蚯蚓原位堆肥与生物炭对黄瓜根结线虫及根际微生物的影响

doi:10.16258/j.cnki.1674-5906.2023.01.011

生态环境学报 ›› 2023, Vol. 32 ›› Issue (1): 99-109.DOI: 10.16258/j.cnki.1674-5906.2023.01.011

蚯蚓原位堆肥与生物炭对黄瓜根结线虫及根际微生物的影响

游宏建(), 张文文, 兰正芳, 马兰, 张宝娣, 穆晓坤, 李文慧, 曹云娥^*()

宁夏大学农学院，宁夏银川 750021

收稿日期:2022-08-01 出版日期:2023-01-18 发布日期:2023-04-06
通讯作者: *曹云娥（1977年生），女，教授，主要从事设施蔬菜营养和土壤微生态调控。E-mail: caohua3221@163.com
作者简介:游宏建（1997年生），男，硕士研究生，主要从事设施蔬菜营养和土壤微生态调控。E-mail: 2215157672@qq.com
基金资助:
宁夏回族自治区重点研发计划项目(2021BBF02005)

Effects of Earthworm in-situ Composting and Biochar on Cucumber Root-knot Nematodes and Rhizosphere Microorganisms

YOU Hongjian(), ZHANG Wenwen, LAN Zhengfang, MA Lan, ZHANG Baodi, MU Xiaokun, LI Wenhui, CAO Yune^*()

College of Agriculture Ningxia University, Yinchuan 750021, P. R. China

Received:2022-08-01 Online:2023-01-18 Published:2023-04-06

摘要/Abstract

摘要：

随着保护地蔬菜连年连茬种植，土壤中的根结线虫大量繁殖，根结线虫病的发生普遍，已经严重危害保护地蔬菜的安全生产。生物防治因其不危害土壤安全被认为是防治根结线虫病的最优措施。目前关于蚯蚓原位堆肥和生物炭联合作用下对黄瓜根结线虫及根际微生物的影响鲜有报道。为探明蚯蚓原位堆肥和生物炭联合作用下对根结线虫和土壤微生物的影响机制，本试验以黄瓜连作土壤为研究对象，以连续3年种植黄瓜的设施土壤为供试土壤，供试黄瓜品种为“德尔99”，试验设计4个处理，分别为对照（CK）；V（蚯蚓原位处理）；B（生物炭3 t·hm^-2）；VB（蚯蚓原位+生物炭3 t·hm^-2）。结果表明：蚯蚓原位+生物炭处理在整个时期对黄瓜根结数量有显著的防治作用，其中在盛果期和拉秧期虫口减退率分别达66.51%、80.42%，植株发病率仅13.33%，防治效果达78.57%。另外，蚯蚓原位堆肥处理、生物炭处理对根结线虫的防治效果分别为71.42%、-35.71%，可见单独施用生物炭并不能防治黄瓜根结线虫，但是蚯蚓原位+生物炭处理对根结线虫防治效果显著。采用高通量测序技术结果表明，对于黄瓜连作土壤根际细菌而言，蚯蚓原位+生物炭与对照相比，VB处理的Ace指数、Simpson指数、Shannon指数均显著高于CK处理，分别高44.84%、45.63%和15.01%（P<0.05）。对于黄瓜连作土壤根际真菌而言，与对照相比，各处理也增加了土壤真菌群落的丰富性和多样性。相关性分析结果表明，细菌中放线菌门（Actinobacteriota）、髌骨菌门（Patescibacteria）与黄瓜根结数量呈显著负相关（P<0.05）；真菌中丝壳属（Kernia）、未分类的小囊菌属（unclassified-f-Microascaceae）黄瓜根结数量呈显著负相关（P<0.05）。

关键词: 蚯蚓, 黄瓜, 生物炭, 根结线虫, 生物防治, 微生物菌落

Abstract:

