转BtCry1Ac与API基因107杨对节肢动物群落多样性及稳定性的影响

doi:10.11707/j.1001-7488.20220412

摘要/Abstract

摘要：

目的: 研究转基因107杨的生态安全性, 为其进一步推广应用提供科学依据。方法: 对嫁接转基因107杨(BtCry1Ac与API基因)和对照107杨, 连续5年调查其节肢动物群落的组成、结构、多样性与稳定性。结果: 共获得节肢动物54091头, 分属13个目, 其中鳞翅目、鞘翅目、双翅目、膜翅目与蜘蛛目为主要类群, 个体数量上鳞翅目占优势。与对照107杨相比, 转基因107杨的害虫亚群落个体数量显著降低(P < 0.05), 靶标害虫杨扇舟蛾、美国白蛾的个体数量明显减少(P < 0.05);但天敌、中性节肢动物亚群落的物种及个体数无明显差异(P>0.05);瓢虫、蜘蛛类天敌及家蚁和蝇类等中性昆虫的个体数量随年份增加表现出一定程度的增多, 转基因107杨亚群落物种及个体数年际差异显著(P < 0.05)。转基因107杨的节肢动物群落的特征指数表现为多样性、均匀度指数高于对照107杨, 优势度指数低于对照107杨; 转基因107杨群落特征指数的年际差异均显著(P < 0.05)。稳定性分析表明, 转基因107杨的总群落物种数与群落总个体数比值S_s/S_i、植食性昆虫物种数与个体数比值S_p/S_b、捕食性天敌与植食性昆虫个体数比值S_a/S_b均显著高于对照107杨(P < 0.05), 表明转基因107杨物种数多, 个体数量少, 在种间数量上制约作用强于对照107杨; 但转基因107杨的天敌物种数与个体数比值S_n/S_a、捕食性天敌与植食性昆虫物种数比值S_n/S_p与对照107杨差异不显著(P>0.05), 表明转基因107杨捕食性天敌物种数与个体数与对照107杨无明显差异(P>0.05), 转基因107杨群落结构相对稳定。结论: 转基因107杨具有持续抗虫效果, 对天敌及中性节肢动物亚群落未产生明显不利影响, 生态安全性较高。

关键词: 转基因107杨, 节肢动物群落, 多样性, 稳定性, 生态安全性

Abstract:

Objective: This study aims to investigate the ecological safety of transgenic poplar 107(Populus×euramericana 'Neva'), and to provide scientific basis for further popularization and application. Method: In this study, the composition, structure, diversity and stability of arthropod community in grafted transgenic 107 poplar (Btcry1Ac and API gene) and control 107 poplar were investigated for five consecutive years. Result: A total of 54 091 arthropods belonging to 13 orders were collected, of which, Lepidoptera, Coleoptera, Coleoptera, Diptera, Hymenoptera and Araneae were the main groups. Lepidoptera is dominant in individual number. Compare with the control 107 poplar, the quanity of pest sub-communities in transgenic 107 poplar was significantly lower (P < 0.05). The number of target pests Clostera anachoreta and Hyphantria cunea decreased significantly (P < 0.05). But there was no significant difference (P>0.05) in species and individual number of natural enemies and neutral arthropod community. The natural enemies of Coccinellidae, Araneae and the other neutral insects of Monamorium pharaonis and flies showed increased to a certain degree year by year. There were significant interannual differences in species and individual numbers of transgenic 107 poplar sub-community (P < 0.05). The characteristic index of arthropod community in transgenic 107 poplar forest showed that the diversity and evenness index was higher than that in the control 107 poplar forest, and the dominance index was lower. There were significant interannual differences in the community characteristic index of transgenic 107 poplar forest (P < 0.05). Stability analysis showed that the ratio of total community species to total community individual (S_s/S_i), the ratio of herbivorous insects species to herbivorous insects individuals (S_p/S_b), and the ratio of predatory natural enemies individuals to herbivorous insects individuals (S_a/S_b) in transgenic 107 poplar forest were significantly higher than those in the control 107 poplar forest (P < 0.05), indicating that the transgenic 107 poplar had more species and fewer individuals, and the restriction on interspecific number was stronger than that of the control 107 poplar. But the S_n/S_a in transgenic 107 poplar forest and S_n/S_p were not significantly different from the control 107 populus (P>0.05), indicating that the number of predatory natural enemies species and individuals in the transgenic 107 poplar forest were not significantly different from those in the control 107 poplar forest (P>0.05), and the community structure of arthropod in the transgenic 107 poplar forest was relatively stable. Conclusion: Transgenic 107 poplar has a continuous insect resistance, and has no obvious adverse effects on natural enemies and neutral arthropod sub-communities. The transgenic 107 poplar has high ecological safety.

