Chinese leek (Allium tuberosum Rottler ex Sprengel) reduced

Transcription

Journal of Integrative Agriculture
Advanced Online Publication 2015
Doi：10.1016/S2095-3119(15)61032-2
Chinese leek (Allium tuberosum Rottler ex Sprengel) reduced
disease symptom caused by root knot nematode1
HUANG Yong-hong1, 2, MAO Zhen-chuan2, XIE Bing-yan2
1
College of Horticulture, Qingdao Agricultural University, Qingdao 266109, P.R.China
2
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081,
P.R.China
Abstract:
Root-knot nematodes (RKNs, Meloidogyne spp.) are obligate biotrophic parasites that settle close
to the vascular tissues in plant roots. The diseases resulting from RKN infections cause serious
damage to agricultural production worldwide. In the present paper, the resistance of Chinese leek
against RKNs, its suppressive effect on nematode disease, its nematicidal activity and its
component profile were studied to identify an efficient disease control method. In soil heavily
infected by nematodes, Chinese leek showed strong resistance to RKNs. Additionally, the gall
indexes of cucumber plants rotated with Chinese leek and of tomato plants intercropped with
Chinese leek were reduced by 70.2% and 41.1%, respectively. In a pot experiment, the gall indexes
of Chinese leek extract-treated tomato and cucumber plants were reduced by 88.9% and 75.9%,
respectively. In an in vitro experiment, the mortality rate of a RKN (Meloidogyne incognita J2)
treated with Chinese leek extract was significantly higher than that of the control. The Gas
chromatography -mass spectrometry (GC-MS) analysis revealed that glycosides, carboxylic acids,
ketones and organic sulfides are the main components in the Chinese leek extract. This study
revealed that Chinese leek possesses a high resistance to RKNs, has strong nematicidal activity
against M. incognita and can significantly reduce the incidence of disease caused by nematodes.
Keywords: Meloidogyne spp., Allium tuberosum Rottler ex Sprengel, root gall index, GC-MS,
biocontrol
韭菜(Allium tuberosum Rottler ex Sprengel)对根结线虫的防控研究
摘要：根结线虫（RKNs，Meloidogyne spp.）是一种专性活体寄生虫，固着在植物根系的维管组织中诱导寄
主形成“根结”
，阻碍寄主对水分和养分的吸收，导致作物营养匮乏，产量下降，品种低劣，对世界农业
生产造成了严重的危害。
【目的】为探索一条高效的线虫病害防控措施，
【方法】本研究结合田间试验，盆
Huang Yong-hong, E-mail: [email protected]; Correspondence XIE Bing-yan, Tel: +86-10-82105979, E-mail: [email protected]
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Doi：10.1016/S2095-3119(15)61032-2
栽实验及离体实验验证了韭菜对根结线虫的抗性，韭菜对根结线虫病害的抑制作用以及韭菜对线虫的致死
效用，并利用气相色谱-质谱联用仪（GC-MS）对韭菜成分进行了分析。【结果】实验结果显示：在感染根
结线虫严重的田间土壤中，韭菜没有表现出根结症状，根结指数为 0，而空心菜和番茄根结症状明显，根
结指数分别为 84.7 和 92.0（F=360.14，P<0.0001）；同样在感染根结线虫严重的田间土壤中，韭菜轮作的
黄瓜根结指数为 28.3，相对对照下降了 70.2%（F=400，P<0.0001）
；间作韭菜的番茄根结指数为 55，比对
照下降 41.1% （F=40.69，P=0.0031）
；盆栽实验中，韭菜提取液处理也显著减轻了南方根结线虫对番茄及
黄瓜的危害。未用韭菜提取液处理的番茄及黄瓜根结数量多，体积大，甚至多个根结连在一起形成棒状或
者念珠状，而韭菜提取液处理的植株根系根结症状明显减轻。统计分析表明，韭菜提取液处理的番茄植株
根结指数为 8.3，比对照降低了 88.9（F =420.25，P<0.0001）
；韭菜提取液处理的黄瓜苗的根结指数为 21.7，
比对照下降了 75.9%（F =420.25，P<0.0001）
。离体条件下，在 100µL 反应体系中，5～25µL 韭菜提取液处
理南方根结线虫 2 龄幼虫。12h 时，线虫校正死亡率为 18.3～88.9%（F=67.71，P <0.0001）
