Soybean (Glycine max (L.) Merr.) is one of the most important crops in Korea as a source of traditional food, especially as a protein source. Soybean seed quality can be reduced by several diseases, one of them is phomopsis seed decay (PSD) (Kmetz et al., 1978), which is extremely common on senescent plants in soybean production areas (Cho & Kim, 2000; Denis, 1992). PSD of soybean is caused by Diaporthe-Phomopsis complex (DPC), Phomopsis longicolla T.W. Hobbs and Diaporthe phaseolorum (Cooke & Ellis) Sacc., which has three varieties, D. phaseolorum var. sojae (S.G. Lehman) Wehmeyer, D. phaseolorum var. caulivora K.L. Athow & R.M. Caldwell, and D. phaseolorum var. meridionalis F.A. Fernandez (Sinclair & Backman, 1989).
Soybean seeds infected by phomopsis have a great effect on soybean grade components and composition as well as on the quality of soybean flour and oil (Hepperly & Sinclair, 1978; Sinclair & Backman, 1989). The diseased seeds may have undesired appearance and can lead to lower seed viability (Fig. 1.), deterioration, and lower prices for producers (RDA, 2006; Sinclair & Backman, 1989).
Environmental conditions are important in the development and severity of PSD (Zimmerman & Minor, 1993). When high humidity and warm temperatures persist during soybean maturation period, pod infection by DPC can increase and it leads to PSD (McGee, 1983). Delayed harvest can also increase the infection of PSD (Balducchi & McGee, 1987; Duncan et al., 1989; Kmetz et al., 1978; Spilker et al., 1981; Yoon & Chu, 2003). Therefore, PSD is a large problem in soybean production areas with these environmental conditions.
Although chemicals and cultivation methods have been suggested as a means of controlling and escaping PSD, to develop resistant cultivar is considered to be the most effective method (Denis, 1992; Thomison et al., 1987; Thomison et al., 1988; Wilcox et al., 1985). Control of PSD by genetic resistance in soybean has not been well studied. Plant introduction (PI) 417479 and PI 80837 which is resistant to PSD have received the most study of the genotypes reported with PSD resistance (Brown et al., 1987; Jackson et al., 2005; RDA, 2006). The resistance to PSD of PI 417479 is conferred by a single dominant gene under nuclear control and that of PI 80837 is conferred by two different dominant genes (Jackson et al., 2005). The objective of our study is to identify novel fungal isolates obtained from infected stems. And we tried to find out suitable growth conditions of these isolates to be used as a disease agent for resistance screening of breeding lines and to evaluate the disease reaction of phomopsis seed decay.
MATERIALS AND METHODS
Pathogen Isolation and Optimum Condition for Developing Conidia
For a study of the PSD, three samples with black fruiting structures in linear rows on senescent soybean stem were collected from the soybean research fields at AVRDC-The World Vegetable Center in Taiwan in 2008. Small pieces of infected tissues including healthy parts were sliced off and put on a stainless metal mesh and then covered with another metal mesh. They were dipped into 1% sodium hypochlorite solution for 1 min to disinfect surface and washed excess 1% NaClO3 with sterilized water. Each small piece of disinfected tissues was put on 3 different media such as potato dextrose agar (PDA), V8 (10% filtered V8 juice, pH 6.0), and water agar (2% WA) to find out adequate media to develop pycnidia. They were incubated at 28°C with 24 hr photoperiod until mycelia occupied fully the three different petri dishes. Three days later, colony on PDA were placed on 2% water agar plates and then three single conidia were cleaned on the agar plate under a stereomicroscope and inoculated on PDA media. These isolates obtained from incubation of the three single conidia were used in this experiment.
When mycelia grew well on the three types of media, each media was punched on the petri dish using sterilized wood borer No. 2 in a diameter of 5 mm and one piece was transferred to the three kinds of media plate. To determine the optimum conditions (temperature and photoperiod) for developing pycnidia and conidia, the plates were put into 3 temperature condition (20, 24, 28°C), 2 photoperiod condition (24 hr, 15 hr), and 24 hr dark condition as a control condition.
