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Effects of varying temperature on the reproduction and damage potential of Heterodera schachtii to Chinese cabbage (Brassica rapa pekinensis)
Accepted Manuscript Effects of varying temperature on the reproduction and damage potential of Heterodera schachtii to Chinese cabbage (Brassica rapa pekinensis)
Md. Faisal Kabir, Jae-Kook Lee, Mun Gi Jeong, Mwamula Abraham Okki, Young Hwa Choi, Dong Woon Lee PII: DOI: Reference:
S1226-8615(17)30529-0 doi:10.1016/j.aspen.2017.11.004 ASPEN 1090
To appear in:
Journal of Asia-Pacific Entomology
Received date: Revised date: Accepted date:
23 August 2017 1 November 2017 2 November 2017
Please cite this article as: Md. Faisal Kabir, Jae-Kook Lee, Mun Gi Jeong, Mwamula Abraham Okki, Young Hwa Choi, Dong Woon Lee , Effects of varying temperature on the reproduction and damage potential of Heterodera schachtii to Chinese cabbage (Brassica rapa pekinensis). The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aspen(2017), doi:10.1016/j.aspen.2017.11.004
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ACCEPTED MANUSCRIPT Effects of varying temperature on the reproduction and damage potential of Heterodera schachtii to Chinese cabbage (Brassica rapa pekinensis)
Md. Faisal Kabir1+, Jae-Kook Lee2+, Mun Gi Jeong1, Mwamula Abraham Okki1, Young Hwa Choi3
Department of Ecological Science, Kyungpook National University, Sangju, 37224, Korea
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and Dong Woon Lee1*
Protection Division, National Academy of Agricultural Science, Rural Development Administration, Wanju,
of Ecology and Environmental System, Kyungpook National University, Sangju, 37224, Korea
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3School
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55365, Korea
*Corresponding
author: Dong Woon Lee
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E-mail: [email protected]
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Major of Applied Biology, School of Ecological Environment and Tourism, Kyungpook National University, Sangju, Gyeongsangbuk-do, 37224, Republic of Korea
authors contributed equally to this work. Conflicts of interest: none.
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+These
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Telephone: +82-54-530-1212
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ACCEPTED MANUSCRIPT Abstract. Heterodera schachtii is a well-known, destructive pathogen of Chinese cabbage (Brassica rapa pekinensis) in Korea, and several studies have attempted to find a potential control measure against it. This study is the first to investigate the effects of varying temperature on the reproduction and damage potential of H. schachtii to Chinese cabbage. Chinese cabbage plants were inoculated with H. schachtii at different densities (1, 2, or 4 juveniles per gram of soil) and grown under three temperature regimes: constant (15, 20, or 25℃), increasing (10, 14, and 18°C), and fluctuating (positive, 16.7-22.0°C; negative, 21.5-11.5°C). At a constant
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temperature after 30 days of inoculation, both Chinese cabbage and H. schachtii performed best at 20℃.
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However, after 60 days of inoculation, H. schachtii had a significantly higher population at 20℃, whereas cabbage growth was best at 25℃. With increasing temperature, the numbers of cysts and females did not change
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significantly, and reached maxima at an initial temperature of 14°C. However, the number of leaves and weights of the Chinese cabbage plants significantly differed at 14°C. Under fluctuating temperatures, temperature
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decreases reduced the H. schachtii population.
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Key words: Brassica rapa pekinensis, climatic condition, cyst nematode, population density, yield loss
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ACCEPTED MANUSCRIPT Introduction Chinese cabbage, Brassica rapa pekinensis (Lour.) Rupr., is one of the most popular Brassica vegetables and has a high market value, and is the third-most-produced crop in Korea (Chang et al., 2008; KREI, 2012; Kim et al., 2014). It is ranked as the most important processed foodstuff, and is an important native side dish in the form of Kimchi (FAO, 2013; Park et al., 2014). Chinese cabbage is widely grown in Korea, and for the best vegetative growth, it needs a temperature range of 15-20°C (KMA, 2011). Extended periods of higher or lower
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temperatures result in premature flowering and reduced foliar weight.
Heterodera schachtii, commonly known as sugar beet cyst nematode, causes severe damage to Brassicaceae
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species (Whitehead, 1998). It has a wide range of host plants, and causes significant losses of Chinese cabbage, even up to 50% yield losses (McCann et al., 1981). The pathogen was first recorded in Chinese cabbage fields in
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the highland area of Korea in 2011 (Lee et al., 2013). Since then, numerous fields have been infested with it. Recent reports have revealed that the pest is steadily spreading within the highland Chinese cabbage growing areas of Gangwon-do province (from 11.2 ha in 2011 to 70 ha in 2015), and crop production is being affected
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(Kwon et al., 2015). Therefore, knowing the pathogen’s potential at different temperatures is crucial. In general, hatching starts when the adult female dies and its body forms a dark brown cyst (Wallace, 1955;
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Kabir et al., 2015). Successful hatching ensures a high number of 2nd stage juveniles (J2) that could severely
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damage a single cabbage field. Temperature is one of the main factors in regulating the development of beet cyst nematodes (Griffin, 1981a; Trudgill, 1995).
