A Practical Method for Forecasting Strawberry Anthracnose Caused by Glomerella cingulata Using an Ethanol-spray Treatment in Nara Prefecture, Japan

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1 関西病虫研報 (57):25-29(2015) 原著論文 Ann. Rept. Kansai Pl. Prot. (57): (2015) 25 A Practical Method for Forecasting Strawberry Anthracnose Caused by Glomerella cingulata Using an Ethanol-spray Treatment in Nara Prefecture, Japan Yoshihiko Hirayama*, Shunsuke Asano*, Masaharu Kubota**, Motoaki Tojo*** and Satoshi T. Ohki*** Summary An ethanol-spray treatment was evaluated as a practical method for forecasting strawberry anthracnose caused by Glomerella cingulata in Nara Prefecture, Japan. Evaluations were conducted on latent infections of the pathogen to determine the disease occurrence in commercial strawberry nurseries from 2010 to The latent infection was highest in early July and corresponded highly to disease occurrence throughout the experiments. The results showed that the ethanol-spray treatment was a reliable and useful method for forecasting anthracnose in strawberry nurseries. Introduction Glomerella cingulate (Stonem.) Spauld. & Schrenk (anamorph Colletotrichum gloeosporioides (Penz.) Penz & Sacc. in Penz.) is a devastating pathogen that causes anthracnose disease of strawberry throughout the world (Howard and Albregts, 1984). Distribution of the pathogen has been extended due to an increase in the use of disease-susceptible cultivars of strawberry in Japan. In Nara Prefecture, this disease in strawberry nurseries has been managed by rain shedding, ebb flow irrigation and drip watering (Okayama, 1993). These are effective because the pathogen spores disperse by water on the ground (Ntahimpera et al., 1999). Scheduled fungicide applications are also effective in suppressing the disease (Inada et al., 2005; MacKenzie et al., 2009; Hirayama, 2009). To increase the effectiveness of these control measures, reliable and useful forecasting systems for the disease have been required. MacKenzie and Peres (2012) developed an effective forecasting system of strawberry anthracnose using leaf wetness and temperature in Florida, U. S. This system, however, may be difficult to use in predicting strawberry anthracnose in Japan. This is because the disease usually occurs in the nursery stage and rain shedding, which controls leaf wetness, has been widely introduced in the country. Therefore, several other methods have been proposed to predict the disease. They are, for example, an ethanol immersion treatment (Ishikawa, 2003), a selective medium (Okayama et al., 2007) and PCR (Mills et al., 1992, Hirayama et al., 2008). All these methods can detect both obvious and latent G. cingulate in strawberry plants. Among all of them, the ethanol immersion treatment is the easiest and can be performed without any special equipment. This method facilitates the production of salmon-pink conidial masses on strawberry leaves due to sterile effects on other fungi. We recently developed an ethanol-spray treatment which is modified from the ethanol immersion treatment. This treatment is easier to perform than the ethanol immersion treatment since only ethanol spraying is required. In this study, we evaluated the ethanol-spray treatment as a practical forecasting of strawberry anthracnose in nurseries of commercial strawberry productions. Materials and Methods Plant materials Strawberry plants (Fragaria ananassa Duch.) cultivar Asukaruby was used in this study. The cultivar was used because it was the most popular cultivar of commercial strawberry production in Nara Prefecture and was suscep- *Nara Prefectural Agricultural Research and Development Center, Kashihara, Nara , Japan, **Institute of Vegetable and Tea Science, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, , Japan, ***Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka , Japan Corresponding author: Yoshihiko Hirayama (Tel: ) Accepted February 12, 2015

