摘要
为了探索仿生类、内吸性化学药剂乙蒜素能否用于甘蔗病害防控及是否具有促生效果,通过室内抑菌试验、毒力测定、桶栽施药试验,评价乙蒜素对白条黄单胞菌(Xanthomonas albilineans (Ashby) Dowson)引起的甘蔗白条病的防控效果,同时通过桶栽施药试验对不同浓度乙蒜素处理的甘蔗进行农艺性状、生物量等表型测定,并结合光合、根系及抗逆等生理特性评价其促生作用。毒力测定结果显示,乙蒜素毒力回归方程为y=26.29x-30.29,相关系数
甘蔗(Saccharum officinarum L.)是重要的糖料作物和绿色可再生生物质能源作
化学防治是作物病害防控的重要途径。有研究表明,甘蔗白条病发病初期用2 000~3 500倍液的72%农用硫酸链霉素、500倍液的50%代森铵、4 000倍液的新植霉素或350倍液的14%络氨铜水剂等药剂进行喷施有一定的防治效
乙蒜素是一种仿生类内吸性化学药剂,可防治多种作物病害,兼具促生作
供试甘蔗白条黄单胞菌菌株为Xa-HN1(华南农业大学甘蔗研究室分离鉴定),80%乙蒜素(C4H10O2S2)原液由河南比赛尔农业科技有限公司提供,供试甘蔗品种为甘蔗白条病易感品种桂糖46号。
XAS液体培养基(250 mL):蔗糖5 g、细菌学蛋白胨1.25 g、磷酸二氢钾0.125 g、七水合硫酸镁0.062 5 g、亚硫酸钠0.012 5 g、溴化钾1.25 g,pH值调至7。
XAS固体培养基(250 mL):琼脂3.75 g,其他成分同XAS液体培养基,pH值调至7。
采用牛津杯抑菌圈法,在无菌条件下,挑取Xa-HN1单菌落于XAS液体培养基中28 ℃培养24 h,用紫外分光光度计测定菌液浓度,加入适量灭菌ddH2O将菌液稀释至OD590≈0.8。用移液枪吸取100 μL菌液至XAS固体培养基上,用玻璃涂布器涂布均匀,用灭菌镊子将无菌牛津杯放置于培养基表面并轻压使牛津杯与培养基紧紧贴合,每个培养皿放置3个牛津杯。用ddH2O将80%乙蒜素原液稀释成2 000、3 000、4 000、5 000、6 000倍及7 000倍稀释液,各选用1个培养皿,用移液枪吸取200 μL稀释液至牛津杯中。以牛津杯中加入等量灭菌ddH2O作为CK。将培养皿用封口膜封好,放置于恒温培养箱28 ℃培养2 d,取出拍照并记录结果,测量所形成的抑菌圈直径以进行毒力回归方程的计算。
药剂毒力回归方程Y=aX+b,其中X为药剂质量浓度(mg/L)的对数,Y为抑菌率(抑菌率=抑菌圈直径/培养皿直径×100%)。有效中质量浓度EC50的值为当Y=50%时X的
在无菌条件下,挑取Xa-HN1单菌落于XAS液体培养基28 ℃培养36 h,将菌液稀释至OD590≈0.6,将桂糖46新鲜单芽在上述菌液中浸泡45 min,将其转移至塑料方形盆中28 ℃孵育侵染12 h后加土覆盖育苗,待蔗苗长至3~4片真叶时采样进行甘蔗白条病菌PCR检测,选取阳性且大小一致的蔗苗移入桶中进行桶栽试验。每处理种植3桶,每桶定植5个阳性蔗苗,共12桶,放置于温室中。用灭菌ddH2O将80%乙蒜素原液稀释成8 000倍液(A1)、5 000倍液(A2)、1 000倍液(A3)。桶栽第7天喷施药剂,每处理喷施1 000 mL,以喷施等量ddH2O作对照(CK)。施药后14、28、42 d进行农艺性状调查、样品采集及叶片光合参数测定等。
采用CTAB法提取病样总DNA,白条黄单胞菌特异性引物XAF1:CCTGGTGATGACGCTGGGTT和XAR1:CGATCAGCGATGCACGCAG
超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、脂氧合酶(LOX)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)等酶活性及超氧阴离子产生速率、总酚含量、类黄酮含量测定参考曹建康
根系活力测定采用TTC法,参考Liu
1)乙蒜素毒力回归方程及有效中质量浓度。检测结果显示,80%乙蒜素2 000、3 000、4 000、5 000、6 000及7 000倍稀释液的抑菌圈直径依次为3.87、3.17、2.90、2.73、2.53、2.20 cm,抑菌率依次为48.33%、39.58%、36.25%、34.17%、31.67%、27.50%。毒力回归方程为y=26.29x-30.29,相关系数
2)室内桶栽防治效果。带菌蔗苗移栽后早期并未观测到甘蔗白条病症状,在施药后34 d甘蔗叶片才开始陆续出现较为明显的疑似甘蔗白条病症状(