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Interestingly, the optimal growth temperatures for Chinese cabbage and H. schachtii hatchlings are very similar, which probably affects the nematode’s attack on Chinese cabbage in Korea. Plant host-nematode relationships
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could be affected, because environmental temperature plays a key role in regulating the biology of plant-parasitic nematodes such as H. schachtii (Griffin, 1988; Trudgill et al., 2005). The relationship between nematode development and the host is mainly influenced by three thermal parameters: base, optimum, and maximum
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temperatures. These parameters are measured in accumulated heat units (Jones, 1975). The optimum temperature for infective H. schachtii juvenile hatching is 25°C, and lower temperatures have a negative effect (Kabir et al., 2015). Chinese cabbage in the Korean highlands is planted after winter, when the temperature remains below 15°C, which is well below the optimum hatching temperature of H. schachtii. Thereafter, the temperature increases from spring to summer, when it is favorable for H. schachtii reproduction (KMA, 2011). In the Korean highlands, spatial temperature fluctuations occur. During the cabbage’s vegetative stage in Taebaek, the temperature fluctuates between 15 and 22°C, whereas in Haenam, another main cabbageproducing area, the temperature fluctuates between 22 and 11°C from the planting to the harvesting periods
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ACCEPTED MANUSCRIPT (Korea Meteorological Administration, retrieved 2011-05-04). Therefore, evaluating the effects of fluctuating temperatures on the reproduction and damage potential of H. schachtii to Chinese cabbage in highland areas is a vital issue. Both the cabbage and the pathogen are highly sensitive to temperature. Infective H. schachtii J2 damage is affected by temperature (Banyer & Fischer, 1971; Shepherd & Clarke, 1971) and the pathogen’s initial population density. Abwi and Mai (1980) reported that at high densities, an initial nematode population can
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cause considerable economic losses to beet production. Barker and Olthof (1976) also reported that the initial H.
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schachtii population density before crop planting is the main factor in the amount of damage inflicted to host
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crops. Of course, other abiotic and biotic factors are also responsible for successful nematode hatching, which can lead to a high final population density and severe damage to the Chinese cabbage crop.
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Therefore, the economic damage caused by H. schachtii is a major production problem, which requires reliable control and management strategies. The objectives of this study were to investigate the following:
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i) The effect of a constant temperature on H. schachtii emergence;
ii) The effect of increasing temperature on H. schachtii emergence;
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iii) The optimum temperature for the best vegetative/marketable Chinese cabbage growth with minimum damage
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by the nematode;
iv) The effect of fluctuating temperatures on the reproduction and damage potential of H. schachtii to Chinese
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Materials and methods
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cabbage, which could minimize yield losses.
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Nematode extraction, cyst counting, and inoculation Nematodes were extracted from the soil in early 2015 from a highly infested Chinese cabbage field in Taebaek, Korea. H. schachtii cysts were isolated by sieving {(20-, 60-, and 400-mesh sieve (850, 250, and 30 µm, respectively)}. The 60-mesh-sieved particles were further filtered through Whatman® 100-µm filter paper. The filter paper was then placed in a Petri dish (6 X 6 cm diameter) and observed under a stereomicroscope (Nikon SM2 1000) to count the number of cysts (with and without eggs) and females, and to determine the total population. Five randomly chosen healthy cysts (undamaged, with egg cysts) were transferred to a small vial with 5 ml of water. The cysts were sonicated at 8000 rpm using a Polytron® PT 1300D sonicator (Kinematica,
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ACCEPTED MANUSCRIPT Switzerland). Sonicated eggs containing water were then transferred to a Petri dish and placed under the microscope to count the number of eggs. J2 inoculation was conducted on 25th March 2015, when the selected cysts were kept for 24 h in a Baermann funnel with water until successful hatching occurred. Hatched J2 were collected from the funnel tube and diluted to make a nematode suspension with a concentration of 150 J2/ml water. Using a pipette, the desired volume (until the suspension contains 800, 1600 or 3200 juveniles) was then inoculated into different pots. For egg
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inoculation, healthy cysts were crushed using a sonicator (Polytron® PT 1300D, Kinematica), and water
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containing the eggs was diluted to make an egg suspension in the same manner as the J2 inoculation. For the
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increasing temperature regime, only 2 g of soil was inoculated. The final nematode population was counted after
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30 and 60 days of inoculation.
Chinese cabbage
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Twenty-day-old Chinese cabbage plants were purchased from the local market and transplanted into plastic pots (10.5 × 13.5 cm) filled with 800 g of autoclaved soil collected from the field, to ensure that no contamination
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with other plant-parasitic nematodes occurred. J2 inoculation was conducted on the day after planting. Pots in a growth chamber that was maintained at a specific temperature were watered (50-75 ml) daily according to their
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needs (visual inspection, and by touching the soil). After 30 and 60 days of inoculation, the lengths and widths of
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the first five leaves from the ground were measured using a ruler. Shoot and root weights were also recorded.
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Temperature effect experiment
Three temperature regimes were used in this experiment: constant, increasing, and fluctuating. Each temperature
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regime was based on the last five years’ meteorological data for Taebaek and Haenam (KMA, 2011).
Constant temperature regime Chinese cabbage ‘Chungwang’ plants were inoculated with 1, 2, or 4 J2 and kept for 60 days in a growth chamber (GC-1000TLH, Jeio Tech, Korea) at 15, 20, or 25℃ with a 11:13 h light: dark photoperiod. The experiment included 20 replicates at each temperature.
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ACCEPTED MANUSCRIPT Increasing temperature regime Three initial temperatures (10, 14, and 18℃) were set in three growth chambers. At 10-day intervals, the temperature was increased by 2℃, and the plants were allowed to grow until the temperature reached 24℃. The plants were inoculated with 2 J2/g soil. Each of the three temperature experiments included 16 replicates.
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Fluctuating temperature regime
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Based on the meteorological data, Taebaek was treated as positive and Haenam as negative fluctuations.
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Taebaek is known for its summer Chinese cabbage production, and Haenam for its fall production. For a positive fluctuation, the initial temperature was set at 16.7°C, and at 10-day intervals, the temperature was increased to 17.8, 18.8, 19.9, 20.6, and 22°C. For a negative fluctuation, the initial temperature was set at 21.5°C, and at 10-
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day intervals, the temperature was decreased to 19.4, 17.4, 15.7, 13.4, and 11.5°C. The photoperiod was 11:13 h
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light: dark.
