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枫香(Liquidambar formosana Hance)是一种优良的抗逆树种选育材料,具有良好的生态适应性(抗风、耐水湿等)[1-4]。枫香作为常用的优良彩叶树种,在我国沿海平原地区防护林建设和城镇绿化中发挥了重要作用。北美枫香(L. styraciflua L.)原产于北美地区,生长速度快,树干挺直,在长江流域地区引种栽培成功。但枫香和北美枫香幼苗对盐胁迫均比较敏感[5],一定程度上制约了该类树种在我国沿海滩涂的大面积推广栽培。培育耐盐枫香是进一步提高沿海防护林体系生态效益的重要途径。利用基因工程技术培育耐盐转基因枫香植株取得了一定进展,如利用甘露糖筛选体系和根癌农杆菌介导法获得了转AtNHXI基因的耐盐植株[6]。然而,林木耐盐性是由多个数量性状基因控制的复杂性状,且育种周期长[7]。因此,在林木耐盐常规育种和分子育种研究中仍遇到很多难题。
根系共生菌对提高植物生产力和适应性至关重要[8]。内生真菌是一类栖息在健康植物组织内部、不引起宿主明显症状的共生菌类群。研究表明,在干旱、盐渍化等极端环境中生长的植物,所蕴含的内生真菌往往具有提高宿主生长、营养吸收和抗逆能力等生理生态功能[9-11]。如日本碱蓬(Suaeda japonica Makino.)中分离到的青霉(Penicillium sp.)菌株可显著增加水稻的株长和茎长[12];Qin等发现,从多种盐生植物根系分离的格孢腔目(Pleaporales)内生真菌,在有机氮条件下能有效促进植株生长[11]。作者前期对内陆和沿海滩涂多种盐生植物根系内生真菌资源进行了调查分析,发现镰刀菌(Fusarium spp.)也是常见的内生菌,且遗传多样性丰富。Maciá-Vicente等对地中海地带滩涂和内陆盐碱地24种植物根系内生真菌进行研究[13],发现尖孢镰刀菌(F. oxysporum Schl.)、茄病镰刀菌(F. solani (Mart.) Sacc.)和木贼镰刀菌(F. equiseti (Corda) Sacc.)等都是优势种。国内学者的一些研究也得出类似的结论,在翅碱蓬(S. heteroptera Kitag.)和盐角草(Salicornia bigelovii Torr.)等盐生植物中鉴定出多种内生镰刀菌[14-15]。一般而言,镰刀菌是一类重要的植物病原菌,严重影响经济林木和作物生产。目前对分布在盐碱地环境中的内生镰刀菌的生物学效应(对植株致病、有益,还是中性等)尚缺乏深入认识。本研究以分离自盐生植物滨麦(Leymus mollis (Trin.) Hara)的3株内生镰刀菌为材料,初步研究其基础生物学特性及其对北美枫香幼苗生长和耐盐性的影响,为进一步挖掘共生菌资源应用于林木抗性育苗提供理论基础和技术依据。
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从图 1可以看出:3株镰刀菌菌株在PDA平板上培养后均能产生红色色素,其中,Class2-1B和Class2-1C生长速度较快,Class2-1B气生菌丝极其发达,Class2-3生长速度最慢。Class2-1B大型分生孢子较短,无明显弯曲,成熟孢子具34个隔膜,未观察到小型分生孢子。Class2-1C分生孢子较长无明显弯曲,成熟孢子具5~6个隔膜,未观察到小型分生孢子。Class2-3孢子顶端细胞明显变窄或呈喙状,3~5个隔膜,未观察到小型分生孢子。
图 1 3株内生镰刀菌菌落特征及孢子形态
Figure 1. Colony and conidia morphology of the three endophytic Fusarium isolates
为进一步探明3株内生镰刀菌的分类学地位,基于TEF-1α基因构建了部分镰刀菌物种的系统发育树。从图 2可以看出:Class2-1B和Class2-1C的遗传关系较近,而Class2-3与Class2-1B、Class2-1C遗传关系较远,并形成了一个独立的分支。其Class2-1B与2株黄色镰刀菌在同一分支,且bootstrap值(自展值)达到100;Class2-1B与假禾谷镰刀菌遗传关系最近,bootstrap值也为100;同时,参考镰刀菌物种鉴定手册[16],发现Class2-1B和Class2-1C分别与黄色镰刀菌和假禾谷镰刀菌的培养特征和产孢结构吻合,故将Class2-1B鉴定为黄色镰刀菌,Class2-1C鉴定为假禾谷镰刀菌。虽然BLAST搜索结果表明,与Class2-3的TEF-1α序列匹配度较高为禾谷镰刀菌与假禾谷镰刀菌,形态学特征与禾谷镰刀菌也较相近,但在系统发育树中,Class2-3处在禾谷镰刀菌、假禾谷镰刀菌和黄色镰刀菌进化分支的基部,且分支较长,推测可能是潜在的新种,故在本研究中,尚未将其鉴定到种。
