JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE) ›› 2024, Vol. 59 ›› Issue (11): 40-50.doi: 10.6040/j.issn.1671-9352.0.2023.488

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Staining methods on arbuscular mycorrhizal fungi in Lycium barbarum roots and the relationship between colonization rate and soil factors

Mu YANG1,2,3(),Shenglian JI1,2,3,Huan GUO1,2,3,Guozhen DUAN1,3,*(),Guanghui FAN1,3,Jianling LI1,3,Zhanlin WANG1,3   

  1. 1. Academy of Agricultural and Forestry Sciences, Qinghai University, Xining 810016, Qinghai, China
    2. College of Agriculture and Animal Husbandry, Qinghai University, Xining 810016, Qinghai, China
    3. Qinghai Plateau Key Laboratory of Tree Genetics and Breeding, Xining 810016, Qinghai, China
  • Received:2023-11-19 Online:2024-11-20 Published:2024-11-29
  • Contact: Guozhen DUAN E-mail:19847372115@163.com;18848110959@163.com

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Abstract:

Understanding the infection and colonization of arbuscular mycorrhizal fungi (AMF) is the basis for mobilizing indigenous AMF to improve plant resistance. Lycium barbarum in nine fields of the Qaidam Basin were the subject. Based on the basic staining method of AMF, previous studies were optimised to investigate the main factors influencing the observation of AMF on the roots of perennial field-grown L.barbarum. The influence of different regions, soil pH, soil physicochemical properties and the colonization rate of AMF on L.barbarum were also compared. The results showed that the optimal observation effect of AMF colonization rate on the roots of Perennial field-grown L.barbarum in the Qaidam Basin was achieved by the following steps. First, the fixed root segments were placed in a 10% KOH 90 ℃ for 90 min, then in the boiling alkaline peroxide (3 mL NaOH+30 mL 10% H2O2+H2O to 600 mL) for 15-20 min, followed by 5 min of H2O2 bleaching, 5 min of lactic acidification, 5 min of acetone ink staining and 30 min of lactic acid glycerol 90 ℃ decolourisation. This method can clearly observe the structures of vesicles and hyphae in the roots of perennial field-grown L.barbarum. The average AMF colonization rate of perennial field-grown L.barbarum in the Qaidam Basin was about 41.32%. RDA analysis showed that the AMF colonization rate in L.barbarum roots was mainly affected by factors such as soil pH, available phosphorous content(xAP), and available potassium content(xAK), among which the soil pH showed a significant positive correlation with the AMF colonization rate, while xAP and xAK showed a significant negative correlation with the colonization rate.

Key words: Lycium barbarum, arbuscular mycorrhizal fungi, colonization rate, redundancy analysis

CLC Number: 

  • Q948.12

Table 1

Sample collection information"

采样地 英文缩写 纬度 经度 海拔/m 样品个数
都兰县宗加镇南沙滩 XY 36°39′N 96.46′E 2 768.33 6
诺木洪农场一大队 NY 36°44′N 96.48′E 2 752.84 6
诺木洪农场二大队 NE 36°45′N 96.46′E 2 718.97 6
诺木洪农场三大队 NS 36°44′N 96.35′E 2 736.39 6
诺木洪农场四大队 NSI 36°44′N 96.31′E 2 718.31 6
诺木洪农场五大队 NW 36°43′N 96.27′E 2 719.58 6
格尔木市大格勒乡 KP 36°44′N 95.72′E 2 733.58 6
格尔木市郭勒木德镇 GEM 36°42′N 94.30′E 2 767.54 6
德令哈市怀头他拉镇 WSJ 37°34′N 96.70′E 2 812.01 6
总计 54

Table 2

Method for determining colonization rate"

步骤 条件 方法1 方法2 方法3 方法4
透明 试剂 KOH KOH KOH KOH
温度/℃ 90 90 90 90
时间/min 60 15 90 90
质量分数/% 10 10 10 10
脱色 试剂 H2O2、NaOH
温度/℃ 100
时间/min 15~20
组成 3 mL NaOH+30 mL 10% H2O2溶液+H2O至600 mL
漂白 试剂 H2O2 H2O2
温度 室温① 室温
时间/min 5 5
质量分数/% 30 30
酸化 试剂 HCl HCl 乳酸 乳酸
温度 室温 室温 室温 室温
时间/min 3~4 5 5 5
质量分数/% 5 1
染色 试剂 苯胺蓝 台盼蓝 醋酸墨水 醋酸墨水
温度/℃ 90 90 室温 室温
时间/min 30 20 3~5 5
质量分数/% 0.05 0.05
褪色 试剂 乳酸、甘油 乳酸、甘油 乳酸、甘油 乳酸、甘油
温度/℃ 25 25 25 90
时间/min 过夜② 30
质量分数/% 50 50 50 50

