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' {.33 if ,9 " KIAA. ..LL'.§- t2 &. if 9 W ‘13: "My This is to certify that the dissertation entitled THE EFFECTS OF CULTURAL PRACTICES AND PETIOLE CALCIUM CONTENT ON SUMMER PRODUCTION AND INCIDENCE OF BACTERIAL SOFT ROT (ERWINIA CAROTOVORA SPP. CAROTOVORA) IN CHINESE CABBAGE (BRASSICA CAMPESTRIS L. SPP. PEKINENSIS) presented byt Vincent A. Fritz has been accepted towards fulfillment of the requirem Ph.D. degree in Horticulture AM / Date ?’£4~ [X /7yé MS U i: an Affirmative Action/Equal Opportunity Institution ents for £21,: i MWJMA Major professor 0-12771 MSU RETURNING MATERIALS: Place in book drop to remove this checkout from LIBRARIES _ - your record. FINES W111 be charged if book is returned after the date stamped below. OCT 1 7 2000 3‘0050 THE EFFECTS OF CULTURAL PRACTICES AND PETIOLE CALCIUM CONTENT ON SUMMER PRODUCTION AND INCIDENCE OF BACTERIAL SOFT ROT (ERWINIA CAROTOVORA SPP. CAROTOVORA) IN CHINESE CABBAGE (BRASSICA CAMPESTRIS L. SPP. PEKINENSIS) BY Vincent A. Fritz A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1985 ABSTRACT THE EFFECTS OF CULTURAL PRACTICES AND PETIOLE CALCIUM CONTENT ON SUMMER PRODUCTION AND INCIDENCE OF BACTERIAL SOFT ROT (ERWINIA CAROTOVORA SPP. CAROTOVORA) IN CHINESE CABBAGE (BRASSICA CAMPESTRIS L. SPP. PEKINENSIS) BY Vincent A. Fritz Raised bed culture was used to reduce the incidence of bacterial soft rot (Erwinia carotovora ssp. carotovora) during summer production of Chinese cabbage (Brassica campestris ssp. pekinensis) in Michigan. Tropical (heat tolerant) cultivars were evaluated for summer production potential. Various between and within row spacing effects on disease incidence and plant growth were studied. Foliar applications of calcium and sand nutrient culture were used to modify the petiole calcium content. Petiole sections (6.45 cmz) were also inoculated with an antibiotic (Rifampin) resistant strain of Erwinia carotovora ssp. carotovora to evaluate resistance to soft rot incidence and progression. Xylem exudates collected from the plants in the sand nutrient culture were analyzed for calcium accumulation. Vincent A. Fritz Raised beds slightly reduced the amount of soft rot when compared to flat culture. Most trOpical cultivars bolted when smm1on June 16; 'Tropical Pride' and 'Asveg #1' formed compact heads without bolting. Within row spacing of 30cm reduced plant growth and decreased the number of harvestable plants. Seed sown after June 16 resulted in an increase in incidence of soft rot. Foliar CaCl2 applications did not increase petiole calcium levels; however, the various calcium levels used in the sand nutrient culture effected petiole calcium accumulation. Relatively higher petiole calcium content had some inhibitory effects on soft rot. Plant age and petiole position (inner and outer) did not consistently effect soft rot incidence and progression. There was a definite trend of reduced soft rot in petiole (inner) sections of Hakuran with increased xylem calcium levels. DEDICATION The author wishes to express his deepest love to his wife Barb for her continuous encouragement, assistance, and understanding during his life as a graduate student. Her support was ever present which played an integral part in the completion of this dissertation. ii ACKNOWLEDGMENT S The author wishes to express sincere appreciation to Dr. S. Honma for his valuable assistance in experi- mentation and in the preparation of the manuscript. Appreciation is also extended to Dr. H. C. Price, Dr. D. Warncke, Dr. I. Widders, and Dr. D. Fulbright for their suggestions and constructive criticism of the manuscript. Finally, the writer wishes to express gratitude to his parents for their continuous support and encourage- ment during his graduate studies. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . viii INTRODUCTION . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . 4 Spring Production . . . . . . . . . 4 Summer Production . . . . . . . . 5 Etiology of Soft Rot . . . . . 6 Effects of Raised Beds/Spacing on Produc- tion . . . . . . . . . . 8 Effects of Calcium on Disease Occurrence . 11 Other Functions of Calcium . . . . . . 14 Calcium Deficiency Symptoms . . . . . . 16 MATERIALS AND METHODS . . . . . . . . . . 19 Field Studies: Cultural Practices . . . l9 Experiment No. l, 1982 . . . . . . . 20 Experiment No. 2, 1983 . . . . . . . 21 Experiment No. 3, 1983 . . . . . . . 22 Experiment No. 4, 1984 . . . . . . . 22 Calcium Effects . . . . . . . . . . 23 Tissue Analysis Procedures . . . . . 23 Bacterial Inoculation . . . . . . . 24 Foliar Calcium Application: General Practices . . . . . . . . . 25 Foliar Calcium Application: Field . . . 28 Foliar Calcium Calcium Application: Green— house . . . . . . . . . . . 30 Sand Nutrient Culture . . . . . . . 30 Experiment No. l . . . . . . . . . 35 Experiment No. 2 . . . . . . . . . 37 Experiment No. 3 . . . . . 38 Petiole Lignin and Cuticle Observations . . 38 iv Page RESULTS AND DISCUSSION . . . . . . . . . 40 Field Studies: Cultural Practices . . . 40 Experiment No. 1, 1982 . . . . . . . 40 Experiment No. 2, 1983 . . . . . . . 47 Experiment No. 3, 1983 . . . . . . . 51 Experiment No. 4, 1984 . . . . . . . 53 Calcium Effects . . . . . . . . 60 Foliar Calcium Application: Field . . 60 Foliar Calcium Application: Greenhouse . 65 Sand Nutrient Culture . . . . . . . . 69 Experiment No. 1, 1984 . . . . . . . 69 Experiment No. 2, 1984 . . . . . . . 75 Experiment No. 3, 1985 . . . . . . 85 Petiole Lignin and Cuticle Observations . . 101 SUMMARY . . . . . . . . . . . . . . 105 BIBLIOGRAPHY . . . . . . . . . . . . 109 10. LIST OF TABLES Chinese cabbage cultivars and their Source . . . . . . . . . . . Concentrations of CaC12 and surfactants used in greenhouse foliar application experiment . . . . . . . . . . . Concentration of nutrient solution (less calcium) used in nutrient culture experi- ments . . . . . . . . . . . . . The effect of sowing date on the incidence of soft rot and head weight of the Chinese cabbage cultivar, 'Nagaoka No. 2' . . . The effect of planting date and within row spacing on % of Chinese cabbage 'Nagaoka No. 2' harvested plants . . . . . . . The percent of immature heads remaining after harvest for within row spacings at different planting dates of the Chinese cabbage cultivar, 'Nagaoka No. 2' . . . Mean maximum/minimum air temperatures for the first and second half of the growing period for various sowing dates (°C) . . The effects of within row spacing on head weight of the Chinese cabbage cultivar 'Nagaoka No. 2' . . . . . . . . . The responses of six Chinese cabbage culti- vars to summer production conditions . . The effect of within row (between plant) spacing of Chinese cabbage cultivar 'TrOpi- cal Pride' on head weight, rot, and marketability . . . . . . . . . . vi Page 21 28 29 41 42 44 45 46 49 52 Table 11. 12. 13. 14. 15. 16. 17. Page The effect of cultivar and culture type on the incidence of soft rot on three Chinese cabbage cultivars . . . . . . . . . 54 Mean of ten daily afternoon air tempera- tures (°C) within the phyllosphere of culture types and ambient air temperature during the last two weeks prior to harvest of Chinese cabbage . . . . . . . . 56 Percentage of bolters produced by three cultivars of Chinese cabbage under summer growing conditions . . . . . . . . 57 Mean maximum/minimum daily air temperatures (°C) for the 1984 growing season . . . . 57 The effect of culture type on the percent- age of marketable heads of three Chinese cabbage cultivars . . . . . . . . . 59 The effect of culture type of headweights of three Chinese cabbage cultivars . . . 59 The effects of foliar applied calcium chloride on inner petiole calcium content (% dry weight) . . . . . . . . . . 64 vii Figure 1. 10. 11. LIST OF FIGURES Rating scale used to evaluate the incidence and progression of soft rot in inoculated petiole sections of Chinese cabbage . . . Sand nutrient culture system used to increase petiole calcium accumulation in Chinese cabbage and Hakuran . . . . . The effect of X-77 surfactant on Chinese cabbage seedlings . . . . . . . . . The effect of foliar spray calcium concen- tration on inner petiole percent calcium of Chinese cabbage . . . . . . . . The effect of nutrient solution calcium concentration on outer petiole percent calcium of Chinese cabbage . . . . . . The effect of nutrient solution calcium concentration on inner petiole percent calcium of Chinese cabbage . . . . . . The effect of nutrient solution calcium concentration on outer petiole percent calcium of Chinese cabbage (30 days) . . The effect of nutrient solution calcium concentration on inner petiole percent calcium of Chinese cabbage (30 days) . The effect of nutrient solution calcium concentration on outer petiole percent calcium of Chinese cabbage (60 days) . . The effect of nutrient solution calcium concentration on inner petiole percent calcium of Chinese cabbage (60 days) . . The effect of nutrient solution calcium concentration on outer petiole percent calcium of Hakuran (30 days) . . . . viii Page 26 32 62 66 71 73 77 79 81 83 86 Figure 12. l3. 