牛尾山药病毒病危害情况调查与茎段脱毒方法

doi:10.3969/j.issn.1004-1524.20230860

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (6): 1329-1338.DOI: 10.3969/j.issn.1004-1524.20230860

牛尾山药病毒病危害情况调查与茎段脱毒方法

程园^a(), 徐升胜^a, 段延碧^b, 徐馨蕾^a, 郭凤根^c, 王仕玉^a, 龙雯虹^a^,^*()

a.云南农业大学园林园艺学院,云南昆明 650201
b.云南农业大学农科教学实验中心,云南昆明 650201
c.云南农业大学农学与生物技术学院,云南昆明 650201

收稿日期:2023-07-13 出版日期:2024-06-25 发布日期:2024-07-02
作者简介:程园(1999—),女,云南红河人,硕士研究生,研究方向为蔬菜学。E-mail:1404551023@qq.com
通讯作者: *龙雯虹,E-mail:lwh57@sina.com
基金资助:
国家自然科学基金(32260770);云南省曲靖市专家工作站项目(2021年)

Investigation on virus disease in ‘Niuwei’ yam and removal of virus with stem culture

CHENG Yuan^a(), XU Shengsheng^a, DUAN Yanbi^b, XU Xinlei^a, GUO Fenggen^c, WANG Shiyu^a, LONG Wenhong^a^,^*()

a. College of Landscape and Horticulturey, Yunnan Agricultural University, Kunming 650201, China
b. Agricultural Science Teaching and Experiment Centery, Yunnan Agricultural University, Kunming 650201, China
c. College of Agriculture and Biotechnology, Yunnan Agricultural University, Kunming 650201, China

Received:2023-07-13 Online:2024-06-25 Published:2024-07-02

摘要/Abstract

摘要：

为探究云南特色品种牛尾山药携带的病毒及其危害情况,明确病毒抑制剂、热处理和低温处理对山药病毒的脱毒效果,以期得到较好的脱毒方法。研究调查了牛尾山药病毒病发病率、植株病症,采用分子生物学方法检测了病毒种类,研究了不同脱毒处理下山药斑驳病毒的脱毒效果。结果表明:牛尾山药携带山药斑驳病毒,叶片可见明显的褪绿斑,发病率为41.67%~43.33%。RT-PCR检测结果显示,60 mol·L^-1盐酸吗啉胍处理、60 mol·L^-1利巴韦林药剂处理能降低山药成活率和生长量,但能有效脱除山药斑驳病毒。采用高温处理(45 ℃ 12 h、40 ℃ 24 h、45 ℃ 24 h)结合顶芽培养能有效脱除山药斑驳病毒,40 ℃处理24 h的山药生长量显著降低,而45 ℃处理12 h、45 ℃处理24 h的山药生长量和成活率均显著降低。低温处理(0 ℃ 24 h、5 ℃ 24 h)结合顶芽培养能显著降低山药成活率和生长量,但能有效脱除山药斑驳病毒。综合考虑成活率、生长量和脱毒效果,建议采用60 mol·L^-1盐酸吗啉胍处理或40 ℃高温处理24 h。

关键词: 山药斑驳病毒病, 高温, 脱毒培养, 茎段培养

Abstract:

To investigate the virus carried by Yunnan’s special variety of ‘Niuwei’ yam and its harmful effects, clarify the effectiveness of viral inhibitors, heat treatment, and low temperature treatment on removing virus from yam, with a view to obtaining better methods for devirusing. The study investigated the incidence of yam virus disease and plant symptoms in ‘Niuwei’ yam, and detected the type of virus using molecular biological detection method, and examined the detoxification effect of different treatments on yam mosaic virus. The results showed that ‘Niuwei’ yam carried yam mosaic virus, and obvious yellowing spots could be seen on its leaves. The incidence rate was 41.67%-43.33%. RT-PCR results showed that treatments with 60 mol·L^-1 moroxydine hydrochloride or 60 mol·L^-1 ribavirin could reduce the survival rate and growth of the yam but effectively remove the yam mosaic virus. High-temperature treatments (12 h at 45 ℃, 24 h at 40 ℃, 24 h at 45 ℃) combined with apical bud culture effectively removed yam mosaic virus. The treatment for 24 h at 40 ℃ significantly reduced the growth of yam, while treatments for 12 h at 45 ℃ and 24 h at 45 ℃ significantly reduced both the growth and survival rate of yam. Low-temperature treatment (24 h at 0 ℃ and 24 h at 5 ℃) combined with apical bud culture significantly reduced the survival rate and growth of yam but were effective in removing the yam mosaic virus. Considering the survival rate, growth, and detoxification effect, 60 mol·L^-1 moroxydine hydrochloride treatment or 40 ℃ high-temperature treatment for 24 h was recommended.