图1 甘蔗白条病症状(A)及喷施乙蒜素后甘蔗叶片白条病菌检出率(B)
Fig.1 Symptoms of sugarcane leaf scald (A) and the detection rate of sugarcane leaf scald pathogen after spraying ethylicin (B)
相同时期柱形图上不同小写字母代表差异达显著水平(P<0.05),下同。On the column chart of the same period, different lowercase letters represent a difference at the 0.05 level, the same as below.
通过对施药后14、28、42 d甘蔗叶片白条病菌检测,上述3个时期CK的甘蔗白条病阳性检出率均为100%,而施药处理甘蔗白条病阳性检出率均呈不同程度的降低。施药后14 d,处理A1、A2、A3阳性检出率分别为66.67%、60.00%、33.33%,较CK依次下降33.33、40.00、66.67百分点;施药后28 d及42 d,A1、A2、A3阳性检出率分别为26.67%、13.33%、13.33%和6.67%、0%、0%(
1) 对主要农艺性状的影响。施药后14、28、42 d各处理株高均显著高于CK,A3显著高于A1、A2,A1和A2无显著差异;施药后42 d A1、A2、A3株高依次较CK增加36.49%、40.82%、53.41%(

图2 喷施乙蒜素后甘蔗植株农艺性状的变化
Fig.2 Changes of agronomic characters of sugarcane plants after spraying ethylicin
A:株高 Plant height; B:茎径 Stem diameter; C:叶长 Leaf length; D:叶宽 Leaf width.
2)对植株鲜质量、干质量的影响。喷施乙蒜素后42 d甘蔗苗鲜质量、根鲜质量、苗干质量、根干质量的3个处理均有不同程度的增加。3个处理中,A3效果最显著,苗鲜质量、根鲜质量、苗干质量、根干质量均显著高于CK,而A1效果最差,与对照比增加量均未达显著水平(

图3 喷施乙蒜素甘蔗幼苗及根系鲜(A、B)、干质量(C、D)变化
Fig.3 Changes of fresh(A,B) and dry weight(C, D) of sugarcane shoots and root plants after spraying ethylicin
1)对光合参数的影响。施药后14、28、42 d,A1、A2、A3 3个处理的甘蔗叶片光合参数均较CK显著提高(

图4 喷施乙蒜素甘蔗叶片光合参数变化
Fig.4 Changes of photosynthetic parameters of sugarcane leaves after spraying ethylicin
A: Pn;B: Gs;C: Ci;D: Tr.
2)对叶绿素含量的影响。由

图5 喷施乙蒜素甘蔗叶片色素含量变化
Fig.5 Changes of chlorophyll content in sugarcane leaves sprayed with ethylicin
A:叶绿素a Chlorophyll-a; B:叶绿素b Chlorophyll-b; C:类胡萝卜素 Carotenoid; D:叶绿素a+叶绿素b Chlorophyll-a+chlorophyll-b.
1)对抗氧化指标的影响。A1、A2、A3 3个处理的叶片SOD活性在3个时期均较CK有不同程度的提高,其中,14 d时,3个处理均与CK差异不显著;28 d时,A3显著高于CK,其余两处理与CK差异不显著;42 d时,各处理与CK间的差异类似于28 d,其中,A3较CK提高21.32%(

