Guohui ZHANG , Long WANG, Yongquan REN, Dingxiang HE, Caiwu LAN, Chengming FAN, Yuhong CHEN, Zanmin HU

1. Department of Environmental and Life Sciences, Kaili College, Kaili 556000, China;

2. Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;

3. Guizhou Changhao Traditional Chinese Medicine Development Co., Ltd., Kaili 556000, China;

4. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

Pseudostellaria heterophylla(Caryophyllaceae)refers to the roots of radix pseudostellariae,and it has functions of tonifying qi and nourishing blood, promoting salivation and strengthening the spleen and stomach. Pseudostellaria heterophylla is a commonly-used tonic herb. Pseudostellaria heterophylla has recently made some new progresses in the treatment of hepatitis, diabetes, coronary heart disease,angina,secondary aplastic anemia, leukopenia, hyperthyroidism, lymphatic and other incurable diseases in clinics[1]. In recent years,functional health products of Pseudostellaria heterophylla have been constantly developed, such as Pseudostellaria heterophylla compound oral liquid and capsules.Moreover,the extract of Pseudostellaria heterophylla has been applied in the development of cosmetics. The market demand for Pseudostellaria heterophylla is increased year by year, so its price also rises[2]. Currently, Pseudostellaria heterophylla has been included in the list of "list of herbs that can be used for health food" by the Ministry of Health,and it is a kind of expensive medicine.

Pseudostellaria heterophylla,as a vigorously promoted and developed variety in Guizhou Province during the Twelfth Five-Year Plan,has huge economic benefits.Due to increased market demand, the acreage of Pseudostellaria heterophylla has been rapidly increased in Qiandongnan Prefecture, and its continuous cropping area has also been increased.The harm and species of fungal diseases that occur in Pseudostellaria heterophylla are increased year by year, resulting in yield of Pseudostellaria heterophylla reduced by 15%-50%[3]. Therefore, it is critical to carryout investigation on fungal diseases in Pseudostellaria heterophylla and studies on relevant prevention techniques. It has been found that viral disease, leaf spot, southern blight,root rot and spot disease are common diseases in Pseudostellaria heterophylla[4-8]. In addition, there are also some reports on damping-off disease in Pseudostellaria heterophylla, but they do not introduce pathogen pictures and relevant bio-control measures. Damping-off disease occurs more and more frequently in Pseudostellaria heterophylla in Qiandongnan Prefecture of Guizhou Province,and its onset period is extended gradually. To reduce the harm of disease and use of chemicals, the pathogen and bio-control measures of dampingoff disease in Pseudostellaria heterophylla were detailedly introduced in this paper.

Materials and Methods

Test time and site

The outdoor survey was carried out in the cultivation fields of Guizhou Changhao Traditional Chinese Medicine Development Co., Ltd. in Shipo Village, Yiwanshui Town, Huangping County, Qiandongnan Prefecture from May, 2011 to May, 2014. The indoor test was carried out in the laboratory of Institute of Genetics and Development Biology, Chinese Academy of Sciences.

Materials

Sample collection The samples were also collected from the cultivation fields of Guizhou Changhao Traditional Chinese Medicine Development Co., Ltd. in Shipo Village, Yiwanshui Town, Huangping County, Qiandongnan Prefecture from May, 2011 to May,2014.The occurrence characteristics and symptoms of diseases in Pseudostellaria heterophylla were recorded.Fresh samples were collected for pathogen isolation and identification.

Test medium The used medium was PSA medium,and its components were as follows:potato 200 g,sucrose 15-20 g, agar 17 g and water 1 000 ml. PSA medium was sterilized at 121°C for 20 min before use.

Apparatus and instruments and reagents The used main apparatus and instruments included high-press steam sterilizer, clean bench, stove,microwave oven, thermostatic incubator, microscope and transplantation needles.

The used main reagents included industrial alcohol(70%),mercuric chloride solution (0.1%) and streptomycin(purity of 95%).

The used bio-control strain was GH 18(Bacillus spp.).

Methods

Observation and description of symptoms The morphology, color,size and harm extent of lesions on tissue surface of Pseudostellaria heterophylla were observed, and the diseased parts were photographed.

Isolation,purification and culture of pathogen The pathogen in diseased parts of Pseudostellaria heterophylla was isolated using tissue isolation method. The diseased tissues (4-5 mm) on the edge of lesions were cut off. They were soaked in 70% ethanol for 2-3 s, and then soaked in 0.1%mercuric chloride solution for 2 min.Subsequently, the diseased tissues were rinsed three times with sterile water. Finally, they were incubated in PSA medium at 28 ℃for 3-5 d. After the culture, the mycelia around the tissues were picked and inoculated on medium plate added with streptomycin and cultured for another 5-7 d. Thus the pure culture of pathogen was obtained. The pathogen was photographed under a microscope, and the sizes of hyphae and spores were measured and recorded. The pathogen in diseased soil was isolated using the dilution plate method. The soil dilution was first prepared. It was inoculated on beef extract peptone plate. The plate was inverted and placed in an incubator at 37 ℃for 2-3 d. The colonies were picked and purified until the pure culture of pathogen was obtained.The pathogen was photographed under a microscope, and the sizes of hyphae and spores were measured and recorded.