Characterization of Isolated Pathogens
Mycelia, pycnidia and conidial spores of isolated fungi were observed under a stereomicroscope. To analyze genotypic characteristics with morphological characteristics, DNA was extracted and amplified by the internal transcribed spacer (ITS) primer set, ITS4 (5'-TCCTCCGCTTATTGATATGC- 3') and ITS5 (5'-GGAAGTAAAAGTCG TAACAAGG-3') primers, and then patterns by restriction fragments length polymorphisms with AluI, AfaI were observed (Zhang et al., 1999; Zhang et al., 1998).
Preparation of Media and Inoculums
Mature conidia were harvested with 15 ml of distilled water (0.5% Tween 20). The surface of the colonies was gently rubbed with an autoclaved slide glass to dislodge conidia. The mycelia/ conidia suspension was filtered through two layers of cheesecloth to remove mycelia fragments. The conidia suspension concentration was counted by a hemacytometer counter (Hausser Scientific, Horsham, PA).
Artificial Inoculation
Two soybean cultivars as plant materials, Taiwan cultivar, TK-5 and Korean relatively early maturing cultivar “Seonyu” were used to artificial inoculation. Inoculation was performed at 7 week-old-plant after sown. The inoculation of two parts with the novel isolates was performed. For inoculation on leaf-stem part, the first trifoliate leaf of each plant was labeled with paper tags and stem was marked at the lowest three nodes from the ground. For pod inoculation, the oldest pod in its growth stage was labeled with tags in each plant, and all pods of node with tagged pod and pods at one upper node of tagged pod were inoculated, when pods of Seonyu reached to R5~R6 growth stage (GS), but pods of TK-5 reached to R4~R5 growth stage (GS).
Because of the slow growth speed of isolate1 among three isolates, isolate2 and isolate3 except isolate1 were used to inoculation at 1.55 × 107 conidia/ ml concentration with an atomizer until runoff. Inoculated plants were incubated in a dark dew chamber at 25°C and 100% RH (relative humidity) for 2 days after inoculation. The plants were then moved into the greenhouse. Disease severity of leafstem was assessed at 3 weeks after inoculation based on percent leaf area infected, and disease severity of pod was evaluated based on the infected area proportion when they reached harvest time. To investigate phomopsis seed decay infection according to the inoculation part, pods were threshed at mature stage and the number of seed with phomopsis seed decay symptom was counted.
RESULT AND DISCUSSION
Optimum Condition for Developing Conidia
Among 3 kinds of media, PDA, V8 and WA (2%), mycelia growth rate on WA media was very slow, which may have resulted from insufficient nutrients. Mycelia on V8 media grew well as much as that of PDA media, but pycnidia didn't develop. Finally, PDA media was chosen to adequate media for inducing pycnidia. From different temperature conditions (20, 24, 28°C), the 28°C induced too many water drops on the lid of the petri-dish in comparison with the others, which fell down onto the media surface and then media over dried with time. It was important that the media plates did not dry out quickly, as they were required to incubate for more than 10 days to induce pycnidia. In both PDA and V8 media, growth of mycelia at 24°C was faster than it of 20°C. For this reason, 24°C was chosen for culturing fungi in these experiments. For the light conditions we expected that photoperiod would have an effect on pycnidia growth but photoperiod did not have a critical effect on development of pycnidia. Different degree of developing mycelia and pycnidia between 24 hr and 15 hr (15 hr light, 9 hr dark) photoperiod was not found. In dark condition, however, mycelia were induced but pycnidia were not induced. This was carried out once the mycelia covered the plates uniformly, to induce conidia development, it was necessary to rub the surface of the plate.
Morphological and Genotypic Characteristics
Unidentified three fungal isolates were isolated from stems with black fruiting structures in linear rows on senescent soybean. The fungal isolates obtained by surface disinfection of infected stems were cultured on PDA media. Colonies of the isolates showed ropelike white mycelia with yellowish tone (Fig. 2(a)). Under a stereomicroscope (400 X) alpha conidia and beta conidia were observed (Fig. 2(b), 2(c)). All isolates produced alpha conidia which size were 8.78 × 3.32 (7.00 to 11.00 × 3.00 to 4.00) μm, and sporadically, beta conidia of 30.58 × 0.85 (26.00 to 33.00 × 0.60 to 1.20) μm but perithecia were not observed in the collected stem samples or the fungal cultures on PDA (Chen et al., 2009).