Key words: transgenic 107 poplar, arthropod community, diversity, stability, ecological safety

中图分类号:

S763.42

任敏霞,李探,张子恒,曾月霞,王利峰,杨敏生,刘军侠. 转BtCry1Ac与API基因107杨对节肢动物群落多样性及稳定性的影响[J]. 林业科学, 2022, 58(4): 110-118.

Minxia Ren,Tan Li,Ziheng Zhang,Yuexia Zeng,Lifeng Wang,Minsheng Yang,Junxia Liu. Effects of Transgenic BtCry1Ac and API gene in Poplar 107 on Diversity and Stability of Arthropod Community[J]. Scientia Silvae Sinicae, 2022, 58(4): 110-118.

图/表 11

表1

图1

图2

图3

图4

图5

图6

表2

表3

表4

表5

参考文献 0

	陈辉惶, 温俊宝, 朱银飞, 等. 转基因杨树棉复合系统对节肢动物群落结构的影响. 新疆农业大学学报, 2012, 35 (3): 189- 195. doi: 10.3969/j.issn.1007-8614.2012.03.004
	Chen H H , Wen J B , Zhu Y F , et al. Effects of transgenic poplar-cotton compound ecosystem on the stucture of arthropod community. Journal of Xinjiang Agricultural University, 2012, 35 (3): 189- 195. doi: 10.3969/j.issn.1007-8614.2012.03.004
	陈盼飞, 任亚超, 张军, 等. 8年生嫁接转基因杨树Bt毒蛋白的表达与运输. 林业科学, 2016, 52 (7): 46- 52.
	Chen P F , Ren Y C , Zhang J , et al. Expression and transportation of Bt toxic protein in 8-year-old grafted transgenic poplar. Scientla Silvae Sinicae, 2016, 52 (7): 46- 52.
	丁莉萍, 王宏芝, 魏建华. 杨树转基因研究进展及展望. 林业科学研究, 2016, 29 (1): 124- 132. doi: 10.3969/j.issn.1001-1498.2016.01.018
	Ding L P , Wang H Z , Wei J H . Progress and prospect of research in transgenic poplar. Forest Research, 2016, 29 (1): 124- 132. doi: 10.3969/j.issn.1001-1498.2016.01.018
	胡建军, 李淑梅, 卢孟柱, 等. 转Bt基因欧洲黑杨抗虫稳定性及其对天敌昆虫的影响. 林业科学研究, 2007, 20 (5): 656- 659. doi: 10.3321/j.issn:1001-1498.2007.05.011
	Hu J J , Li S M , Lu M Z , et al. Stability of insect resistance of Bt transformed Populus nigra plantation and its effects on the natural enanies of insects. Forest Research, 2007, 20 (5): 656- 659. doi: 10.3321/j.issn:1001-1498.2007.05.011
	胡建军, 卢孟柱, 杨敏生. 我国抗虫转基因杨树生态安全性研究进展. 生物多样性, 2010, 18 (4): 336- 345.
	Hu J J , Lu M Z , Yang M S . Research progress on ecological safety of insect-resistant transgenic poplar in China. Biodiversity Science, 2010, 18 (4): 336- 345.
	胡金林. 中国农林蜘蛛. 天津: 天津科学技术出版社, 1983.
	Hu J L . Chinese agricultural and forestry spiders. Tianjin: Tianjin Science and Technology Press, 1983.
	姜韬. 2018. 三种转基因玉米对害虫天敌种群动态及生长发育的影响. 扬州: 扬州大学.
	Jiang T. 2018. Effects of three kinds of transgenic maize on population dynamics and growth of natural enemies of pests. Yangzhou: Yangzhou University. [in Chinese]
	姜文虎, 刘军侠, 李超丽. 区组混栽转基因杨树林节肢动物群落结构研究. 山东农业大学学报(自然科学版), 2011, 42 (3): 383- 387. doi: 10.3969/j.issn.1000-2324.2011.03.011
	Jiang W H , Liu J X , Li C L . Study on community structure of arthropod in transgenic poplar with mixed cultivating. Journal of Shandong Agricultural University(Natural Science Edition), 2011, 42 (3): 383- 387. doi: 10.3969/j.issn.1000-2324.2011.03.011
	姜文虎, 张德健, 刘军侠, 等. 转Bt基因和非转基因杨树-棉花复合系统中节肢动物群落比较分析. 林业科学, 2018, 54 (10): 73- 79. doi: 10.11707/j.1001-7488.20181009
	Jiang W H , Zhang D J , Liu J X , et al. Comparative analysis of arthropod communities in transgenic Bt and non-transgenic poplar-cotton composite systems. Scientia Silvae Sinicae, 2018, 54 (10): 73- 79. doi: 10.11707/j.1001-7488.20181009
	李立. 2009. 转抗虫基因三倍体毛白杨成年树木外源基因表达稳定性研究. 保定: 河北农业大学.
	Li L. 2009. Expression stability of a Bt+API construct in transformed triploid populus tomentosa plants infulence of isogenic lines and plant age. Baoding: Agricultural University of Hebei. [in Chinese]
	李探, 姜文虎, 郭萌萌, 等. 转抗虫基因杨树林节肢动物群落与多样性变化研究. 河北农业大学学报, 2018, 41 (3): 84- 88.
	Li T , Jiang W H , Guo M M , et al. Study on the changes in biodiversity of arthropod community in transgenic insect-resistant poplar. Journal of Hebei Agricultural University, 2018, 41 (3): 84- 88.
	凌闵. 浅谈转基因植物在我国农业上的应用现状及未来. 上海农业科技, 2020, (6): 10- 13. doi: 10.3969/j.issn.1001-0106.2020.06.004
	Ling M . Talking about the application status and future of transgenic plants in agriculture in my country. Shanghai Agricultural Science & Technology, 2020, (6): 10- 13. doi: 10.3969/j.issn.1001-0106.2020.06.004
	刘冬艳, 张斌, 曹杨宇, 等. 转抗虫基因杨树外源基因表达的研究进展. 河北林果研究, 2015, 30 (3): 33- 37.
	Liu D Y , Zhang B , Cao Y Y , et al. The advances in expression of foreign genes in transgenic insect-resistance poplar. Hebei Journal of Forestry and Orchard Research, 2015, 30 (3): 33- 37.
	刘洁. 2015. 转抗虫基因741杨对节肢动物群落结构及动态的影响. 保定: 河北农业大学.
	Liu J. 2015. Effect of transgenic insect resistant gene poplar 741 on arthropod community structure and dynamics. Baoding: Agricultural University of Hebei. [in Chinese]
