摘要
【目的】明确长枝木霉(SC5)代谢粗提物对极细链格孢(ABL2)产毒抑制机制。【方法】以富士苹果的叶片为供试材料,通过生长速率法、LC-MS和RT-qPCR技术测定了SC5代谢粗提物对ABL2菌落生长、6种非寄主选择性毒素产生及产毒相关基因表达的抑制作用。【结果】质量浓度为0.5 mg·mL^(-1)的SC5代谢粗提物对ABL2菌落生长和致病力具有显著的抑制作用,第6天时抑制率分别为38.08%和76.96%;同时,0.5 mg·mL^(-1) SC5代谢粗提物对ABL2菌株产生的非寄主选择性毒素TEN、ALT和TeA均具有显著抑制作用,其中处理2d后对毒素的抑制作用最显著,其含量分别降低69.39%、98.51%和48.99%,并且其合成相关基因TES、TES(1)、PksA和PksJ的表达量显著下调,分别降低89.02%、98.20%、46.49%和40.13%,但是对ABL2菌株非寄主选择性毒素ATX-I含量和参与编码其合成的PksF基因表达量具有提升作用。【结论】质量浓度为0.5mg·mL^(-1)的SC5代谢粗提物对ABL2菌株生长和致病力具有抑制作用,可能通过调控ABL2菌株TES、TES(1)、PksA和PksJ基因下调表达,进而降低TEN、ALT和TeA的毒素的产生量及致病力。
【Objective】Alternaria spp.can cause a variety of apple leaf diseases,which occur in all ma-jor apple producing areas in the world.It can lead to brown disease spots in apple leaves and even result in early defoliation,which seriously affects the development of apple industry and causes huge econom-ic losses.Apple leaf blight caused by Alternaria tenuissima was found for the first time in apple produc-ing areas of Gansu province.This fungus can damage apple leaves and petioles,causing leaves to die and fall off.A.alternata mainly damages plants by producing Alternaria toxins.The Alternaria toxins that have been found can be divided into five categories,i.e,diphenyl-α-pyrones and their derivatives,perylenequinones and their derivatives,tetraamino acids and their derivatives,long-chain amino polyols of glycerol tricarboxylate compounds,and hybrid structures.All of them have obvious toxicity,which can cause serious harm to plants and endanger the food safety of agricultural products.At present,chem-ical control is still the main means to prevent and control the diseases caused by Alternaria fungi.How-ever,due to the influence of Alternaria resistance and environmental pollution,it is of great significance to find a series of biocontrol agents with higher controlling effect on Alternaria.Biocontrol of Tricho-derma is safer and greener than the traditional chemical control methods.Trichoderma metabolites are also good antifungal substances,with broad-spectrum and efficient antibacterial activity,inhibiting the growth and metabolism of pathogenic fungi.The growth and metabolism of A.tenuissima ABL2 strain were inhibited by Trichoderma longibrachiatum SC5 metabolites.The content of non-host selective tox-ins in the metabolites of A.tenuissima ABL2 strain and the relative expression of genes related to the synthesis of A.tenuissima toxin were determined.The inhibition mechanism of T.longibrachiatum SC5 metabolites on A.tenuissima ABL2 strain production was clarified.The aim was to provide a reference and theoretical basis for the prevention and control of apple leaf blight caused by A.tenuissima.【Methods】T.longibrachiatum SC5 and A.tenuissima ABL2 strains were cultured on PDA medium for 7 days.The PDB liquid medium was made,and the SC5 strain was cultured on the medium for 15 days.The fer-mentation broth was filtered with filter paper and the broth was retained.The liquid was added with 3-fold-volume-ethyl acetate and oscillated for 1 hour.After standing extraction,the organic phase was evaporated in a rotary evaporator,and 1 ml of methanol was added to dissolve and evaporate.The SC5 metabolites were made into an orginal liquid with a concentration of 200.00 mg·mL^(-1),and added to the PDA medium to make a drug-containing medium with different concentrations of SC5 metabolites(0.01,0.05,0.10,0.25,0.50,1.00 