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Statistical analysis
The data were analyzed by an analysis of variance using SAS 9.4 (SAS Institute Inc., NC, USA). Numbers of
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cysts, females, eggs, juveniles, and the total population were compared among the temperature regimes (constant, increasing, and fluctuating) and inoculation concentrations (1, 2, and 4 J2/g soil). Fisher’s least significant
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difference test was conducted with significance set at P≤0.005. A percent weight reduction graph was prepared
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Results
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by calculating weight differences between the control and treated plants.
Constant temperature regime H. schachtii population size
After 30 days of inoculation, the largest population was at 20℃, whereas it was small at 15℃ (Fig. 1A). A similar result was obtained after 60 days of inoculation (Fig. 1B). The highest inoculation density (4 J2/g) had the largest population at all temperatures, after both 30 and 60 days of inoculation. The total population was smaller after 60 days of inoculation than after 30 days, possibly because of intraspecific competition.
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ACCEPTED MANUSCRIPT Chinese cabbage growth The greatest growth (number of leaves and plant weight) was recorded at 25℃ (Fig. 2) after 60 days of inoculation. At 15℃, the number of leaves was similar as at 25℃. Only a few of the comparisons were significantly different. The overall trend was of increasing weight with increasing temperature.
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Increasing temperature regime
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H. schachtii population size
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The numbers of cysts and females were significantly higher at 14℃ than at the other temperatures (Fig. 3). At
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10℃, the pathogen population was extremely small, and was slightly larger at 18℃.
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Chinese cabbage growth
Figure 4A shows that the number of leaves at 10, 14, and 18℃ was similar, with the most at 14℃ (Fig. 4A). The
Fluctuating temperature regime
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H. schachtii population size
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Chinese cabbage plants weighed the most at 14℃, and the least at 10℃ (Fig. 4B).
Both in the positive and negative temperature fluctuation experiments, the H. schachtii populations were very
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small, and there were no significant differences. The populations were counted after 60 days of inoculation. Interestingly, the positive temperature fluctuation experiment had significantly more eggs and cysts than the
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negative temperature fluctuation experiment (almost 20-fold) (df=1, F=37.96, p=0.0001), and significantly more J2 (df=2, F=11, p=0.03).
Number of Chinese cabbage leaves There were significantly more leaves in the positive temperature fluctuation experiment than in the negative temperature fluctuation experiment (df=1, F=10.89, p=0.005), according to the results of a Duncan’s multiple range test.
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ACCEPTED MANUSCRIPT Chinese cabbage weight Percent yield losses were calculated based on the control and treated plants’ shoot weights. The positive temperature fluctuation experiment had significantly less weight reduction than the negative temperature fluctuation experiment (Fig. 5) (df=1, F=6.80, p=0.02).
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Discussion
We found that cysts hatched fastest at a constant temperature of 20℃ at both 30 and 60 days after inoculation,
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which supports the results of KMA (2011), who reported that Chinese cabbage needs 15-20°C for the best vegetative growth. The optimum temperature requirement for juvenile emergence from eggs may depend on the
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size of the nematode population (Sharma & Renu, 1998). A previous study found that H. schachtii needs a temperature range of between 21 and 27℃ for hatching (Franklin, 1972), but we found that the best hatching temperature was 20℃. Banyer and Fisher (1971) stated that the optimum temperature for hatching is 20℃, but
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most studies state that it ranges between 20 and 30℃. In the present study, we not only investigated the number of hatched J2, but also the numbers of cysts and females, as well as cabbage growth. As cabbage completes its
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vegetative growth at 15-20℃ (KMA, 2011), the nematode population might complete hatching within this range. Apart from temperature, hatching is also affected by other factors, such as soil moisture, day length, and relative
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humidity (Sharma, 1998). Root extracts facilitate egg hatching, and J2 prefer 25℃ when using root extracts as a hatching medium (Kabir et al., 2015). In the present study, the potted plants had little chance to produce root
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diffusate, as the plants were still alive at cyst extraction. However, some of the plants were almost dead, and the roots had started to rot before cyst extraction, but this was rare.
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Under the increasing temperature regime, the highest numbers of cysts and females were at an initial temperature of 14℃, although it is unclear why this was the case. Wang and Yan (1993) reported that in China, the cyst
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nematode hatching rate is greater at 15℃ than at 5, 10, or 20℃. However, in the Korean highlands, the greatest J2 emergence is in late May (data not published), when the average air temperature ranges from 14 to 20°C (KMA, 2011). This may be why our H. schachtii population was largest at 14°C. Under the constant temperature regime, the Chinese cabbage plants were heaviest at 20℃, which agrees with the findings of KMA (2011). After 30 days of inoculation at 20℃, an initial inoculation concentration of 4 J2/g soil resulted in the highest numbers of cysts and females and the largest total population. Surprisingly, all of the untreated control plants had fewer leaves and lower weights than the treated plants. We assumed, as the time span was only 30 days, that as there was little or no chance of overcrowding in the initial population, the same would be true in the final population. The nematode reproductive rate depends upon the initial population
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ACCEPTED MANUSCRIPT density (Weischer et al., 1973), and after 60 days of inoculation, the largest final H. schachtii population was recorded at 20℃ with a 4 J2/g soil initial inoculation density. This supports Weischer et al.’s (1973) assertion that if the initial population is large, then the final population will also be large and cause extensive damage. The expected final population growth rate of many cyst species, including H. schachtii, can be described by the logistic model dN/dT=rN[(K-N)/K], where r is the intrinsic population growth rate, N is the size of the population, and K is the carrying capacity. Initial population density is the most important factor that affects the
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carrying capacity.
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Chinese cabbage leaf number and plant weight painted a vivid picture of the effects of initial inoculation density.