	吕威, 卢楠, 侯荣轩, 等. 12年生转基因毛白杨外源基因转移及其对土壤微生物的影响. 东北林业大学学报, 2018, 46 (9): 1- 6. doi: 10.3969/j.issn.1000-5382.2018.09.001
	Lü W , Lu N , Hou R X , et al. Exogenous gene transfer of 12 a transgenic populus[(Populus tomentosa×P.bolleana) ×P.tomentosa] and its effect on soil microbial quantity. Jouranl of Northeast Forestry University, 2018, 46 (9): 1- 6. doi: 10.3969/j.issn.1000-5382.2018.09.001
	任亚超. 2013. 多基因植物转化载体对烟草和欧美杨107杨的遗传转化. 保定: 河北农业大学.
	Ren Y C. 2013. Genetic transformation of poplar 107 from tobacco and European and American poplars by multigene plant transformation vectors. Baoding: Agricultural University of Hebei. [in Chinese]
	睢闫瑞仙, 傅利红, 等. 杨树对鳞翅目害虫抗性的研究进展. 园林科技, 2019, (1): 34- 38.
	Sui W , Yan R X , Fu L H , et al. Advances in studies on resistance of poplar to Lepidoptera pests. Garden of Science and Technology, 2019, (1): 34- 38.
	田亚坤. 2014. 转基因741杨树对节肢动物群落及土壤微生物的影响. 保定: 河北农业大学.
	Tian Y K. 2014. Effects of transgenic hybrid poplar 741 on arthropod community and soil microorganism. Baoding: Agricultural University of Hebei. [in Chinese]
	王璞, 郭同斌, 魏辉, 等. 转Bt基因'南林895'杨对美国白蛾和杨小舟蛾抗虫性分析. 分子植物育种, 2020, 18 (14): 4645- 4656.
	Wang F , Guo T B , Wei H , et al. Analysis of insect resistance of Bt-transgenic poplar (NL-895) to Hyphantria cunea and Micromelalopha troglodyte. Molecular Plant Breeding, 2020, 18 (14): 4645- 4656.
	王智, 张军, 王连荣, 等. 转抗虫基因741杨嫁接苗对目标昆虫的抗性分析. 河北农业大学学报, 2014, 37 (1): 74- 79.
	Wang Z , Zhang J , Wang L R. , et al. Poplar seedlings grafted carring the insect resistance gene resistance analysis of the target insects. Journal of Hebei Agricultural University, 2014, 37 (1): 74- 79.
	武二忠. 2011. 转基因抗虫棉对棉大卷叶螟及其主要天敌的影响. 扬州: 扬州大学.
	Wu E Z. 2011. Effect of transgenic cotton cultivars on sylepta derogate fabricius and its main natural enemies. Yangzhou: Yangzhou University. [in Chinese]
	夏兰芹, 王远, 郭三堆. 外源基因在转基因植物中的表达与稳定性. 生物技术通报, 2000, (3): 8- 12.
	Xia L Q , Wang Y , Gou S D . The stability of the expression of foreign genes in transgenic plants. Biotechnology Bulletin, 2000, (3): 8- 12.
	夏敬源, 崔金杰, 马丽华, 等. 转Bt基因抗虫棉在害虫综合治理中的作用研究. 棉花学报, 1999, 11 (2): 2- 9.
	Xia J J , Cui J J , Ma L H , et al. Study on the effect of transgenic Bt cotton on integrated pest management. Cotton Science, 1999, 11 (2): 2- 9.
	杨双. 转基因作物发展状况及趋势. 中国种业, 2018, 278 (5): 36- 40.
	Yang S . Development status and trend of transgenic crops. China Seed Industry, 2018, 278 (5): 36- 40.
	杨艳丽, 刘兴菊, 刘桂林, 等. 双抗虫基因转化欧美杨107的研究. 北方园艺, 2012, (7): 120- 122.
	Yang Y L , Liu X J , Liu G L , et al. Study on two insect-resistance gene transformation of poplar 107. Northern Horticulture, 2012, (7): 120- 122.
	姚丽, 高宝嘉. 转基因741杨对节肢动物群落结构的影响. 福建农业学报, 2016, 31 (5): 480- 486.
	Yao L , Gao B J . Effect of arthropod community structure in transgenic hybrid poplar 741. Fujian Journal of Agricultural Sciences, 2016, 31 (5): 480- 486.
	张芳. 2004. 转双抗虫基因741杨对节肢动物群落及动态的影响. 保定: 河北农业大学.
	Zhang F. 2004. Effects on dynamics in arthropod community and population of transgenic hybrid Poplar 741 with two insect-resistance. Baoding: Agricultural University of Hebei. [in Chinese]
	张巍巍, 李元胜. 中国昆虫生态大图鉴. 重庆: 重庆大学出版社, 2011.
	Zhang W W , Li Y S . Great ecological illustrations of China. Chongqing: Chongqing Uiversity Press, 2011.
	赵蓬晖, 李淑梅, 晏增, 等. 转基因欧洲黑杨土壤微生物状况和酶活性分析. 河南林业科技, 2010, 30 (4): 1- 4.
	Zhao P H , Li S M , Yan Z , et al. The Affection on soil microorganism and enzyme activity of transgenic Populus nigra. Journal of Henan Forestry Science and Technology, 2010, 30 (4): 1- 4.
	周晓静, 申坚定, 李金玲, 等. 转基因抗虫植物的发展现状. 农业科技通讯, 2018, (9): 16- 18.
	Zhou X J , Shen J D , Li J L , et al. Development status of transgenic insect-resistant plants. Bulletin of Agricultural Science and Technology, 2018, (9): 16- 18.
	Wang G Y , Dong Y , Liu X J , et al. The current status and development of insect resistant genetically engineered poplar in China. Frontiers in Plant Science, 2018, (9): 1- 15.
	Zhang B Y , Chen M , Zhang X F , et al. Expression of Bt-Cry3A in transgenic Populus alba × P. glandulosa and its effects on target and non-target pests and the arthropod community. Transgenic Research, 2011, 20 (3): 523- 532.
	Zuo L , Yang R , Zhen Z , et al. A 5-year field study showed no apparent effect of the Bt transgenic 741 poplar on the arthropod community and soil bacterial diversity. Scientific Reports, 2018, 8 (1): 1- 13.