Key words: yam mosaic virus disease, high-temperature, detoxification, stem culture

中图分类号:

S632.1

程园, 徐升胜, 段延碧, 徐馨蕾, 郭凤根, 王仕玉, 龙雯虹. 牛尾山药病毒病危害情况调查与茎段脱毒方法[J]. 浙江农业学报, 2024, 36(6): 1329-1338.

CHENG Yuan, XU Shengsheng, DUAN Yanbi, XU Xinlei, GUO Fenggen, WANG Shiyu, LONG Wenhong. Investigation on virus disease in ‘Niuwei’ yam and removal of virus with stem culture[J]. Acta Agriculturae Zhejiangensis, 2024, 36(6): 1329-1338.

图/表 11

表1 PCR所用引物信息

Table 1 Primer information for PCR

检测病毒 Detect virus	引物名称 Primer name	序列(5'→3') Sequence (5'→3')	退火温度 Annealing temperature/℃
YMMV	YMMVDetF1 8716	TTCCTCGCATCAAAGCACCAATGAG	59.58
	YMMVDetR1 9254	TGCTGCYTTCATYTGCATGTG	53.66
	YMMVDetR1a9254	TGCCGCTTTCATTTGCATGTG	55.61
JYMV	JYMVF8430	TACGCATGGCTTCTTGGGCAAGC	61.33
	JYMVR9256	TTCCTCGAAGTCCATAGCGTGGC	61.33
	JYMVR9256a	TCCTCTGAAGTCCATAGCGTGGC	61.33
YCNV	YCNMVF604	TTAACGTCATAGTAGCTCTCAGC	55.99
	YCNMVR1269	TAGTCGAGCTGAACCCTAACCAC	59.55
YMOV	YMOVF4146	ATTGTGGTTCATGCAGTTGCAGG	57.77
	YMOVR5056	ATATACAACAAGTGCAACTAACACG	54.66

图1 山药带病叶片与健康叶片图A与图B均为山药生长旺盛时期叶片。图A为患病毒病山药叶片,图B为山药健康叶片。

Fig.1 Diseased leaves and healthy leaves of Chinese yam Fig. A and B were the leaves of yam during its vigorous growth. Fig. A showed the leaves of Chinese yam infected with virus disease, and Fig. B showed the healthy leaves of Chinese yam.

图2 YMOV引物PCR检测结果 YMOV的PCR产物大小为910 bp。M,DL 2 000 DNA marker。泳道1、16,阳性对照组外植体样品;泳道2、17,阴性对照组外植体样品;泳道3~15和泳道18~29,随机检测田间疑似病毒病植物叶片样品。

Fig.2 PCR detection results of YMOV primer The PCR product length of YMOV is 910 bp. M, DL 2 000 DNA marker. Lane 1 and 16, Positive control explant samples; Lane 2 and 17, Negative control explant samples; Lanes 3-15 and Lanes 18-29, Random detection of leaf samples of plants suspected of viral diseases in the field.

表2 盐酸吗啉胍处理后组培苗的生长状况

Table 2 Growth of tissue culture seedlings treated with moroxydine hydrochloride

质量浓度 Mass concentration/ (mg·L^-1)	成活率 Survival rate/%	侧芽萌发数 Number of sprouting lateral buds	侧芽长出数 Number of elongating lateral buds	株高 Plant height/cm	叶色 Leaf color
20	95.53±0.91 a	2.68±0.67 a	1.30±0.98 ab	2.13±0.35 a	绿Green
40	96.63±2.91 a	2.34±0.28 c	1.20±0.05 b	1.87±0.40 a	绿Green
60	70.30±0.95 b	2.14±0.53 d	0.64±0.05 c	1.00±0.26 b	绿Green
0(CK)	94.63±1.24 a	2.59±0.65 ab	1.42±0.53 a	2.23±0.15 a	绿Green

图3 盐酸吗啉胍对组培苗的脱毒效果 M,DL 2 000 DNA marker。泳道1、2,20 mg·L-1盐酸吗啉胍处理的下部样品、上部样品;泳道3、4,40 mg·L-1盐酸吗啉胍处理的下部、上部样品;泳道5、6,60 mg·L-1盐酸吗啉胍处理的上部、下部样品;泳道7、8,对照组外植体下部、上部样品。

Fig.3 Detoxification effect of moroxydine hydrochloride on tissue culture seedlings M, DL 2 000 DNA marker. Lanes 1-2, Lower sample and upper sample of explants treated with 20 mg·L-1 moroxydine hydrochloride; Lanes 3-4, Lower sample and upper sample of explants treated with 40 mg·L-1 moroxydine hydrochloride; Lanes 5-6, Lower sample and upper sample of explants treated with 60 mg·L-1 moroxydine hydrochloride; Lanes 7-8, Lower sample and upper sample of control explants.