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从表 1可以看出:测试的5株镰刀菌都能产生毒素,且毒素类型不同,主要检测到8种毒素,痕量的毒素种类未列出。Class2-1B能产生4种毒素,包括3-乙酰基脱氧雪腐镰刀菌烯醇、15-乙酰基脱氧雪腐镰刀菌烯醇、玉米赤酶烯酮和腾毒素;Class2-1C只产生前2种毒素,除Class2-1B产生的玉米赤酶烯酮浓度接近100 μg·L-1外,其余毒素含量都极低(少于10 μg·L-1)。相反,Class2-3虽然产生毒素类型较少,但恩镰孢菌素含量极高,浓度接近8 000 μg·L-1,同时也产生少量的串珠镰刀菌素。2株病原镰刀菌均产生极高量的玉米赤酶烯酮,其中,WZ2-8A毒株产生的毒素种类较多。
表 1 5株镰刀菌产毒素类型及含量测定
Table 1. In vitro toxin production of five Fusarium isolates
毒素种类
Types of mycotoxinClass2-1B Class2-1C Class2-3 FG-1 WZ2-8A 脱氧雪腐镰刀菌烯醇
Deoxynivalenol/(μg·L-1)- - - - 77.91±45.18 3-乙酰基脱氧雪腐镰刀菌烯醇
3-Acetyldeoxynivalenol/(μg·L-1)9.90±0.64 4.07±2.65 - - 44.43±23.45 15-乙酰基脱氧雪腐镰刀菌烯醇
15-Acetyldeoxynivalenol/(μg·L-1)3.14±2.10 4.68±0.91 - 2.93±1.80 146.41±62.35 玉米赤酶烯酮Zearalenone/(μg·L-1) 95.79±35.22 - - 4 549.43±432.65 8 249.15±1 763.04 伏马毒素B1Fumonisin B1/(μg·L-1) - - - 138.78±4.13 - 恩镰孢菌素Enniatin/(μg·L-1) - - 7 892.20±348.53 - - 腾毒素Tentoxin/(μg·L-1) 1.095±0.54 - - 5.96±0.97 5.09±0.77 串珠镰刀菌素Moniliformin/(μg·L-1) - - 47.75±25.41 - - -
图 3为利用台盼蓝组织染色法观察3株内生镰刀菌侵染幼苗根系后形成的结构特征。从图 3可以看出:Class2-1B、Class2-1C能顺利进入根部皮层细胞并形成明显侵染结构,且侵染强度高,菌丝分化成串珠状的膨大细胞;而Class2-3菌丝多数只在根表定殖,在皮层细胞组织中几乎观察不到菌丝的结构,且根系组织出现褐化现象。
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从接种试验结果(图 4、图 5A~E)可以看出:共培养25 d后,3株内生镰刀菌对北美枫香幼苗生长的调控存在正负效应,其中,Class2-1B和Class2-1C对幼苗表现出明显的促生效应,而Class2-3对幼苗的生长具有明显的抑制作用,表现出强致病性。在培养后期,Class2-3菌丝可逐渐蔓延至幼苗地上部分,最终导致幼苗萎蔫死亡,这与Class2-3产生大量恩镰孢菌素有直接关系。Class2-1B和Class2-1C虽然菌丝生长旺盛(在MS培养基上产生红色色素),却不能侵染幼苗地上部分。混合孢子接种的幼苗长势也良好,推测混合接种后,Class2-3未能成为优势菌,可能与Class2-1B和Class2-1C较快的生长速度有关。
图 4 共培养体系下不同处理组幼苗生长情况比较
Figure 4. Comparison of seedling growth between five treatments under co-culture system
图 5 盐胁迫前后各处理组幼苗生长情况对比
Figure 5. Comparison of seedling growth between treatments under normal and saline conditions.