； 24h 时，线虫
的校正死亡率为 32.4%～96.7%（F=43.01，P <0.0001）
； 48h 时，线虫校正死亡率为 64.0～98.7%（F=32.7，
P <0.0001）
。在韭菜提取液中，通过 GC-MS 鉴定到 21 种主要物质成分，包括糖苷类（38.06%）
、羧酸类
（15.36%）
、酮类（14.49%）
、有机硫化物（8.88%）及酚类（5.34%），其中乙酸（Acetic acid）
、2，3-二氢
-3，5-二羟基-6-甲基-4H-吡喃-4-酮（4H-Pyran-4-one，2，3-dihydro-3，5-dihydroxy-6-methyl-）和二甲基三硫
（Dimethyl trisulfide）是相对含量最高的三种化合物，分别为 15.36%、8.29%和 5.51%。
【结论】该研究表
明：韭菜对根结线虫具有很高的抗性；在根结线虫病害严重的土壤中，通过与韭菜间作或者轮作能有效抑
制黄瓜或番茄的病害的发生；离体条件下，韭菜提取液对南方根结线虫具有很强的致死效应。韭菜提取液
中的主要成分为糖苷类、羧酸类及酮类化合物。【创新点】该研究成果为根结线虫的防控提供了一种高效
的生物防控途径，并为利用韭菜防控根结线虫提供了理论支撑。
1. Introduction
Root-knot nematodes (RKNs, Meloidogyne spp.) are sedentary biotrophic parasites. They can infest
more than 5000 plant species (Jaouannet et al. 2013) and cause considerable quantitative and
qualitative crop plant losses (Echeverrigaray et al. 2010; Dang et al. 2011; Naz et al. 2013a),
resulting in an estimated $100 billion in annual losses worldwide (Ibrahim et al. 2011).
In the last decade, most of the effective nematicides, such as methyl bromide,
dibromochloropropane, and ethylene dibromide, have been banned in many regions because of
environmental and human health concerns (Andrés et al. 2012; Ntalli et al. 2011a). Instead, less
toxic, environmentally friendly and effective control measures using natural products are in great
demand (Ntalli et al. 2011b; Echeverrigaray et al. 2010). Phytochemicals have a great potential in
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nematode control since they can be developed for use as nematicides themselves or can serve as
model compounds for the development of derivatives with enhanced activity (Andrés et al. 2012).
In addition, they have little toxicity to human and are rapidly degraded into non-toxic substances
under environmental conditions (Dang et al. 2012).
In recent years, essential oils and extracts from many plant species have been reported to
have nematicidal activities. Extract from Myrtus communis (Oka et al. 2012), Fumaria
parviflora(Naz et al. 2013a,b), Arisaema erubescens (Du et al. 2011), Ailanthus altissima (Caboni et
al. 2012) and essential oil from Verbesina encelioides (Oka 2012), Kadsura heteroclite (Li et al.
2011), Origanum vulgare, Mentha pulegium (Ntalli et al.2010), Foeniculum vulgare and Pimpinella
anisum (Ntalli et al. 2011a) exhibited high nematicidal activity against RKNs. These studies
suggested that developing botanical nematocides bacame a research hotspot and it may provide
potential alternatives to the currently used nematicides.
Chinese leek (Allium tuberosum Rottler ex Sprengel) is used as a food, herbal remedies and
spices worldwide (Shim 2012; Chung et al. 2010). Recently, we observed that in fields heavily
infested with nematodes, tomato (Solanum lycopersicum L.) plants were heavily infected; however,
Chinese leek showed no disease symptoms when planted in similarly infested fields. Additionally,
the disease symptoms on tomato plants were minor when cropped with Chinese leek. Thus, we
hypothesized that Chinese leek may be immune to nematodes or have a nematicidal activity.