14. 15. 16. 17. 18. The effect of nutrient solution calcium concentration on inner petiole percent calcium of Hakuran (30 days) . . . . . The effect of outer petiole percent calcium on the incidence and progression of soft rot in inoculated petiole sections of Hakuran (30 days) . .. .. . . .. . . . The effect of nutrient solution calcium concentration on outer petiole percent calcium of Hakuran (60 days) . . . . . The effect of nutrient solution calcium concentration on inner petiole percent calcium of Hukuran (60 days) . . . . . The effect of outer petiole percent calcium on the incidence and progression of soft rot in inoculated petiole sections of Hakuran (60 days) . . . . . . . . . The effect of inner petiole exudate calcium concentration on the incidence and progres— sion of soft rot in inoculated petioles of Hakuran (60 days) . . . . . . . . Cuticle thickness of (a) head cabbage, (b) Chinese cabbage, and (c) Hakuran . . ix Page 88 91 93 95 97 99 102 INTRODUCTION Chinese cabbage (Brassica campestris ssp. pekinensis) was an important food source as early as the fifth century as portrayed in agricultural art works (Sturtevant, 1919). Cultivation of the crop was not reported in the United States until 1884 (Boswell, 1949). The slow growh in popularity until recently may have been due to the lack of knowledge concerning its culinary uses. Chinese cabbage is commonly served as a fresh salad, cole slaw, boiled cabbage, or stir fried with other vegetables. Due to influx (of Asian migration, in the last five years there has been increased interest in growing spring and summer Chinese cabbage on organic and upland soils, espe- cially in the northern United States. With the increase in the popularity of the Chinese cabbage, information on cultural practices to extend and provide a continuous spring to fall production is not available in the western world. Harvest of Chinese cabbage in Michigan is limited to the late summer and fall, when cool temperatures pre- vail during the heading stage. For an early crop the possibility of vernalization remains as late as mid June. Minimum daily temperatures (113°C) for a two week period will vernalize the plants (Yamasaki, 1956). Once vernali- zation occurs, protrusion of the flower stalk will occur before the completion of heading. The three-week-old plants under protective row covers for spring production significantly reduced the incidence of premature bolting (Fritz and Honma, 1984). Plantings made in late April yielded a marketable crop by the last week in June. Crops harvested after the 10th of July until early September encounter maximum daily air temperatures that promote the incidence and progression of bacterial soft rot (Erwinia carotovora ssp. carotovora). Once the bacteria infect the plant, progression of rot is rapid and death of the plant can occur in as little as three days. The bacteria enter the petioles in the phyllosphere via insect wounds or natural openings. The time of maximum occurrence of the disease correlated with the heading stage of the plant, approximately two weeks prior to harvest (Tsuyama and Sakamoto, 1953). In Asia soft rot incidence during summer production is usually avoided by producing a crop in the highlands wehre moderate maximum daily air temperatures prevail. Modification of cultural practices to alter the environment within the phyllosphere may be a viable approach in inhibiting the incidence of soft rot during summer production. Between and within row spacing studies on the incidence of diseases have been limited in many crops. Spacing effects on soft rot incidence in Chinese cabbage have not been reported. One reason may be the Asian practice of using every available piece of land for maximum crop productivity. Raised bed culture has been used for several crops primarily to facilitate drainage in poorly drained soils or in areas of high rain- fall (Ryder, 1979; Nakagawa, 1957). Preliminary experimentation on potato has suggested an inhibitory effect of tuber calcium concentration on the progression of bacterial soft rot (Anon., 1983; Stapp and Hartwich, 1957). The objective of this study was to develop cultural practices and to determine if the use of calcium treat- ments would reduce the incidence of bacterial soft rot to facilitate continuous production of Chinese cabbage. The following cultural practices were investigated: planting dates, use of raised beds, between and within row spacings, and tropical (heat tolerant) cultivars. LITERATURE REVIEW Spring Production Chinese cabbage (Brassica campestris ssp. pekinensis) is usually grown as a fall crop in the northern areas of the U.S.A. It has a very large leaf area and shallow root system and, therefore, is very sensitive to low soil moisture content and temperature extremes (cold and hot). It is frequently vernalized when early (spring) production is attempted in the northern U.S.A., which results in premature bolting. Yamasaki (1956) formulated a workable mathematical model that would determine if vernalization has occurred in many spring cultivars. Nakamura (1976) simplified the formula to read: (13 - x) y _>_ 87°C where X temperature below 13°C Y number of days with a minimum daily temperature below 13°C When the heat sum of the minimum daily temperatures reaches or exceeds 87°C, vernalization will occur. Approximately two weeks exposure to minimum daily temperatures of 13°C or less will cause vernalization. Guttormsen and Moe (1985) reported that certain heat tolerant cultivars ('Tropical Pride' and "Saladeer') can be vernalized when grown at temperatures below 23°C. Subsequent long days and relatively warmer temperatures will hasten flower stalk development (Lorenz, 1946; Mori, Eguchi, and Matsul, 1979). Some attempts have been made to inhibit vernalization and produce an early season crop in Michigan using protective row tunnels (Fritz and Honma, 1984). Summer Production The major limiting factor in growing summer Chinese cabbage is the incidence of bacterial soft rot (Erwinia carotovora ssp. carotovora). Many bacteria cause soft rot and can infect a wide range of host plants. However, the soft rotting (Erwinias are considered to be the most important disease problem for summer production of Chinese cabbage (Perombelon and Kelman, 1980). The disease can rapidly cause death to the plant. Since Chinese cabbage is extremely susceptible to soft rot, "summer" production is usually avoided because of the absence of any viable control methods (Nakamura, 1974). The use of antibiotics to control soft rot bacteria show some beneficial effects on summer production of Chinese cabbage (Mazzucchi and Svampa, 1972); however, the use of antibiotics for soft rot control in Chinese cabbage in the U.S.A. is cost pro- hibitive. The maximum daily air temperatures (>25°C) can also inhibit head formation in many cultivars. However, satis- factory heads can be obtained by tying the foliage similar to the method used with cauliflower at the onset of head- ing (Sajjapongse and Roan, 1981). Etiology of Soft Rot Erwinia carotovora ssp. carotovora is facultatively anaerobic, peritrichously flagellated, rod shaped, and gram- negative (Kelman and Dickey, 1980). This pathogen is a soil borne bacterium and multiplies in the rhizosphere of Chinese cabbage and other vegetables and weeds. The bacter- ium overwinters in decaying organic matter or in many weeds. It is not easily detected in soil samples (Togashi, 1971). The bacterium can be introduced into an uninfected cropped field via insect transmission (Phillips and Kelman, 1982). Three main factors affecting the susceptibility of Chinese cabbage to the disease are stage of development of the host plant, temperature, and relative humidity in the immediate phyllosphere (Togashi, 1972). The phyllosphere is the abaxial surface of the older leaves near the point of attachment