2 26 関西病虫研報 (57)2015 tible to G. cingulate. Inoculum preparation and application Inoculum preparation and inoculation were conducted in September 2010 as follows. Glomerella cingulata isolate Nara-gc5 from a strawberry plant in Nara Prefecture was used (Okayama et al., 2007). The fungal isolate was cultured in an Erlenmeyer flask containing 100 ml of potato sucrose broth on a rotary shaker at 120 rpm at 28 C for 10 days under an ambient light conditions. Conidia of the fungal isolate were suspended in sterile distilled water at a concentration of conidia/ml. Five ml of the conidial suspension was sprayed on to the whole surface of strawberry plants which were grown from runner shoots for 60 days. The inoculated plants were maintained in a greenhouse at 15 C to 30 C. The lowest leaves of the plant with no visible symptom of anthracnose were collected two weeks after inoculation. Experiments were repeated three times with twenty leaflets for each treatment. An ethanol-spray treatment An ethanol-spray treatment for the detection of a latent G. cingulate infection was performed using the following method. The lowest leaves were washed thoroughly in running tap water and air dried. The whole leaf was sprayed with 70% ethanol, kept for 30 sec and rinsed in running tap water. The leaves were placed in plastic bags with a zippertype closure, moistened with distilled water and incubated at 28 C in the dark. The number of leaves forming conidial masses that could be seen with the naked eye was determined 14 days after the treatment. At the same time, a portion of colonies were examined microscopically to confirm the identity of the fungus. Percentage of conidial mass formation (the number of leaves with conidial mass / the number of leaf tested 100) was calculated as the infection rate of G. cingulate. An ethanol-immersion treatment An ethanol-immersion treatment was conducted by Ishikawa (2003). Strawberry leaves were dipped into 70% ethanol for 30 seconds. Other procedures were performed as described above. Field investigation Strawberry plants were investigated at a total of 82 commercial strawberry production fields in Nara Prefecture distributed across three cities and one village including 26 locations in Yamato-Koriyama City (34 39 N, E) from 2010 to 2014, 28 locations in Asuka Village (34 28 N, E) from 2010 to 2014, 19 locations in Tenri City (34 36 N, E) from 2011 to 2014, and 9 locations in Sakurai City (34 31 N, E) from 2010 and Twenty strawberry plants were randomly selected from each of eighty-two fields (Fig. 1) in the nursery stage. One of the lowest leaves from each of the 20 plants was collected. The presence of a latent G. cingulate infection was examined by the ethanol-spray method described above. The research was conducted in mid-june, early July and late July from 2010 to Occurrence of anthracnose disease was examined in the field-grown stage in the 82 strawberry fields (Fig. 1) from mid-june to early December every year from 2010 to The examination was performed on all the plants which were shown to have a latent infection of G. cingulate in the nursery stage by the ethanol-spray treatment. Five hundred strawberry plants were examined in each field through the five years. Statistical analyses of data Pearson s correlation coefficient analysis and simple regression were used to assess the relations between the mean of the latent infection rate and the mean proportion of the diseased strawberry plants. Results Comparison between ethanol-spray treatment and immersion treatment for the detection of a latent Glomerella cingulata infection The detection rate of the latent pathogen infection in leaves was 80.5% and 86.1% in the ethanol-spray treated and ethanol-immersion treated plants, respectively (Fig. 2). There were no significant differences in detection rates between the treatments in Student s t-test. Latent infection rate of Glomerella cingulata in strawberry in the nursery stage Rates of the latent infection varied among the years tested (Table 1). The infection rate during those five years was always highest in early July except in 2010 and 2011 when the test was conducted only in early July. Relationships between the latent infection rate of Glomerella cingulate in the early-nursery stage and anthracnose symptom development in the latenursery and the field-grown stages Anthracnose occurrences were initially observed in the early-nursery stage in late July and incidence increased in

3 Hirayama, Asano, Kubota, Tojo and Ohki: A practical forecasting method of strawberry anthracnose 27 Fig. 1. Locations investigated (circles) for latent Glomerella cingulata infection and anthracnose occurrence in strawberry production fields in Nara Prefecture, Japan. Table 1. Latent infection rate of Glomerella cingulate detected using an ethanol-spray treatment in commercial strawberry cultivation in Nara Prefecture from 2010 to Years Latent infection rates (%) Mid Jun Early Jul Late Jul 2010 ND 41.0 ND 2011 ND 44.2 ND Av Fig. 2. Comparison between ethanol-spray treatment and immersion treatment for detection of Glomerella cingulate on strawberry leaves under artificial inoculation conditions. Error bar measures standard error of mean. There were no significant differences between the treatments in Student s t-test (p > 0.05). late August (Fig. 3). This high level was kept until early November, although it varied among the examined years. The disease occurrence decreased in early December. The correlation coefficient analysis showed that there was a statistically significant (p < 0.002) correlation (R 2 = ) N = 300 (15 fields 20 leaves) in 2010, 380 (19 fields 20 leaves) in 2011, 320 (16 fields 20 leaves) in 2012, 2013 and ND: Not determined. Av.: Average from 2012 to between the latent infection rate of G. cingulata estimated using the ethanol-spray treatment in early July and the disease occurrence in late August to early November (Fig. 4). The disease readily developed in the fields where the high latent infection was observed. Discussion Since a labor-saving forecasting method was needed for strawberry anthracnose detection, the ethanol-immersion treatment (Ishikawa 2003) was modified simply by using