Therefore, in this present study, two field experiments was designed to identify any resistance of
Chinese leek to nematodes, and the effects of the Chinese leek on the nematode disease
symptoms of other crop plants in naturally infected soil. Additionally, two greenhouse pot
experiments were performed to accurately test the inhibitory effects of Chinese leek on gall
formation caused by a RKN (Meloidogyne incognita (Kofoid and White 1919) Chitwood 1949) on
plant roots. Then the nematicidal activity of Chinese leek extract against M. incognita was
evaluated in in vitro experiments. Finally, the main components of Chinese leek extract were
identified by GC-MS system.
2. Results
2.1. Chinese leek possesses high resistance to nematodes
To test the resistance of Chinese leek to nematodes, Chinese leek was cultivated in heavily infested
fields, and water spinach (Ipomoea aquatica Forsk) and tomato were grown as negative controls.
Ten months later, the roots of the Chinese leek were healthy, showing no disease symptoms (Fig.
1-A). However, the roots of the two negative controls were infected by nematodes. For the water
spinach at 10 months, the galls were numerous and some were clustered together, forming a
moniliform shape (Fig. 1-B). For the tomato plants, the disease symptoms appeared to be more
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severe. Almost all of the roots were galled and had a rod-like shape. They were swollen, hard and
inflexible only three months after transplanting (Fig. 1-C). The statistics showed that the gall
indexes of water spinach and tomato were 84.7 and 92.0, respectively, but that of Chinese leek was
0 (F=360.14, P<0.0001) (Fig. 2), suggesting Chinese leek was immune to RKNs in heavily infested
field soil.
2.2. Chinese leek suppressed root galls in field trials using intercropping and rotating systems
To determine the suppressive effect of Chinese leek on disease symptoms, field trials were carried
out using a Chinese leek-tomato intercropping system and a Chinese leek-cucumber (Cucumis
sativus L.) rotation. The two cultivation measures significantly reduced disease symptoms on
tomato and cucumber plants (Fig. 3). According to the statistics, the gall index of the tomato plants
intercropping with Chinese leek was reduced by 41.1% compared with the control plants (F=40.69,
P=0.0031), and the gall index of the cucumbers rotating with Chinese leek was reduced by 70.2%
compared with the control plants (F=400, P<0.0001) (Fig. 4). This showed the Chinese leek could
reduce nematode disease on other crops in naturally infested field soil.
2.3. The suppressive effect of Chinese leek extract against nematode disease caused by M.
incognita on potted plants
The suppressive effects of Chinese leek extract on gall formation were determined using potted
tomato and cucumber seedlings. For both plants, 45 days after the Chinese leek extract treatment,
galls induced on the treated roots were fewer and smaller than the untreated roots (Fig. 5).
Compared with the corresponding untreated controls, the gall indexes were reduced by 88.9% for
the Chinese leek extract-treated tomato plants (F=420.25, P<0.0001) and by 75.9% for the Chinese
leek extract-treated cucumber plants (Fig. 6) (F=420.25, P<0.0001), showing the Chinese leek
extract could suppress the gall information caused by M. incognita.
2.4. Nematicidal activity of Chinese leek extract against M. incognita J2
Chinese leek extract has a strong nematicidal activity against the nematode M. incognita J2. After
48 h, nematodes treated with 25 μL of Chinese leek extract in a 100-μL test system were straight,
resting, and did not move even when touched with a micro-needle (Fig. 7-A). However, the
untreated control nematodes were continuously moving (Fig. 7-B). According to the statistics, the
mortality increased with the increasing Chinese leek extract concentrations and the treatment
times (Fig. 8). At the three recording time points, 12, 24 and 48 h, the mortality rates of J2
deposited in the 100-μL test system containing various concentration of Chinese leek extract were
significantly different (F12 h=67.71, P12 h<0.0001 for 12 h; F24 h=43.01, P24 h<0.0001 for 24 h; and F48
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h=32.7,
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P48 h<0.0001).