所属目	2 a			3 a			4 a			5 a			6 a			总比Total
Orders	NS	NI	R(%)	NS	NI	R(%)	NS	NI	R(%)	NS	NI	R(%)	NS	NI	R(%)	R(%)
鳞翅目 Lepidoptera	11	3 318	48.7	13	5 246	48.8	13	7 584	67.3	17	6 578	55.8	18	3 639	25.4	48
半翅目 Hemiptera	9	459	6.7	10	1 516	14.1	11	1 216	10.8	16	849	7.2	21	4 188	29.3	15.0
膜翅目 Hymenoptera	6	1 080	15.8	5	1 256	11.7	6	901	8.0	9	2 005	17.0	9	2 577	18.0	14.2
蜘蛛目 Araneida	7	173	2.5	8	515	4.8	11	269	2.4	17	1 274	10.8	20	2 455	17.2	8.5
鞘翅目 Coleoptera	11	964	14.1	14	1 562	14.5	12	973	8.6	18	365	3.1	17	730	5.1	8.4
双翅目 Diptera	11	742	10.9	8	345	3.2	10	193	1.7	7	579	4.9	11	403	2.8	4.1
脉翅目 Neuroptera	1	68	1.0	1	89	0.8	1	31	0.3	1	60	0.5	1	196	1.4	0.8
直翅目 Orthoptera	2	14	0.2	3	198	1.8	4	43	0.4	3	59	0.5	5	78	0.5	0.7
其他 Other	0	0	0.0	2	14	0.1	1	51	0.5	5	13	0.1	3	33	0.2	0.2
合计 Total		6 818		10 741			11 261			11 782			14 299