由表 2中表型指标可以看出:接种Class2-1B、Class2-1C和混合接种的幼苗生物量显著高于对照组幼苗和Class2-3接种的幼苗。与对照组相比,Class2-1B、Class2-1C接种幼苗的株高、根长和单株的叶片数量均有增加,多数差异显著(P < 0.05)。Class2-1B促进幼苗生物量积累的能力最优,Class2-1C主要促进幼苗叶片数、株高和根长的增加,混合接种对幼苗生长有一定的促进作用,但不如Class2-1B和Class2-1C处理组效果明显。
表 2 盐胁迫前接种内生镰刀菌对北美枫香幼苗表型的影响
Table 2. Phenotypic indicators of L.styraciflua seedlings under normal condition after inoculation with three endophytic Fusarium isolates
处理
Treatments生物量
Fresh weight /mg叶片数
Number of leaves株高
Seedling height/mm根长
Root length/mm对照组Control 114.00±23.99 c 7.43±1.43 b 34.71±5.29 b 19.43±8.20 b Class2-1B 158.43±36.01 a 7.75±1.45 ab 39.98±5.50 a 20.23±4.91 b Class2-1C 147.90±26.66 ab 8.10±0.81 a 41.08±5.52 a 24.43±6.77 a Class2-3 87.28±19.42 d 5.72±1.61 c 31.01±4.84 c 13.81±3.45 c 混合接种Mixed inoculation 142.74±29.19 b 7.87±1.38 ab 35.97±6.28 b 18.67±6.66 b 注:同列数据后不同小写字母表示处理间在0.05水平差异显著,下同。
Note: values followed by different letters in the same column are significantly different among the treatments at the 0.05 level, the same below.由于接种Class2-3的北美枫香幼苗后期全部死亡,故在盐胁迫试验中并未设置Class2-3接种处理组。盐胁迫处理10 d后,测定植株表型指标、盐害指数和叶绿素含量(表 3、4)。经盐胁迫处理后,Class2-1B接种的幼苗株高显著高于对照组和混合接种组,比对照组株高增加了约22%。3个处理组幼苗的整株生物量和地上生物量均高于对照组,但差异不显著。Class2-1B处理组幼苗的盐害指数最低,与对照组相比,差异显著(P=0.016 6)。叶绿素含量的测定结果(表 4)显示,各处理组与对照组叶片叶绿素含量并无显著性差异。
表 3 盐胁迫后接种内生镰刀菌对北美枫香幼苗表型的影响
Table 3. Phenotypic indicators of Liquidambar styraciflua seedlings under saline condition after inoculation with three endophytic Fusarium isolates
组别 全株生物量
Total freshweight/mg地上生物量
Abovegroundfresh weight/mg地下生物量
Belowgroundfresh weight/mg根冠比
Root-canopy ratio株高
Seedling height/mm根长
Root length/mm叶片数
Numberof leaves盐害指数
Salt injuryindex对照组
Control126.95±55.05 a 94.59±42.98 a 32.36±18.87 a 0.38±0.24 a 40.95±7.82 c 22.09±7.41 b 6.86±1.81 a 59.02±0.54 a Class2-1B 151.26±58.84 a 115.19±51.14 a 36.06±16.68 a 0.35±0.17 a 50.16±7.97 a 23.29±10.75 b 6.87±2.25 a 33.22±3.23 b Class2-1C 140.73±53.14 a 116.20±42.13 a 24.53±17.46 b 0.21±0.13 b 47.63±9.60 ab 21.07±10.65 b 7.23±1.75 a 36.20±5.59 b 混合接种
Mixed inoculation140.96±60.63 a 105.00±45.75 a 35.96±19.61 a 0.35±0.19 a 45.39±7.58 bc 33.61±15.23 a 7.17±2.04 a 66.90±12.86 a 表 4 盐胁迫后接种内生镰刀菌对北美枫香幼苗叶绿体色素含量的影响
Table 4. Chloroplast pigment contents of L. styraciflua seedlings between four treatments under saline condition
组别 叶绿素a含量
Chl a content /(mg·g-1)叶绿素b含量
Chl b content /(mg·g-1)类胡萝卜素含量
