms(Mortierella,Geobacillus and Brevibacillus)in A.panax infection(i),were significantly upregulated in A.panax infection after 2,3-butanediol induction(Ei).Tumebacillus and Streptomyces showed a downward trend in A.panax infection(i),but significantly upregulated in A.panax infection after 2,3-butanediol induction(Ei).The strains isolated from soil were used for antagonism and induction test.The results showed that Penicillium ochrochloron,T.koningiopsi,M.alpina,C.globosum,Bacillus sp.and Streptomyces sp.could effectively antagonize the growth of I.destructans,F.solani and F.oxysporum,the pathogens of P.notoginseng root rot.Except Streptomyces sp.,the other five antagonists could induce P.notoginseng resistance from the root to significantly reduce the lesion areas on the leaves caused by A.panax.These results showed that 2,3-butanediol could induce resistance of P.notoginseng to black spot disease by recruiting beneficial microorganisms and suppressing the harmful microorganisms in the rhizosphere soils.4.2,3-butanediol induces Panax notoginseng to change root exudates to regulate rhizosphere microorganisms.In analyses by UPLC-MS / MS showed that 2,3-butanediol induction with or without A.panax infection(E and Ei)could significantly upregulate the contents of L-leucine,L-isoleucine,L-phenylalanine,L-tyrosine,2 ’-deoxyguanosine,2’-deoxycytidine and 2 ’-deoxyadenosine in the two pathways of glucosinolate biosynthesis and ABC transporter.Correlation analyses showed that the seven root exudates had significant positive correlations with the above beneficial rhizosphere microorganisms(Trichoderma,Chaetomium,Fitibacillus,Bacillus,Paenibacillus,Brevibacillus,Tumebacillus and Geobacillus),while significant negative correlations with harmful microorganisms(Fusarium,Ilyonectria,Sphingobium and Sphingopyxis).The results of joint transcriptional and metabolic analysis showed that the phenylpropanoid biosynthesis pathway was co-enriched by 2,3-butanediol induction with or without A.panax infection(E and Ei),and L-phenylalanine and L-tyrosine were coenriched in this pathway.These results suggest that these two root exudates may be key exudates after 2,3-butanediol induction.Bioassay results showed that L-phenylalanine and L-tyrosine could promote the growth of beneficial microorganisms(T.koningiopsi,C.globosum,P.ochrochloron,Bacillus sp.,Fitibacillus sp.,Streptomyces sp.and Paenibacillus sp.)and inhibit the growth of pathogens(I.destructans and F.solani).These analyses revealed that 2,3-butanediol could promote the growth of beneficial microorganisms and inhibit the growth of pathogens by changing exudations of Lphenylalanine and L-tyrosine in the pathways of glucosinolate and phenylpropanoid biosynthesis.To sum up,the leachates and 2,3-butanediol from Pinus armandii needles could produce the allelopathic effects of promoting growth and inducing resistance to Panax notoginseng.On the one hand,2,3-butanediol could directly activate the related defense genes and transcription factors in PTI and ETI to enhance the disease resistance of P.notoginseng;On the other hand,it could recruit beneficial microorganisms by inducing the change of root exudates of P.notoginseng to indirectly activate microorganismsmediated disease resistance from the root of Panax notoginseng.This study deciphered the interaction mechanism between understory species from the allelopathic effect of pine and P.notoginseng mediated by leachates,which provided theoretical support for the organic planting of understory P.notoginseng.