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After 30 days of inoculation, the maximum number of leaves was recorded on the control plants at 20℃. Interestingly, at 15℃, the untreated plants had fewer leaves than treated plants that had been inoculated with 4 J2/g soil. After 60 days of inoculation, there were far fewer leaves on plants that had been inoculated with high
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J2 densities. There were fewer leaves than on the control plants, with the maximum number at 25℃, and there was little difference in leaf number between the different temperatures. This may be explained by the seedlings’
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initial condition before they were transplanted, as well as other factors. It is possible that the nematodes had no effect on the number of leaves. Initially, the number and size of the leaves increased through photosynthesis,
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which increased the plants’ weights. If the pathogen attacks the roots, there would be insufficient nutrient uptake and a reduction in weight.
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Under the increasing temperature regime, the highest number of leaves and greatest weight were recorded at 14℃, but there were few differences between 14 and 18℃. This suggests that in field conditions, both nematodes
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and cabbages require the same temperature for growth. Therefore, changing the cabbage planting time may decrease the nematode population, but simultaneously could have a negative impact on cabbage weight.
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However, field conditions are much more challenging than laboratory conditions, and both hosts and nematodes are affected by several other factors, including soil temperature and moisture, day length, and relative humidity (Sharma, 1998). Plant age also plays a significant role in J2 hatching: young Chinese cabbage plants are more
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susceptible to nematode attack than older ones (data not published), and old plants have a greater carbon dioxide gradient capacity than younger ones and more leachate, which attracts nematodes (Dusenburry, 1987). Fluctuating temperatures result in different population densities, and often depend on planting time. Koenning and Anand (1991) reported that if planting is delayed, J2 soybean cyst nematodes continue hatching, and many of them die due to the absence of a host. But H. schachtii cyst can survive without a hot until 5 years (Sharma, 1998). There were no significant differences in the numbers of cysts or females, or the total population, between the positive and negative temperature fluctuation experiments. This supports the local meteorological data, because until cyst formation, the temperature ranges are favorable for cyst formation. However, the numbers of
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ACCEPTED MANUSCRIPT eggs and J2 differed between the two temperature fluctuation experiments: the negative temperature fluctuation experiment had significantly fewer eggs and J2 than the positive temperature fluctuation experiment, possibly because the average hatching temperature lies between 20.5 and 27.5℃ (Kakaiere et al., 2012). The final temperature in the negative temperature fluctuation experiment was 11.5℃, which is not favorable for successful hatching. Lewis and Mai (1960) reported that the moisture level alters the effect of temperature on the survival of H. rostochiensis eggs and larvae within cysts, and that alternating temperatures have a more negative effect
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than a constant temperature. Increasing temperatures caused by global warming could result in faster nematode
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development and a shorter life cycle (Curi & Zmoray, 1966; Griffin, 1981b; Kakaire et al., 2012). In the negative temperature fluctuation experiment, the plants had fewer leaves and lighter shoots than in the positive
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temperature fluctuation experiment, and they exhibited a lower percent weight reduction in the positive temperature fluctuation experiment than in the negative temperature fluctuation experiment (Fig. 5). Low
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temperature inhibits egg hatching, and the optimal hatching temperature for H. schachtii ranges between 15 and 30°C (Vandenbossche, 2015). J2 emergence in H. schachtii is inhibited by a period of warmth following low temperatures (Banyer & Fischer, 1971; Shepherd & Clarke, 1971), which is known as delayed dormancy (Zheng
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& Ferri, 1991). Futai (1980) reported that the optimal temperature for hatching lies between 25 and 30°C. In the
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positive temperature fluctuation experiment, the increasing temperature facilitated hatching at the expected time.
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Conclusion
The optimum temperature for H. schachtii is 20°C, but this is dependent on its initial population density.
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Positive temperature fluctuations (summer cultivation) are beneficial to both pathogen and cabbage growth, and the cabbage plants lost less weight than under negative temperature fluctuations (fall cultivation). Therefore, we
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suggest planting Chinese cabbage when the field temperature is below 10°C, so that the nematode population will take a relatively long time to cause economic damage. Consequently, the cabbage can reach its
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vegetative/marketable stage before the nematode causes any economic damage. Because this study was mainly conducted in laboratory conditions, it cannot precisely reflect what would happen in field conditions, but the results are a good foundation for empirical studies of H. schachtii on Chinese cabbage.
Acknowledgements We appreciate the technical assistance of Kim Jeong Eun, Kim Hyun Gook, An Hyeon Jeong, and Na Hee Bin. The study was conducted with the support of the Rural Development Administration, Republic of Korea under the project “Research Program for Agriculture, Science, and Technology Development, Project No. PJ010774”. 10
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ACCEPTED MANUSCRIPT Figure Legends
Fig. 1. Mean number of Heterodera schachtii cysts after 30 (A) and 60 (B) days of inoculation at different inoculation densities in potted Chinese cabbage under a constant temperature regime. Bars represent standard deviations. The same lowercase letters on the bars indicate that there was no significant
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difference among means.
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Fig. 2. Mean number of leaves (A) and weight (B) of potted Chinese cabbage under a constant temperature regime after 60 days of inoculation at varying Heterodera schachtii inoculation densities.
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Bars represent standard deviations. The same lowercase letters on the bars indicate that there was no significant
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difference among means.
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Fig. 3. Mean numbers of Heterodera schachtii cysts and females at different initial temperatures after inoculation with 2 J2/g soil in potted Chinese cabbage.
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Bars represent standard deviations. The same lowercase letters on the bars indicate that there was no significant
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difference among means.
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Fig. 4. Mean number of leaves (A) and weight (B) of potted Chinese cabbage under an increasing temperature regime after Heterodera schachtii inoculation with 2 J2/g soil.
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Bars represent standard deviations. The same lowercase letters on the bars indicate that there was no significant difference among means.