A genotypic characteristic was observed by restriction fragment length polymorphism (RFLP) patterns of the PCR products of three isolates. After they were amplified with ITS primer sets, ITS4 and ITS5, the PCR products were cut by two restriction enzymes, AluI and AfaI (Fig. 3). Three isolations were cut into two pieces by AluI, but were not cut by AfaI. P. longicolla should be cut two pieces by AfaI. PCR-RFLP patterns by ITS4 and ITS5 for all isolates were identical to the patterns reported for Diaporthe phaseolorum var. sojae (Zhang et al., 1998). Based on morphological and genotypic characteristics, three isolations that we isolated from infected soybean stems were regarded as D. phaseolorum var. sojae.
Severity of Disease Symptom on Leaf and Pod and Effect of the Inoculation Part on Phomopsis Seed Decay
Leaf symptom was assessed at 3 weeks after inoculation. Disease severity of leaf was evaluated based on percent leaf area infected within five scales (Fig. 4(a) ~ (e)): 0 = no symptom (a), 1 = less 5% (b), 2 = 5 ~ 25% and/or small stem (petiole) symptom less than 2 mm (c), 3 = 25 ~ 50% and/or extensive stem (petiole) symptom more than 2 mm (d), 4 = over 50% (e).
Disease lesions on soybean pods were assessed when they were the time of harvest as the disease symptom on pods could not develop until it reached mature stage. Five scales of disease severity were developed based on the infected area proportion (Fig. 5(a) ~ (d)): 0 = no symptom (not shown), 1 = less 5 % (a), 2 = 5 ~ 25% (b), 3 = 25 ~ 50% (c), 4 = over 50% (d).
To investigate degree of PSD infection according to the inoculation part, seeds were removed from the pods which obtained by the each inoculation part at the same time with harvest and disease severity of phomopsis seed decay was evaluated as percent of infected seed number.
Pycnidia could be found after they were dry enough to thresh. We could only harvest the Seonyu cultivar to evaluate PSD, because TK-5 didn't reach the mature stage. The incidence of soybean PSD of Seonyu was assessed in two parts, inoculated on leaves-stem and on pods at GS R5. In the result of two inoculation parts with two isolates, there was no significant difference in degree of pod infection and seed infection (%) between isolate2 and isolate3, but there was a tendency that pod inoculation than leaf and stem inoculation caused higher level of seed infection (Table 1.).
적 요
미이라병은 Diaporthe/Phomopsis complex에 의해 유발되는 병으로 콩 재배기간 중 따뜻하고 습한 환경에서 종자가 성숙 되면 감염률이 높아지며 감염된 콩 종자는 외관상 품질뿐만 아니라 종자 활력이 저하된다. 미이라병에 대한 연구를 수행 하기 위하여 대만에 위치한 아시아채소개발연구센터(AVRDC) 의 콩 시험포장에서 미이라병 병징을 보이는 콩 줄기를 채집 하고 이로부터 3개의 곰팡이 균주(isolate)를 분리하였다. 배지 위에서의 곰팡이 균사의 생육특성, 현미경하에서 관찰된 알파, 베타 분생자(conidia)의 모양 그리고 PCR-RFLP 분석으로, 3 개의 균주는 Diaporthe phaseolorum var. sojae 으로 확인되 었다. 한편, 미이라병 저항성 육종을 위해서는 유전자원과 계 통의 검정이 선행되어야 하는데, 인공접종을 위해서 분생자의 최적 배양조건을 탐색하였다. 그 결과 배지는 PDA, 온도는 24°C에서 잘 배양되었으며, 일장은 암조건에서는 균사체만 유 도되고 분생자는 유도되지 않았으며, 24시간과 15시간의 일장 에서는 균사체 유도 및 분생자의 유도 정도에 차이가 없었다. 또한 잎-줄기와 꼬투리, 두 개의 접종 부위에 따른 미이라병 감염률을 조사하였는데, 두 접종 부위에 따른 미이라병 감염 정도는 통계적인 유의차는 나타나지 않았으나 잎-줄기에 접종 한 개체 보다 꼬투리에 접종한 개체의 종자 감염률이 높은 경 향을 보였다.
