Sprout Rot Research Articles

Investigation of soybean sprout rot and the elimination of improper seeds for quality control on soybean seedlots

Soybean sprouts have been a considered a nutrient-rich vegetable for hundreds of years. To evaluate the seedlot quality of soybean sprouts grown, and to evaluate a method for reducing the presence of improper seeds in soybean seedlots, microbes associated with soybean sprout rot were isolated from samples collected. Morphological characteristics and gas chromatography profiles of the cultured fungal and bacterial strains were identified. Eight types of improper seeds were identified: purple stain(Ps), black rot(Br), seed coat black spot(Cb), wrinkled seed(Ws), brown hilum(Bh), seed coat fracture(Cf), unripe seed(Us), and brown seed coat(Bc). The improper seeds were also dipped into 15%, 20%, and 25% NaCl solutions, as well as a saturated solution of NaCl, for 1min. As the NaCl concentration increased, the number of floating improper seeds increased as well. The highest floating rates were observed for the Cf seeds.

Evaluation of biological seed treatments in combination with management practices for the control of Fusarium dry rot of potato

Seed-borne diseases of potato represent a significant constraint to potato production in the US. The use of an effective fungicide in combination with good management practices during cutting and storage, prior to planting, is essential to reducing disease. The efficacy of two biocontrol agents (Bacillus subtilis and Trichoderma harzianum), and a commercially formulated mixture of the chemicals fludioxonil plus mancozeb, applied as seed treatments in combination with different management practices, were evaluated over two years for the control of seed piece decay and sprout rot caused by Fusarium sambucinum. Treatments were made 10days prior to planting and at planting, and tubers were re-stored at either 18°C and 95% RH with forced air ventilation at 5950lmin−1 (optimal conditions), at 25°C in the dark without ventilation (sub-optimal), or not stored at all prior to planting. Seed piece and sprout health were evaluated in vitro and agronomic impacts evaluated in field experiments. Results showed that the biological control agents B. subtilis and T. harzianum provided good control of sprout rot and seed piece decay caused by F. sambucinum, when seed was re-stored under optimal conditions or not re-stored at all. Under optimal conditions, treatment with B. subtilis reduced sprout rot and seed piece decay on average by 66% and 84%, respectively. Treatment with T. harzianum reduced sprout rot and seed piece decay on average by 70% and 81%, respectively. Treatment with fludioxonil+mancozeb reduced sprout rot and seed piece decay under both re-storage regimes. Under optimal conditions, disease incidence and severity was reduced on average by 81% and 97%, respectively. Neither biological control agent reduced seed piece decay incidence under either re-storage regime compared to the untreated controls.

Pseudomonas sp. 유래 녹두 부패병의 병 저항성 녹두 계통 검정

녹두나물(숙주나물)은 국내뿐만 아니라 세계적으로도 널리 이용되고 있는 채소다. 그런데 녹두나물 재배를 하는 과정에서 발생되는 녹두나물 무름병은 녹두나물 생산량은 물론 품질을 심각하게 저하시킨다. 본 연구에서는 녹두나물 부패 조직으로부터 70계통의 병원균을 분리하였으며, 각 병원균의 병원성을 검정하였다. 그 가운데 강한 병원성을 가진 Pseudomonas 균류의 계통 YV-St-033를 확인하여 선발하였으며, 분리된 병원균계의 16S rRNA 유전자 염기서열을 분석하고 유전학적 유연관계를 분석하였다. YV-St-033는 P. mosselii, P. putita, P. fluorescens, P. entomophila, P. lecoglossicida 등의 종이 속한 그룹으로 확인이 되었으며, Pseudomonas mosselii R10 strain과 가장 높은 염기서열 identity (약 99%)를 보였다. 또한 YV-St-033 strain을 이용하여 영남대학교에서 보유하고 있는 녹두 유전자원들에 대해 녹두나물 무름병 저항성을 검정하였다. 3일간 배양한 녹두에 병원균을 접종하고 녹두의 생장율을 비교한 결과 YV148 line에서 높은 저항성이 확인되었으며, 그 외 녹두 계통에서도 부분적인 저항성을 나타내었다. 숙주나물 무름병에 저항성을 보인 YV 148 계통은 나물이 가늘고 연하고 생장율이 우수하여 앞으로 품종 육종의 좋은 재료로 활용될 수 있을 것으로 판단되었다. Mungbean sprout rot is one of the most serious problems of the commercial mungbean sprout industry. In this study, 70 strains of mungbean sprout rot pathogens were isolated from rotten sprouts at different time intervals. The pathogenicity of the isolated pathogens was tested. The highly pathogenic strain (YV-St-033) was identified as Pseudomonas sp. by 16S rRNA gene sequencing. In phylogenetic analysis, the YV-St-033 strain was grouped with P. mosselii, P. putita, P. fluorescens, P. entomophila, and P. lecoglossicida. The results of the 16S rRNA gene sequence analysis revealed that the YV-St-033 strain shared the highest sequence identity (more than 99%) with the P. mosselii R10 strain. The mungbean lines of Yeungnam University germplasm were screened against the YV-St-033 strain. Based on the growth rate of the sprouts after 3 days of inoculation with the pathogen, the YV148 line was highly resistant to the pathogen. The remaining lines were either partially or fully infected. The highly resistant line YV 148 is suitable for future breeding programs due to their thin sprouts and fast growing nature.