亚群落 Sub-community	指标 Index	株系Strains		林龄Stand age		株系×林龄Strains×Stand age
亚群落 Sub-community	指标 Index	F	P	F	P	F	P
害虫亚群落	NS	3.547	0.016	112.502	< 0.001	1.225	0.270
Pest sub-community	NI	38.215	< 0.001	4.250	0.004	0.927	0.543
天敌亚群落	NS	0.687	0.063	99.021	< 0.001	0.936	0.533
Natural enemy sub-community	NI	0.875	0.483	44.786	< 0.001	0.364	0.987
中性节肢动物亚群落	NS	0.440	0.779	10.701	< 0.001	1.154	0.325
Neutral arthropod sub-community	NI	0.930	0.451	19.480	< 0.001	0.408	0.976

株系Strains	指数Index	2 a	3 a	4 a	5 a	6 a	均值Mean
CK	H′	2.10±0.32b	1.51±0.32b	1.55±0.22b	1.98±0.07b	2.49±0.16b	1.93±0.18b
#1		2.47±0.05ab	2.45±0.16a	2.20±0.17a	3.08±0.08a	3.06±0.08a	2.65±0.18a
#2		2.80±0.06a	2.79±0.08a	2.18±0.13a	3.06±0.10a	3.04±0.14a	2.77±0.16a
#3		2.52±0.13ab	2.49±0.08a	2.47±0.20a	2.90±0.03a	2.89±0.14a	2.65±0.10a
#9		2.53±0.32ab	2.62±0.17a	2.02±0.17a	3.13±0.07a	3.16±0.06a	2.69±0.21a
CK	J	0.60±0.10a	0.41±0.08b	0.45±0.07b	0.51±0.02b	0.60±0.04c	0.51±0.04b
#1		0.72±0.01a	0.69±0.04a	0.66±0.03a	0.80±0.01a	0.77±0.02ab	0.73±0.03a
#2		0.80±0.02a	0.77±0.02a	0.64±0.04a	0.78±0.02a	0.76±0.03ab	0.75±0.03a
#3		0.73±0.04a	0.69±0.02a	0.70±0.06a	0.76±0.01a	0.71±0.03b	0.72±0.01a
#9		0.74±0.09a	0.74±0.05a	0.58±0.04a	0.81±0.01a	0.79±0.01a	0.73±0.04a
CK	C	0.44±0.12a	0.68±0.09a	0.64±0.07a	0.56±0.03a	0.41±0.07a	0.55±0.05a
#1		0.32±0.03a	0.29±0.04b	0.34±0.06b	0.18±0.01b	0.16±0.02b	0.26±0.04b
#2		0.21±0.03a	0.24±0.03b	0.42±0.08b	0.19±0.04b	0.20±0.05b	0.25±0.04b
#3		0.31±0.05a	0.34±0.02b	0.32±0.07b	0.18±0.02b	0.27±0.06b	0.28±0.03b
#9		0.30±0.12a	0.26±0.04b	0.50±0.04b	0.14±0.01b	0.14±0.02b	0.27±0.07b