2.5. Main components of Chinese leek extract
To know the possible nematocidal components, Chinese leek extract was preliminary analyzed by
using GC-MS system. Twenty-one main components were identified from the extract, including
glycosides (38.06%), carboxylic acids (15.36%), ketones (14.49%), organic sulfide (8.88%), and
phenols (5.34%), etc. (Table 1). Some components highly matched those from the NIST mass
spectra database, but their relative peak areas were small. On the contrary, some components’
relative peak areas were large, but they poorly matched the standard database. Among these
identified components, acetic acid, 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- and
Dimethyl trisulfide, matched the standard database well and accounted for the three highest
relative peak areas.
3. Discussion
In the paper, we proved that the Chinese leek was immune to nematodes in heavily infested fields.
We also found the disease symptoms on other vegetable plants were significantly inhibited by
adopting a rotating/intercropping system with Chinese leek in naturally infested fields or when
treated with Chinese leek extract in potted experiments. Additionally, Chinese leek showed a
strong nematicidal activity against M. incognita, the most threatening RKN. This showed Chinese
leek may provide a potential effective way to control the disease caused by RKNs.
Chinese leek and water spinach are herbaceous perennials and can be harvested several times
in one growing season. Tomato is a popular vegetable with a high economic benefit. Therefore, we
selected water spinach and tomato as negative controls to assay the resistance of Chinese leek to
nematodes in a field experiment. To increase the opportunity for nematode infestation, the
Chinese leek was sown six months earlier than the tomato. Thus, in the experiment, Chinese leek
and water spinach were cultured for 10 months and tomato was cultured for only three months in
the field. In the end, no galls were found on Chinese leek roots, but water spinach and tomato
showed severe disease symptoms. This result suggested that the Chinese leek has a strong
resistance to nematodes in heavily infested field soil.
Allium species, such as onion, garlic, and chive, possess antibacterial and antifungal activities
(Rattanachaikunsopon & Phumkhachorn 2009). They have been extensively studied in food science
(Najjaa et al. 2007; Lazarevic et al. 2011; Yin and Tsao 1999) and medicine (Chen et al. 2011; Seyfi
et al. 2010). In recent years, Allium species were reported to have significant inhibitory effects on
plant pathogens (Ho et al. 2007; Balestra et al. 2009). Chinese leek significantly inhibited the
growth of Fusarium oxysporum f. sp. cubense W. C. Snyder and H. N. Hansen 1940, a causal agent
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of Fusarium wilt in banana (Huang et al. 2012; Zhang et al. 2013). In the present study, Chinese
leek showed very strong nematicidal activity against M. incognita. This is a new finding. It explains
why water spinach and tomato plants developed severe disease symptoms, but no sign of disease
was present on the Chinese leek plants in the field trials. And all these results positively proved our
previous hypothesis.
Since Chinese leek showed high toxicity to RKNs in in vitro bioassay and reduced nematode
disease symptom in field trials and potted experiments, it is necessary to understand the main
component profile of the Chinese leek. The GC-MS results showed the main components of
Chinese leek were glycosides (38.06%), carboxylic acids (15.36%), ketones (14.49%), organic sulfide
(8.88%), and phenols (5.34%). The previous showed that glycosides (Du et al. 2011), carboxylic
acids (Browning et al. 2006) and ketones (Ntalli et al. 2011b) showed strong nematocidal activity
against RKNs. The phenols improved the resistance of plants to M. incognita (Bajaj and Mahajan,
1977). The organic sulfides included in Chinese leek showed high antibacterial (Pongsak et al. 2009)
and insecticidal activities (Zhang et al. 2013). Seo reported that 0.1% acetic acid caused 100%
mortality of M. incognita (Seo and Kim, 2014). In our study, acetic acid, accounting for 15.36%, was
the most abundant substance of the 21 components identified from Chinese leek. Therefore, acetic
acid, together with other components, was probably the factors of the nematocidal activity of
Chinese leek against RKNs. This preliminary analysis would accelerate the research to isolate and
identify the exact nematicidal components in Chinese leek.
