Studies on storage rot in sugar beet [Beta vulgaris]

In 1980s, the area cultivated with sugar beet totaled 70,000ha, and root yeild was maintained at more than three million tons in Hokkaido. However, storage rot, occurring on large amounts of roots piled outdoor for long periods during winter, has become increasingly economically important. This paper describes the studies on the causal fungi and bacteria of storage rot, the changes in fungal flora in a large pile, and the measures of reducing the storage rot damage and sugar loss of stored roots. 1. Causal agents of storage rot and the changes in quality of sugar beet roots 1. Fungi isolated from stored sugar beet roots and their pathogenicity Four families comprising 19 genera including 28 species of fungi were found in sugar beet roots stored in piles, and among these, 12 genera including 16 species were isolated from rotten tissues. Botrytis sp. was most frequently isolated at the rate of 71.7%. The mycelia predominantly colonizing the root surface were classified into five types: the Botrytis, Cladosporium, Fusarium, Geotrichum and Penicillium types. The Botrytis, Cladosporium and Penicillium types were frequently observed on rotten tissues. Botrytis sp. was frequently isolated from internal tissues of rotten beet roots colonized by these three mycelial types, but Cladosporium sp. was less frequently isolated from them. Botrytis sp. was the most pathogenic among the 22 species tested by inoculation to the root tissue of sugar beet. Fusarium sp., Penicillium sp. and Phoma sp. also formed clear lesions on root tissues following inoculation. The four species were identified as Botrytis cinerea Pers. ex Fr., Fusarium culmorum (W. G. Smith) Sacc., Penicillium expansum Link ex Gray emend. Thom and Phoma betae Frank. 2. Characteristics of B. cinerea: pathogen of storage rot in sugar beet Mycelia of B. cinerea isolated from sugar beet, kidney bean, statice, strawberry and tomato could grow on PDA at the temperature range of -2 to 30 deg C, but not at 35 deg C. Their optimum growth temperature was 20 deg C except for one isolate from strawberry. All B. cinerea isolates, regardless of host plants from which they were isolated, rapidly infected the root tissues of sugar beet. The incidence of storage rot of sugar beet roots harvested by a machine was more than twice of that harvested manually. Storage rot lesions appeared on all parts of the bruised beet roots harvested by a machine, whereas the lesions were mainly observed on the upper part of beet roots dug up manually. The mycelia of B. cinerea were incapable of cuticular infection through the periderm of sugar beet roots. Rot lesions were only observed on tissues when mycelia were inoculated on peeled roots. Conidia of B. cinerea were also capable of wound infection through the peeled root tissue at the spore concentration ranging from 8 * 10**1 to 8 * 10**4. The size of rot lesions resulting from conidial inoculation, however, was much smaller than that from mycelial inoculation. 3. Changes in qualities of sugar beet roots due to fungal storage rot A significant decline in sugar content and pH, as well as a marked increase in the concentration of reducing sugar, was observed in the sugar beet roots exhibiting decay in approximately half of the tissues due to fungal storage rot. Concentrations of raffinose, brix and total nitrogen also declined, but those of potassium and sodium were almost constant. The sugar content and pH of sound tissues separated from rotten beet roots remained at the same level as those of healthy roots; however, the sugar content was nearly 0% and pH was markedly low in rotten tissues separated from the same rotten roots. Regarding the relationship between storage rot and root qualities, it was found that the rotten area of the storage roots had a significant positive quadratic correlation with sugar content and a significant negative quadratic correlation with the concentration of reducing sugar. A significant decline in sugar content and pH, as well as a marked increase in the concentration of reducing sugar, was also found in juice pressed from sugar beet roots inoculated with B. cinerea or P. expansum. The concentration of raffinose was not reduced, and among the carboxylic acids, citric acid concentration was markedly increased in the pressed juice. Sucrose was almost completely reduced, whereas significant amounts of glucose and fructose were produced in the artificial media containing sucrose as the sole carbon source and inoculated with B. cinerea or P. expansum. Con cerning the carboxylic acids, citric acid was markedly produced in those media. 4. Pathogenic bacteria isolated from stored sugar beet roots showing soft rot and the identification Bacterial soft rot was found at the center of the top surface of storage piles under continuously high humid conditions applied immediately after piling at Kamikawa district. Three types of field soil samples (clay, clay loam and sandy loam soils) and three samples of "separated soils", which were removed by a piler from the beet roots harvested in those fields, were collected from Kamikawa and other districts. The washed healthy sugar beet roots were coated with the six types of soil samples and then stored in a warehouse. Severe bacterial soft rot was only observed on the beet roots which were coated with the separated clay soil collected from Kami kawa district. Bacterial soft rot was observed irrespective of the quantity of the soil coated, and the sugar content and pH of beet roots were significantly decreased. The pathogenicity of 12 among 18 gram-negative rod-shaped bacterial isolates from sugar beet roots stored in piles and exhibiting bacterial soft rot was confirmed by the tobacco hypersensitive reaction test. All these isolates were identified to be Erwinia carotovora (Jones) Bergey, Harrison, Breed, Hammer and Huntoon. Concerning gram-positive coccoid bacterial isolates from beet roots with bacterial soft rot in storage piles and from the roots exhibiting soft rot symptoms in the soil-coating trials, eight out of ten isolates were found to be pathogenic by the tobacco hypersensitive reaction test. Of the 25 isolates which were inoculated to potato tubers and sugar beet roots by the puncturing method, 17 isolates were pathogenic to potato, 13 to sugar beet and 10 to both potato and sugar beet. All these isolates were identified to be Leuconostoc mesenteroides (Tsenkovskii) van Tieghem. The designation "Leuconostoc storage rot" of sugar beet was proposed for this soft rot caused by L. mesenteroides. 5. Changes in qualities of stored sugar beet roots due to bacterial storage rot A significant decline in sugar content and pH, as well as a marked increase in the concentration of reducing sugar, was found in the stored sugar beet roots exhibiting decay of approximately half of the tissues due to bacterial soft rot. A decline in the concentration of raffinose and total nitrogen was also found in the same roots. Among the carboxylic acids, the concentrations of acetic acid and lactic acid also decreased in those roots. A significant decline in sugar content and pH, as well as a marked increase in the concentration of reducing sugar was found in the juice pressed from sugar beet roots inoculated with L. mesenteroides or E. carotovora. Raffinose was markedly reduced in the juice pressed from beet roots inoculated with E. carotovora. Concerning carboxylic acids, only D(-)-lactic acid was significantly increased in the juice pressed from beet roots inoculated with L. mesenteroides, and formic acid, D(-)-lactic acid, L(+)-lactic acid and succinic acid were slightly increased in the juice pressed from beet roots inoculated with E. carotovora under the same culture conditions. Acetic acid and lactic acid were markedly produced in the artificial media containing sucrose as the major carbon source and inoculated with L. mesenteroides. Productions of succinic, acetic and lactic acids were found in the artificial media inoculated with E. carotovora subsp. carotovora. 2. Fungi occurring in storage piles of sugar beet roots and conditions suitable for their proliferation 1. Changes in fungal flora in large outdoor piles of sugar beet roots during the storage period Mainly five mycelial types were found on the surface of the storage pile of sugar beet roots. The Botrytis type was frequently observed in all parts of a storage pile. The average percentage of moldy beet roots of this type was the highest among the five mycelial types. The Penicillium type was also observed in all parts of a pile. The average percentage of moldy beet roots of this type was lower than that of the Botrytis type; but on the top surface of storage pile, the percentage of the Penicillium type was higher than that of the Botrytis type. The Fusarium type was also observed in all parts of a pile. However, the average percentage of this type in every part of a storage pile was markedly lower than those of the Botrytis and Penicillium types. The Cladosporium type was mainly found on the dry surface along the sides, while the Geotrichum type, on the humid top surface of storage piles. Moldy roots were observed on the top surface of a storage pile in the middle of November, 15 days after piling, and the incidence increased gradually until late in February, the end of the storage period. The incidence of the Botrytis type increased gradually from the middle of November to late February. The Penicillium type was observed from the early stage of storage, but the occurrence of this type increased gradually from January. A low incidence of the Fusarium type was observed in the middle of February. The Cladosporium type was abundantly observed in the early stage of storage from the middle of November, but was scarcely observed from the middle of December. The Geotrichum type was not observed in the early stage of storage, but was observed in the middle of January, and then rapidly increased, particularly on the top surface of storage piles. Cladosporium, Botrytis and Penicillium were the main genera found as fungal spores in space above the top surface of a storage pile. The component ratio of Cladosporium in space (1.8m height) above the sugar beet pile was approximately 50% immediately after piling, but gradually decreased during the storage period. On the contrary, the component ratio of Botrytis spores gradually increased during the storage period, though no spores were collected from the atmosphere immediately after piling. It was found that the component ratio of Botrytis spores in the atmosphere had a high positive correlation between the rate of moldy roots, the rate of rotten roots and the severity of storage rot in sugar beet piles. The highest correlation coefficient was found between the component ratio of Botrytis spores and the rate of rotten roots. 2. Relationship between temperature of a storage pile and rot severity or increase in sugar loss in stored sugar beet roots Pile temperature, rot severity and sugar loss in stored sugar beet roots in a large outdoor pile (22m in width, 4.5m in height, approximately l00m in length) were measured for three years and presented as averages of storage periods of ll5days. The average of daily mean temperature of a pile was 3.5 deg C, and the average of storage rot severity was 0.20 based on a scale from 0 to 5. The average of daily sugar loss was 79 g/ t/day, equivalent to 5.5% of the total sugar yield contained in the stored sugar beet roots. The values for these three factors were markedly different, depending on the portion of a storage pile from which samples were obtained. It was, however, found that the daily mean temperature had a significant positive correlation with rot severity (P = 0.01), and with sugar loss (P = 0.05) in storage piles. 3. Controlling storage rot of sugar beet 1. Relationship between water content of beet roots and incidence of storage rot A severer storage rot, and a more marked decrease in sugar content and increase in the concentration of reducing sugar were found in more dehydrated roots dried with a ventilator immediately after harvest. On the other hand, less severe storage rot and sugar content decline, as well as the suppression of increase in the concentration of reducing sugar, were found in roots with higher water content due to moist conditions during storage. 2. Relationship between storage temperature and incidence of storage rot After harvesting manually, untreated and the chemically treated sugar beet roots were stored at two-grade, relatively low temperatures during a long storage period. Sugar losses due to respiration and that due to storage rot were calculated to be 59g/t/day and 16g/t/day, respectively, at the average storage temperature of 93 deg C. The losses were decreased when the storage temperature was close to the freezing point, and were calculated to be 37g/t/day and 12g/t/day, respectively, at the average storage temperature of 4.5 deg C. 3. Prevention of storage rot using chemical compounds Dicloran and thiabendazole were effective against sugar beet storage rot among nine fungicides and seven chemical compounds tested. Particularly, dicloran stably prevented the occurrence of storage rot and sugar loss. 4. Biological control of storage rot using antifungal bacteria A total of 3,114 antifungal bacteria were isolated from 133 soil samples and 36 sugar beet root tissue specimens. Five isolates of Pseudomonas cepacia were selected as biological agents exhibiting the following features:1) excellent antifungal activity against both sugar beet storage rot pathogens, B. cinerea and P. expansum, 2) ability to grow below 8 deg C, 3) avirulent to sugar beet roots and 4) their application results in a small amount of sugar loss. They successfully reduced the incidence of storage rot even during long-term storage trials. Particulary, isolate D-202 was the most effective in suppressing the rate and severity of root rotting, one-third to those of untreated roots. D-202 treatment, however, was unable to prevent sugar loss as compared with nontreatment.