With the continuous cropping of vegetables in protected areas, the root-knot nematodes in the soil have proliferated extensively, causing root-knot nematode disease widely, which has seriously endangered the vegetables production in protected areas. Biological control is the best measure to control root knot nematode disease because it does not endanger soil health. At present, there are few reports about the effects of earthworm in situ composting and biochar on cucumber root-knot nematodes and rhizosphere microorganisms. To explore the mechanism of the combined effect of earthworm in-situ composting and biochar on root-knot nematodes and soil microorganisms, this study targeted continuous cropping soil of cucumber as the research object, and the greenhouse soil planted with cucumber for three consecutive years as the tested soil. The cucumber variety was ‘Del 99’, and four treatments were designed as control (CK); V (earthworm in-situ treatment); B (biochar 3 t·hm^-2); VB (earthworm in-situ+biochar 3 t·hm^-2). The results showed that earthworm in situ+biochar treatment had significant effects on biological control of the number of cucumber root knots throughout the period. Among them, the population decline rates at the full fruit stage and the seedling stage were 66.51% and 80.42%, respectively. The plant incidence rate was only 13.33%, and the control efficiency was 78.57%. In addition, the control efficiency of earthworm compost and biochar on root-knot nematodes were 71.42% and -35.71%, respectively. The application of biochar alone could not control cucumber root-knot nematodes, but earthworm compost+biochar treatment had a significant effect on root-knot nematodes. High-throughput sequencing results showed that the Ace, Simpson and Shannon indices of earthworm in situ+biochar for cucumber rhizosphere bacteria were significantly higher (44.84%, 45.63% and 15.01%, respectively) than those of CK treatment (P<0.05). As for cucumber rhizosphere fungi, each treatment increased the richness and diversity of soil fungal communities compared with the control. Correlation analysis showed that Actinobacteriota and Patescibacteria bacteria were significantly and negatively correlated with the number of cucumber root nodules (P<0.05). Kernia and unclassified-f-Microascaceae fungi were significantly and negatively correlated with the number of cucumber root knots (P<0.05).

Key words: earthworm, cucumber, biochar, root knot nematode, biological control, microbial colonies

中图分类号:

S154.1
X17

游宏建, 张文文, 兰正芳, 马兰, 张宝娣, 穆晓坤, 李文慧, 曹云娥. 蚯蚓原位堆肥与生物炭对黄瓜根结线虫及根际微生物的影响[J]. 生态环境学报, 2023, 32(1): 99-109.

YOU Hongjian, ZHANG Wenwen, LAN Zhengfang, MA Lan, ZHANG Baodi, MU Xiaokun, LI Wenhui, CAO Yune. Effects of Earthworm in-situ Composting and Biochar on Cucumber Root-knot Nematodes and Rhizosphere Microorganisms[J]. Ecology and Environment, 2023, 32(1): 99-109.

图/表 21

表1 试验地土壤基础性质

Table 1 Basic properties of soil in the test site

pH	有机质 w_{(Organic matter)} (g·kg^-1)	全氮 w_{(Total N)} (g·kg^-1)	全磷 w_{(Total P)} (g·kg^-1)	速效氮 w_{(Available N)} (g·kg^-1)	速效磷 w_{(Available P)} (mg·kg^-1)	速效钾 w_{(Available K)} (mg·kg^-1)
7.94	9.43	0.78	0.31	24.2	67.4	185.9

表2 试验设计

Table 2 Experimental design

处理	土壤处理
CK	未处理
V	蚯蚓原位
B	生物炭 (3 t·hm^-2)
VB	蚯蚓原位+生物炭 (3 t·hm^-2)

表3 PCR 扩增引物设计

Table 3 Primer design for PCR amplification

测序区域	引物	引物序列
515F_806R	515F	GTGCCAGCMGCCGCGG
515F_806R	806R	GGACTACHVGGGTWTCTAAT
ITS1F_ITS2R	ITS1F	CTTGGTCATTTAGAGGAAGTAA
ITS1F_ITS2R	ITS2R	CTTGGTCATTTAGAGGAAGTAA

表4 PCR反应体系

Table 4 PCR reaction system

试剂	快速PFU DNA 聚合酶 5×Fast Pfu Buffer 5 U·µL^-1	脱氧核糖核苷三磷酸 2.5 mmol·L^-1	正向引物 5 µmol·L^-1	反向引物 5 µmol·L^-1	快速PFU DNA 聚合酶 5 U·µL^-1	牛血清蛋白 5 mg·mL^-1	模板 DNA 100 µg·mL^-1	无菌水补ddH₂O至 1 g·mL^-1
体积 V/µL	4	2	0.8	0.8	0.4	0.2	10	20

表5 不同土壤处理对黄瓜土壤线虫的防效变化

Table 5 Control effects of different soil treatments on cucumber root-knot nematodes