Unlike in the past, agricultural reagents are now required not only to possess potent activity
but also to be safe for animals, humans and ecosystems in general. Obviously, Chinese leek is a
valuable material for the development of such reagents. In ancient China, Allium plants were
recognized not only as a traditional medicine, but also a safe food additive (Zhang et al. 2013),
imposing no harm to humans or the environment. Nowadays, Chinese leek is still a common and
popular vegetable in China. More importantly, the Chinese leek has a broad spectrum of
antimicrobial and antifungal activities against many genera of plant pathogens (Yu, 1999; Tsao &
Yin, 2001; Zhang et al. 2013), and also has insecticidal activities (Zhang et al. 2013). Thus, we
believe that making use of the Chinese leek, its production or by-production to control nematode
disease, together with other important diseases and pests, has the potential to be an efficient,
environmentally friendly agricultural management tool.
4. Conculsion
Chinese leek has highly resistance to RNKs. Chinese leek can significantly reduce nematode disease
symptoms in naturally infected soil and in potted experiments. In in vitro condition, Chinese leek
extract has highly nematicidal activity against M. incognita J2, one of the RKNs. Glycosides,
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carboxylic acids, ketones and organic sulfide are the highest amount of components in the Chinese
leek extract.
5. Materials and Methods
5.1. Plant materials
Chinese leek (Allium tuberosum Rottler ex Sprengel) cv. “Fujiu 12”, water spinach cv. Zhuye, tomato
cv. Zhongza 9, cucumber cv. Zhongnong 16 were used in the experiments. Chinese leek and water
spinach seeds were directly sown into field soil. Seeds of tomato and cucumber were heat-shocked
in 80°C hot water for 30 s, immediately dipped in cool water, and then soaked in warm water for 6
h. For germination, they were placed on sterilized filter paper in a Petri dish at 28°C in the dark.
Germinated seeds were sown in culture medium (peat soil:vermiculite:perlite=2:1:1) to produce
seedlings.
5.2. Nematode collection
M. incognita J2 were obtained from a pure culture that was previously initiated by egg masses and
propagated on tomato seedlings in a greenhouse. Egg masses were handpicked using sterilized
forceps from heavily infected roots (40 d after inoculation). These egg masses were
surface-sterilized with 0.5% NaOCl for 1 min, washed with sterile distilled water three times, and
then placed on 15-mesh sieves (8 cm in diameter) in Petri-dishes with water just deep enough to
contact the egg masses. They were incubated 25 to 26°C to obtain freshly hatched M. incognita J2s.
Emerged RKNs were collected daily and stored at 4°C. M. incognita J2s up to 4 days old were used
in the experiments (Li et al. 2011).
5.3. Preparation of Chinese leek extract
For in vitro experiments, the aerial parts of the Chinese leek were cut into pieces 5 cm in size, and
ground into powder using liquid nitrogen. Distill water (100 ml) was added to 10 g of the powder
and the active ingredients leached for 24 h. The extract was filtered through four layers of gauze,
then centrifuged at 10000 r min-1 for 3 min. The supernatant was sterilized using a 0.22-μm filter
and stored at 4°C.
For the greenhouse pot experiment, the aerial parts of the Chinese leek were cut into 5-cm
pieces, 200 g of which was mixed with 200 mL water and mashed into a smooth paste using a juicer.
The paste was transferred into an Erlenmeyer flask, supplemented with distilled water to a total
volume of 2000 mL, and the active ingredients leached for 24 h. The supernatant was filtered
through two layers of gauze before use.
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5.4. Field experiments
5.4.1. Field site
To identify any resistance of Chinese leek to nematodes and the control effects of Chinese leek on
the disease symptoms of other vegetable plants in fields heavily infested with RKNs, two field
experiments were carried out at the Shunyi experimental station, Beijing, China. The experiments
were performed in a solar greenhouse that were specially used for the field trials related to M.
incognita. The field soil was heavily infested with nematodes for intermittent inoculation with M.
incognita and for continuous culturing vegetables susceptible to nematodes, such as tomato and
cucumber for years. The average density of nematodes in the field was identified to be 350
individuals/100 g fresh soil by using shallow dish method (Mao et al. 2004).