Carotenoid content/(mg·g-1)叶绿素含量
Chl content /(mg·g-1)类胡萝卜素/
叶绿素Car/Chl叶绿素a /叶绿素b
Chl a/Chl b对照组Control 1.837 5±0.185 7 a 0.836 1±0.113 6 a 0.320 8±0.029 3 a 2.673 6±0.299 1 a 0.120 2±0.003 2 a 2.205 1±0.072 5 a Class2-1B 1.719 2±0.213 2 a 0.775 9±0.126 3 a 0.294 4±0.034 5 a 2.495 1±0.338 5 a 0.118 2±0.002 4 a 2.227 0±0.096 6 a Class2-1C 1.780 3±0.197 5 a 0.812 6±0.118 1 a 0.306 9±0.033 2 a 2.592 9±0.315 4 a 0.118 6±0.001 9 a 2.198 9±0.076 3 a 混合接种
Mixed inoculation1.992 9±0.217 2 a 0.903 0±0.142 3 a 0.341 4±0.031 6 a 2.896 0±0.359 6 a 0.118 2±0.004 2 a 2.218 6±0.098 5 a
3株滨麦内生镰刀菌毒素积累及对北美枫香幼苗生长和耐盐性的影响
Toxin Accumulation of Three Leymus mollis-associated Endophytic Fusarium Isolates and Their Effects on Growth and Salt Tolerance of Liquidambar styraciflua Seedlings
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摘要:
目的 探讨3株源于滨麦的内生镰刀菌对北美枫香幼苗生长和耐盐性的调控效应,为开发极端环境中内生菌资源应用于林木抗性育苗提供理论基础。 方法 结合形态学和分子系统学方法鉴定内生镰刀菌并体外检测其毒素种类及含量,建立内生镰刀菌与无菌北美枫香实生幼苗共培养体系;利用台盼蓝染色观察菌株在根系组织中形成的侵染结构;在正常和盐胁迫条件下,研究内生镰刀菌对幼苗生长和耐盐性的影响。 结果 3个菌株分别为黄色镰刀菌(Class2-1B)、假禾谷镰刀菌(Class2-1C)和1株未鉴定到种的镰刀菌(Class2-3)。与2株强毒力农作物病原镰刀菌相比,Class2-1B、Class2-1C产毒素类型和含量极少,而Class2-3能产生大量恩镰孢菌素。Class2-1B、Class2-1C菌丝能顺利进入根部皮层细胞并形成串珠状的膨大细胞,Class2-3菌丝几乎只在根表定殖。接种试验表明:Class2-1B、Class2-1C接种幼苗的生物量、株高、根长和叶片数等多数指标显著高于对照组(P < 0.05),而Class2-3对幼苗生长有明显的抑制和毒害作用。3个菌株孢子混合接种也对幼苗表现出一定的促生效应。在海盐溶液胁迫处理下,Class2-1B处理组幼苗盐害指数显著低于对照组(P < 0.05),但处理组幼苗叶绿素体色素含量与对照组相比并无显著差异。 结论 3株内生镰刀菌菌株对北美枫香幼苗生长和耐盐的调控存在显著差异,正负效应可能与镰刀菌产毒素类型及含量有密切关系。 Abstract:Objective To evaluate the potential use of endophytes from extreme conditions as novel probiotics in improving salt tolerance of tree seedlings. Method The effects of three endophytic Fusarium isolates, recovering from the halophytic Leymus mollis, on the growth and salt tolerance of Liquidambar styraciflua seedlings were investigated. The three isolates were identified through morphological description and molecular phylogeny. In vitro toxin production of the endophytic Fusarium isolates were analyzed. The co-culturing system was used to investigate the binary interactions between gnotobiotic seedlings and Fusarium isolates under normal and saline conditions. Tissue staining method was applied to examine the fungal infection structures formed in roots. Result Two Fusarium isolates were identified to F. culmorum (Class2-1B) and F. pseudograminearum (Class2-1C), and the