Microbial Communities in Potato Roots and Soil in Organic and Integrated Production Systems Compared by the Plate Culturing Method

AbstractMicrobial communities in roots, rhizoplane, rhizosphere soil and non‐rhizosphere soil in potato were compared in organic and integrated production systems in 2005–2007. Identification of microorganisms was based on morphotyping. The density (number of colony‐forming units in a sample) of Fungi and Oomycota was significantly greater in the integrated system. Greater densities of Arthrobacter and Streptomyces occurred more often in organic and integrated systems, respectively. The dominant fungal taxa (with frequency >5% in at least one habitat) included Aspergillus, Clonostachys + Gliocladium, Colletotrichum coccodes, Fusarium + Gibberella + Haematonectria + Neonectria, Gibellulopsis nigrescens, Paecilomyces, Penicillium, Phoma and Trichoderma. The subdominant taxa (with frequency 1–5%) included species from 16 genera. In the rhizoplane, rhizosphere and non‐rhizosphere soil, the total density of pathogens was greater in the organic system, and of antagonists in the integrated system. Dominant pathogens, that is, C. coccodes, Fusarium culmorum, Haematonectria haematococca and G. nigrescens, and dominant antagonists, that is, Clonostachys + Gliocladium and Trichoderma, occurred at greater density in the organic system. Subdominant pathogens, that is, Alternaria + Ulocladium, Pythium and Thanatephorus cucumeris, and subdominant antagonists, that is, Mortierella and Umbelopsis vinacea, occurred at significantly greater density in the integrated system. Incidence of sprout rot was more frequent in the organic system, and of Fusarium dry rot and black scurf in the integrated system. The organic system provided a less disease‐suppressive environment than the integrated system and resulted in smaller potato yield. An integrated system of potato production based on 4‐year rotation, white mustard as a cover crop, inorganic fertilizers including ammonium nitrate and chemical control of insects and diseases may be promoted in Poland.

First Report of Anthracnose Caused by Colletotrichum acutatum on Mung Bean Sprouts in Taiwan.