Fig. 5. Original weight and % weight reduction of Chinese cabbage plants under a fluctuating temperature regime.
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ACCEPTED MANUSCRIPT Highlights 1. A range of 6-20°C was the best for successful growth and development of Chinese cabbage and nematode. 2. Fall cultivation suppressed the nematode population, followed by less yield loss. 3. Cabbage gave the best vegetative growth and less attacked by the nematode at a range of 10-
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Md. Faisal Kabir, Jae-Kook Lee, Mun Gi Jeong, Mwamula Abraham Okki, Young Hwa Choi, Dong Woon Lee PII: DOI: Reference:
S1226-8615(17)30529-0 doi:10.1016/j.aspen.2017.11.004 ASPEN 1090
To appear in:
Journal of Asia-Pacific Entomology
Received date: Revised date: Accepted date:
23 August 2017 1 November 2017 2 November 2017
Please cite this article as: Md. Faisal Kabir, Jae-Kook Lee, Mun Gi Jeong, Mwamula Abraham Okki, Young Hwa Choi, Dong Woon Lee , Effects of varying temperature on the reproduction and damage potential of Heterodera schachtii to Chinese cabbage (Brassica rapa pekinensis). The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aspen(2017), doi:10.1016/j.aspen.2017.11.004
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ACCEPTED MANUSCRIPT Effects of varying temperature on the reproduction and damage potential of Heterodera schachtii to Chinese cabbage (Brassica rapa pekinensis)
Md. Faisal Kabir1+, Jae-Kook Lee2+, Mun Gi Jeong1, Mwamula Abraham Okki1, Young Hwa Choi3
Department of Ecological Science, Kyungpook National University, Sangju, 37224, Korea
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and Dong Woon Lee1*
Protection Division, National Academy of Agricultural Science, Rural Development Administration, Wanju,
of Ecology and Environmental System, Kyungpook National University, Sangju, 37224, Korea
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3School
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55365, Korea
*Corresponding
author: Dong Woon Lee
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E-mail: [email protected]
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Major of Applied Biology, School of Ecological Environment and Tourism, Kyungpook National University, Sangju, Gyeongsangbuk-do, 37224, Republic of Korea
authors contributed equally to this work. Conflicts of interest: none.
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Telephone: +82-54-530-1212
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ACCEPTED MANUSCRIPT Abstract. Heterodera schachtii is a well-known, destructive pathogen of Chinese cabbage (Brassica rapa pekinensis) in Korea, and several studies have attempted to find a potential control measure against it. This study is the first to investigate the effects of varying temperature on the reproduction and damage potential of H. schachtii to Chinese cabbage. Chinese cabbage plants were inoculated with H. schachtii at different densities (1, 2, or 4 juveniles per gram of soil) and grown under three temperature regimes: constant (15, 20, or 25℃), increasing (10, 14, and 18°C), and fluctuating (positive, 16.7-22.0°C; negative, 21.5-11.5°C). At a constant
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temperature after 30 days of inoculation, both Chinese cabbage and H. schachtii performed best at 20℃.
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However, after 60 days of inoculation, H. schachtii had a significantly higher population at 20℃, whereas cabbage growth was best at 25℃. With increasing temperature, the numbers of cysts and females did not change
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significantly, and reached maxima at an initial temperature of 14°C. However, the number of leaves and weights of the Chinese cabbage plants significantly differed at 14°C. Under fluctuating temperatures, temperature
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decreases reduced the H. schachtii population.
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Key words: Brassica rapa pekinensis, climatic condition, cyst nematode, population density, yield loss
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ACCEPTED MANUSCRIPT Introduction Chinese cabbage, Brassica rapa pekinensis (Lour.) Rupr., is one of the most popular Brassica vegetables and has a high market value, and is the third-most-produced crop in Korea (Chang et al., 2008; KREI, 2012; Kim et al., 2014). It is ranked as the most important processed foodstuff, and is an important native side dish in the form of Kimchi (FAO, 2013; Park et al., 2014). Chinese cabbage is widely grown in Korea, and for the best vegetative growth, it needs a temperature range of 15-20°C (KMA, 2011). Extended periods of higher or lower
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temperatures result in premature flowering and reduced foliar weight.
Heterodera schachtii, commonly known as sugar beet cyst nematode, causes severe damage to Brassicaceae
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species (Whitehead, 1998). It has a wide range of host plants, and causes significant losses of Chinese cabbage, even up to 50% yield losses (McCann et al., 1981). The pathogen was first recorded in Chinese cabbage fields in
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the highland area of Korea in 2011 (Lee et al., 2013). Since then, numerous fields have been infested with it. Recent reports have revealed that the pest is steadily spreading within the highland Chinese cabbage growing areas of Gangwon-do province (from 11.2 ha in 2011 to 70 ha in 2015), and crop production is being affected
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(Kwon et al., 2015). Therefore, knowing the pathogen’s potential at different temperatures is crucial. In general, hatching starts when the adult female dies and its body forms a dark brown cyst (Wallace, 1955;
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Kabir et al., 2015). Successful hatching ensures a high number of 2nd stage juveniles (J2) that could severely
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damage a single cabbage field. Temperature is one of the main factors in regulating the development of beet cyst nematodes (Griffin, 1981a; Trudgill, 1995).
