Inhibition and control effect of nano-iron and copper on Ascochyta medicaginicola and spring black stem disease

doi:10.11686/cyxb2024157

Abstract

Abstract:

Alfalfa （Medicago sativa）， the most extensively cultivated leguminous forage in China and globally， is susceptible to spring black stem disease caused by Ascochyta medicaginicola， one of the five most devastating diseases of alfalfa in China. Among various nanoparticles tested for their antifungal properties against A. medicaginicola， copper nanoparticles （Cu NPs） demonstrated the most pronounced inhibitory effect， with an inhibition rate of 74.1% at a concentration of 400 mg·L^-1. The application of Cu NPs also significantly mitigated the impact of spring black stem disease on alfalfa， reducing the relative lesion area to 5.95% when applied prior to inoculation with a spore suspension， and to 8.75% when applied after inoculation with a spore suspension. When alfalfa leaves were pre-treated with a Cu NPs suspension， the relative conductivity was only 18.11%， contrasting sharply with the 82.27% relative conductivity of alfalfa leaves inoculated with a spore suspension only. The enzymatic responses in Cu NPs-treated alfalfa were notable， with a significant decrease in the activities of superoxide dismutase and phenylalanine ammonia-lyase compared with the spore-inoculated control group. Conversely， the activity of polyphenol oxidase was increased by 42.6%， while catalase activity decreased by 45.0% in the first Cu NPs inoculation treatment. Ultrastructural analyses revealed that Cu NPs punctured and caused visible damage to the cell membrane of A. medicaginicola within alfalfa leaves. In contrast， the chloroplast thylakoids in the treated leaves remained clearly layered， indicating that alfalfa cell membrane integrity remained intact. These findings offer valuable insights into the potential use of Cu NPs as a control measure for alfalfa spring black stem disease.

Key words: Medicago sativa, Cu NPs, Ascochyta medicaginicola, inhibition, control effect

Tuo-xuan DONG, Xun-feng CHEN, Da-hai MEI, Yong-sha GUO, Xu-hong WEI, Qiu-yan SONG. Inhibition and control effect of nano-iron and copper on Ascochyta medicaginicola and spring black stem disease[J]. Acta Prataculturae Sinica, 2025, 34(4): 201-211.

Figures/Tables 15

Table 2 Formulations of Cu NPs medium with different concentration gradients

浓度 Concentration （mg·L^-1）	马铃薯 Potatoes （g）	无水葡萄糖 Anhydrous glucose （g）	琼脂 Agar （g）	纳米材料悬浮液 Nanomaterial suspension （1000 mg·L^-1， mL）	无菌水 Sterile water （mL）
0（空白CK）	4.0	0.4	0.4	0	20.0
25	4.0	0.4	0.4	8.0	12.0
50	4.0	0.4	0.4	4.0	16.0
100	4.0	0.4	0.4	2.0	18.0
200	4.0	0.4	0.4	1.0	19.0
400	4.0	0.4	0.4	0.5	19.5

Table 3 Blade treatment

处理方式Treatment	接种顺序Vaccination sequence
空白CK	无菌水Sterile water （5 mL）→无菌水Sterile water （5 mL）
接菌Fungal inoculation	孢子悬浮液Spore suspensions （5 mL）→无菌水Sterile water （5 mL）
先注射菌→再注射纳米铜Inoculation of fungi→reinoculation with Cu NPs	孢子悬浮液Spore suspensions （5 mL）→纳米铜悬浮液Cu NPs suspension （5 mL）
先注射纳米铜→再注射菌Inoculation of Cu NPs→reinoculation with fungi	纳米铜悬浮液Cu NPs suspension （5 mL）→孢子悬浮液Spore suspensions （5 mL）

Fig.1 Mycelial growth after treatment with different nanomaterials on 9th day

Fig.2 Diameter of mycelium growth after treatment with different nanomaterials

Fig.3 Inhibition rate of mycelial growth of different nanomaterials on 9th day

Fig.4 Mycelial growth after treatment with different concentrations of Cu NPs on 9th day

Fig.5 Diameter of mycelium after treatment with different concentrations of Cu NPs

Fig.6 Inhibition rate of mycelial growth of Cu NPs at different concentrations on 9th day

Fig.7 Fitting curves of growth inhibition rate of Cu NPs hyphae at different concentrations on 9th day

Fig.8 Leaf lesions under different treatments

Fig.9 Relative lesion area of leaves under different treatments

Fig.10 Relative conductivity of blades under different treatments

Fig.11 Superoxide dismutase （SOD）， L-phenylalanine ammonia-lyase （PAL）， catalase （CAT）， polyphenol oxidase （PPO）， peroxidase （POD） enzyme activity in leaves under different treatments

Fig.12 Transmission electron microscopy （TEM） images of A. medicaginicola spores of inoculated leaves under different treatments

Fig.13 Chloroplast transmission electron microscopy （TEM） images of inoculated leaves under different treatments

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