Mung bean sprouts (Vigna radiata (L.) R. Wilczek) are a commonly consumed vegetable in Asian countries. Anthracnose lesions on mung bean sprouts (cv. You-lu-dou from Mayamar) were found in an indoor sprouting facility in Taichung County, Taiwan in May of 2009. The incidence of disease exceeded 90% in some lots. Infected hypocotyls had smooth, diamond-shaped to fusiform brown spots, which became further depressed and enlarged with age. A fungus was isolated on potato dextrose agar (PDA) from symptomatic hypocotyls after surface sterilization in 0.6% NaOCl. Fungal colonies were initially salmon to orange in color and became greenish gray on the surface within a week. Setae were not produced in acervuli that developed on PDA. Conidia in the acervuli were one-celled, cylindrical, and hyaline with an average length and width of 14.8 (7.0 to 23.7) × 4.9 (3.3 to 6.7) μm (n = 53). The internal transcribed spacer (ITS) region was amplified with primers ITS1 and ITS4 (4). The sequence was deposited in GenBank (Accession No. GQ889269). The sequence was 99% identical to that of ATCC 56816 strain of Glomerella acutata (Guerber & J. C. Correll), the teleomorph of Colletotrichum acutatum (J. H. Simmonds) over a 522-bp alignment. Thus, the mung bean pathogen was identified as C. acutatum based on morphological (1) and molecular characters. Pathogenicity of the strain was determined by inoculating mung bean seeds from I-Mei Foods Co. (imported from Australia). The seeds were disinfested in 0.6% NaOCl for 10 min, rinsed with sterile distilled water twice, and immersed in a conidial suspension (1.5 × 104 conidia/ml) of C. acutatum for 10 min. Fifteen inoculated seeds were placed on moistened paper towels, distributed into three flasks, and stored in the dark at 32°C. Symptoms similar to those observed on the original sprouts appeared 3 days later on the hypocotyls of the seedlings and all seedlings were infected by day five. Conidia of C. acutatum were produced on all lesions and colonies of C. acutatum were recovered from symptomatic tissues, fulfilling Koch's postulates. Controls (seed immersed in water) remained symptomless. The pathogenicity test was repeated with similar results. The pathogen has been recorded on mung bean sprouts in Korea (2) and on other Vigna spp. in India (3). To our knowledge, this is the first report of sprout rot of mung bean caused by C. acutatum in Taiwan.

Effective Heat Treatment Techniques for Control of Mung Bean Sprout Rot, Incorporable into Commercial Mass Production

Seedlot disinfection techniques to control mung bean sprout rot caused by Colletoricum acutatum and C. gloeosporioides were evaluated for commercial production scheme. Soaking seedlots in propolis (100 X) and ethanol (20% for 30 min) appeared promising with control values of 85.5 and 80.8 respectively, but still resulted in up to 20% rot incidence. None of the C. acutatum conidia survived through hot water immersion treatment (HWT) for 10 min at temperatures of 55, 60 and , whereas the effective range of the dry heat treatment (DHT) was . Tolerance of mung bean seedlot, as estimated by hypocotyl elongation and root growth, was lower for HWT than for DHT. Germination and growth of sprouts were excellent over the range of intervals, except for HWT at for 5 min. At this marginal condition, heat damage appeared so that approximately 2% of seeds failed to sprout to normal germling and retarded sprouts were less than 5% with coarse wrinkled hypocotyls. These results suggested that DHT would be more feasible to disinfect mung bean seedlots for commercial sprout production. Heat treatment at above ranges was highly effective in eliminating the epiphytic bacterial strains associated with marketed sprout rot samples. HWT of seedlot at 55 and for 5 min resulted in successful control of mung bean sprout rot incidence with marketable sprout quality. DHT at 60 and for 30 min also gave good results through the small-scale sprouting system. Therefore, we optimized DHT scheme at 60 and for 30 min, considering the practical value of seedlot disinfection with high precision and accuracy. This was further proved to be a feasible and reliable method against anthracnose incidence and those bacterial strains associated with marketed sprout rot samples as well, through factory scale mung bean sprout production system.

Seed treatment application-timing options for control of fusarium decay and sprout rot of cut seedpieces

The efficacy of application timings of a commercially formulated mixture of fludioxonil plus mancozeb (Maxim MZ) applied prior to planting for the control of seedpiece decay and rotting of sprouts, caused by the dry rot pathogenFusarium sambucinum, was evaluated over two years. Cut potato seedpieces were inoculated with a virulent strain ofF. sambucinum and either treated with the fungicide mixture or not. Treatment applications were made 10, 5 or 2 days prior to planting. Seedpiece and sprout health were evaluatedin vitro and agronomic impacts were evaluatedin vivo in field experiments. Overall, thein vitro experiments indicated that inoculation withF. sambucinum did not have an effect on the mean number of sprouts per seedpiece but did affect the incidence of rotting sprouts and seedpiece decay. However, treatment of seedpieces with the fungicide mixture 10, 5 or 2 days before planting significantly reduced the percentage of diseased sprouts per seed-piece and seedpiece decay. Inoculated seedpieces treated with the fungicide mixture produced similar numbers of healthy sprouts as did the non-inoculated seedpieces. The experiment, conducted in 2003, showed that final plant stand, RAUEPC and RAUCCC were similar for non-inoculated seedpieces and inoculated fungicide-treated seedpieces. Overall, there were no significant differences among treatment timings, and these results suggest that applying a fungicide seed treatment up to 10 days prior to planting can provide effective control of both Fusarium seedpiece decay and sprout rot.