Dual test between bio-control strain and pathogenic strain The pathogenic strain was first inoculated in a plate, and the bio-control strain broth was then inoculated on both sides of the strain using a sterilized toothpick. The plate was inverted and placed in an incubator at 37 ℃for 2-3 d.

Results and Analysis

Symptoms of damping-off disease in Pseudostellaria heterophylla

The lesions of damping off were mainly shown at the base of young stems. After pathogen invaded the stems, water-soaked brown spots first appeared at the base of stems. The spots were extended rapidly around the stems,depressed gradually and finally constricted into waspish shape,resulting in lodging and death of young seedlings. The diseased parts, in the length of about 1-2 cm, were brown,and there was apparent boundary between the healthy and diseased parts.Damping-off disease developed rapidly,and the leaves were still green and fresh when the young seedlings got flattened. The recent surveys show that the onset period of damping-off disease is relatively long in fields, and it occurs in a large scale from February to May of every year. When damping off occurs in fields, the spots extend around rapidly,resulting in lodging and death of a large number of seedlings(Fig.1,Fig.2).

Morphological characteristics of pathogen

The newly-generated hyphae were colorless and transparent, and they were distributed at a right or nearly right angle. The hyphae were constricted slightly at the branch. There were clear diaphragms among the mycelia. The mature mycelia were brown with a diameter of about 5-7 μm(Fig.3).

According to the morphological characteristics, the pathogen was identified as Rhizoctonia solani Kuhn,which belongs to Eumycota, Deuteromycotina, Agonomycetales and Rhizoctonia.

The pathogen isolated from the diseased soil was identified as Alternaria alternate, which belongs to Eumycota, Deuteromycotina, Hyphomycetes and Hyphomycetales.The conidiophores had single or multiple branches in diameter of 3-6 μm.They were knee-shaped, brown and separated. The conidia were dark brown,bunchy and pour clavate.They had obtuse tops, smaller bases, but had no beaks. The conidia had 1-4 thoracic diaphragms and 1-2 longitudinal diaphragms(Fig.4).

Bio-control test of GH18

The GH18 strain (Bacillus spp.)isolated from soil was used in the dual test. The results showed that GH18 strain had significant inhibitory effect on Rhizoctonia solani(Fig.5).The dual test between Alternaria alternate and GH18 strain showed that GH18 strain could produce clear inhibition zone,and it had significant inhibitory effect on Alternaria alternate(Fig.6).

Conclusions and Discussion

Pseudostellaria heterophylla is one of expensive medicinal plants,and its nutritional and medicinal value has been utilized by medicine. Pseudostellaria heterophylla occupies an important position in the traditional Chinese medicine market. However,diseases and pests have been one of constraints to high yield and quality of Pseudostellaria heterophylla[10].Fungal diseases directly reduce yield and quality of Pseudostellaria heterophylla.Even worse, pathogens may produce a variety of toxins and secondary metabolites harmful to humans and animals during their invasion, greatly threatening people’s health and security[11-12]. In addition, prevention and control of major fungal diseases in plants often depends on chemicals.The use of chemical pesticides not only increases the cost of production,but also leads to environmental pollution and pesticide residues in agricultural products, resulting in serious harm to livestock and the environment. Therefore, bio-control measures should be adopted according to occurrence regularity and pathogenesis of fungal disease and resistance mechanism of host plant to further reduce the adverse effects of viral disease on yield and quality of Pseudostellaria heterophylla,as well as environmental pollution and pesticide residues in agricultural products.

In China,there are rare reports on damping-off disease in Pseudostellaria heterophylla, let alone detained description and photographs. In this study, the damping-off disease and its pathogen Rhizoctonia solani in Pseudostellaria heterophylla were described in detail, and the biological control measures were discussed.