to the main stem which is in contact with the soil surface. The outbreak of the disease is positively correlated with the wrapping stage of the plant. The bac- terium can enter the plant petioles by natural openings, abrasions, or insect wounds. From the time heading begins until harvest, the bacteria level in the soil which is in contact with the outer petioles, steadily increases via nutrients leached from the petioles and roots (Tsuyama and Sakamoto, 1953). As the temperature increases (>23°C), the invasion of the bacterium is promoted (Koda et al., 1974). Eguchi (1979) found Chinese cabbage was susceptible to soft rot bacteria at temperatures above 15°C at the heading stage. Powelson (1980) found that the predominant pathogen in potatoes was Erwinia carotovora ssp. carotovora when the mean daily air temperatures reached 22°C which corresponded with the months of July and August. Lund and Kelman (1977) determined that potato tubers that were mechanically harvested and passed through a flume and rinsing system had a higher incidence of soft rot than hand harvested tubers that were not passed through the flume system and rinsed. Since the outer petioles of Chinese cabbage cover the soil surface in the phyllosphere, evaporation of water from the soil is inhibited, thus creating a humid environment for disease development. Due to the humid environment, the bacteria grow rapidly and can cause death of the plant in as little as three days. The disease progresses more rapidly under anaerobic condi- tions which could become important on poorly drained soils. This is primarily due to the loss of resistance to the bacterium by the host plant (DeBoer and Kelman, 1975). Once inside the petiole, the bacteria enzymatically digest the plant's cell walls and middle lamella by producing pectolytic enzymes. The disease symptoms are characterized by water soaked, slimy lesions on the abaxial side of phyllosphere petioles of Chinese cabbage which turn brown and progress basipetally towards the crown and eventually rot out the entire plant. The bacterium can exist in the host plant (potato) and yet cause no visible symptoms (Perombelon and Kelman, 1980). This is referred to as the bacterial latent stage. When certain conditions arise, this latency is broken and the bacteria become pathogenically active (Perombelon and Kelman, 1980). Bartz (1981) also observed a latency period in tomato fruits inoculated with Erwinia carotovora ssp. carotovora. The variable latency phenomenon was suggested to be due to the host plant temporarily localizing the pathogen rather than an artifact caused by a method of inoculation. This latency phenomenon has not been reported in Chinese cabbage. Effects of Raised Beds/Spacing on Production The use of raised beds in the production of vege- table crOps has recently received a lot of attention. There is evidence which suggests that the use of the beds creates a nonconducive environment for disease organism growth and infestation. The use of raised beds in the production of tomatoes reduced the incidence of soft rot caused by E. carotovora when compared with a raised bed with plastic mulch (Segall and Dow, 1977). The use of beds has also resulted in a reduction of bottom rot disease in lettuce caused by the fungus, Rhizoctonia solani (Pleczarks and Lorbeer, 1974). Any cultural practice which results in good air circulation and lower air and soil temperatures might effectively control diseases that flourish in high temperature and high humidity conditions. Tumuhairwe and Gumbus (1983) found that the use of raised flat beds had the lowest temperatures in the afternoon when compared with single row ridges, double row ridges, and cambered beds. Since Erwinia infection is facilitated by insect or mechanical damage, efforts to prevent injury to the petioles should be taken. Chinese cabbage production in Hawaii involves the use of raised beds primarily to aid in the draining of the heavy, poorly drained soils (Ryder, 1979; Nakagawa, 1957). Leal (1974) found no significant effects of the use of raised beds on cabbage production; Gutierrez (1938) found that the use of ridges increased yields of cabbage when compared to the use of raised flat beds. In soils which are well drained, Chinese cabbage is sown in sunken or flat beds to minimize low water stress (Nakagawa, 1957). Raised beds used in conjunction with black plastic mulch increased marketable yield of sweet potatoes in New England when compared with production without mulch and raised 10 beds (Hochmuth and Howell, 1983). It was suggested that the increase in root yield was due to increased soil tem- peratures and less soil compaction under the mulch. Tumuhairwe and Gumbs (1983) evaluated the effect of bed type and mulch on cabbage growth. It was suggested that the use of bed type effected earliness of yield. The order of earliness was as follows: double row ridges > single row ridges > raised flat beds > cambered beds. Average head weights were also significantly effected by bed type. Head weight was in the order: double row ridges > single row ridges > raised flat beds > cambered beds. Soil mois- ture content was also highest in the raised flat beds and lowest in the single row ridges. The use of bagasse (sugar cane) mulch had no effect on soil temperatures, headweight, or earliness. Mulching increased the soil moisture content, but did not increase the occurrence of diseases. Plant spacing also has some effects on the inci- dence of diseases. Minton (1980) found that the number of cotton seedlings with Verticillium wilt symptoms increased with increasing plant density. Thomson, Hills, Whitney, and Schroth (1981) found that when in row spacing increased from 15 to 45 cm in sugar beet production, the incidence of bacterial vascular necrosis and rot (Erwinia carotovora ssp. betavasculorum) linearly increased. It was suggested that the less dense population allowed the roots to grow 11 rapidly and develop more cracks than the more dense plant- ing. The resulting cracks became entry portals for the bacteria. Plant spacing has been extensively investigated, primarily for determining the optimal spacing combination (between and within row) for new cultivars or extending the growing season. Total production of cabbage was higher with a plant density of 71700/ha when compared to 53700/ha; however, the average head weight was higher in the less dense planting (Molero and Perez, 1978). Similar results were also reported when varying cabbage plant densities in no-till systems (Knavel and Herron, 1981). Effects of Calcium on Disease Occurrence Several studies have been conducted to evaluate various effects of calcium on the incidence of diseases and cell wall softening. Plant tissue or prepared sub- strates are used to study pectolytic enzyme activity by which the bacteria are able to macerate plant tissue. Hancock and Stanghellini (1968) found that the pectolytic enzyme, pectate lyase, is dependent on calcium for its activity in Fusarium infested squash hypoctyls. They also found that as calcium concentrations of a sub- strate (pectate) preparation increased to 10.4 M, the rate of pectate degradation was accelerated; however, at rates 3 of 10- M calcium, maceration of tissue was inhibited. 