other one (Class2-3) was not currently identified to the species level. In comparison to two pathogenic Fusarium species, the Class2-1B and Class2-1C only produced very low level of several toxins, while the Class2-3 produced a large amount of enniatine. Both Class2-1B and Class2-1C exhibited an extensive colonization pattern in roots, but the hyphae of the Class2-3 seldom entered the inner root tissues and just distributed around the root surface. Inoculation experiment confirmed that the biomass, seedling height, root length and number of leaves in plants treated with either Class2-1B or Class2-1C were higher than the control group, although not all differences reached the significant level. In contrast, the plant growth was obvious inhibited in the presence of the Class2-3. Under salinity stress, it appeared that most seedlings inoculated with either the Class2-1B or Class2-1C were more salt tolerant, particularly for the Class2-1B treatment, than the control and mixed inoculation groups as evidence by the salt injury index. However, the chlorophyll contents in all treatments did not differ significantly. Conclusion The degree of endophytic Fusarium-mediated plant growth and salt tolerance mainly depends on different Fusarium species. The result of the study, at least in part, demonstrates that the positive or negative effect of endophytic Fusarium on plants might be related to the type and content of the toxins they produced. -
Key words:
- endophytic fungi
- / salt tolerance
- / symbiosis
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表 1 5株镰刀菌产毒素类型及含量测定
Table 1. In vitro toxin production of five Fusarium isolates
毒素种类
Types of mycotoxinClass2-1B Class2-1C Class2-3 FG-1 WZ2-8A 脱氧雪腐镰刀菌烯醇
Deoxynivalenol/(μg·L-1)- - - - 77.91±45.18 3-乙酰基脱氧雪腐镰刀菌烯醇
3-Acetyldeoxynivalenol/(μg·L-1)9.90±0.64 4.07±2.65 - - 44.43±23.45 15-乙酰基脱氧雪腐镰刀菌烯醇
15-Acetyldeoxynivalenol/(μg·L-1)3.14±2.10 4.68±0.91 - 2.93±1.80 146.41±62.35 玉米赤酶烯酮Zearalenone/(μg·L-1) 95.79±35.22 - - 4 549.43±432.65 8 249.15±1 763.04 伏马毒素B1Fumonisin B1/(μg·L-1) - - - 138.78±4.13 - 恩镰孢菌素Enniatin/(μg·L-1) - - 7 892.20±348.53 - - 腾毒素Tentoxin/(μg·L-1) 1.095±0.54 - - 5.96±0.97 5.09±0.77 串珠镰刀菌素Moniliformin/(μg·L-1) - - 47.75±25.41 - - 表 2 盐胁迫前接种内生镰刀菌对北美枫香幼苗表型的影响
Table 2. Phenotypic indicators of L.styraciflua seedlings under normal condition after inoculation with three endophytic Fusarium isolates
