Study on Pathogenicity Difference of Plasmodiophora brassicae Under Different Temperature and pH Conditions

Weihua PEI, Xiangdong LI, Peiwen YANG, Jifen CAO, Yunqing BI, Zilin YANG, Wen RUI,Guiming MA, Xinghua LIN, Lifeng ZHOU, Mingying YANG*

1. Institute of Agricultural Environment and Resources, Yunnan Academy of Agricultural Sciences, Kunming 650205, China;

2. Lincang Plant Protection Station, Lincang, 677000, China;

3. Supervision and Testing Center for Agricultural Product Quality of Lijiang City, Lijiang 674100, China;

4. Lijiang Plant Protection Station, Lijiang 674100, China

Clubroot on cruciferous plants is a worldwide disease caused by Plasmodiophora brassicae Woron, which is also one of the serious soil-borne diseases. The disease occurred in North America, South America, Europe, South Africa, East Asia and other places in the world. It was first found in Taiwan as early as in 1936, and then it also occurred Heilongjiang, Liaoning, Shandong, Xinjiang, Gansu, Shaanxi, Jiangsu, Zhejiang, Fujian, Jiangxi, Hubei, Hunan,Guangdong, Guangxi, Sichuan, Yunnan,Tibet and other provinces and autonomous regions. The losses in the areas attacked by P. brassicae are generally about 20%,even up to 70%-80%,or 100%in some areas.So it is a threat to the development of the vegetable industry[1-3].

In recent decades,much progress has been made in the occurrence,prevalence, forecasting and detection of pathogens and pathogenic differentiation of clubroot with the applications of molecular biology in plant pathology. Yang et al.[4]carried out rapid detection on clubroot of cabbage by PCR, and found that the germination of resting spores was a prerequisite for pathogenic infection.The study of Guo et al.[5]revealed that the germination rate of resting spores was affected by the environment temperature and pH,and significantly inhibited by light. So,temperature and soil pH are two important factors affecting the germination of spores in vitro. In this study,the cabbage plants were grown inthe soil mixed with resting spores of Plasmodiophora brassicae at 1 ×108(spores/g soil) in pot experiment, at seven different temperature sets and six soil pH values to investigate the correlation of pathogenicity of P.brassicae with temperature and soil pH,with an attempt to provide theoretical basis for the prevention and control of disease in field by adjusting planting pattern and soil pH.

Materials and Methods

Materials

The P. brassicae was collected from fresh cabbage samples infected by clubroot, and its pathogenicity was tested on Chinese cabbage cultivar 83-1.

Preparation of soil sample

The Chinese cabbage samples infected by clubroot were soaked in water for 10 d. The spore concentration in the mixture of vermiculite and disinfected humus at a ratio of 1∶1 was adjusted to 1×108spores/g soil,before the soil sample was covered by film for 10 d,during which the soil sample was flipped once every 5 d, so that the pathogens and the soil could fully mixed.

Cabbage cultivation under different temperature and soil pH conditions Cabbage cultivation under different temperature conditions The cabbage seeds were sown in the soil samples prepared in above steps in pots of 30 cm in diameter and 20 cm in height,and cultivated in a light incubator at 25 ℃till germinating. Then, the pots were placed into different light incubators and cultivated at 5, 10, 15,20, 25, 30 and 35 ℃. Pathogen-free soil was used in control group. Three pots were prepared for each temperature treatment, and 12 plants were remained in each pot.

Cabbage cultivation under different soil pH condition The soil samples were placed in pots of 30 cm in diameter and 20 cm in height, and their pH was respectively adjusted to 4,5,6,7,8 and 9 before cabbage seeds were sown. Pathogen-free soil was used in control group. Three pots were prepared for each soil pH treatment, and 12 plants were remained in each pot.Then, the pots were cultivated in a light incubator at 25 ℃.

Other management measures The plants were watered with sterile water regularly, and no fungicides were applied during the entire experimental stage.

Measurement items and methods

The plant survival rate in every treatment was calculated 90 d after seed sowing,while the cabbages were uprooted for clubroot grading, according to DB53/T 442-2012"Technological Process for Identification of Rape Resistance to Clubroot"(Table 1).

Results and Analysis

Pathogenicity of P. brassicae on Chinese cabbage at different temperature sets

The results showed that the average survival rate was 55.0%, 75.2%,80.6%,90.0%,95.2%,82.0%and 45.3%;and the average plant height was 5.5,8.6,9.4,15.6,12.2,9.2 and 6.0 cm for cabbage grown at 5,10,15,20,25,30 and 35 ℃, all indicating that the cabbage plant grew better at 20-25 ℃than at other temperature conditions(Fig.1).

The incidence rate of clubroot on cabbage grown at 5,10,15,20,25,30 and 35 ℃was 0%,3.8%,15.2%,61.8%,89.6%,90.2%and 53.6%,with the disease index of 0, 0.8, 3.5, 6.5, 15.6,12.0 and 4.2, indicating that the incidence of clubroot on cabbage plants was higher at 25-30 ℃than at other temperature conditions, namely P.brassicae had stronger pathogenicity at 25-30 ℃(Fig.2).

Table 1 The grading standards of clubroot(at seedling stage)

Pathogenicity of P. brassicae on Chinese cabbage at different soil pH values

As shown in Fig.3, the average survival rate was 25.6%,76.4%,85.5%,90.6%, 78.9%, 58.7 % and the average plant height was 6.5, 8.8, 16.5,17.8,15.2 and 13.6 cm at soil pH 4,5,6, 7, 8 and 9, respectively, suggesting that the cabbage plant grew better when the soil pH was 6,7 and 8.

The incidence rate of clubroot on cabbage grown in soil with pH value of 4, 5, 6, 7, 8 and 9 was 80.4%, 100%,65%,10.8%,3.2%and 0.0%, with the disease index of 6.8, 15.9, 14.2, 3.5,1.5 and 0.0.The incidence rate of clubroot was higher, but the cabbage growth was inhibited in the soil with pH 4. The incidence rate of clubroot was lower at soil pH 7 and 8,and was zero at pH 9.When the soil pH was 5 and 6,the cabbage plant grew well, and the incidence rate of clubroot was high,suggesting that soil pH 5-6 was most suitable for the survival and reproduction of P. brassicae. So, the occurrence of clubroot in field could be reduced through increasing the application of organic fertilizers,plant ash and alkaline fertilizers to adjust the soil pH.

Discussion

Clubroot pathogen on cruciferous plant is an obligate parasite,and it has strong vitality as it can survive for 7 to 9 years. Many factors can affect the incidence of clubroot infection,such as soil moisture, amount of pathogen source, susceptible host, especially soil temperature and pH. The study of Yang et al.[6]revealed that the incidence rate of clubroot was lower in soil with water content of 20%, 30% and 80%,but higher in soil with water content of 70%-80%. By measuring the vertical distribution of resting spores in soil infected by clubroot, Yang et al.[6]found that the amount of resting spore of clubroot pathogen in soil of 10 cm deep was higher than in surface soil,both higher than in soil of 20 cm deep.The study of Yang et al.[7]showed that,if the infected tissue of cabbage was soaked in water or exposed to sunlight for 5, 10 and 15 d before inoculation,the incidence rates of clubroot on cabbage were 55.2%, 79.2%, 89.6%and 85.7%,100%,100%,respectively;and the incidence rate was 84.9% for the cabbage inoculated with fresh infected root. The study of Lv[8]showed that resting spores of clubroot pathogen could germinate at temperature from 6 to 30 ℃,and 21-22 ℃was the most suitable soil temperature.Guo et al.[5]found that the germination rate of the resting spores of clubroot pathogen at different temperature in a descending order was 24 ℃＞20 ℃＞28 ℃＞16 ℃＞32 ℃＞8 ℃＞36 ℃＞4 ℃＞40 ℃. All the above studies revealed the effects of temperature and pH on the germination of resting spores of clubroot pathogen in vitro.The present study simulated the natural field conditions to investigate the effects of temperature and pH on the pathogenicity of clubroot pathogen, and to find out the main factors affecting clubroot occurrence, which could provide a scientific basis for developing measure to prevent and control clubroot.The results showed that the incidence rate of clubroot on cabbage increased with soil temperature increasing from 15 to 30 ℃but decreased at 35 ℃.The pathogenicity of P. brassicae was weak at 5 and 10 ℃.So,soil temperature from 25 to 30 ℃was most suitable for the propagation of clubroot pathogen. In summary, 20-30 ℃was the most suitable temperature for the occurrence of clubroot infection in soil and the germination of resting spores in vitro. Besides, clubroot could occur at temperature from 15 to 30 ℃,which covered the major vegetable producing areas in Yunnan, so clubroot occured widely here. The occurrence of clubroot was less when the temperature was below 15 ℃ or above 30 ℃[9-10].

Our results also showed that among the soil pH values of 4,5,6,7,8 and 9, the incidence rate of clubroot was higher at pH 4, but the growth of cabbage was inhibited and its survival rate was reduced.The incidence rates of clubroot were low at pH value of 7 and 8, and was 0% at pH 9. The Chinese cabbage grew better at pH value of 5 and 6, but had high incidence rates of clubroot. Thus, the soil pH at 5-6 was most suitable for the occurrence and infection of clubroot on cruciferous plants. However, the soil pH in most vegetable growing areas in Yunnan Province was 5-6, so, clubroot widely occurred in these areas and even very severely in same of the areas. So, the application of alkaline fertilizers can reduce the occurrence of clubroot[11].

Sustained and comprehensive measures are necessary for the prevention and control of clubroot on cruciferous plants. Suitable measures should be conducted according to the climatic and soil situation, such as rotation of different crops,introduction or screening of disease-resistant (toler-ant) cultivars, application of fermented composting manures or alkaline fertilizers, cleaning the pathogen resource timely to reduce the occurrence of clubroot on cruciferous crops[12].

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