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Interestingly, the optimal growth temperatures for Chinese cabbage and H. schachtii hatchlings are very similar, which probably affects the nematode’s attack on Chinese cabbage in Korea. Plant host-nematode relationships
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could be affected, because environmental temperature plays a key role in regulating the biology of plant-parasitic nematodes such as H. schachtii (Griffin, 1988; Trudgill et al., 2005). The relationship between nematode development and the host is mainly influenced by three thermal parameters: base, optimum, and maximum
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temperatures. These parameters are measured in accumulated heat units (Jones, 1975). The optimum temperature for infective H. schachtii juvenile hatching is 25°C, and lower temperatures have a negative effect (Kabir et al., 2015). Chinese cabbage in the Korean highlands is planted after winter, when the temperature remains below 15°C, which is well below the optimum hatching temperature of H. schachtii. Thereafter, the temperature increases from spring to summer, when it is favorable for H. schachtii reproduction (KMA, 2011). In the Korean highlands, spatial temperature fluctuations occur. During the cabbage’s vegetative stage in Taebaek, the temperature fluctuates between 15 and 22°C, whereas in Haenam, another main cabbageproducing area, the temperature fluctuates between 22 and 11°C from the planting to the harvesting periods
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ACCEPTED MANUSCRIPT (Korea Meteorological Administration, retrieved 2011-05-04). Therefore, evaluating the effects of fluctuating temperatures on the reproduction and damage potential of H. schachtii to Chinese cabbage in highland areas is a vital issue. Both the cabbage and the pathogen are highly sensitive to temperature. Infective H. schachtii J2 damage is affected by temperature (Banyer & Fischer, 1971; Shepherd & Clarke, 1971) and the pathogen’s initial population density. Abwi and Mai (1980) reported that at high densities, an initial nematode population can
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cause considerable economic losses to beet production. Barker and Olthof (1976) also reported that the initial H.
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schachtii population density before crop planting is the main factor in the amount of damage inflicted to host
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crops. Of course, other abiotic and biotic factors are also responsible for successful nematode hatching, which can lead to a high final population density and severe damage to the Chinese cabbage crop.
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Therefore, the economic damage caused by H. schachtii is a major production problem, which requires reliable control and management strategies. The objectives of this study were to investigate the following:
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i) The effect of a constant temperature on H. schachtii emergence;
ii) The effect of increasing temperature on H. schachtii emergence;
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iii) The optimum temperature for the best vegetative/marketable Chinese cabbage growth with minimum damage
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by the nematode;
iv) The effect of fluctuating temperatures on the reproduction and damage potential of H. schachtii to Chinese
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Materials and methods
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cabbage, which could minimize yield losses.
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Nematode extraction, cyst counting, and inoculation Nematodes were extracted from the soil in early 2015 from a highly infested Chinese cabbage field in Taebaek, Korea. H. schachtii cysts were isolated by sieving {(20-, 60-, and 400-mesh sieve (850, 250, and 30 µm, respectively)}. The 60-mesh-sieved particles were further filtered through Whatman® 100-µm filter paper. The filter paper was then placed in a Petri dish (6 X 6 cm diameter) and observed under a stereomicroscope (Nikon SM2 1000) to count the number of cysts (with and without eggs) and females, and to determine the total population. Five randomly chosen healthy cysts (undamaged, with egg cysts) were transferred to a small vial with 5 ml of water. The cysts were sonicated at 8000 rpm using a Polytron® PT 1300D sonicator (Kinematica,
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ACCEPTED MANUSCRIPT Switzerland). Sonicated eggs containing water were then transferred to a Petri dish and placed under the microscope to count the number of eggs. J2 inoculation was conducted on 25th March 2015, when the selected cysts were kept for 24 h in a Baermann funnel with water until successful hatching occurred. Hatched J2 were collected from the funnel tube and diluted to make a nematode suspension with a concentration of 150 J2/ml water. Using a pipette, the desired volume (until the suspension contains 800, 1600 or 3200 juveniles) was then inoculated into different pots. For egg
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inoculation, healthy cysts were crushed using a sonicator (Polytron® PT 1300D, Kinematica), and water
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containing the eggs was diluted to make an egg suspension in the same manner as the J2 inoculation. For the
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increasing temperature regime, only 2 g of soil was inoculated. The final nematode population was counted after
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30 and 60 days of inoculation.
Chinese cabbage
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Twenty-day-old Chinese cabbage plants were purchased from the local market and transplanted into plastic pots (10.5 × 13.5 cm) filled with 800 g of autoclaved soil collected from the field, to ensure that no contamination
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with other plant-parasitic nematodes occurred. J2 inoculation was conducted on the day after planting. Pots in a growth chamber that was maintained at a specific temperature were watered (50-75 ml) daily according to their
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needs (visual inspection, and by touching the soil). After 30 and 60 days of inoculation, the lengths and widths of
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the first five leaves from the ground were measured using a ruler. Shoot and root weights were also recorded.
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Temperature effect experiment
Three temperature regimes were used in this experiment: constant, increasing, and fluctuating. Each temperature
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regime was based on the last five years’ meteorological data for Taebaek and Haenam (KMA, 2011).
Constant temperature regime Chinese cabbage ‘Chungwang’ plants were inoculated with 1, 2, or 4 J2 and kept for 60 days in a growth chamber (GC-1000TLH, Jeio Tech, Korea) at 15, 20, or 25℃ with a 11:13 h light: dark photoperiod. The experiment included 20 replicates at each temperature.
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ACCEPTED MANUSCRIPT Increasing temperature regime Three initial temperatures (10, 14, and 18℃) were set in three growth chambers. At 10-day intervals, the temperature was increased by 2℃, and the plants were allowed to grow until the temperature reached 24℃. The plants were inoculated with 2 J2/g soil. Each of the three temperature experiments included 16 replicates.
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Fluctuating temperature regime
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Based on the meteorological data, Taebaek was treated as positive and Haenam as negative fluctuations.
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Taebaek is known for its summer Chinese cabbage production, and Haenam for its fall production. For a positive fluctuation, the initial temperature was set at 16.7°C, and at 10-day intervals, the temperature was increased to 17.8, 18.8, 19.9, 20.6, and 22°C. For a negative fluctuation, the initial temperature was set at 21.5°C, and at 10-
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day intervals, the temperature was decreased to 19.4, 17.4, 15.7, 13.4, and 11.5°C. The photoperiod was 11:13 h
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light: dark.
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Statistical analysis
The data were analyzed by an analysis of variance using SAS 9.4 (SAS Institute Inc., NC, USA). Numbers of
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cysts, females, eggs, juveniles, and the total population were compared among the temperature regimes (constant, increasing, and fluctuating) and inoculation concentrations (1, 2, and 4 J2/g soil). Fisher’s least significant
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difference test was conducted with significance set at P≤0.005. A percent weight reduction graph was prepared
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Results
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by calculating weight differences between the control and treated plants.
Constant temperature regime H. schachtii population size
After 30 days of inoculation, the largest population was at 20℃, whereas it was small at 15℃ (Fig. 1A). A similar result was obtained after 60 days of inoculation (Fig. 1B). The highest inoculation density (4 J2/g) had the largest population at all temperatures, after both 30 and 60 days of inoculation. The total population was smaller after 60 days of inoculation than after 30 days, possibly because of intraspecific competition.
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ACCEPTED MANUSCRIPT Chinese cabbage growth The greatest growth (number of leaves and plant weight) was recorded at 25℃ (Fig. 2) after 60 days of inoculation. At 15℃, the number of leaves was similar as at 25℃. Only a few of the comparisons were significantly different. The overall trend was of increasing weight with increasing temperature.
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Increasing temperature regime
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H. schachtii population size
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The numbers of cysts and females were significantly higher at 14℃ than at the other temperatures (Fig. 3). At
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10℃, the pathogen population was extremely small, and was slightly larger at 18℃.
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Chinese cabbage growth
Figure 4A shows that the number of leaves at 10, 14, and 18℃ was similar, with the most at 14℃ (Fig. 4A). The
Fluctuating temperature regime
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H. schachtii population size
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Chinese cabbage plants weighed the most at 14℃, and the least at 10℃ (Fig. 4B).
Both in the positive and negative temperature fluctuation experiments, the H. schachtii populations were very
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small, and there were no significant differences. The populations were counted after 60 days of inoculation. Interestingly, the positive temperature fluctuation experiment had significantly more eggs and cysts than the
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negative temperature fluctuation experiment (almost 20-fold) (df=1, F=37.96, p=0.0001), and significantly more J2 (df=2, F=11, p=0.03).
Number of Chinese cabbage leaves There were significantly more leaves in the positive temperature fluctuation experiment than in the negative temperature fluctuation experiment (df=1, F=10.89, p=0.005), according to the results of a Duncan’s multiple range test.
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ACCEPTED MANUSCRIPT Chinese cabbage weight Percent yield losses were calculated based on the control and treated plants’ shoot weights. The positive temperature fluctuation experiment had significantly less weight reduction than the negative temperature fluctuation experiment (Fig. 5) (df=1, F=6.80, p=0.02).
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Discussion
We found that cysts hatched fastest at a constant temperature of 20℃ at both 30 and 60 days after inoculation,
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which supports the results of KMA (2011), who reported that Chinese cabbage needs 15-20°C for the best vegetative growth. The optimum temperature requirement for juvenile emergence from eggs may depend on the
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size of the nematode population (Sharma & Renu, 1998). A previous study found that H. schachtii needs a temperature range of between 21 and 27℃ for hatching (Franklin, 1972), but we found that the best hatching temperature was 20℃. Banyer and Fisher (1971) stated that the optimum temperature for hatching is 20℃, but
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most studies state that it ranges between 20 and 30℃. In the present study, we not only investigated the number of hatched J2, but also the numbers of cysts and females, as well as cabbage growth. As cabbage completes its
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vegetative growth at 15-20℃ (KMA, 2011), the nematode population might complete hatching within this range. Apart from temperature, hatching is also affected by other factors, such as soil moisture, day length, and relative
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humidity (Sharma, 1998). Root extracts facilitate egg hatching, and J2 prefer 25℃ when using root extracts as a hatching medium (Kabir et al., 2015). In the present study, the potted plants had little chance to produce root
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diffusate, as the plants were still alive at cyst extraction. However, some of the plants were almost dead, and the roots had started to rot before cyst extraction, but this was rare.
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Under the increasing temperature regime, the highest numbers of cysts and females were at an initial temperature of 14℃, although it is unclear why this was the case. Wang and Yan (1993) reported that in China, the cyst
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nematode hatching rate is greater at 15℃ than at 5, 10, or 20℃. However, in the Korean highlands, the greatest J2 emergence is in late May (data not published), when the average air temperature ranges from 14 to 20°C (KMA, 2011). This may be why our H. schachtii population was largest at 14°C. Under the constant temperature regime, the Chinese cabbage plants were heaviest at 20℃, which agrees with the findings of KMA (2011). After 30 days of inoculation at 20℃, an initial inoculation concentration of 4 J2/g soil resulted in the highest numbers of cysts and females and the largest total population. Surprisingly, all of the untreated control plants had fewer leaves and lower weights than the treated plants. We assumed, as the time span was only 30 days, that as there was little or no chance of overcrowding in the initial population, the same would be true in the final population. The nematode reproductive rate depends upon the initial population
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carrying capacity.
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Chinese cabbage leaf number and plant weight painted a vivid picture of the effects of initial inoculation density.
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After 30 days of inoculation, the maximum number of leaves was recorded on the control plants at 20℃. Interestingly, at 15℃, the untreated plants had fewer leaves than treated plants that had been inoculated with 4 J2/g soil. After 60 days of inoculation, there were far fewer leaves on plants that had been inoculated with high
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J2 densities. There were fewer leaves than on the control plants, with the maximum number at 25℃, and there was little difference in leaf number between the different temperatures. This may be explained by the seedlings’
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initial condition before they were transplanted, as well as other factors. It is possible that the nematodes had no effect on the number of leaves. Initially, the number and size of the leaves increased through photosynthesis,
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which increased the plants’ weights. If the pathogen attacks the roots, there would be insufficient nutrient uptake and a reduction in weight.
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Under the increasing temperature regime, the highest number of leaves and greatest weight were recorded at 14℃, but there were few differences between 14 and 18℃. This suggests that in field conditions, both nematodes
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and cabbages require the same temperature for growth. Therefore, changing the cabbage planting time may decrease the nematode population, but simultaneously could have a negative impact on cabbage weight.
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However, field conditions are much more challenging than laboratory conditions, and both hosts and nematodes are affected by several other factors, including soil temperature and moisture, day length, and relative humidity (Sharma, 1998). Plant age also plays a significant role in J2 hatching: young Chinese cabbage plants are more
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susceptible to nematode attack than older ones (data not published), and old plants have a greater carbon dioxide gradient capacity than younger ones and more leachate, which attracts nematodes (Dusenburry, 1987). Fluctuating temperatures result in different population densities, and often depend on planting time. Koenning and Anand (1991) reported that if planting is delayed, J2 soybean cyst nematodes continue hatching, and many of them die due to the absence of a host. But H. schachtii cyst can survive without a hot until 5 years (Sharma, 1998). There were no significant differences in the numbers of cysts or females, or the total population, between the positive and negative temperature fluctuation experiments. This supports the local meteorological data, because until cyst formation, the temperature ranges are favorable for cyst formation. However, the numbers of
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ACCEPTED MANUSCRIPT eggs and J2 differed between the two temperature fluctuation experiments: the negative temperature fluctuation experiment had significantly fewer eggs and J2 than the positive temperature fluctuation experiment, possibly because the average hatching temperature lies between 20.5 and 27.5℃ (Kakaiere et al., 2012). The final temperature in the negative temperature fluctuation experiment was 11.5℃, which is not favorable for successful hatching. Lewis and Mai (1960) reported that the moisture level alters the effect of temperature on the survival of H. rostochiensis eggs and larvae within cysts, and that alternating temperatures have a more negative effect
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than a constant temperature. Increasing temperatures caused by global warming could result in faster nematode
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development and a shorter life cycle (Curi & Zmoray, 1966; Griffin, 1981b; Kakaire et al., 2012). In the negative temperature fluctuation experiment, the plants had fewer leaves and lighter shoots than in the positive
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temperature fluctuation experiment, and they exhibited a lower percent weight reduction in the positive temperature fluctuation experiment than in the negative temperature fluctuation experiment (Fig. 5). Low
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temperature inhibits egg hatching, and the optimal hatching temperature for H. schachtii ranges between 15 and 30°C (Vandenbossche, 2015). J2 emergence in H. schachtii is inhibited by a period of warmth following low temperatures (Banyer & Fischer, 1971; Shepherd & Clarke, 1971), which is known as delayed dormancy (Zheng
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& Ferri, 1991). Futai (1980) reported that the optimal temperature for hatching lies between 25 and 30°C. In the
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positive temperature fluctuation experiment, the increasing temperature facilitated hatching at the expected time.
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Conclusion
The optimum temperature for H. schachtii is 20°C, but this is dependent on its initial population density.
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Positive temperature fluctuations (summer cultivation) are beneficial to both pathogen and cabbage growth, and the cabbage plants lost less weight than under negative temperature fluctuations (fall cultivation). Therefore, we
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suggest planting Chinese cabbage when the field temperature is below 10°C, so that the nematode population will take a relatively long time to cause economic damage. Consequently, the cabbage can reach its
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vegetative/marketable stage before the nematode causes any economic damage. Because this study was mainly conducted in laboratory conditions, it cannot precisely reflect what would happen in field conditions, but the results are a good foundation for empirical studies of H. schachtii on Chinese cabbage.
Acknowledgements We appreciate the technical assistance of Kim Jeong Eun, Kim Hyun Gook, An Hyeon Jeong, and Na Hee Bin. The study was conducted with the support of the Rural Development Administration, Republic of Korea under the project “Research Program for Agriculture, Science, and Technology Development, Project No. PJ010774”. 10
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Fig. 1. Mean number of Heterodera schachtii cysts after 30 (A) and 60 (B) days of inoculation at different inoculation densities in potted Chinese cabbage under a constant temperature regime. Bars represent standard deviations. The same lowercase letters on the bars indicate that there was no significant
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difference among means.
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Fig. 2. Mean number of leaves (A) and weight (B) of potted Chinese cabbage under a constant temperature regime after 60 days of inoculation at varying Heterodera schachtii inoculation densities.
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Bars represent standard deviations. The same lowercase letters on the bars indicate that there was no significant
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difference among means.
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Fig. 3. Mean numbers of Heterodera schachtii cysts and females at different initial temperatures after inoculation with 2 J2/g soil in potted Chinese cabbage.
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Bars represent standard deviations. The same lowercase letters on the bars indicate that there was no significant
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difference among means.
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Fig. 4. Mean number of leaves (A) and weight (B) of potted Chinese cabbage under an increasing temperature regime after Heterodera schachtii inoculation with 2 J2/g soil.
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Bars represent standard deviations. The same lowercase letters on the bars indicate that there was no significant difference among means.
Fig. 5. Original weight and % weight reduction of Chinese cabbage plants under a fluctuating temperature regime.
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Fig. 2.
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Fig. 5
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Graphical abstract
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ACCEPTED MANUSCRIPT Highlights 1. A range of 6-20°C was the best for successful growth and development of Chinese cabbage and nematode. 2. Fall cultivation suppressed the nematode population, followed by less yield loss. 3. Cabbage gave the best vegetative growth and less attacked by the nematode at a range of 10-
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18°C.
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