图6 喷施乙蒜素甘蔗叶片抗氧化酶活性(A、B、C)及O
Fig.6 Changes of antioxidant enzyme activity(A,B,C) and O
2)对脂氧代谢指标的影响。对于LOX活性(

图7 喷施乙蒜素甘蔗叶片脂氧合酶(LOX)活性(A)及丙二醛(MDA)含量(B)变化
Fig.7 Changes of LOX activities(A) and MDA content(B) in sugarcane leaves sprayed with ethylicin
3)对次生代谢指标的影响。对于PPO活性(

图8 喷施乙蒜素甘蔗叶片多酚氧化酶(PPO)活性(A)、苯丙氨酸解氨酶(PAL)活性(B)及总酚含量(C)、类黄酮含量(D)变化
Fig.8 Changes of PPO activities (A), PAL activities (B), total phenol content (C) and flavonoid content (D) in sugarcane leaves sprayed with ethylicin
4)对渗透调节物质含量的影响。A1、A2、A3处理的叶片可溶性糖含量在3个时期均较CK有不同程度的提高。其中,14 d时3个处理与CK差异不显著;28 d时3个处理均显著高于CK,增加幅度为12.74%~18.18%;42 d时,仅A3显著高于CK(增加17.40%),其余两处理与CK差异不显著(

图9 喷施乙蒜素甘蔗叶片渗透调节物质可溶性糖(A)、可溶性蛋白(B)及脯氨酸(C)含量变化
Fig.9 Changes of osmoregulation substance content of soluble sugar(A),soluble protein(B) and proline(C) in sugarcane leaves sprayed with ethylicin
1)对甘蔗根系活力的影响。施药42 d 3个处理A1、A2、A3的根系活力均显著高于CK,提高幅度为35.93%~62.44%,A3显著高于A1、A2,A1和A2差异不显著,A3处理较CK增加62.44%(

图10 喷施乙蒜素42 d甘蔗植株根系活力
Fig.10 Root vigor of 42 days sugarcane after spraying ethylicin
2)对甘蔗根系性状的影响。本研究所测量的根系性状包括根长、根表面积、根平均直径及根体积、根尖数及根分支点数等6个指标。由

图11 喷施乙蒜素42 d甘蔗植株根系性状
Fig.11 Root traits of 42 d sugarcane plants after spraying ethylicin
A:根长Root length;B:根表面积Root surface area;C:根体积Root volume;D:根平均直径Root average diameter;E:根尖数Apical number;F:根分支点数Root branch number.
乙蒜素是一类硫代磺酰酯衍生物,具有活性稳定、杀菌谱广、易被作物吸收、易降解等特点,前人研究发现施用量在1.18 kg/h
适宜浓度的乙蒜素可促进种子萌发,提高水稻种子萌芽
光合作用是植物生长发育的基础,光合参数反映植物光合作用的能
PAL、PPO在植物抗病方面起重要作
乙蒜素为乳油状液体,喷施后可短时间内覆盖在叶片表面导致气孔堵塞而影响叶片光合作
有报道认为过量施用乙蒜素或施用乙蒜素浓度过高可能会抑制植物生
综上,乙蒜素对甘蔗白条病菌具有毒力和抑菌效果,喷施适宜浓度的乙蒜素能显著降低甘蔗植株白条病菌检出率,有效防控甘蔗白条病,且能促进甘蔗生长,尤其以80%乙蒜素1 000倍稀释液防病促生效果为佳。喷施乙蒜素能明显提高甘蔗叶片光合能力、根系活力及改善根系性状,促进甘蔗生长。而且,喷施乙蒜素能显著提高甘蔗叶片内源防御酶的活性,增加次生代谢物(总酚和类黄酮)和渗透调节物质(可溶性糖、可溶性蛋白、脯氨酸)的含量,降低膜脂过氧化损伤程度,提高甘蔗抗逆性,有利于甘蔗生长。
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