Strawberry Fusarium wilt disease, caused by Fusarium oxysporum f. sp. fragariae, is the most devastating disease in strawberry production. The pathogen produces chlamydospores which tolerate against harsh environment, fungicide and survive for decades in soil. Development of detection and quantifica...
Strawberry Fusarium wilt disease, caused by Fusarium oxysporum f. sp. fragariae, is the most devastating disease in strawberry production. The pathogen produces chlamydospores which tolerate against harsh environment, fungicide and survive for decades in soil. Development of detection and quantification techniques are regarded significantly in many soilborne pathogens to prevent damage from diseases. In this study, we improved specific-quantitative primers for F. oxysporum f. sp. fragariae to reveal correlation between the pathogen density and the disease severity. Standard curve $r^2$ value of the specific-quantitative primers for qRT-PCR and meting curve were over 0.99 and $80.5^{\circ}C$, respectively. Over pathogen $10^5cfu/g$ of soil was required to cause the disease in both lab and field conditions. With the minimum density to develop the wilt disease, the pathogen affected near 60% in nursery plantation. A biological control microbe agent and soil solarization reduced the pathogen population 2-fold and 1.5-fold in soil, respectively. The developed F. oxysporum f. sp. fragariae specific qRT-PCR protocol may contribute to evaluating soil healthiness and appropriate decision making to control the disease.
Strawberry Fusarium wilt disease, caused by Fusarium oxysporum f. sp. fragariae, is the most devastating disease in strawberry production. The pathogen produces chlamydospores which tolerate against harsh environment, fungicide and survive for decades in soil. Development of detection and quantification techniques are regarded significantly in many soilborne pathogens to prevent damage from diseases. In this study, we improved specific-quantitative primers for F. oxysporum f. sp. fragariae to reveal correlation between the pathogen density and the disease severity. Standard curve $r^2$ value of the specific-quantitative primers for qRT-PCR and meting curve were over 0.99 and $80.5^{\circ}C$, respectively. Over pathogen $10^5cfu/g$ of soil was required to cause the disease in both lab and field conditions. With the minimum density to develop the wilt disease, the pathogen affected near 60% in nursery plantation. A biological control microbe agent and soil solarization reduced the pathogen population 2-fold and 1.5-fold in soil, respectively. The developed F. oxysporum f. sp. fragariae specific qRT-PCR protocol may contribute to evaluating soil healthiness and appropriate decision making to control the disease.
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제안 방법
Those finding also reproduced in strawberry nursey fields. Application of the diagnosis protocol was not only confirmation of control the disease but also contributed reviewing of agronomical control procedure to observe variation of the pathogen density through calculation of the biomass in the soil.
In this study, we developed the strawberry Fusarium wilt pathogen specific qRT-PCR system to increase accuracy of FOF quantification in both lab and field condition. The system demonstrated that correlation between the pathogen density and the disease incidence/severity.
, San Jose, CA, USA). Quantitative detection based on qRT-PCR was conducted to calculate the population in three times (0, 3 and 5 weeks).
Soil sterilization as solar heat method was testified in main cultivation area of the two greenhouses above mentioned. Soil samplings were conducted at before sterilization and after sterilization with 3 replications.
Both types of soil were sterilized two times at 121℃ for 15 min. The pathogen spore density was measured by Haemacytometer (SUPERIOR,Lauda-Konigshofen, Germany) under a microscope and calculated spore density to mix into in the soil media and raw soil, different density of the pathogen spores was amended (102, 104 and 106) with three replications. Chromosomal DNA per ng based on average weight of nucleotides, which is 650 Dalton per nucleotide.
oxysporum. The primer set was designed based on sequences of two transposable elements Han and Skippy, which existed only F. oxysporum strains or races. The transposable elements were reported from F.
Plants were incubated in a growth chamber (25℃; 16 h light/8 h dark cycle), and the severity of disease were observed every 7 days up to 35 days after planting. The severity of the disease was evaluated using a 0-5 scale, every week: 0, healthy; 1, 1 to 3 leaves rolled and yellowed; 2, 3 to 4 leaves rolled and deformed; 3, chlorosis and early plant wilting; 4, necrosis and entire plant wilting; and 5, dead or nearly so. The results were statistically analyzed by Tukey’s HSD test (with significance set at p = 0.
Sulhyang) were planted in each pot. The treatments were arranged in a complete randomized block design and replicated five times. Plants were incubated in a growth chamber (25℃; 16 h light/8 h dark cycle), and the severity of disease were observed every 7 days up to 35 days after planting.
Although various chemical fungicides have been tried to control strawberry Fusarium wilt disease, no reliable fungicide has been registered yet. Therefore, in this study, efficiency of eco-friendly fungicides included Phoscraft, Curamin, NaDCC, and S4-7 were evaluated in terms of the disease control and the pathogen population with the qRT-PCR primer set. A microbial agent, S4-7 was reported by Cha et al.
대상 데이터
, 2016) were evaluated their efficiency in the disease prevent. The agents were treated as Phoscraft (1000 times dilution), Curamin (2000 times dilution), NaDCC (100 ppm), 106 cfu of S4-7 and the pathogen (F9 strain) was inoculated at 105 cfu in each treatment. After 5 weeks of the treatment, the samples randomly collected from 3 pots of each treatment.
To evaluate efficiency of S. griseus S4-7 on the pathogen suppression in field condition, strawberries (cv. Sulhyang) in two greenhouses which place in JinJu, Gyeongnam province were employed. Each greenhouse was divided into two sections: treatment and control areas replicated 3 times for each section, one of replication consist of 15 mother plants and their offspring plants were only treated in treatment area with 106 cfu of S4-7 in Jun 9th and Jul 6th, 2016 and soil sampling also carried out at same days.
데이터처리
The results were statistically analyzed by Tukey’s HSD test (with significance set at p = 0.05) using Sigma Plot ver. 11.0 (Systat software INC., San Jose, CA, USA).
성능/효과
Total 4 different densities of the pathogen were treated in pot which have been planted strawberry plants. (A) Disease index was evaluated 5 weeks after planting and severity of the wilt were evaluated on a 0-5 scale; 0 = healthy, 1 = 1-3 leaves rolled and yellowed, 2 = 3 leaves rolled and deformed, 3 = chlorosis and early plant wilting, 4 = necrosis and entire plant wilting, and 5 = dead. (B) Soil samples were amplified by qRT-PCR and calculate population of the pathogen.
The pathogen was inoculated 105 cfu/gram of soil, the agents were treated only one time when the strawberry planted. (A) Disease index was performed 5 weeks after inoculation and severity of wilt were evaluated on a 0-5 scale; 0 = healthy, 1 = 1-3 leaves rolled, and yellowed, 2 = 3 leaves rolled and deformed, 3 = chlorosis and early plant wilting, 4 = necrosis and entire plant wilting, and 5 = dead. (B) At 5 weeks, soil DNA were extracted and amplified with the qRT-PCR primer set to calculate population of the pathogen.
To check, detection limit in both soil media and raw soil, the F9 spores were inoculated in both soils at 106, 104 and 102 cfu/g of soil concentration. As result, the qRT-PCR with 106 and 104 cfu/g of soil samples detected the pathogen level as 106, 105 cfu/g of soil. However, the qRT-PCR failed to distinguish between 102 and 103 pathogen densities in soils (Fig.
, 2016). Five weeks after the treatments, Phoscraft, Curamin and NaDCC and positive control (F9, 105 cfu/g of soil) showed severe the disease occurrence, the disease severity scores were 4. However, S4-7 treated strawberry plants were showed the disease severity less than score 2 (Fig.
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