5.4.2. Determination of Chinese leek resistance to nematodes
The experiment was designed with three treatments and three replications. Nine plots, 2 m×6 m
each, were arranged in a randomized complete block design. The treatments were Chinese leek,
tomato and water spinach. In November 2012, three to four seeds of Chinese leek or water
spinach were sown at a density of 20 cm × 20 cm in plots. In May 2013, when Chinese leek and
water spinach grew to normal height, tomato seedlings with seven to eight leaves were planted at
a 25×30-cm density in prepared plots. In August 2013, all plants were uprooted to observe disease
symptoms. Root galls were evaluated based on a 0 to 5 rating system: 0=no infection, 1=1 to 20%,
2=21 to 40%, 3=41 to 60%, 4=61 to 80%, and 5=81 to 100% of the roots were galled (Oka et al.
2000). The root gall index was calculated as previously reported (Powell et al. 1971).
5.4.3. Chinese leek-tomato intercropping
For Chinese leek-tomato intercropping, the experiment was designed with two treatments and
three replications. Six plots, 4 m × 6 m each, were arranged in a randomized complete block design.
In November 2012, three to four seeds of Chinese leek were sown at a density of 20 cm×20 cm in
plots. In May 2013, tomato seedlings with seven to eight leaves were transplanted into the six
prepared plots, with or without Chinese leek, at a density of 25 cm×30 cm. Tomato seedlings that
were planted in the plots with the Chinese leek were regarded as Chinese leek treatments. Those
planted in the plots without the Chinese leek were used as controls (untreated controls). In August
2013, all plants were uprooted to observe disease symptoms. Root galls were evaluated based on a
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0 to 5 rating system described in section 5.4.2.
5.4.4. Chinese leek-cucumber rotation
For this experiment, Chinese leek was transplanted in three greenhouses (667 m2/greenhouse) for
commercial production in 2009, and tomato or cucumber were cropped in other greenhouses. In
October 2012, Chinese leek seedlings were removed from these greenhouses, which were
replanted with cucumber (Chinese leek treatment). The cucumber cropped in the similar
greenhouses rotating with tomato were used as controls (untreated controls). All cucumber plants
were managed normally. After harvesting, 100 treated and control plants were randomly uprooted
to record disease symptoms. Root galls were evaluated based on a 0 to 5 rating system described in
section 5.4.2.
5.5. Pot experiments
To accurately investigate suppressive effects of Chinese leek on nematode disease caused by RKNs
(M. incognita), a pot experiment was conducted using tomato and cucumber plants under
greenhouse conditions.
5.5.1. Tomato
For tomato plants, 50 ml of the Chinese leek extract was mixed with 50 g culture medium (peat
soil:vermiculite:perlite=2:1:1) in a 9×9-cm plastic pot. Distilled water (50 mL) was mixed with 50 g
culture medium in a similar plastic pot as a control. The experiment had 10 replications. A total of
1000 M. incognita J2 in 5 mL distilled water were poured into each pot and then covered with a
1-cm-thick layer of the same culture medium. The pots were sealed in a plastic bag and incubated
at 25°C for 3 days. The plastic bags were removed after incubation and one 20-day-old tomato
seedling was planted in each pot. The seedlings were placed in the greenhouse and managed as
usual. The tomato plants were uprooted 45 d after planting. Root galls were counted and recorded
based on a 0 to 5 rating system described in section 5.4.2.
5.5.2. Cucumber
For cucumber plants, 50 ml of the Chinese leek extract was mixed with 50 g culture medium (peat
soil:vermiculite:perlite=2:1:1) in a 9×9-cm plastic pot. As a control, 50 mL of water was mixed with
50 g culture medium in the same type of plastic pot. The experiment had 10 replications. A total of
1000 M. incognita J2 in 5 mL distilled water were poured into each pot and then covered with a
1-cm-thick layer of the same culture medium. Each pot was sealed in a plastic bag and incubated at
25°C for 3 days. Plastic bags were removed after incubation, and one germinated cucumber seed
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was sown in each pot. Seedlings were placed in the greenhouse and managed as usual. The
cucumber plants were uprooted 45 days after planting. Root galls were counted and recorded
based on a 0 to 5 rating system described in section 5.4.2.