处理
Treatments生物量
Fresh weight /mg叶片数
Number of leaves株高
Seedling height/mm根长
Root length/mm对照组Control 114.00±23.99 c 7.43±1.43 b 34.71±5.29 b 19.43±8.20 b Class2-1B 158.43±36.01 a 7.75±1.45 ab 39.98±5.50 a 20.23±4.91 b Class2-1C 147.90±26.66 ab 8.10±0.81 a 41.08±5.52 a 24.43±6.77 a Class2-3 87.28±19.42 d 5.72±1.61 c 31.01±4.84 c 13.81±3.45 c 混合接种Mixed inoculation 142.74±29.19 b 7.87±1.38 ab 35.97±6.28 b 18.67±6.66 b 注:同列数据后不同小写字母表示处理间在0.05水平差异显著,下同。
Note: values followed by different letters in the same column are significantly different among the treatments at the 0.05 level, the same below.表 3 盐胁迫后接种内生镰刀菌对北美枫香幼苗表型的影响
Table 3. Phenotypic indicators of Liquidambar styraciflua seedlings under saline condition after inoculation with three endophytic Fusarium isolates
组别 全株生物量
Total freshweight/mg地上生物量
Abovegroundfresh weight/mg地下生物量
Belowgroundfresh weight/mg根冠比
Root-canopy ratio株高
Seedling height/mm根长
Root length/mm叶片数
Numberof leaves盐害指数
Salt injuryindex对照组
Control126.95±55.05 a 94.59±42.98 a 32.36±18.87 a 0.38±0.24 a 40.95±7.82 c 22.09±7.41 b 6.86±1.81 a 59.02±0.54 a Class2-1B 151.26±58.84 a 115.19±51.14 a 36.06±16.68 a 0.35±0.17 a 50.16±7.97 a 23.29±10.75 b 6.87±2.25 a 33.22±3.23 b Class2-1C 140.73±53.14 a 116.20±42.13 a 24.53±17.46 b 0.21±0.13 b 47.63±9.60 ab 21.07±10.65 b 7.23±1.75 a 36.20±5.59 b 混合接种
Mixed inoculation140.96±60.63 a 105.00±45.75 a 35.96±19.61 a 0.35±0.19 a 45.39±7.58 bc 33.61±15.23 a 7.17±2.04 a 66.90±12.86 a 表 4 盐胁迫后接种内生镰刀菌对北美枫香幼苗叶绿体色素含量的影响
Table 4. Chloroplast pigment contents of L. styraciflua seedlings between four treatments under saline condition
组别 叶绿素a含量
Chl a content /(mg·g-1)叶绿素b含量
Chl b content /(mg·g-1)类胡萝卜素含量
Carotenoid content/(mg·g-1)叶绿素含量
Chl content /(mg·g-1)类胡萝卜素/
叶绿素Car/Chl叶绿素a /叶绿素b
Chl a/Chl b对照组Control 1.837 5±0.185 7 a 0.836 1±0.113 6 a 0.320 8±0.029 3 a 2.673 6±0.299 1 a 0.120 2±0.003 2 a 2.205 1±0.072 5 a Class2-1B 1.719 2±0.213 2 a 0.775 9±0.126 3 a 0.294 4±0.034 5 a 2.495 1±0.338 5 a 0.118 2±0.002 4 a 2.227 0±0.096 6 a Class2-1C 1.780 3±0.197 5 a 0.812 6±0.118 1 a 0.306 9±0.033 2 a 2.592 9±0.315 4 a 0.118 6±0.001 9 a 2.198 9±0.076 3 a 混合接种
Mixed inoculation1.992 9±0.217 2 a 0.903 0±0.142 3 a 0.341 4±0.031 6 a 2.896 0±0.359 6 a 0.118 2±0.004 2 a 2.218 6±0.098 5 a -
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