At the seedling stage, the damping-off disease in Pseudostellaria heterophylla is caused by Rhizoctonia solani.Rhizoctonia solani is widely distributed in nature,and it is mainly fromsoil.Rhizoctonia solani is adapted to a wide range of soil pH,but it is a kind of parasite preferring acidic environment.In soil, Rhizoctonia solani mainly lives in humus and diseased plants, and it can survive 2-3 years in general. Under favorable conditions, damping-off disease caused by Rhizoctonia solani may outbreak. If Pseudostellaria heterophylla is infected with Rhizoctonia solani before the seedling stage,Pseudostellaria heterophylla seedlings may not emerge. After the emergence of Pseudostellaria heterophylla seedlings, Rhizoctonia solani mainly infects the stems of plants, and the spots are mainly shown in basal stems of seedlings. Rhizoctonia solani is distributed in the farming and non-farming soil across the world,and can be easily isolated from infected plants and soil. It has a wide host range, and can cause many plant diseases, such as damping off at seedling stage and sheath blight in cereal crops[13].Damping-off disease in Pseudostellaria heterophylla is mainly caused by Rhizoctonia solani carried in seeds and soil.Moreover, poor growth, poor resistance, bad weather and poor soil drainage at seedling stage aggravate the disease.

Rhizoctonia solani overwinters in the form of mycelium and sclerotia in soil or host residual organizations.When Pseudostellaria heterophylla seeds germinate next year, Rhizoctonia solani invades into young organizations, leading to rot and death of seedlings. Rhizoctonia solani can live a saprophytic life as long as 2-3 years,and it is mainly spread by the rain and farming operations. Under conditions of successive-day high temperature or rain,poor drainage and poor light environment in seedbed, the pathogen would infect seedlings in height about 17 cm. Generally, when it is rainy and cold, the diseases occurs more seriously;when it is rainy and hot,the disease occurs in more adult plants[14].Chen et al.[15]found that regardless of whether the Rhizoctonia solani Kuhncarried seeds were disinfected,damping-off disease would occur at the seedling stage if the soil was not disinfected,and the incidence was 16.2%-18.1%. On the contrary, when the soil was disinfected, the damping-off disease did not occur at the seedling stage. It is confirmed that the infection source of damping-off disease at the seedling stage is from the soil rather than seeds. But in the actual cultivation process, soil disinfection is infeasible unless soil biological disinfection techniques are adopted[15-16].

In addition, fertilization also has great impact on soil microorganisms.Numerous studies have shown that the application of fertilizers, especially of nitrogen fertilizer, can promote the growth of Fusarium, Verticillium, Rhizoctonia and other soil-borne diseases[17-18].Alternaria alternate in soil is spread with the rain in the form of conidia, and their pycnidia overwinter on diseased residues.Next year,conidia cause primary infection, and after the onset, newly-produced conidia cause secondary infection. Especially under conditions of rainy weather,extensive management and poor growth of plants,the disease occurs more seriously. The pathogen lives a saprophytic life, and is distributed in soil,plant residues,industrial materials and air. It can parasitize many plants, and aggravate the occurrence of damping off in Pseudostellaria heterophylla.

Bio-control measures are critical for prevention and control damping-off disease in Pseudostellaria heterophylla due to the following reasons: Alternaria alternate carried in seeds and soil is difficult to be removed; soil disinfection is infeasible; damp and lowtemperature cultivation environment is unlikely to be changed; chemicals may lead to adverse consequences.It is reported that Bacillus circulans and Bacillus polymyxa have antagonistic effect on Rhizoctonia solani,the pathogen of damping-off disease in Pseudostellaria heterophylla[13,19-20].The antagonistic substances produced by Bacillus mainly include antibiotics,bacteriocins, cell wall degrading enzymes and other antimicrobial proteins and volatile antimicrobial substances.Among them, antimicrobial proteins make important contribution to inhibitory effect of Bacillus. Currently,the antagonistic bacteria used as biological fungicides include mainly Bacillus subtilis and Bacillus lichenformis.The development and utilization of antagonistic bacteria and their metabolites,as well as the development of efficient, stable and adaptable new antagonistic bacteria by genetic engineering, is the future development trend of bio-control bacteria[21].The biocontrol test in this study showed that GH18 strain has significant inhibitory effect on Rhizoctonia solani, and has great antagonistic effect, so GH18 strain can be studied in depth.

In short, since damping-off disease occurs seriously in Pseudostellaria heterophylla and it is difficult to be controlled and prevented, comprehensive measures must be adopted to control and prevent this disease. The comprehensive control may focus on bio-control (such as Bacillus spp.) and agricultural prevention. On the basis of disinfection of soil and seeds[22-24],the soil should be avoided from high moisture content and the nursing management should be strengthened under the assistance of chemical and physical control. At appropriate time,safe, low-toxicity and residue, efficient and practical pesticides, along with bio-control agents, should be used to reduce drug resistance of pathogens of diseases and pests in Pseudostellaria heterophylla[19-25]. The adoption of pathogen-free seeds is the most direct and effective way to prevent and reduce the degree of harm. In addition,newly-cultivated fields can be selected,and Pseudostellaria heterophylla can also be intercropped with rice[7-9].The results of this study indicate that bio-control strain of Bacillus spp. is feasible and necessary in control and prevention of damping-off disease in Pseudostellaria heterophylla in Qiandongnan Prefecture of Guizhou Province.

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