处理	定植前	盛果期			拉秧期
处理	线虫数量	线虫数量	虫口减退率/%	虫口防效/%	线虫数量	虫口减退率/%	虫口防效/%
CK	88.67a	104.00a	-17.44c	—	143.00a	-62.06d	—
V	88.67a	37.33c	57.44a	64.10a	49.67c	44.06b	65.27b
B	95.33a	74.00b	22.44b	28.85b	109.33b	-14.68c	23.54c
VB	90.01a	30.33c	66.51a	70.83a	28.00d	68.93a	80.42a

表6 不同土壤处理对黄瓜根结发生程度的防效

Table 6 Control effects of different soil treatments on cucumber root-knot nematodes

处理	发病率/%	根结指数	防治效果/%
CK	66.67b	46.67b	—
V	13.33c	13.33c	71.42a
B	86.67a	63.33a	-35.71b
VB	13.33c	10c	78.57a

表7 质控后的优化数据

Table 7 Display of optimized data after quality control

序列扩增区域	样本	有效序列/bp	测序片段/bp	平均长度/bp
338F_806R	12	1645443	682947425	415

图1 同处理对黄瓜土壤细菌OUT水平Venn图

Figure 1 Venn diagram of OUT level of cucumber soil bacteria under different treatments

表8 质控后的优化数据

Table 8 Display of optimized data after quality control

序列扩增区域	样本	有效序列/bp	测序片段/bp	平均长度/bp
ITS1F_ITS2R	12	2591024	627735197	242

图2 不同处理对黄瓜土壤真菌OUT水平Venn图

Figure 2 Venn diagram of OUT level of cucumber soil fungi under different treatments

表9 土壤样本中细菌和真菌的Alpha多样性分析

Table 9 Alpha diversity analysis of bacteria and fungi in soil samples

种类	处理	覆盖度/ %	Chao1 指数	Ace	香农指数	辛普森指数
细菌 Bacteria	CK	96.22a	3595.22a	3599.6b	6.32c	0.0032b
	V	96.07a	3479.87a	4249.55a	6.67b	0.0049a
	B	95.88a	3874.49a	3917.35ab	6.21c	0.0024b
	VB	96.65a	3490.29a	4236.61a	6.82a	0.0059a
真菌 Fungus	CK	99.96a	307.05b	303.37b	3.35b	0.098ab
	V	99.89b	581.58a	572.93a	3.65b	0.12a
	B	99.96a	314.99b	308.25b	3.62b	0.071b
	VB	99.92b	541.86a	541.81a	4.14a	0.058b

图3 门分类水平上不同土壤细菌物种相对丰度

Figure 3 Relative abundance of different soil bacterial species at phylum classification level

图4 不同处理下黄瓜土壤中细菌群落热图分析

Figure 4 Thermal map analysis of bacterial community in cucumber soil under different treatments

图5 分类水平上不同土壤真菌物种相对丰度

Figure 5 Relative abundance of different soil fungal species at phylum classification level

图6 不同土壤处理黄瓜真菌群落热图分析

Figure 6 Heat map analysis of cucumber fungal community under different soil treatments

图7 不同处理对黄瓜土壤细菌OUT水平主成分分析的影响

Figure 7 Effect of different treatments on OUT level principal component analysis of cucumber soil bacteria

图8 不同处理对黄瓜土壤真菌OUT水平主成分分析的影响

Figure 8 Effect of different treatments on OUT level principal component analysis of cucumber soil fungi

图9 细菌与黄瓜根系中根结数量的相关分析门

Figure 9 Correlation analysis gate between bacteria and the number of root nodes in cucumber roots

图10 细菌与黄瓜根系中根结数量的相关分析属

Figure 10 Correlation analysis genus between bacteria and the number of root nodes in cucumber roots

图11 真菌门水平与黄瓜根系中根结数量的相关分析

Figure 11 Correlation analysis between fungal phylum level and the number of root nodes in cucumber roots

图12 真菌属水平与黄瓜根系中根结数量的相关分析

Figure 12 Correlation analysis between fungal genus level and root knot number in cucumber roots

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蚯蚓原位堆肥与生物炭对黄瓜根结线虫及根际微生物的影响

Effects of Earthworm in-situ Composting and Biochar on Cucumber Root-knot Nematodes and Rhizosphere Microorganisms

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