4 28 関西病虫研報 (57)2015 Fig. 3. Anthracnose occurrence in commercial strawberry cultivation from nursery stage to field-grown stage in Nara Prefecture from 2010 to N = 7,500 (15 fields 500 plants) in 2010, 9,500 (19 fields 500 plants) in 2011, 8,000 (16 fields 500 plants) in 2012, 2013 and Fig. 4. Relationships between the latent infection rates of G. cingulata estimated by ethanol-spray treatment in early July and the disease occurrence in late August to early November in commercial strawberry fields in Nara Prefecture from 2010 to Simple regression analyses were performed by Pearson s correlation coefficient test. an ethanol-spray. This ethanol-spray treatment is easier to apply the plant than the ethanol-immersion treatment. It also requires a smaller amount of ethanol than the ethanolimmersion treatment. Comparisons between the ethanolimmersion and the ethanol-spray treatments showed that they were equivalent in detecting the latent infection of G. cingulate in strawberry leaves. This result suggests that the ethanol-spray treatment can be a reliable and useful method for forecasting anthracnose in strawberry nurseries. In order to confirm the effectiveness of the ethanolspray treatment in forecasting anthracnose development, we examined it under field conditions. The results showed that there was a strong positive correlation between the latent infection rate of G. cingulata estimated by ethanolspray treatment in early July and the disease occurrence in late August to early November in commercial strawberry fields in Nara Prefecture from 2010 to This suggests that the ethanol-spray treatment can serve as a practical forecasting system of strawberry anthracnose caused by G. cingulate. The optimum temperature for the growth of G. cingulata is 28 C (Smith and Black, 1987). Therefore, the pathogen may increase its population in the stages from mid-june to late August in Nara Prefecture. However, the results showed that the latent infection rate of the pathogen was lower in late July than that in early July. This may be due to the timing of routine fungicide application in commercial strawberry fields in Nara Prefecture. In strawberry fields, the interval between fungicide applications has often been shortened in mid-june (Hirayama, 2009). Another reason may be the decrease in humidity at the end of the rainy season in the area in mid-july. Using this ethanol-spray treatment, it takes 2 weeks to complete pathogen detection. These factors suggest that the forecasting method should be performed before early July in Nara Prefecture. In this study, we only used G. cingulate, because all the isolates detected in the investigated nurseries were identified as G. cingulate (data not shown). This result suggests that G. cingulate can be considered as a predominant anthracnose pathogen in strawberry production in the area. C. acutatum, another important pathogen of strawberry anthracnose, widely occurs in other areas of Japan (Ishikawa 2003). G. cingulate and C. acutatum have been known to exist in the same field (Howard et al., 1992). Moreover, G. cingulate has different sensitivity to fungicides with C. acutatum. Ishikawa (2004) reported ethanol immersion method was also useful for diagnosing latent infection of strawberry plants by C. acutatum. These indicate that a research to confirm the efficacy of the ethanol-spray treatment to detect C. acutatum should be required in the future. Fungicides are usually applied according to the plant protection calendar in commercial strawberry fields in Nara Prefecture. Since anthracnose occurrence shows year to year variation, the timing of fungicide application is important to control strawberry anthracnose. The fungicide application s timing has been estimated based on leaf wetness and temperature in strawberry production in Florida, U. S. (MacKenzie and Peres, 2012). This method is unsuitable

5 Hirayama, Asano, Kubota, Tojo and Ohki: A practical forecasting method of strawberry anthracnose 29 for Japan because leaf wetness is usually controlled by rain shedding. The present study demonstrated that the timing of fungicide application can be, at least roughly, estimated by a detection of latent infection using the ethanol-spray treatment. Acknowledgments This study was supported, in part, by the Plant Protection Division of the Ministry of Agriculture, Forestry and Fisheries. We gratefully acknowledge Dr. Seiju Ishikawa, Hosei University, Dr. Shinya Tsuda, the National Agricultural Research Center, the National Agriculture and Food Research Organization, and the late Mr. Masahiro Nishizaki, for their valuable comments, and Kana Ueda, Nara Plant Protection Association, for technical assistance. References Hirayama, Y., T. Suzuki, Y. Ito, K. Okayama, M. Nishizaki and S. Matsutani (2008) Jpn. J. Phytopathol. 74: 198. (Abstr. in Japanese) Hirayama, Y. (2009) Plant Protection 63: (in Japanese) Howard, C. M. and E. E. Albregts (1984) Plant Dis. 68: Howard, C. M., J. L. Mass, C. K. Chandler and E. E. Albregts (1992) Plant Dis. 76: Inada, M., J. Yamaguchi and A. Furuta (2005) Kyushu Pl. Prot. Res. 51: (in Japanese) Isikawa, S. (2003) J. Gen. Plant Pathol. 69: Isikawa, S. (2004) J. Gen. Plant Pathol. 70: MacKenzie, S. J., J. C. Mertely and N. A. Peres (2009) Plant Dis. 93: MacKenzie, S. J. and N. A. Peres (2012) Plant Dis. 96: Mills, P. R., S. Sreenivasaprasad and A. E. Brown (1992) FEMS Microbiol. Lett. 98: Ntahimpera, N., L. L. Wilson, M. A. Ellis and L. V. Madden (1999) Phytopathology 89: Okayama, K. (1993) Ann. Phytopath. Soc. Japan 59: Okayama, K., Y. Hirayama and M. Nishizaki (2007) Jpn. J. Phytopathol. 73: (in Japanese) Smith, B. J. and L. L. Black (1987) Plant Dis. 71: