1. Brief introduction
As one of the most economically important vegetable crops, cucumbers are known worldwide for their flavor and nutritional value. However, diseases caused by plant pathogens can lead to stunted plant growth and significant yield losses.
RKN is a mandatory bionutrient that is able to survive in the soil for many years and parasitizes a variety of crops by stimulating bile production.
With the expansion of greenhouse crop production and the use of continuous cropping systems, the problems caused by RKN are becoming more and more serious, and it is becoming increasingly difficult to control RKN in commercial agricultural operations.
A variety of management systems have been developed to prevent RKN infestation, including chemical control, soil heat treatment, flooding, biological control, and crop rotation, where chemical control and soil heat treatment methods are widely used.
While chemical control can greatly reduce the adverse effects of RKN on vegetable yields, chemical pesticides are expensive, harmful to the environment, or lead to the development of pesticide resistance. As a result, there is a growing trend to implement agronomic practices as a measure to control RKN.
Three common agronomic practices have been developed for the management of RKN. One involves incorporating plant residues into the soil as a green fertilizer. Naz observed that adding purple pansy plants to the soil could reduce the root-knot index of tomato plants, promote tomato growth, and increase productivity.
Monfort confirmed that mustard species produce gluconate and exhibit universal biocidal effects, and the inclusion of these species in green manure prior to transplantation of vegetable crops may significantly reduce nematode populations without adversely affecting the growth and yield of subsequent vegetable crops.
Therefore, green fertilizers can inhibit RKN and other soil-borne plant pathogens by influencing the confrontation and competition between soil microorganisms. Another agronomic practice used to control RKN is related to cropping systems.
For example, interplanting tomatoes with garlic or Spanish clover and acacia species in a crop rotation system can effectively control RKN in the field, probably because of plant secretory characteristics.
The third agronomic practice involves the use of extracts from different plant parts. Olabiyi observed that aqueous extracts from plant roots can effectively reduce the concentration of RKN in the soil, which helps protect tomato plants from RKN infection.
Black pepper leaf extract has been reported to inhibit the growth and development of nematodes in a dose-dependent manner. These extracts penetrate the nematode egg mass matrix, significantly limit egg hatching, kill J2 and promote plant growth. In addition, extracts from neem, marigold, and peppermint are directly lethal to nematodes.
Plant-synthesized compounds in the rhizosphere originating from root exudate or sites of previous nematode infiltration can influence nematode behavior. Some of the compounds released by plants, some of which are present in root exudates, either attract nematodes to the roots, or cause rejection, motor inhibition, and even lead to nematode death.
For example, analysis of tomato root exudate showed that four compounds, namely 2,6-di-tert-butyl-p-cresol, L-ascorbyl 2,6-dipate, dibutyl phthalate, and phthalate, were able to inhibit nematode egg hatching and were associated with increased J2 mortality.
Dutta observed that the presence of small lipophilic molecules in the root exudate of tomato and rice negatively affected the mobility of nematode species. Welsh onions belong to the Lycoris family.
Previous studies have shown that Welsh onions are not infected with RNns when grown as part of a companion plant or crop rotation. In addition, Welsh onions mitigated the severity of RN-induced damage to partner plants or subsequent crops in crop rotations.
Therefore, Welsh onions may have a unique protection mechanism against RKN. We have previously observed that the use of Welsh onions as a companion plant can control RKN.
Elucidating the mechanism behind the inhibitory effect of Welsh onion extract components on RKNs may have important implications for crop production.
In this study, we prepared four Welsh onion root exudate extracts and evaluated their effect on nematode egg hatching. We also screened the dominant compounds in the root exudate and analyzed their effects on egg hatching and J2 growth.
2. Materials and growth conditions
The entire experiment was conducted from 2013 to 2017 at the Yantai Academy of Agricultural Sciences, Shandong, China. The Welsh variety "Tie Gan" was used in the study. The cucumber variety "YU XIU" was bred by the Yantai Academy of Agricultural Sciences, Shandong Province, China.
Stage 2 larvae (J2) and eggs for inoculation were provided by the Key Laboratory of Plant Protection, Shandong Agricultural University, Shandong Province, China. Eggs containing 10,000 eggs/mL and J2 suspension containing 6,000 J2/mL were prepared.
Cucumber and Welsh onion seeds are sown in trays containing turf soil and incubated in a plant accelerator at 16h/8h (light/dark) photoperiod and corresponding 25°C/18°C temperatures. When cucumber seedlings have three to four true leaves, and Welsh onion seedlings are about 20 cm tall, the plants are transferred to the greenhouse.
The greenhouse has been used to grow tomatoes and cucumbers for 5 years, during which time severe RKN infections have occurred. We first ploughed and cultivated the experimental plots.
To account for the possible uneven distribution of RKN in the soil, we randomly set up three test plots of approximately 20 square meters for each treatment.
Approximately 90 cucumber plants were planted in each test plot. Two treatments were applied, cucumber plants grown in monocultures and cucumber plants grown with Welsh onions as companion plants.
The two treatments were applied to each of the 20-square-metre plots, each of which planted approximately 90 cucumbers and 180 Welsh onion plants. The early fruit yield corresponds to the first 25 days after the early fruit stage.
The fruit yield at this stage is indicative of the early growth of the plant. However, total fruit production refers to the entire fruit production period. The experimental results for each treatment are the average of the three test charts.
At the end of cultivation, the cucumber roots are washed without soil and then weighed. The total number of egg masses throughout the root system was calculated, and 10 egg masses were randomly selected. The egg masses are dissolved in 0.5 ml of concentrated NaClO, respectively, to release the eggs.
Dilutions were prepared and the number of eggs per egg mass was calculated; the average number of 10 egg masses was calculated. Each treatment should be repeated at least three times. To estimate the total number of nematodes, roots were stained with the acidic fuchsin method described by Byrd et al.
Observe and count the number of J2 under a microscope. A single juvenile female contains about 200-300 eggs, so the number of juveniles is estimated based on the number of eggs.
3. Effect of Welsh onion as a companion plant on RCN infection of cucumber plants in greenhouse
Cucumber monoculture exhibits typical symptoms of RKN infection. Specifically, the leaves are yellow and wilted. In contrast, cucumber plants grown in combination with Welsh onion plants exhibit superior growth and a healthy appearance.
Compared to the root system of the CK plant, the root system of the T plant has a lower amount of bile and eggs. The roots of Welsh onions also do not have bile. These results suggest that the use of Welsh onion as a companion plant increases the RKN resistance of cucumber plants.
The early fruit yields of CK and T plants were 1.7±0.6 kg/m2 and 2.0±0.4 kg/m2, respectively. The total fruit yields of CK and T plants were 4.3 ± 1.2 kg/m2 and 5.7 ± 1.1 kg/m2, respectively.
The fresh root weight of T plants was higher than that of CK plants, but it was not significant. We observed 522.6 ± 36.4 J2 nematodes, 1245.8 ± 58.1 eggs, and 4.2 ± 0.9 to 6.3 ± 1.1 female adult RKN per gram of fresh roots.
It was significantly higher than the recorded 121.5 ± 23.4 J2 nematodes, 290.4 ± 38.1 eggs and 1.3 ± 0.2 female adult RKN per gram of fresh roots.
The roots of T plants had 77.0% less RKN and eggs than those of CK plants. These results suggest that Welsh onion root exudate inhibits the growth and activity of RKN.
4. Effect of Welsh onion root exudate extract on RKN egg hatching
The hatchability of eggs treated with the four root exudate extracts increased over time, and the hatchability of eggs generally decreased as the concentration of the extract increased. Of the four treatments, the final egg hatching rate of the control treatment ranged from 80% to 90%.
In chloroform and ether extract treatments, the inhibition was not evident at the four lowest concentrations, and egg hatchability ranged from 75 to 98%. Higher concentrations of the extract resulted in egg hatching capacity ranging from 42% to 62%.
These results suggest that the composition of chloroform and ether extracts is able to inhibit the hatching of RKN eggs at sufficiently high concentrations. Ethyl acetate extract is more effective than chloroform and ether extract in inhibiting egg hatching. Volumes of 50 μL or higher reduce egg hatchability to 40% to 50%.
5. Effect of exogenous 4-hydroxyphenylethanol on RKN egg hatching and J2 survival
N-butanol root extract inhibits the hatching of RKN eggs, and one of the main components of this extract is 4-hydroxyphenyethanol. A subsequent experiment showed that 4-hydroxyphenylethanol inhibited the hatching of RKN eggs, and the inhibition of hatching capacity increased with the increase in the concentration of 4-hydroxyphenylethanol.
At 1.2 mM and above, the egg hatching capacity is less than 50%. At 9.6 mM, the hatchability of the eggs is less than 10%, and at 19.2 mM, almost no eggs are able to hatch.
Studies of J2 nematode response to different concentrations of 4-hydroxyphenyl ethanol have shown that treatment with 0.3 or 0.6 mM 4-hydroxyphenylethanol for 48 h has little effect on the survival of J2 nematodes.
However, exposure to concentrations of 1.2 mM or higher results in slow-moving J2 nematodes with abnormally stretched bodies. Higher concentrations of 4-hydroxyphenylethyl alcohol (9.8 and 19.2 mM) are somewhat fatal to RKN, with a 48-hour mortality rate of more than 50%.
VI. Conclusions
Root-knot nematodes are obligatory endoparasites that infect many crops and cause severe yield losses. In this study, we investigated the effect of Welsh onions grown as companion plants on the resistance of cucumber plants to RKN infection and analyzed the components of the Welsh onion root exudate with the richest concentration.
The results showed that when planted with Welsh onion as a companion plant, the root knot and egg mass of cucumber root were 77.0% less than that of control cucumber root. Welsh onion root exudate was collected and extracted with chloroform, ether, n-butanol, and ethyl acetate.
High concentrations of Welsh onion root extract reduced the hatchability of RKN eggs. In particular, the inhibitory effect of n-butanol extract is significant, and the hatchability of RKN eggs does not exceed 10%.
Gas chromatography-mass spectrometry analysis showed that the most abundant component of n-butanol extract was 4-hydroxyphenyl ethanol. Treatment with 1.2 mM 4-hydroxyphenyl ethanol reduced egg hatchability to 40%, while treatment with 9.6 mM or higher concentrations of 4-hydroxyphenyethanol reduced egg hatchability to less than 10%.
In addition, concentrations of 1.2 mM or higher 4-hydroxyphenylethyl alcohol reduced the activity of the second stage. Higher concentrations of 4-hydroxyphenylethanol are somewhat fatal to RKN, with a mortality rate of more than 50% with 48 hours of treatment.
The current results suggest that the cultivation of Welsh onions as a companion plant may be an alternative to the application of synthetic nematode agents with fewer side effects. We confirm that 4-hydroxyphenylethanol is a naturally occurring and potent nimalist.