| Fungal diseases resulted in significant economic losses of fruits.The fungal disease caused by Phomopsis amygdali has become one of the main diseases in the major peach producing areas in China.Now,the application of fungicides is the most effective approach for disease management of the disease.However,it is necessary to develop alternative control methods to control fungal disease due to the residue of fungicides,environment pollution,the fungicides-resistant strains.Plant activators could induce the plant’s immunity to pathogen in vivo.L-glutamic acid(Glu),6-benzylaminopurine(BAP),gamma-aminobutyric acid(GABA),methyl jasmonate(MeJA)and salicylic acid(SA)could induce most immunity of plants to pathogen by activating the defense response.SA as a signal molecule for inducing plant resistance to abiotic stress and plant disease resistance not only plays a key role in the growth and development of plants,but also regulates systemic acquired resistance(SAR)and its signal transduction pathways.SA could induce changes in resistance-related defense enzyme activities,delay the occurrence of chilling injury caused by low temperature of postharvest peaches,and effectively avoid natural rot during storage.However,it is unclear whether these five chemicals could regulate systemic plant resistance in response to infection of P.amygdali.A series of experiments were carried out using these five chemicals:Glu,BAP,GABA,MeJA and SA after spraying the peach fruit.First,peaches were treated with the same concentrations(1.0 mM)to detect the effect of them on P.amygdali.We found that treatment with SA,BAP and GABA separately was most effective in inhibiting the occurrence and expansion of P.amygdali lesions.In addition,the electrical conductivity tests showed that both SA and BAP could significantly reduce the relative electrical conductivity of the peach.The results of resistance-related defense enzyme activities showed that SA,Glu and BAP significantly induced superoxide dismutase(SOD)activity in peaches,while catalase(CAT)activity was significantly inhibited through separately treatment by SA,MeJA,BAP,Glu and GABA.These results indicate that SA and BAP could reduce the relative electrical conductivity of the peach fruits,regulate the activity of resistance-related defense enzymes in the peach fruits,and increase the disease resistance to P.amygdali.In order to further study whether SA could respond to P.amygdali infection and its molecular mechanism by regulating the systemic resistance of peach fruit.First,we detected SA pathway-related genes(PpICS2,PpPAL,PpWRKY2 and PpNPR1)and pathogenesis-related protein(PR)genes(PpPR1,PpPR2 and PpPR5)expression after inoculation with P.amygdali using qRT-PCR.The results showed that the expressions of PpICS2,PpPAL,PpWRKY2,PpNPR1,PpPR1,PpPR2 and PpPR5 could be significantly enhanced after infection,indicating that the SAR pathway of peach fruits were rapidly activated.In addition,we selected the SA analogue benzothiadiazole(BTH),SA synthesis inhibitor 1-aminobenzotriazole(ABT)and SA to further confirm the results.We studied the effects of different concentrations of BTH,ABT and SA on the growth of P.amygdali in vitro.The experiment showed that high concentration of BTH,ABT and SA significantly inhibited the hyphae expansion of P.amygdali,and same concentration of ABT significantly induced the formation of conidia of the pathogen.However,low concentration of BTH,ABT and SA had no significant effect on the hyphae expansion and conidia formation of P.amygdali.Moreover,fruit inoculation and qRT-PCR experiments were did to detect the lesion diameter of P.amygdali and the fungal content in peach fruits after different concentrations of SA treatment on peach fruits.It was found that the diameter of peach fruit lesions was the smallest after 1.0 mM SA treatment,and the fungal content in the peach fruits was the lowest.While 0.3 mM SA treatment had no obvious effect.It showed that 1.0 mM SA could most significantly improve the resistance of peach fruit to P.amygdali.We also studied the infectivity of P.amygdali after treatment with 1.0 mM SA,BTH and ABT on peach branches and peach fruits.The inoculation test of peach branches showed that both BTH and SA treatments significantly reduced the diameter of the disease spots on the branches,while the diameter of the lesions increased after the ABT treatment.Peach fruits inoculation test showed that BTH and SA treatment significantly increased the endogenous SA content of peach fruit,reduced the diameter of the lesion and fungal content.ABT inhibits the content of endogenous SA,increases the diameter of lesions and fungal content.This indicates that BTH and SA increased the resistance of peach fruits to P.amygdali.The results of the oxidative damage level showed that SA and BTH significantly reduced the relative conductivity and content of MDA in the peach fruits,while treatment of ABT had opposite results,indicating that SA and BTH could reduce the oxidative damage of the peach fruit.Therefore,we detected the effects of 1.0 mM SA,BTH and ABT on reactive oxygen species(ROS),resistance-related defense enzymes activities,SA pathway-related genes and pathogenesis-related protein genes in peach fruits.ROS detection results showed that SA and BTH significantly increased content of H2O2 and superoxide,and induced ROS accumulation in peach fruits,while ABT reducing the content of ROS in peach fruits.The results of the resistance-related defense enzymes activities found that both S A and BTH could increase PAL,POD,CHI,β-1,3-GA and SOD in peach fruits,while inhibiting CAT activity.ABT could induce CAT activities in peach fruits,while inhibit the activities of PAL and SOD.The results of qRT-PCR showed that both SA and BTH could enhance the expression of PpICS2,PpPAL,PpWRKY2,PpNPRl,PpPRl,PpPR2 and PpPR5.ABT could inhibit the expression ofPpPAL,PpWRKY2,PpPR1,PpPR2 and PpPR5 in peach fruits.It showed that ABT might reduce the resistance of the peach fruits by inhibiting the expression of PpPAL.Furthermore,silencing PpICS2 and PpNPR1 in peach fruits respectively by virus-induced gene silencing(VIGS)technology.The results showed that silencing PpICS2 and PpNPR1 could significantly inhibit the endogenous SA content of peach fruit,increase the diameter of lesions,and significantly inhibit the expression of PpPR1,PpPR2 and PpPR5 in peach fruits.At the same time,the expression of histone H3 was significantly increased.However,re-spraying 1.0 mM SA after silencing peach fruits could increase the expression of PpPR1,PpPR2 and PpPR5.Finally,overexpressing PpICS2 and PpNPR3 in peach fruits through agrobacterium-mediated transient overexpression technology.The results showed that overexpress PpICS2 could significantly increase the endogenous SA content and reduce pathogenicity of P.amygdali.Significantly enhanced the expression of PpPR1,PpPR2 and PpPR5,while the expression of histone H3 gene was significantly inhibited.However,overexpressing PpNPR3 significantly inhibited the endogenous SA content,increased the diameter of lesions,inhibited the expression of PpPR1,PpPR2 and PpPR5,and fungal content in peach fruits.These results indicated that PpICS2 and PpNPR1 could positively regulate the resistance of peach fruits to P.amygdali,while PpNPR3 negatively regulate the resistance of peach fruit.In summary,SA could increase the resistance of peach fruit to P.amygdali by reducing the level of oxidative damage,inducing the ROS accumulation,regulating the activity of resistance-related defense enzymes,increasing the content of endogenous SA,and thus activating the SAR pathway.PpICS2 and PpNPR1 play a positive regulatory role in this pathway.Our research could provide theoretical support for green control of P.amygdali and activating systemic acquired resistance(S AR)of peach fruit. |
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