| Atractylodes Lancea (Thunb.) DC. is a perennial medicinal herb in the Compositae family. The source plant mainly includes Atractylodes lancea and Atractylodes japonica, which are used dried roots as medicine. A. japonica is genuine medicinal materials of Liaoning Province. With the increase of market demand and the depletion of wild resources, the cultivated area is gradually expanding. Over the disease survey of A. japonica in 2013, a new leaf spot disease was found, which infected leaves with round or oval, resulting in leaves withered early. The disease occurrence of the area showed a rising trend year by year, seriously influence the harvest and quality of A. japonica. Access to domestic and international literature, the relevant report about leaf spot disease of A. japonica in Liaoning area has not been found. Aimed to identify the causal agent, the morphological characteristics, culture character and multiple gene sequence analysis was combined. The biological characteristics, infection conditions, host resistance enzyme response, fungicides screening and control were studied.1. Damage and pathogen identification of leaf spot disease occurred in A. japonica After the investigation of field disease, the disease were founded in Fushun, Shenyang, Benxi, Xinbin, Qingyuan, Kuandian area,1-3 years old plants could be infected plants with. The agent mainly infect the bottom leaves with greywhite lesions and brown or dark brown edge, 2-3 diameters. Subsequently, lesion gradually expanded, merged with semi-buried dark brown pycnidia. Prematurely withered leaves with lesions crowded in the field, which seriously affected the seed maturity and root yield. The survey of 2014 showed the disease field incidence rate was 90-100%, disease plant rate was 69-88%, disease index was 32.3-41.9. According to the morphological characters of pathogens, culture characteristics, pathogenicity determination and the ITS and LSU gene sequence analysis and the phylogenetic tree constructed, the agent was identificated as Paraphoma chrysanthemicola (Hollos).2. Study on the biological characteristics of the pathogen. This paper systematically studied the biological characteristics of the pathogen, the determination of nutrition, temperature, pH value and light conditions on mycelial growth, spore production and conidium germination respectively. Results showed that the most suitable medium for colony growth was MEA medium; the optimum carbon source was soluble starch; the optimum nitrogen source was peptone and NaNC>3.Pathogen was strongly resistanted to acid and alkali, it could grown in pH 4-11, the optimum pH value is 5, the optimum temperature of 25℃, the lethal temperature to 48℃; urea on mycelial growth and conidia production has obvious inhibitory effect. Most suitable for spore production medium for PNA medium; the optimum carbon source was soluble starch and lactose; the optimum nitrogen source for yeast extract; the optimum pH was 7; the optimum temperature of 30℃; dark conditions favorable for spore production. Conidia in the relative humidity%could germinate, humidity is high, germination rate is high; the optimum temperature of 25℃; the optimum carbon source was mannitol; the optimum nitrogen source for (NH4)2SO4；the optimum pH value was 6; illumination for spore germination; lethal temperature is 46℃.3. Infection conditions and epidemic dynamics of leaf spot of A. japonica For inquiry the optimum condition of infection, the were determined incubation period, diseased leaf rate, lesion diameter and disease index at different temperatures, humidity, fungus age and leaf position. The results showed that, the formation of germ tube extension of hyphae invaded the stomas when conidia germination of 48 h; 72 h, hyphae was rapidly extended out from the stomas. In the range of 5-25℃, with the increase of temperature, leaf spot disease, the illness is aggravating gradually, pathogen under the condition of 25℃ showed high pathogenicity with 3 days of incubation period,52.5 of disease index and lesion with 3.29 cm; under the condition of 30℃, lesion growth was restricted. The occurrence of disease was 85% in the lowest humidity condition, when humidity is 100%, the disease index was 57.5 and the lesion diameter was 3.26 cm. Incubation period was 5 d when mycelium age were 7 d and 14 d.35 d age hyphae have a high pathogenicity, the incubation period was 3 d. Under the same conditions, the top leaves were difficult to be infected; the incidence rate was 20%. The bottom leaves of morbidity were 90%.To clear the field epidemic dynamics of A. japonica leaf spot disease, we continuous monitored the disease leaf rate, diseased plant rate and disease index of A. japonica leaf spot, under the conditions of natural infection in field in the growing season of 2015, and analyze the data. The results showed that the initial stage June 25, the major onset was from June 25 to September 21,over the September 21 was recession period. The disease index rose sharply in August, up to 55.9, the largest growth rate for the 0.76 disease index. In late September, leaves withered prematurely. Gompertz model can better reflect the dynamic of disease index with the time, and prediction the epidemic dynamics.4 Response of host resistance enzymes during pathogen infection In order to study the influence of resistance enzymes during the infection of the pathogen, the activities of PPO, PAL, CAT and POD were determined in this paper. The results showed that PPO activity respectively of the top leaves, middle leaves and bottom leaves were 1.48 U·mol-1·mg-1,12.87 U·mol-1·mg-1,5.69U·mol-1·mg-1; POD activity respectively were16.57 U·mol-1·mg-1,12.94 U·mol-1·mg-1,25.33U·mol-1·mg-1; CAT activity respectively were 0.86 U·mol-1·mg-1,0.48 U·mol-1·mg-1,0.50 U·mol-1·mg-1; PAL activity respectively were 1.58 U·mo-1·mg-1,0.85 U·mol-1·mg-1,0.97 U-mol-1·mg-1。Infection could lead to activity of CAT decended firstly and then ascended, the activity of PPO ascended. PAL activity of top leaves and middle leaves decended, the activity of POD ascended firstly and then decended. In conclusion, there were differences of the resistance responses rate and enzymes activity with different age of leaves.Binding of the pathogen conidia germination time and infection period shows that, at 12 h after inoculation, the pathogen infection caused by PPO activity increased, decreased the activity of POD, CAT and PAL, resistance enzyme of top leaves changed obviously than the middle leaves and bottom leaves, synergetic enhancement of enzyme activity, peak value advancement. The pathological reaction of external symptoms after inoculation of different leaf positions was consistent with the internal reaction.5. Fungicide screening and field control of A. japonica leaf spot Toxicity test results showed that the tested 8 fungicides of A. japonica leaf spot fungus mycelial growth and conidial germination had different degrees of inhibition.250 g/L propiconazole EC on the mycelial toxicity strongest EC50 values 1.2221 mg/L,300 g/L azoxystrobin SC bisection conidial virulence had the strongest effect and EC50 values for 0.0185mg/L. The drug test results showed that the concentration of the tested agents have a significant effect on the field A. japonica leaf spot of field control. Integrated medicine defence effect,300g/L benzoyl propiconazole EC 3000 times liquid control effect best, achieve 80.53%; 300 g/L azoxystrobin SC by 1500-6000 control effect were above 70%; 70% thiophanate methyl wettable powder 600 times,80% mancozeb wettable powder 800 times,430 g/L the diniconazole suspending agent 3000 times the control effect were in more than 70%, control effect respectively 76.09%,73.1% and 71.37%. |
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