12 Research on Rhizoctonia solani in bean hypocotyls by Bateman and Lumsden (1965) indicated that tissue maceration is greatly inhibited by the presence of barium, calcium, and to a slight degree, by magnesium. Bateman and Lumsden (1965) also found that tissue resistant to maceration by polygalacturonase from Rhizoctonia was high in calcium pectate and suggested that an acquired natural resistance to maceration occurs with aging in bean hypocotyls due to the conversion of pectin to calcium pectate. This increase in calcification was confirmed (Stockwell and Hanchey, 1982); however, it was suggested that the acquired resistance by the older plants was not simply due to increased calcification. Instead, a lack of plant exudates on the hypocotyl surface in older plants may decrease the pathogen's ability to form an infection cushion, which preceeds the invasion of the tissue. Older plants have a more extensive cuticle which may result in less exudation by the plant tissue, thereby inhibiting infection. Deshpande (1959) also noticed a decrease in proto— pectinase (from Rhizoctonia) activity after applying both + + +, Mg +) to a monovalent and divalent cations (Na+, Ca protopectinase solution. He noticed a greater loss in enzyme activity with divalent cations. However, when potato discs were soaked in salt solutions, there was no effect on protopectinase activity. Tribe (1955) found that and NaOH concentrations increased in as NaCl, MgSo CaCl 4' 2' l3 cucumber tissue, the resistance to maceration by Botrytis cinerea and Bacterium aroidease enzyme preparations increased. No significant decrease in maceration was observed with the addition of monovalent ions (Na, K). Sladdin and Lynch (1983) found that CaO2 has anti- microbial (fungi and bacteria) properties when used as a seed treatment. Sugars and amino acids exuded by imbibing seed is suspected of stimulating microbial growth which can compete with the seed for oxygen. It was suggested that the antimicrobial properties of CaO2 was due to its ability to provide oxygen to the seed, especially in cold, wet soils. Segall and Dow (1977) found that by using soil applications of calcium chloride, they were able to reduce the incidence of soft rot in tomato caused by Erwinia carotovora; however, the effect was not statistically sig— nificant. Scientists in Wisconsin have also studied cal— cium effects on soft rot infection in potatoes and have reported preliminary studies that suggest the maceration of potato tubers may be inhibited when calcium levels are relatively high (Anonymous, 1983). Bangerth (1979) suggested that in the absence of calcium, cell walls and the middle lamellae may weaken in many plant tissues. McClendon and Somers (1960) found that although the calcium bridges formed with pectic compounds resulted in greater tissue strength, maceration by l4 pectinase (from fungi) does not seem to be inhibited. Cleland (1960) suggested that noncarboxyl organic sub— stance linkages were responsible for cell wall rigidity rather than the bridges formed in the pectic compounds. However, Stapp and Hartwich (1957) found that as the total calcium content in potato plants decreased, the suscepti— bility to Erwinia phytophora increased. Two modes of action for calcium are proposed (Edgington, Corden, and Dimond, 1961): the enzyme of the pathogen is unable to hydrolyze the calcium/pectate linkage or the calcium released as a result of hydrolysis is inhibitive to the enzyme of the pathogen. Other Functions of Calcium Calcium has been associated with several other metabolic processes. After calcium forms a complex with the protein calmodulin in the cytoplasm, several enzymes within the cell are activated, thereby regulating those metabolic processes associated with these enzymes (Marx, 1980). It is currently thought that calmodulin serves as an intracellular calcium receptor which readily binds to calcium (Marx, 1980). This theory is supported by Leshen, Sridhara, and Thompson (1984) who found that the presence of an internal calciumzcalmodulin complex enhanced senescence in pea foilage by activating the enzyme, phospho— lipase, which degrades membrane structure. 15 It has been suggested that calcium in apple tissue rigidifies the membrane, thereby maintaining membrane integ— rity and ethylene biosynthesis (Legge, Thompson, Baker, and Kieberman, 1982). The preservation of the membranes by calcium maintains activity of a membrane bound enzyme necessary for conversion from 1—aminocyclopropane— carboxylic acid (ACC) to ethylene. Lieberman and Wang (1982) observed similar results with apple tissues with extremely high levels of calcium (lOOmM) which inhibited deteriorative changes in the cell membranes. After a six— hour incubation of the tissue, calcium and magnesium treat- ments prevented the deterioration of membranes which can cause lipid perioxidation and protein leakage. Christian— sen and Foy (1979) also suggested that calcium plays a role in membrane stabilization. Jones and Lunt (1967) sug- gested that calcium may play an important role in membrane structure, chromosome structure, and enzyme activation. Poovaiah and Leopold (1973) also found that calcium inhibited the increase in free space and membrane permea— bility in leaf discs of corn and Rumex which were corre- lated with senescence. Calcium was shown to inhibit leaf abscission in bean petiole explants. Poovaish and Leopold (1973) sug- gested that the inhibition of abscission by calcium may, in part, be due to the salt bridges formed in the cell 16 walls between pectic compounds. However, the ability to inhibit senescence by reducing the tissue response to and production of ethylene may also be a major contributing factor. It has also been inferred that calcium ions are essential for the maintenance of the selective ion absorp- tion mechanism in the cell membranes (Epstein, 1961). In the presence of calcium, potassium and rubidium are mutually competitive. It has also been found that applications of benzyl- adenine or kinetin to young apple trees resulted in increased movement of calcium into the mature leaves (Shear and Faust, 1970). Possible explanations are that either the rate of senescence was retarded or the transpiration rate was increased which resulted in more influx of calcium. Calcium also seems to play a role in chlorophyll synthesis. Calcium can induce the production of chlorophyll b from chlorophyll a in the dark (Tanaka and Tsuji, 1981). Calcium Deficiency Symptoms Due to the poor phloem translocation of calcium within Chinese cabbage, calcium deficiency is generally characterized by extensive marginal tipburn in the young, expanding leaf tissue (Juo, Tsay, Tsai, and Chen, 1981); Hori, Yamasaki, and Kamihama and Aoki, 1960). The older, 17 more fully expanded leaves only show tipburn symptoms under severe deficiencies. Transpiration of the mature leaves of cabbage serves as a strong sink for most of the calcium contained in the xylem sap which results in a localized calcium deficiency in the margins of young leaves due to their low rate of transpiration (Wiebe, Schatzier, and Kuhn, 1977). Collier and Huntington (1983) found low levels of calcium in young leaves of lettuce, which developed tip burn, and higher levels of calcium in the older leaves, which were free of tip burn. Similar con— clusions were made concerning many other crop plants (Kirby, 1979). This calcium disorder is often referred to as heart rot of cabbage (Hori, Yamasaki, Kamihama, and Aoki, 1959), and is more likely to occur in low relative humidity con- ditions. The low humidity promotes a high rate of trans- piration and calcium accumulation in the older leaves. As a result, a localized deficiency in the young expanding leaves develop. However, high humidity promotes a more uniform distribution of calcium throughout the plant. Transpiration rates are greatly decreased in high humidity and calcium in the xylem sap is more readily transported to the young growing leaf. The ability of the young grow— ing tissue to receive adequate levels of calcium has been determined to be dependent on the presence of a positive root pressure which can result in guttation. It is sug— gested that the establishment of high relative humidity 18 at night and rather dilute nutrient solutions favor the development of guttation and effective transport of cal- cium to the apex of strawberry plants (Bradfield and Guttridge, 1979; Guttridge, Bradfield, and Holder, 1981). Root pressure has also been determined to be required to transport adequate levels of calcium to cabbage leaves which are not actively transpiring (Palzkill and Tibbitts, 1977; Palzkill, Tibitts, and Williams, 1976). Similar observations were also observed in tomatoes (Bradfield and Guttridge, 1984). In many heading vegetables, a localized deficiency of calcium can eventually result in internal browning of the head. When extensive tip burn occurs, pathogens are then able to invade the plant and cause extensive rotting. Sonneveld and Mook (1983) found that increasing levels of sodium and magnesium in irrigation water resulted in increased tip burn in lettuce. The addition of calcium to the irrigation water resulted in a reduction of tip burn. The cation ratios were determined to be very impor— tant in the development of tip burn. In high Mg/Ca ratios, magnesium may substitute calcium in the cell membrane which may have deleterious effects on membrane integrity. MATERIALS AND METHODS Field Studies: Cultural Practices Between and within row spacing and raised beds were used in an effort to increase air circulation and lower the air temperature in the immediate phyllosphere of the Chinese cabbage plant and eliminate the environment for the