外源褪黑素提高草莓黑斑病抗性的效果和作用机制初探

Effect and Mechanism of Exogenous Melatonin on Improvement of Black Rot Disease Resistance in Strawberry

该研究以黑斑病抗性较弱的草莓主栽品种‘红颊’为试验材料,先从病株中分离和鉴定草莓黑斑病的致病菌,然后进行外源喷施褪黑素和接种黑斑病菌处理,通过统计病菌致病性、褪黑素的抑菌效果和促进草莓的抗菌性能力,并测定各处理草莓发病过程中叶片相关酶活性和抗性基因的表达水平,初步探讨外源褪黑素提高草莓对黑斑病抗病性的作用机理。结果表明:(1)通过病菌分离、序列分析和侵染试验结果证明,‘红颊’草莓黑斑病致病菌为链格孢菌。(2)在含有不同浓度(1、2、4和8 mmol/L)褪黑素的PDA培养基上链格孢菌菌丝的生长速度受到不同程度的抑制,褪黑素浓度越高病菌生长速度越慢,并在8 mmol/L褪黑素的培养基上链格孢菌菌丝的抑制率达68.9%。(3)外源褪黑素预处理草莓叶片和匍匐茎24 h后接种致病菌(链格孢菌),侵染的草莓叶片和匍匐茎的黑斑病发病进程得到有效延缓,抑制效果随着褪黑素浓度的升高而升高,并以0.5 mmol/L褪黑素抑制病原菌侵染的效果最佳。(4)0.5 mmol/L外源褪黑素预处理可显著提高接菌草莓叶片抗病相关酶CAT、POD、PAL和PPO的活性,其中PPO活性变化最大,比对照显著提高了23.8%。(5)0.5 mmol/L外源褪黑素处理可显著提高草莓抗病相关基因PR1A-like、PR10、WRKY1、PPO和CCR等的表达量。研究发现,外源褪黑素能有效抑制黑斑病致病菌链格孢菌的菌丝生长,延缓其发病进程,并以0.5 mmol/L浓度最佳;WRKY1转录因子在提高草莓黑斑病抗性的过程中起到了重要的调控作用,外源褪黑素可能通过激活该转录因子的表达调控相关抗病基因的表达和相关抗氧化酶和防御酶的活性,从而提高草莓对黑斑病的抗性。

The materials used in this research is‘Benihoppe’,a popular cultivar in strawberry production with weak resistance to black rot disease.We isolated and identified the pathogen of black rot disease from sick strawberry plants.Then,we further studied on the effect and mechanism of exogenous melatonin on improving the resistance of strawberry to black rot.We detected the pathogenicity of strawberry black rot,the bacteriostatic effect,the antibacterial ability of melatonin on strawberry,the changes of related enzymes activities and expression level of resistance-related genes in the leaves with spraying exogenous melatonin and inoculating the pathogen of strawberry black rot.The results showed that:(1)the separation,sequence analysis and infection test proved that the pathogen was Alternaria alternate,which can result in black rot disease in‘Benihoppe’strawberry.(2)The hyphae growth rate of A.alternate was inhibited to different degrees on PDA media containing different concentrations of melatonin 1,2,4 and 8 mmol/L.It had slower growth rate with higher melatonin concentration.The inhibition rate of hyphae of A.alternate on the medium containing 8 mmol/L melatonin was 68.9%.(3)We found that exogenous melatonin had the function of delaying the infected onset process of pathogen(A.alternate)in leaves and stolons after they were pretreated for 24 hours.The inhibitory effect increased with the increase of melatonin concentration and 0.5 mmol/L melatonin was the most effective in inhibiting pathogen infection.(4)Biochemical analysis showed that 0.5 mmol/L exogenous melatonin pretreatment could improve the activities of disease-related enzymes such as CAT,POD,PAL and PPO after the strawberry leaves infected with pathogen,among which the activity of PPO changed most significantly,with a 23.8%higher inhibition rate than that of the control group.(5)0.5 mmol/L exogenous melatonin treatment could significantly increase the expression levels of disease-related genes such as PR1A-like,PR10,WRKY1,PPO and CCR in strawberry.It suggested that exogenous melatonin could effectively inhibit the hyphae growth rate of A.alternate and delay the onset process.The best concentration was 0.5 mmol/L.And WRKY1 transcription factor plays an important regulatory role during the process of improving strawberry resistance to black rot disease.Exogenous melatonin may improve strawberry resistance to black rot by activating the expression of this transcription factor to regulate the expression of related genes and the activities of related antioxidant enzymes and defense enzymes.