First Report of Potato Tuber Sprout Rot Caused by Fusarium sambucinum in Michigan.

Fusarium dry rot is one of the most important diseases of potato (Solanum tuberosum L.), affecting tubers in storage and whole seed or seed pieces after planting (2). Fusarium sambucinum Fuckel (teleomorph Giberella pulicaris) is the most common pathogen causing dry rot of stored tubers in North America. (4). Cut seed potato tubers of cvs. FL1879 and Pike with severe sprout rot were collected in Michigan during May 2006. As well as having rotted sprouts, all diseased tubers had dry rot. When diseased sprouts were cut in half, brown, necrotic lesions could be seen spreading down the center of the sprout in vascular tissue and at the base of the sprout in tuber tissue. Pathogen isolations were made from both infected tuber tissue and diseased sprouts on potato dextrose agar (PDA). In both cases, only F. sambucinum was isolated from diseased sprout and tuber tissue. Identification of the pathogen was based on colony and conidial morphology. This included white, fluffy mycelium on the surface and crimson coloration of the colonies viewed from the underside of PDA plates and large distinctive macroconidia (3). Identification was confirmed by comparison of ITS (internal transcribed spacer) sequence data with reference isolates. The ITS region of rDNA was amplified by polymerase chain reaction (PCR) with primers ITS1/ITS4 and sequenced. BLASTn analysis (1) of the sequence obtained showed a 100% homology with F. sambucinum Fuckel. For inoculum production, isolates were grown on PDA at 8°C for 14 days prior to inoculation. Pathogenicity was tested in potato tubers of cv. FL1879 with a single isolate collected from diseased sprouts. Whole seed tubers with 4 mm long sprouts were cut in half longitudinally with a sterile knife to ensure that seed pieces had viable sprouts. The cut surfaces of seed pieces were spray inoculated with 200 ml of conidial suspension (1 × 104 conidia ml-1) over the entire cut surface to give a final dosage of approximately 1 ml per seed piece. Care was taken to limit inoculum spray to the cut surface so that sprouts were not inoculated. Seed pieces (40 per replicate × 4 replicates) were then placed in plastic boxes (30 × 15 × 10 cm) and incubated in the dark at 18°C and 95% relative humidity for 30 days in a controlled environment chamber. As a control, cut seed pieces were spayed with sterile distilled water and incubated as above. All tubers inoculated with the pathogen developed typical Fusarium dry rot symptoms consisting of a brown, dry decay of tuber tissue with mycelial lined cavities. Sprouts on inoculated tubers developed symptoms that were observed in the initially collected seed pieces, and F. sambucinum was reisolated from all infected sprouts. The noninoculated control tubers did not develop any symptoms of dry rot. The results of the pathogenicity tests indicate that F. sambucinum caused sprout rot on potato seed pieces. Since only the cut surfaces of tubers were inoculated, it is assumed that infection of sprouts is systemic through the tuber. To our knowledge, this is the first report of F. sambucinum causing a sprout rot of developing sprouts on seed tubers in the United States.

Developing Polyclonal Antibody-based Indirect-ELISA to Detect Anthracnose Inocula Prior to Soybean Sprout Rot