proliferation by the soft rotting bacterium, Erwinia carotovora ssp. carotovora. Heat tolerant cultivars were also examined for summer production and soft rot resistance. All field studies were conducted on a Spinks loamy sand that was fertilized with 908 kg/ha of 16-16-16. Following bed formation using a single moldboard plow and shaper, the beds were allowed to settle and weed seeds to germinate. A contact herbicide (paraquat) was applied to the weed seedlings one week before planting. The beds were 91 cm wide and 15 cm high, with two rows of plants per bed. Spacing between beds was 198 cm from the center of one bed to the center of the other. Land without beds, hereafter referred to as flat culture, was prepared by plowing, disking, fertilizing with 908 kg/ha of 16-16- 16, and then harrowing. One week after sowing, the emerged Chinese cabbage seedlings were thinned by hand and watered with 120 ml per 19 20 plant of a starter fertilizer (10—52-17) and insecticide (diazinon) mixture to promote early growth and protect against maggot infestation. Following thinning, side- dressings of 172 kg/ha ammonium nitrate (NH4N03) were applied biweekly. At the onset of heading, a sidedressing of 172 kg/ha of potassium nitrate (KNO3) was applied. Insect and fungal disease control was maintained with a weekly spray program. COpper or bacterial inhibitory compounds were not applied at any time. All experiments were irrigated with overhead sprinklers to insure 3 cm of water per week. Mean headweight, percent harvested, percent rotted of total, and percent bolted of total were the parameters measured. Percent bolted was not recorded in Experiment No. 1. Data from all experiments were analyzed statisti- cally using analysis of variance (AOV) and least signifi- cant difference (LSD) after apprOpriate transformations were made. Experiment No. l, 1982 Seed of the spring/summer cultivar, 'Nagaoka No. 2' (Wong Bok type) were sown at two week intervals on beds starting on June 2 with the last planting on July 12. The objective was to correlate a sowing date with a maximum amount of soft rot occurrence. This would provide a 21 guideline as to the proper time to sow the seeds for the evaluation of the various treatment effects. SpaCing between rows was 30, 46, and 61 cm while spacing within rows was 30, 46, 61, and 76 cm. Mature heads were har- vested on August 7, August 22, September 6, and Septem- ber 23, respectively. The experimental design was a completely randomized design with three replicates. Each experimental unit was comprised of 16 plants. Experiment No. 2, 1983 Several tropical (heat tolerant) cultivars which have been reported to have resistance to high temperatures, were evaluated for their raction to soft rot. These culti- vars are smaller in size at maturity than spring and fall cultivars, producing heads which weigh 1-2 kg as compared to 3-4 kg. 'Nagaoka No. 2' was also used to compare a spring/summer cultivar to tropical cultivars. The culti- vars evaluated and their seed source are shown in Table 1. Table 1. Chinese cabbage cultivars and their source Variety Classification Source Nagaoka TrOpicana TrOpic Takii TrOpical Pride Tropic Sakata Saladeer Tropic Takii Asveg No. 1 TrOpic AVRDC 77M(3)-26 Tropic AVRDC Tropical Delight Tropic Sakata Nagaoka No. 2 Spring/Summer Takii 22 The cultivars were grown on beds and flat culture land to determine if the beds significantly effected the environment in the phyllosphere and inhibited rot progres- sion. The seed were sown on June 16 in a split plot design with three replicates and 18 plants/experimental unit. The beds were the main plots and the cultivars, the subplots. The seeds were sown in paired rows (flat cultue and bed) with between and within row spacing of 61 and 46 cm, respectively. Harvesting began on August 21 and continued until September 1. EXperiment No. 3L 1983 Between and within row spaCing treatment combina- tions were conducted on beds to evaluate the effects on soft rot occurrence in beds with the cultivar, 'TrOpical Pride'. The seed were sown on June 17 in a randomized complete block design with three replicates and twin rows on the bed. The treatments were comprised of combinations of 30, 46, and 61 cm between and within rows for a total of 9 treatment combinations each containing 18 plants/ replicate. Harvesting began on September 3 and continued until September 15. Experiment No. 4, 1984 The efficacy of beds in combination with between and within row spacing and cultivars in reducing the incidence of bacterial soft rot (Erwinia carotovora ssp. 23 carotovora) was evaluated. Three cultivars were used: 'Nagaoka No. 2', 'Tropical Pride', and 'Asveg No. 1'. Between and within row spacing consisted of combinations of 30, 46, 61 cm. Seeds were sown in bed and flat culture land in paired rows on June 17. The experimental design was a split plot with three replicates and twenty plants/ experimental unit. The beds were the main plots and factorial combinations of between/within row spacing and cultivar, the subplots. Afternoon air temperatures in the phyllosphere of all treatment combinations were measured daily between 3 and 4 p.m. with a portable thermograph at the beginning of heading until harvest. Harvesting began on August 30 and continued until September 17 since the close spacing treatments matured later. Calcium Effects The effects of calcium levels in the Chinese cabbage petiole on the incidence and progression of soft rot were evaluated by modifying the calcium content of the petiole. Two methods were used: foliar applications of CaClz and a nutrient sand culture system. Petiole sections were inoculated with E. carotovora ssp. carotovora and rated for the incidence and progression of disease symptoms. Tissue Analysis Procedures At each sampling date, the third and ninth petioles were removed from each sampled plant. Prior to tissue H- —- L ..'.3. ‘_&..—. _'. ._ 24 analysis, a 6.45 cm2 section 2.54cm from the base of the petiole was removed for bacterial inoculation. The remain- ing portion of the petiole was rinsed in deionized water for two minutes and forced air oven dried at 130°C. The dried tissue was groumdwith a Wiley mill. A .250g dry weight sample of each petiole was wet ashed in 5ml of con- centrated (70%) HNO3 and heated until completely digested. Hydrogen peroxide (30%) was added (approximately 2ml) drop by drop following digestion to complete the oxidation. Final dilutions were made with 1000ppm of LaCl3 and stored in polyethylene scintillation vials. The diluted samples were measured for total calcium content using an atomic absorption spectrophotometer. Bacterial Inoculation A colony of E. carotovora ssp. carotovora was trans- ferred from a 48 hr. Rifampin (antibiotic) resistant culture (Weller and Saettler, 1978) to 50ml nutrient broth (Difco) and incubated on a rotary shaker at 28°C for 8 hours. The Rifampin resistant culture was obtained by inoculating petri plates with nutrient agar containing Rifampin with 2ml of an Erwinia culture that had incubated in 50ml of nutrient broth (Difco) for 12 hours at 22°C. Two days after the Rifampin nutrient agar inoculation, growing Rifampin resistant colonies were transferred to another Rifampin nutrient agar petri plate. The use of 25 an antibiotic resistant mutant of Erwinia facilitated the maintenance of a pure culture and the reisolation from 4 CFU/ml inoculated tissue. After incubation, a 10 inoculum was prepared by injecting .065ml of the nutrient broth into a serile 100ml volumetric flask and brought to volume with sterile saline (.09% NaCl). Sterile toothpick tips (Williams, 1983) were soaked in inoculum for five minutes. The 6.45 cm2 section of petiole was prepared for inoculation by soaking it for one minute in a 10% sodium hypochlorite (bleach) followed by a rinse in sterile saline for one minute. The petiole section was placed in a sterile polystyrene petri dish on a sterile moistened filter paper (12.5cm dia.). Each petri dish contained an additional petiole section to serve as a control. The toothpick tips were then inserted into the petiole sections and incubated at 30°C for 36 hours and evaluated for rotting symptoms (Fig. 1) (1 = no rot symptoms, 5 = completely rotted). The control petiole section in each petri dish was inserted with a toothpick tip soaked only in sterile saline. Foliar Calcium Application: General Practices The plants were sprayed to runoff with a C02 pressurized sprayer equipped with a single 80/0/4 flat fan nozzle. Combinations of various concentrations of CaCl2 and 26 Figure 1. Rating scale used to evaluate the incidence and progression of soft rot in inoculated petiole sections of Chinese cabbage (1 = no rotting symptoms, 5 = completely rotted). g-"-’l"l|||l'$|n..|l|'| .ll.|l|ul.c- i“'§'.'-l--'0ll' lllllllll ’ llllll ‘i':-’.:'-""-’{cl'io'I’uI-il'U-IIII IIIIIIIII | é'---".""-'l'lrllpslll’I-"xlxrnu)--.-I\\0.|I.\t E:‘.‘|--'.""".¢a"l.-ll-Iu'|-‘l.l.-|.I"---I II - 1Hiihlnl.‘-""‘""”'.’"“-"--Il'"- ........ ‘ 33%."-0. n70-10. 'iltrlr-I'Iu‘l"-’8’--"--I'II‘-I. iz'i1’13,,c."j““cli.l.0"!I-I'II\I.I¢\..I‘I.OI'|-\I. ik.t'ii:."ul'S“!"’tufi'-"""'-"'l-..-.----. ii‘:.‘§i.-i’l-‘~l|Ilml.".lrul.l_.3l\‘5‘].‘0'0030‘000I'. .i-’.‘£’r‘tl..ffllml"2'.IQ-tl"3 IIIIII 4 llllll 5 IIIIII . i-“‘n|'.-a\".lslb’lxli lllllllll I..."f..l.r|li||-...l..¢ol. ....--‘I‘--‘u'r.fl lllll 5....“1 .¥.0H.“"“.f I! . .. I .,-‘ 1“. . "’ ... “ if 7;... m 2". l‘ i 1.3.9...- 1. J. 1 1.1 ...4 ...... , I I. at--. i....IL. .. .9) .....-“lv‘u'fl 1“--"l¢."-y.wl.l .. 1"“‘-‘ll"‘s‘m.. 0 28 surfactants were applied (Table 2). The plants were sprayed weekly with fungicides and insecticides. Biweekly sidedressings of 172kg/ha ammonium nitrate (NH4NO3) were applied. Table 2. Concentrations of CaCl2 and surfactants used in greenhouse foliar application experiment Grams CaClZ/liter Surfactants 7.06 Ortho X-77 (.5%) 14.12 Crop Oil Concentrate (.6%) 21.18 Foliar Calcium Application: Field Seeds of 'Tropical pride' were sown in the green- house on July 5, 1984, in sterilized black plastic tray cells (6.35 x 7.00cm) containing vermiculite. Upon germination, each cell was thinned to one plant and watered daily with complete fertilizer (Table 3) (Epstein, 1972, p. 39) containing 0.5mM CaClZ. The treatments were applied weekly. The seedlings received weekly foliar applications of CaCl2 until transplanting on July 26 for a total of three applications. The field experimental design was a randomized complete block with two replicates and 10 plants per experimental unit. The plants were grown on a 29 Spinks loamy sand which had been preplant incorporated with a herbicide (trifluralin) at 908 gms of active ingredient per hectare and fertilized with 1135 kg/ha of 16-16—16. At the onset of heading, petioles were removed from five plants of each experimental unit and evaluated for total calcium content and degree of resistance to soft rot incidence and progression. Table 3. Concentration of nutrient solution (less calcium) used in nutrient culture experiments* Concentration Salt KNO3 6.0mM Mg(NO3) 2 1 5mM NaH2P04 1.5mM MgSO4 0.5mM Micronutrients Fe (Sequestrene) 1.4mM H3BO3 25.0UM MnSO4 H20 2.0UM ZnSo4 7H20 2.00M CuSo4 SHZO 0.SUM H2M004 (85% M003) 0.5UM *Modified from Epstein (1972), p. 39. .ra-z ‘9‘ a..- ... 30 Foliar Calcium Application: Greenhouse Seeds of 'Tropical Pride' were sown in the green- house on October 24 in sterilized wooden flats with No. 3 grade vermiculite and fertilized with nutrient solution (Table 3). The seedlings were transplanted on November 12 into a greenhouse bed. The planting in the beds was staggered over a 30-day period to facilitate proper mainte— nance of plots and the preparation of each sampling for tissue analysis and bacteria inoculation. The experimental design was a randomized complete block with each of three beds comprising the blocks. Spacing within and between rows was 38 and 61 cm, respectively. Combinations of CaCl2 levels and the use of crop oil concentrate (COC) surfactant were applied biweekly after transplanting for a total of six applications (Table 2). Petioles were sampled at 30, 60, and 90 days after transplanting from three plants of each treatment combination. Plants sampled 30 days after transplanting received three treatment applications. Plants sampled 60 and 90 days after trans— planting received six treatment applications. Sand Nutrient Culture In April of 1984, four nutrient culture systems (10 crocks per system) were constructed to increase the calcium levelijithe petiole tissue through the roots. The system was designed for recycling the nutrient solution 31 runoff to the seedlings after pH adjustments. Glazed ceramic crocks (21.6 x 24.1cm), lined with plastic liners to prevent leakage from cracks, were placed on greenhouse benches. The drainage hole in the crock was fitted with a one hole No. 4 rubber stopper. A .32cm teflon tubing was inserted through the stopper so that 2.54cm protruded on each side of the stopper. A 20ml polyethylene scin- tillation vial with the bottom removed, filled with glass wool was fitted to the rubber stopper inside the crock. Silicone sealer was used to prevent nutrient leakage where the vial pierced the plastic liner. Each crock was filled with 9.08 kg of coarse silica sand. A 200 liter plastic barrel was used as a reservoir and collector for each system. The reservoir barrel was placed above ground and the drainage barrel partially burried in the ground beneath the greenhouse bench (Fig. 2). For the drainage system, a .64cm Tygon tubing was attached to the drainage hole Teflon tubing outside the crock and the other end was inserted into 3.8lcm rigid PVC drainage pipe. The exterior of the Tygon tubing was painted silver to inhibit algae growth. This rigid PVC pipe was installed with a grade beneath the bench. The PVC pipe was capped at one end and fitted with an elbow on the other end to help in the collection of the nutrient solution. 32 .cmusxmm Ugo ommbbmo omwcflno ca COHumasfidooo Esfloamo oHofipom ommmuocfl on com: Eoummm munpaso ucmfluwsa ccwm .m musmflm s. ... ... .4 , _ ._ 34 The nutrient solution was delivered to each crock by a 1.5 amp submersible pump placed inside the reservoir barrel. A flexible PVC aflflxm;(1.27cm), which ran the length of the bench, was attached to the pump and the solu- tion was fed to individual crocks through a trickle irri- gation tubing. An air line was installed in each reservoir to continually aerate and maintain uniform concentrations of the nutrient solutions. All four systems were washed, acid rinsed with a dilute HCl solution (.2N), and rinsed thoroughly with deionized water prior to operation. The nutrient solution treatments consisted of a basic nutrient solution (Epstein, 1972, p. 39) minus calcium (Table 3) to which different levels of calcium were added as CaClz. The pH was adjusted with 1n NaOH to 5.0-5.5 for maximum calcium solubility. Two week old seedlings were transplanted into the crocks through a hole in nylon mesh which was placed on the surface of the sand of each crock. After transplanting, perlite was placed on top of the nylon mesh to reduce the light reaching the surface of the sand thus inhibiting algae growth. The nylon mesh facilitated the removal of the perlite at the time the roots were removed for reuse of the nutrient system. Each treatment consisted of 10 crocks each containing one seedling each. The crocks from each of the two treatments on each bench were randomly arranged with two benches comprising the entire experiment. 35 Day and night greenhouse temperature was maintained at 22°C i 5°C and flourescent lighting placed 91cm above the top of the bench to provide a 16-hour-day environment. For the first week after transplanting, the plants were automatically watered three times daily to saturation by the use of time clocks. By the second week, the plants had developed a root system and the frequency of watering was reduced to twice daily. At harvest, xylem exudates were collected from decapitated plants to evaluate the calcium level within the xylem stream. Time of collection was between 10 and 12 a.m. After decapitation at the base of the plant, the plant stumps were rinsed with deionized water. Gum rubber tubing (.64 x 8.00cm) with a thin coating of petroleum jelly on the inside of one end was then slipped onto the plant stump (Noggle and Fritz, 1976). After 20 minutes, exudate samples were collected with transfer pipets and discarded. Exudate samples were collected again, one hour later, and put in preweighed polyethylene scintilla— tion vials. The total weight of the vial and exudate was determined. The exudates were diluted with 1000ppm of LaCl3 and analyzed for calcium concentration. Experiment No. 1 In this study, seeds of 'Tropical Pride' were sown on April 29, 1984, in vermiculite in wooden flats and 36 fertilized until transplanting into the crocks. A basic nutrient solution minus calcium (175 liters) was prepared for each of the 4 reservoir barrels (Table 3). The calcium concentrations used were .25mM, 1.00mM, 4.00mM, and 6.00mM. The pH of the solution was adjusted to between 5.0 and 5.5 with In NaOH for maximum calcium solubility. The plants were harvested on June 29 at the onset of heading. The petioles were removed from each plant and prepared for total calcium analysis and evaluation of soft rot inci- dence and progression. The petiole sections were sprayed with carborundum prior to spraying with an inoculum of 104 CFU/ml Erwinia. The petiole sections were incubated at 30°C and periodically observed until the sections had totally rotted. Differential extraction of ground freeze dried petiole tissue into an acetate (calcium pectate) and hydrocholoric acid (calcium oxalate) fraction was conducted (Ferguson and Turner, 1980). The purpose was to determine if a particular calcium fraction was more closely related to soft rot progression than total calcium. Sequential fractionation of ground petiole tissue (100mg) initiated with the addition of 3ml 80% acetate and agitated for 30 minutes. The sample was centrifuged at 500g for 10 minutes. The supernatant was collected and the whole procedure repeated on the same sample. After collecting the acetate 37 fraction from the sample, 3ml 5% HCl was added. The sample was agitated for 30 minutes and centrifuged at 500g for 10 minutes. The resulting supernatant was collected and the procedure was repeated. The residue remaining after both the acetate and HCl extractions was digested with HNO3 and H202 as described previously. The collected supernatants were placed in 25ml volumetric flasks and heated to evaporate half of the liquid. After evaporation, HNO3 and H202 were added to ensure total digestion. ApprOpriate dilutions were made and analyzed for calcium on an atomic absorption spectrophotometer. Experiment No. 2 This nutrient culture experiment was begun on September 19 to determine if petiole tissue calcium accumu- lation could be enhanced above that observed in the preceeding experiment and to evaluate the effect of a high level of calcium on soft rot incidence and progression. Two seedlings of 'Tropical Pride' were transplanted to each crock on October 10. The concentrations of calcium used were 1.00mM, 3.50mM, 6.00mM, and 8.00mM. The first harvest was made on November 12 and the second harvest on Decem- ber 7 which corresponded to 30 and 60 days after trans— planting, respectively. Xylem exudates were collected on each sampling date. Petiole tissue was prepared for calcium analysis and soft rot resistance evaluation. 38 Experiment No. 3 This planting in the nutrient culture system was conducted using the cultivar ‘Hakuran' (Takii Seed Co.; Kyoto, Japan), a cross between Chinese cabbage and heading cabbage. This cultivar has been reported to show resis- tance to bacterial soft rot (Nishi, 1981). Head cabbage was the source of resistance to soft rot. Two seedlings were transplanted into each crock on February 5, 1985. Calcium concentrations used were 1.00mM, 3.50mM, 6.00mM, and 8.00mM. The first and second samplings were taken on March 10 and April 6; 30 and 60 days after transplanting, respectively. Xylem exudates were collected on both sampling dates. Linear regression was conducted for all experiments to determine the significance of relationships between calcium and disease resistance in young and old tissue. Petiole Lignin and Cuticle Observations Lignin has been reported to be resistant to break- down by many microorganism (Vance, Kirk, and Sherwood, 1980). Differences in lignin content between Chinese cabbage, Hakuran (Chinese cabbage x cabbage), and heading cabbage were investigated to determine if the mode of resistance to soft rot could be due to high lignin content in the vascular system. 39 Thin petiole cross sections were stained with 1% phloroglucinol in 95% ethanol, followed by the application of a few drops of concentrated HCl (37%). The tissue was then rinsed with water and evaluated for lignin content with the use of a light microscope. Differences in cuticle thickness was also examined under the microscope. RESULTS AND DISCUSSION Field Studies: Cultural Practices Experiment No. l, 1982 The purpose of this experiment was (1) to gain an understanding as to when the Optimal field conditions and plant age interact to promote the incidence of soft rot in a field of Chinese cabbage, and (2) to learn whether the between and within row spacing on raised bed culture would modify the environment sufficiently to affect the incidence and progression of soft rot. The susceptible cultivar, 'Nagaoka No. 2"', was used. The first planting was first harvested on August 7, the second planting on August 22, the third planting on September 6, and the last planting on September 23. The incidence of soft rot was significantly higher if the crop was planted after the June 16 planting date (p=.05) (Table 4). Togashi (1979) reported that there was a relationship between the physiological age of the plant and the incidence of bacterial soft rot. He found that the population of Erwinia reaches inoculating levels in the soil as the plant begins to form a head. The data suggest that the relationship between the time of heading and the degree of soft rot infection may have been the strongest 40 $1 ... -...— 41 Table 4. The effect of sowing date on the incidence of soft rot and head weight of the Chinese cabbage cultivar, 'Nagaoka No. 2.‘ Values in % rot rolumn are the arc sine /F and %: respectively. Sowing Date % Rotted Plants Head Weight (95) (1b.) (kg) June 2 16.22 ( 7.8) 3.35 1.52 June 16 13.03 ( 5.1) 2.16 0.98 June 28 28.87 (23.3) 2.84 1.28 July 12 28.41 (22.6) 3.09 1.40 LSD (.05) 6.34 .43 after the June 16 planting for the summer of 1982. However, the data represent only one production season. Between and within row spacing treatments had no significant effect on the incidence of soft rot. The number of harvestable plants was significantly affected by a planting date X within row spacing inter- action (p=.01). The reduction in harvested heads for all within row spacing treatments (30, 46, 61, 76cm) was the greatest for the June 28 planting date, the same planting date that showed the highest rot incidence (Table 5). For the June 28 and July 12 planting dates, the 30cm spacing showed a lower percentage of heads harvested than the 46, 61, or 76cm spacings. This reduction in the percentage of plants harvested may have been due to rot incidence, water 42 .AW\ mcflm oumv mGOmHHmmEoo cEdHoo QHSDHB MOM Umpoadoamo mosam> qu He.n u AHo.I emu mm.m u Amo.v qu Am.mmv vo.>v Am.mvv hm.mv Am.ovv hh.mm A>.mmv Hm.mm NH maze Am.Hmv mo.mv AH.mwv mw.ov Am.Hmv vo.mv Am.mmv mm.mm mm mash Am.mnv mm.mm Am.mmv mm.wm An.vmv vm.mm Ao.mmv v>.mo ma maze AH.mnv «a.mm “a.mwv em.mm An.mnv Hm.mm Av.omv Nn.mm N muse Aw. Awe Awe Awe mama oamvmumn vmammufim mmmovumv unnhmuom mcflucmHm Awumuomn Hem mummHmnaov mcflommm .mao>fluoommou.w can wx ocflm one can mcfidaoo CH mosam> .wucwam woumo>umn .m .02 mxommmz. mmmnbmo mmmnflnu mo m so mcflommm 30H cflnpfls cam dump mcfluamam mo pommmo one .m wanna 43 and nutrient competition, spacial competition between plants, and plant interaction (allelopathy) since a large number of plants did not reach maturity in the 30cm June 28 and July 12 plantings (Table 6) during the experiment. If nitrogen availability is decreased, the rate of leaf differentiation is retarded and prevents the formation of solid mature heads (Koda et al., 1974). If the rate of nitrogen fertilization was increased (> 45 kgs. N/A) in the high density planting (30cm), nitrogen availability should not be a limiting factor in maturity. Plants spaced at 30cm for the June 16 planting showed a higher percentage of heads harvested when com- pared to plants sown on June 2 at the same_spacing. This increase in the number of heads harvested is due to a low number of immature heads left in the field at the conclu— sion of the experiment (Table 6). Bolting did not occur in any of the treatments for the duration of the experi— ment. There was also a decrease in the incidence of soft rot in the June 16 planting; however, it was not signifi- cant. It may be that the competition between plants at the 30cm within row spacing was greater than the other within row spacing treatments (46, 61, and 76cm), assuming that there are no allelopathic interactions between plants. The weight of the marketable heads varied with the planting date. The head weight of the June 16 planting was significantly lower when compared to the June 2 44 Table 6. The percent of immature heads remaining after harvest for within row spacings at different planting dates of the Chinese cabbage cultivar, 'Nagaoka No. 2'. % of Immature Heads Within Row Spacing (cm) Date 30 46 61 76 June 2 21.7 18.5 17.4 13.1 June 16 8.1 27.2 22.4 17.3 June 28 41.1 18.6 28.1 21.9 July 12 43.9 31.7 28.7 17.6 planting (p=.05) (Table 4). Heads harvested form the June 28 and July 12 plantings were heavier when compared to the June 16 planting (p=.05). The temperatures may have been more conducive (cooler) for head development for the plantings before or after June 16 date, although the maximum/minimum temperatures for the latter half of the growing period for those plants sown June 16 and June 2 were very similar (Table 7). Cooler temperatures during the first half of the growth period of 'Nagaoka No. 2' may have promoted more rapid vegetative develOpment in the June 2 planting when compared to the June 16 planting which resulted in a more dense head. Since 'Nagaoka No. 2' was developed as a spring cultivar, it appears that the cultivar grows well vegetatively under cool temperatures. 