Fig.1

Cross method of AMF colonization rate"

Fig.2

Comparison of staining effects between trypan blue and aniline blue"

Fig.3

Staining effects of acetic acid ink"

Table 3

Specific steps for determining colonization rate of AMF in L.barbarum root systems"

步骤 目的 操作
1 透明    自来水冲洗根系后置于试管中, 加入10%的KOH溶液, 将试管在90 ℃条件下水浴加热90 min
2 脱色    颜色较深的根段清洗后, 将根放入组织包埋盒后, 放入沸腾的碱性双氧水15~20 min(3 mL NaOH+30 mL 10% H2O2溶液, 加水至600 mL)
3 漂白    自来水冲洗3次并控干水分, 加入质量分数30%的H2O2溶液漂白5 min
4 酸化    自来水冲洗并控干水分, 加入乳酸于室温下酸化5 min
5 染色    倒去乳酸, 加入醋酸墨水于室温下染色5 min
6 褪色    倒去染色液, 将根段浸泡在乳酸甘油(乳酸与甘油体积比为1∶1)中, 90 ℃水浴锅中加热褪色30 min, 随后取出根段平行放置于载玻片上, 盖上盖玻片, 显微镜观察并统计侵染率

Fig.4

Staining effects of the acetic acid ink modification method"

Table 4

Comparison of AMF colonization rates in inter-root soils of perennial L.barbarum at different sites"

样地 总侵染率/% 菌丝侵染率/% 泡囊侵染率/%
XY 79.13±13.47A 64.93±7.92A 14.20±10.48A
NY 31.71±1.60C 30.49±0.23B 1.22±1.83BC
NE 25.48±3.65C 25.49±7.06B 7.14±8.71C
NS 43.04±26.52BC 41.40±24.05B 1.64±2.56C
NSI 41.87±9.56BC 40.21±9.89B 1.66±1.44C
NW 34.48±1.32BC 34.26±1.53B 0.22±0.38C
KP 30.37±5.34C 28.93±4.58B 1.44±0.77C
GEM 53.14±4.59B 41.95±11.95B 11.19±11.8BC
WSJ 32.67±0C 31.89±0.19B 0.89±0.38C
平均值 41.32±5.07 37.73±4.89 4.40±2.19

Fig.5

RDA of the colonization rate of soil AMF in relation to soil environmental factors"

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[2] XU Chun-hua,GAO Bao-yu,LU Lei,XU Shi-ping,CAO Bai-chuan,YUE Qin-yan and ZHANG Jian . Study of chemically enhanced primary treatment of wastewater received by urban rivers[J]. J4, 2006, 41(2): 116 -120 .
[3] CHEN Hong-yu1, ZHANG Li2. The linear 2-arboricity of planar graphs without 5-, 6-cycles with chord[J]. JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE), 2014, 49(06): 26 -30 .
[4] YANG Ying, JIANG Long*, SUO Xin-li. Choquet integral representation of premium functional and related properties on capacity space[J]. J4, 2013, 48(1): 78 -82 .
[5] XIE Yun-long,DU Ying-ling . Function S-rough sets and integral metric of laws[J]. J4, 2007, 42(10): 118 -122 .
[6] HUANG Xian-li，LUO Dong-mei. Feature impprtance study on  transfer learning of  sentiment  text  classification[J]. J4, 2010, 45(7): 13 -17 .
[7] GAO Zheng-hui, LUO Li-ping. Philos-type oscillation criteria for third-order nonlinear functional differential equations with distributed delays and damped terms[J]. J4, 2013, 48(4): 85 -90 .
[8] LI Zhi-rong . Computational formulae of generalized m-th-order Bell numbers and generalized m-order orderd Bell numbers[J]. J4, 2007, 42(2): 59 -63 .
[9] SONG Yu-dan, WANG Shi-tong*. Minimum within-class variance SVM with absent features[J]. J4, 2010, 45(7): 102 -107 .
[10] SHI Yan-hua1, SHI Dong-yang2*. The quasi-Wilson nonconforming finite element approximation to  pseudo-hyperbolic equations[J]. J4, 2013, 48(4): 77 -84 .
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