We developed a polyclonal antibody based-ELISA system to monitor inocula accurately and rapidly before onset of anthracnose on soybean sprouts. Titer of mouse antisera against conidia of Colletotrichum gloeosporioides, determined by indirect ELISA, was high enough to be detectable up to 25,600 dilutions. Both PAb1 and PAb2 had the highest level of reactivity to Colletotrichum gloeosporioides. Absorbance readings exceeded 0.15. Sensitivity of PAb to C. gloeosporioides was precise enough to detect spore concentration as low as 500 conidia/well by indirect ELISA. Both antibodies are very sensitive and highly specific to the target pathogen Colletotrichum gloeosporioides, apparently discriminating other unrelated pathogen, or epiphytes. This kit fulfills the requirements far detecting inocula before infection and onset of anthracnose. Our ELISA system should also be feasible to detect C. acutatum (Mungbean sprouts rot) and G. cingulata (C. gleosporioides), (apple, pepper). It was remarkable that absorbance value was not reduced even after 4 consecutive washings (Fig.4), suggesting that antigenic determinants are on the surface of conidia. Antigenic determinant was characterized by heating and enzyme treatment: Both PAb1 and PAb2 bind to protein epitope that does not contain residue of amino acid, arginine, and Iysine, even though more work needs to be done.

Control of Soybean Sprout Rot Caused by Pythium deliense in Recirculated Production System

A soybean-sprout rot epidemic occurred in a mass production soybean sprout factory in 2000 and 2001 in Korea, which caused up to 20% production loss. Among the causal pathogenic bacteria and fungi, Pythium deliense was found to be the dominant pathogen of severe root and hypocotyls rot, particularly in recirculating water system. An average of 90% of the isolated fungi from the rotted sprout on potato dextrose agar were Pythium sp. The fungal density of Pythium in the sampled water was monitored in the recycled water system for 1 year using a selective medium (com meal agar with Pimaricin, 10 mg； Rifampicin, 10 mg； and Ampicillin, 100 mg per 1 liter). The drained water from the soybean-sprout cultivation always had a certain amount of fungus in it. The removal of Pythium from the recycling water system must be thorough, safe, and environment friendly. However, the pathogen in the water was easily found even after ozone and chlorine treatments, which were devised on the recycling system for the removal of microorganisms. 5-<TEX>$\mu\textrm{m}$</TEX> pore size filter was applied and was able to successfully control the disease. As the sprout industry increasingly shifts into mass production, the demand for water will increase continuously. Recycling water for sprout production is eco-friendly. However, a process must be devised to be able to first decompose organic matters before Pythium zoospores are filtered.

Colletotrichum species에 의한 콩나물 부패

Two novel casual agents of soybean sprout rot occurred at soybean sprouts cultivated under structure in Suwon area in 1997 were isolated and their pathogenicity was tested in vivo. An isolate formed crowed, black acervuli which were oval to elongated with numerous black, needlelike, intermixed long and short setae, 65~1103.5~6.6 . Conidia were curved, lunate, unicellular and hyliane and measured 21.5~22.53.5~4.0 . The other isolate produced conidia with straight and cylindrical, and measured 14.0~17.53.5~4.5 . Apressorium size was measured 6.3~8.54.5~5.0 . The agents were identified as Colletotrichum truncatum and C. gloeosporioides based on their morphological characteristics. There was a large difference in pathogenicity between two isolates. C. gloeosporioides caused dark brownish discoloration of whole plants. It showed high pathogenicity with severe disease development. Meanwhile C. gloeosporiodes caused light brown spots on cotyledon and its pathogenicity was not strong. The soybean sprout rot occurred by the two Colletotrichum species was firstly reported in soy-bean sprout in Korea, and we suggest it as “Colletotrichum rot of soybean sprout”.

006 Effects of Chitosan on Growth and Rot of Soybean Sprouts

The practicality of utilizing chitosan (MW = 5000-10,000) as a natural antimicrobial compound to reduce soybean sprout rot was tested. Soybean seeds were soaked for 6 h in solutions containing different levels of chitosan and acetic acid (glacial), and cultivated at 25 °C for 5 days. Soaking seeds with 1000 ppm chitosan increased germination percentage, hypocotyl thickness, total length, and fresh weight of sprouts by 4%, 5%, 2%, and 1%, respectively. The total sprout yield was increased by chitosan in a concentration-dependent manner in that 1000 ppm chitosan resulted in 8% increment of total yield (7.47 kg sprouts/kg seed). Compared to control (13.8%), chitosan significantly reduced sprout rot percentage to 7.0%, and consequently enhanced the marketable sprout yield by 39%. Although 100 ppm acetic acid also decreased sprout rot percentage to 11.8%, its yield-increasing effects were not as prominent as chitosan.