5.6. In vitro experiment
An in vitro bioassay was performed to determine the nematicidal activity of Chinese leek against M.
incognita J2. Various amounts of Chinese leek extract (0, 5, 10, 15, 20, and 25 μL) were added to
wells of a 96-microwell plate, and supplemented with sterile distilled water to a volume of 80 μL.
Then, 100 M. incognita J2 in 20 μL water were introduced into each well. A total of four
replications for each Chinese leek extract concentration were performed and the experiment was
repeated. The microwell plate was incubated in the dark at 28°C. The total number of inactive J2 in
each well was counted at 12, 24 and 48 h after incubation. Juveniles that were straight and did not
move even after mechanical prodding were defined as dead. The percentages of dead J2 in the
microwell assays were corrected by eliminating the natural death in control according to the
following (Ntalli et al. 2011b).
5.7. GC-MS analysis of Chinese leek extract
The prepared extracts were analyzed with an Agilent 6980 N gas chromatograph coupled with
an Agilent 5975B mass selective detector (Agilent technologies, Santa Clara, CA, USA). A 1 μL
aliquot of the extracted samples were injected into a HP-5MS capillary column (with 30.0 m length,
0.32 mm internal diameter, and 0.25 μm particle size). Helium was used as carrier gas at a constant
flow rate of 1.0 mL min-1 and a 1:10 split ratio. The column temperature was initially kept at 60°C
for 1 min and then increased from 60°C to 280°C at 10°C min −1, where it was held for 5 min. The
injector temperature was set at 270°C. Ionization was effected by electron ionization (EI) at 70 eV.
Masses were acquired from m/z 50 to 550. The MS quadrupole temperature was set at 150°C and
the ion source temperature at 230°C. The mass charge ratios and the abundance of which were
compared with a standard mass chromatogram in the NIST 11 (National Institute of Standards and
Technology) mass spectra library by the ChemStation Software.
5.8. Data statistics
All data were analyzed using SAS version 8.0 software. All data were submitted to a one-way
ANOVA. The significance of the treatments was determined using Fisher’s protected least
significant difference (LSD) test (P≤0.05).
10
Doi：10.1016/S2095-3119(15)61032-2
Acknowledgements
The research was supported by the National Natural Science Foundation of China (31272151,
31471864), Agro-scientific Research in the Public Interest (201103018) and China Agriculture Research
System (CARS-25).
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AA
B
B
CC
Fig. 1 The underground part of the Chinese leek, water spinach and tomato plants in the soil
heavily infected by nematodes. No gall was found on the Chinese leek roots (A), while many galls
were formed on the water spinach roots (B) and tomato roots (C). The Chinese leek and the
water spinach were cropped in the fields for 10 months, but the tomato was only cropped for 3
months.
Fig. 2 In fields heavily infested with nematodes, the root gall indexes of the Chinese leek were
significantly lower than that of water spinach and tomato plants. The Chinese leek and the water
spinach were cropped in the fields for 10 months, but the tomato was only cropped for three
months. Error bars represent one standard error. Bars labeled with the same letter are similar at
the 5% level according to Fisher’s protected least significant difference test.
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10.1016/S2095-3119(15)61032-2
A
A
BB
C
C
DD
Fig. 3 The underground part of the Chinese leek treated tomato and cucumber plants in the fields
heavily infested with namatodes. The galls on the tomato plants intercropped with Chinese leek
(A) were larger in size and more in quantity compared to the control plants (B) three months after
treatment. The galls on the cucumber plants rotated with Chinese leek (C) were also more and
larger than the untreated control plants (D) after harvesting.
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10.1016/S2095-3119(15)61032-2
Fig. 4 In fields heavily infested with nematodes, the gall indexes of tomato and cucumber plants
were significantly reduced by adopting Chinese leek-tomato intercropping system (A) and Chinese
leek-cucumber rotating system (B). Error bars represent one standard error. Bars labeled with the
same letter are similar at the 5% level according to Fisher’s protected least significant difference
test.