表3 利巴韦林处理的组培苗生长状况

Table 3 Growth of tissue culture seedlings treated with ribavirin

质量浓度 Mass concentration/ (mg·L^-1)	成活率 Survival rate/%	侧芽萌发数 Number of sprouting lateral buds	侧芽长出数 Number of elongating lateral buds	株高 Plant height/cm	叶色 Leaf color
20	82.20±3.05 b	1.23±0.53 b	0.42±1.15 b	0.96±0.15 b	绿Green
40	63.57±3.23 c	1.18±0.20 b	0.41±0.58 b	0.50±0.10 c	绿Green
60	55.43±0.75 d	0.87±0.53 c	0.15±0.95 c	0.19±0.79 d	绿Green
0(CK)	94.63±1.24 a	2.59±0.65 a	1.42±0.53 a	2.23±0.15 a	绿Green

图4 利巴韦林对组培苗的脱毒效果 M,DL 2 000 DNA marker。泳道1~2,利巴韦林20 mg·L-1下部、上部样品;泳道3~4,利巴韦林40 mg·L-1下部、上部样品;泳道5~6,利巴韦林60 mg·L-1上部、下部样品;泳道7~8,对照组外植体下部、上部样品。

Fig.4 Detoxification effect of ribavirin on tissue culture seedlings M, DL 2 000 DNA marker. Lanes 1-2, Ribavirin 20 mg·L-1 lower and upper samples;Lanes 3-4, Ribavirin 40 mg·L-1 lower and upper samples; Lanes 5-6, Ribavirin 60 mg·L-1 upper and upper samples; Lanes 7-8, Control explant lower and upper samples.

表4 高温处理组培苗对顶芽生长状况的影响

Table 4 Effects of high temperature on shoot tip growth of tissue culture seedlings

处理 treatment	成活率 Survival rate/%	侧芽萌发数 Number of sprouting lateral buds	侧芽长出数 Number of elongating lateral buds	株高 Plant height/cm	叶色 Leaf color
40 ℃ 12 h	86.20±2.89 a	2.09±0.97 a	1.16±0.98 a	2.10±0.30 a	绿Green
45 ℃ 12 h	63.00±4.11 b	1.62±0.62 b	0.80±0.53 b	1.46±0.21 b	绿Green
40 ℃ 24 h	81.37±3.25 a	1.96±0.87 b	1.00±0.77 b	1.96±0.06 b	绿Green
45 ℃ 24 h	53.90±3.53 c	1.15±0.25 c	0.43±0.53 c	0.90±0.10 c	嫩绿Light green
对照组Control	87.13±4.59 a	2.16±0.79 a	1.22±0.15 a	2.27±0.15 a	绿Green

图5 高温处理对组培苗的脱毒效果 M,DL 5 000 DNA marker;泳道1,45 ℃处理24 h;泳道2,40 ℃处理24 h;泳道3,45 ℃处理12 h;泳道4,40 ℃处理12 h;泳道5,对照组。

Fig.5 Detoxification effect of high temperature on tissue culture seedlings M, DL 5 000 DNA marker; Lane 1, 45 ℃ for 24 h; Lane 2, 40 ℃ for 24 h; Lane 3, 45 ℃ for 12 h; Lane 4, 40 ℃for 12 h; Lane 5, Control.

表5 低温处理组培苗对顶芽生长状况的影响

Table 5 Effects of low temperature on shoot tip growth of tissue culture seedlings

处理 Treatment	成活率 Survival rate/%	侧芽萌发数 Number of sprouting lateral buds	侧芽长出数 Number of elongating lateral buds	株高 Plant height/cm	叶色 Leaf color
0 ℃ 12 h	80.83±1.40 b	1.17±1.00 b	8.33±1.77 b	0.68±1.23 b	嫩绿Light green
-5 ℃ 12 h	60.63±2.15 c	1.09±0.08 c	5.00±1.00 c	0.55±0.09 bc	嫩绿Light green
0 ℃ 24 h	55.87±1.79 d	1.05±0.53 c	5.33±0.58 bc	0.45±0.05 c	嫩绿Light green
-5 ℃ 24 h	45.73±0.50 e	1.01±0.33 d	4.03±0.98 d	0.45±0.05 c	嫩绿Light green
对照组Control	87.13±1.15 a	2.16±0.79 a	12.00±0.15 a	2.27±0.15 a	绿Green

图6 低温处理对组培苗的脱毒效果 M,DL 2 000 DNA marker;泳道1,-5 ℃处理24 h;泳道2,0 ℃处理24 h;泳道3,-5 ℃处理12 h;泳道4,0 ℃处理12 h;泳道5,对照组。

Fig.6 Detoxification effect of low temperature on tissue culture seedlings M, DL 2 000 DNA marker; Lane 1,-5 ℃ for 24 h; Lane 2, 0 ℃ for 24 h; Lane 3,-5 ℃ for 12 h; Lane 4, 0 ℃ for 12 h; Lane 5, Control.

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牛尾山药病毒病危害情况调查与茎段脱毒方法

Investigation on virus disease in ‘Niuwei’ yam and removal of virus with stem culture

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