and 2.00 mg·mL^(-1)).The ABL2 strain was inoculated on the drug-con-taining medium and cultured for 24 hours in a light incubator.The diameter of ABL2 colonies was mea-sured and the colony inhibition rate was calculated on the 2,4,6,8 and 10 days by cross method.On the 6th day,5 mycelial plugs were prepared with a sterile puncher and placed in a 10 mL of EP tube,with 3 replicates per concentration.After adding 8 mL of ethyl acetate,ultrasonic oscillation was performed for 1.5 hours,centrifuged and filtered.After the ethyl acetate phase was evaporated to dryness,1 mL of methanol was added to dissolve it,and 5 mm circular sterile filter paper was placed in it for later use.In-oculate healthy leaves after disinfection and rinsing using stab inoculation method.A circular filter paper soaked in ABL2 metabolites was placed at each wound,and placed in an artificial climate box for mois-turizing culture for 7 days.The lesion size was measured and the pathogenic activity of the metabolites was calculated.The optimal inhibitory concentration of SC5 metabolites on the growth of ABL2 strain was screened.The total RNA and the metabolites of ABL2 strain growing in the optimal concentration of drug-containing medium was extracted on 2,4,6,8 and 10 days,and the quantitative standard curves of six non-host-selective toxins were made respectively.Six toxin-producing related gene primers were designed and verified for specificity.Liquid Chromatograph Mass Spectrometer and Real Time Quantita-tive PCR were used to determine the content of toxins in ABL2 metabolites and the relative expression of toxin-producing related genes.【Results】The crude extract of SC5 metabolism at a concentration of 0.5 mg·mL^(-1) had a significant inhibitory effect on the growth and pathogenicity of ABL2,and the inhibi-tion rates were 38.08%and 76.96%on the 6th day,respectively.0.5 mg·mL^(-1) SC5 metabolic crude ex-tract had a significant inhibitory effect on the non-host selective toxins TEN,ALT and TeA produced by ABL2 strain.The inhibitory effect was the most significant after 2 days treatment,and its content was reduced by 69.39%,98.51%and 48.99%,respectively.The expression levels of TES,TES(1),PksA and PksJ were significantly down-regulated by 89.02%,98.20%,46.49%and 40.13%,respectively.However,the content of non-host selective toxin ATX-I of ABL2 strain and the expression of PksF gene in-volved in its synthesis increased.【Conclusion】The SC5 metabolites of T.longibrachiatum at a concen-tration of 0.5 mg·mL^(-1) had an inhibitory effect on the growth and pathogenicity of A.tenuissima ABL2 strain.It could reduce and inhibit the content of TEN,ALT and TeA by down-regulating the expression of TES,TES(1),PksA and PksJ genes in ABL2 strain,thereby reducing its pathogenicity.This study can provide a theoretical basis for the biological control of ABL2 strain.
作者
邵学辉
张树武
徐秉良
SHAO Xuehui;ZHANG Shuwu;XU Bingiang(College of Plant Protection,Gansu Agricultural University/Engineering Laboratory for Biological Control of Crop Diseases and Pests,Lanzhou,730070,Gansu,China)
出处
《果树学报》
CSCD
北大核心
2024年第1期133-142,共10页
Journal of Fruit Science
基金
甘肃省重点研发计划项目(23YFNA0020)
甘肃省科技重大专项(22ZD6NA045)
甘肃农业大学“伏羲杰出人才培育计划”项目(Gaufx-03J03)
兰州市科技计划项目(2021-1-39)。
关键词
苹果叶片
极细链格孢
长枝木霉
毒素
基因表达
生物防治
Fuji apple leaves
Alternaria tenuissima
Trichoderma longibrachiatum
Toxin
Gene ex-pression
Biological control