指数	株系Strains		林龄Stand age		株系×林龄Strains×stand age
Index	F	P	F	P	F	P
H′	22.493	< 0.001	22.117	< 0.001	1.209	< 0.001
J	24.308	< 0.001	7.658	< 0.001	1.106	0.365
C	25.088	< 0.001	11.613	< 0.001	1.511	0.118

株系Strains	指标Index	2 a	3 a	4 a	5 a	6 a
CK	S_s/S_i	0.07±0.02b	0.04±0.01b	0.04±0.01b	0.04±0.00c	0.05±0.01c
#1		0.11±0.01ab	0.09±0.01a	0.07±0.01a	0.11±0.01ab	0.10±0.01ab
#2		0.15±0.01a	0.12±0.02a	0.08±0.01a	0.12±0.01a	0.09±0.01ab
#3		0.09±0.00ab	0.08±0.01a	0.09±0.01a	0.08±0.00b	0.09±0.01b
#9		0.13±0.03a	0.11±0.01a	0.07±0.01a	0.12±0.01a	0.11±0.00a
CK	S_p/S_b	0.05±0.02b	0.02±0.01c	0.03±0.01a	0.03±0.00c	0.04±0.01c
#1		0.09±0.00b	0.08±0.02b	0.06±0.01a	0.10±0.01a	0.11±0.00ab
#2		0.15±0.02a	0.12±0.01a	0.06±0.02a	0.12±0.01a	0.11±0.01ab
#3		0.08±0.01b	0.07±0.01b	0.06±0.01a	0.07±0.00b	0.09±0.01b
#9		0.12±0.03a	0.10±0.01ab	0.04±0.01a	0.11±0.02a	0.13±0.01a
CK	S_n/S_a	0.15±0.02a	0.13±0.01a	0.14±0.01a	0.18±0.02a	0.11±0.01a
#1		0.14±0.03a	0.16±0.01a	0.13±0.03a	0.19±0.02a	0.10±0.01a
#2		0.14±0.02a	0.13±0.01a	0.17±0.04a	0.15±0.03a	0.09±0.02a
#3		0.11±0.02a	0.13±0.03a	0.17±0.03a	0.16±0.03a	0.11±0.02a
#9		0.13±0.02a	0.11±0.02a	0.18±0.02a	0.15±0.02a	0.10±0.01a
CK	S_n/S_p	0.64±0.05a	0.65±0.03a	0.52±0.07ab	0.77±0.04a	0.62±0.02a
#1		0.69±0.08a	0.65±0.07a	0.46±0.04b	0.76±0.02a	0.70±0.07a
#2		0.68±0.04a	0.72±0.02a	0.64±0.10ab	0.66±0.06a	0.66±0.05a
#3		0.63±0.04a	0.68±0.07a	0.75±0.11a	0.75±0.07a	0.61±0.06a
#9		0.67±0.06a	0.80±0.08a	0.73±0.06a	0.73±0.02a	0.72±0.04a
CK	S_a/S_b	0.11±0.01b	0.11±0.03c	0.10±0.03a	0.11±0.01c	0.22±0.02b
#1		0.36±0.04a	0.35±0.11bc	0.29±0.13a	0.36±0.04ab	0.75±0.10a
#2		0.54±0.10a	0.59±0.01ab	0.26±0.11a	0.54±0.10a	0.93±0.25a
#3		0.33±0.05a	0.36±0.10bc	0.30±0.11a	0.33±0.05b	0.64±0.23b
#9		0.49±0.07a	0.78±0.11a	0.14±0.02a	0.49±0.07ab	0.87±0.10a