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10.1016/S2095-3119(15)61032-2
A
A
B
B
CC
DD
Fig. 5 The underground part of the Chinese leek treated tomato and cucumber plants inoculated
with Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949. in pot expreiments. Few
galls were found on the tomato (A) and cucumber (B) plants treated with an aqueous extract of
Chinese leek, while numerous galls were formed on the corresponding control plants (C, D) 45
days after transplanting.
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10.1016/S2095-3119(15)61032-2
A
B
Fig. 6 The inhibitory effects of Chinese leek on disease development in tomato (A) and cucumber
(B) plants inoculated with Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949 in pot
experiments. The gall index of plants treated with an aqueous extract of Chinese leek was
significantly lower than that of the untreated plants 45 days after transplanting. Error bars
represent one standard error. Bars labeled with the same letters within the same time point are
similar at the 5% level according to Fisher’s protected least significant difference test.
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10.1016/S2095-3119(15)61032-2
A
B
Fig. 7 Nematocidal activity of Chinese leek extract on the Meloidogyne incognita (Kofoid and
White, 1919) Chitwood, 1949. 48 h after treatment, almost all of the nematodes deposited into
25 μL of Chinese leek extract in a 100-μL test system were straight, and dead (A), while almost all
of the control nematodes were live and move (B).
Fig. 8 The mortality rates of nematodes Meloidogyne incognita (Kofoid and White 1919)
Chitwood, 1949 J2 treated with various concentrations of aqueous extract of Chinese leek were
significantly different at three time points. Error bars represent one standard error. Bars labeled
with the same letters within the same time point are similar at the 5% level according to Fisher’s
protected least significant difference test.
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Table 1 The main components identified from the crude extract of Chinese leek leaf
Peak
Retention
Molecular
Match
Relative area
no.
time
weight
percent (%)
(%)
1
2.497
Silanediol, dimethyl-
C2H8O2Si
92
74.4
0.89
2
3.023
Acetic acid
C2H4O2
60
89.9
15.36
3
3.712
Butanenitrile, 2,3-dioxo-, dioxime, O,O'-diacetyl-
C8H9N3O4
211
10.4
3.20
4
4.200
Cyclohexanone, 3-hydroxy-
C6H10O2
114
9.18
1.54
5
4.816
N-Chloroacetyl-3,6,9,12-tetraoxapentadec-14-yn-1-amine
C13H22ClNO5
307
8.46
1.12
6
5.63
Dimethyl trisulfide
C2H6S3
126
96.7
5.51
7
6.996
1,5-Diazocine, octahydro-1,5-dinitroso-
C6H12N4O2
172
7.81
2.77
8
7.825
Benzeneacetaldehyde
C8H8O
120
78.4
2.23
9
8.506
S-Methyl methanethiosulphonate
C2H6O2S2
126
87.7
3.38
10
9.002
Cyclotrisiloxane, hexamethyl-
C6H18O3Si3
222
47.3
3.87
11
10.833
4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-
C6H8O4
144
96.9
8.29
12
11.811
4H-Pyran-4-one, 3,5-dihydroxy-2-methyl-
C6H6O4
142
9.33
3.71
13
12.739
6-Methylenebicyclo[3.2.0]hept-3-en-2-one
C8H8O
120
15.6
0.95
14
13.054
6-Acetyl-β-d-mannose
C8H14O7
222
15.9
2.28
15
15.04
2-Methoxy-4-vinylphenol
C9H10O2
150
68.5
5.34
16
16.082
R-Limonene
C10H16O3
184
25.8
0.69
17
17.334
Gentamicin a
C18H36N4O10
468
16.1
4.38
18
18.408
[1,1'-Bicyclopropyl]-2-octanoic acid, 2'-hexyl-, methyl ester
C21H38O2
322
36.6
1.70
19
21.242
Desulphosinigrin
C10H17NO6S
279
11.6
14.48
Components
Formula
21
20
25.24
d-Mannose
21
43.459
Doi：10.1016/S2095-3119(15)61032-2
C6H12O6
180
12.7
16.93
C14H17NO9
343
22.5
1.39
Tetraacetyl-d-xylonic nitrile
22

Chinese leek (Allium tuberosum Rottler ex Sprengel) reduced

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