45 Table 7. Mean maximum/minimum air temperatures for the first and second half of the growing period for various sowing dates (°C). First Half Second Half Date Maximum Minimum Maximum Minimum June 2 23 11 28 16 June 16 26 13 28 13 June 28 28 15 23 11 July 12 24 10 23 9 Marukawa (1975) reported that the optimum temperatures for vegetative growth and head formation are generally 20°C and 16°C, respectively. Maximum/minimum daily air temperatures for the June 28 and July 12 planting dates during the second half of the growing period were cooler than for the June date (Table 7). These cool temperatures promoted good head development. Head weight was also affected by within row spac- ing (p=.05) (Table 8). Plants harvested from the 46, 61, and 76cm spacings yielded significantly larger heads when compared to the 300m spacings while the 76cm spacing produced significantly larger heads than the 46 and 61cm spacings. These results suggest a neighboring plant interaction for nutrients, water, and space at the 30 cm spacing due to the overlapping root systems. Closer 46 Table 8. The effects of within row spacing on head weight of the Chinese cabbage cultivar 'Nagaoka No. 2'. Within Row Spacing Head Weight Average Tons/Acre (cm) (1b.) (kg) 30 2.32 1.05 22.42 46 2.78 1.26 17.26 61 2.92 1.32 12.81 76 3.43 1.56 13.76 LSD (.05) .43 spacings may have also resulted in shoot competition for light resulting in reduced plant photosynthetic efficiency. Some of the plant competition may be overcome by increas- ing the total amount of fertilizer applied over the production period. At the 76cm spacing, there may be less between plant interaction (nutrients, space, allelo- pathy, light, etc.) resulting in more unrestricted growth than the closer spacings (30, 46, 61cm). Several researchers have made spacing recommenda- tions. Matsumura (1980) generally recommends 600m between rows and 45cm within rows for early maturing cultivars, and 70cm between rows and 50 and 55cm within rows for midseason and late maturing cultivars, respectively. Nissley (1934) suggested 81-89cm between rows and 46-61cm within rows for most cultivars. Opena (1981) suggested 47 50cm between rows and 40—50cm within rows were optimal spacing conditions for Chinese cabbage production in Taiwan. The resultsfromthis experiment were variable and the optimal spacing treatment changed depending on the sowing date. Between row spacings of 60cm and within row spacings of 30 and 46cm produced the largest yields. Experiment No. 2, 1983 The objective of the experiment was to evaluate several tropical (heat tolerant) cultivars for summer production potential in Michigan. The tropical cultivars were also of interest because of the potential to improve marketability in the United States by growing cultivars that produce small heads. The small head, approximately 1-1.5kg in weight, is more attractive to the American con— sumer than the 5-6kg head. The tropical cultivars were grown on both beds and flat culture land to determine if the raised beds effected the environment in the phyllo- sphere and inhibited rot incidence. The experiment was direct seeded on June 16 and harvested from August 21 to September 1. The cultivars used in this study showed signifi- cant differences in response to summer production condi— tions. There were differences in the performances of the 48 cultivars for the parameters measured; percent bolted, percent rotted, percent harvested, and headweight. The amount of soft rot incidence between cultivars was significant. 'Tropical Pride' and 'Nagaoka Tropicana' showed a higher incidence of soft rot than 'Asveg #1', 'Tropical Delight', 'Salad-er', or 77M(3)-26 (p=.001). 'Tropical Pride' showed a higher incidence of soft rot than 'Nagaoka Tropicana' (p=.001). 'Asveg #1' showed more soft rot than 'Tropical Delight' and 77m(3)-26 (p=.05). Many of the tropical cultivars showed a high percentage of bolting (Table 9). It is apparent that the heat tolerant cultivars are generally more sensitive to low temperatures and can be readily vernalized (Xu, 1983). Guttormsen and Moe (1985) reported that certain heat tolerant cultivars ('Saladeer' and 'Nagaoka Tropicana') can be vernalized when grown at temperatures below 23°C. Two cultivars, 'Tropical Pride' and 'Asveg #l' at maturity did not bolt; however, upon cutting the heads transversely, a short flower stalk was present, indicating they had started to bolt. According to Nakamura (1976), most cultivars are able tx> safely form harvestable heads if 30-35 inner leaves have been developed in the apex prior to vernalization. If the plants become vernalized after the development of the inner leaves, head formation will be completed prior to flower stalk protrusion out of the head. Harvested heads within internal developing flower 49 mv.o hm.m wo.oa he.m AHoo.v omq om.o mm.m mm.w nm.v AHo.V own mm.o Hv.m ve.m mm.m Amo.v omq . . . . . . . . GNIAMVSee Amm ov mm H Aoo Hmv mm mm A00 0 I oo o Ano mmv we we Dam?N Amm.ov NH.N Amm.mmv mm.mh Amm.fi V mo.w Aoo.o I 00.0 H* mmbmm Amn.ov H>.H Amo.mmv on.mm Amm.o V mw.m Amm.Hmv mo.ov Hoocmamm . . . . . . . . ooflum Amm ov mo N on vmv Hm mm Amm mmv ma om A00 0 I oo o Hmoflmoue O O 0 0 0 O O O “CMOHQOIHB Aoa Hy me m Aow vmv Hm mm Aam HHV mm ma Amm any mm mm mxommmz . . . . . . . . pamnamo ANH 0. mm Amm o I no m Ava o v ma m Ame may am an HMOHQQHB Amxv A.QHV w w w Hm>HuHDU panama cmmm manmpmxumz w pom w uaom w .mam>fluoommmn .m can m\ ocwm own one massaoo pmo>umn w can .Dou w .vHOQw CH mosam> .mcofluflpcoo :owuoscoum museum 0» mum>fluaso mmmnnmo mmwuflno xflm mo noncommwu one .m mange 50 stalks are marketable, but quality and head density are reduced. It is possible that both 'Tropical Pride' and 'Asveg #1' grew faster vegetatively than the other tropi- cal cultivars and developed the critical number of inner leaves before vernalization occurred or they may require a longer period of low temperature to become vernalized. The incidence of soft rot and the percentage of bolting are two factors that effect the number of heads harvested. The greater the number of plants that showed soft rot symptoms or bolted, the lesser the number of plants that remained to be harvested. 'Asveg #1' had the highest number of heads harvested when compared to the other cultivars in the study (p=.001) (Table 9). 'Tropical Delight', 'Nagaoka Tropicana', 'Saladeer', and 77M(3)-26 showed a lower incidence of soft rot, but they bolted. It may be possible to grow these cultivars in Michigan if they are direct seeded after June 16 when warm temperatures prevail during the early growth period prior to the development of 30-35 inner leaves. 'Tropical Pride' did not bolt but was susceptible to soft rot. Advantages of these tropical cultivars are that they have a shorter maturity period and smaller headweights than spring and fall cultivars which produce heads that weigh from 1.4kg to as much as 6.8 kg each. 'Nagaoka TrOpicana' produced the heaviest heads and 'Tropical Delight' produced the lightest heads (p=.001) (Table 9). 51 Culture type (raised beds vs. flat culture) and the culture type X cultivar interaction had no significant effects on % bolted, % rot, % harvest, or head weight. 'Tropical Pride' and 'Asveg #1' were used in sub- sequent field experiments because of their resistance to bolting. In addition, 'Tropical Pride' was a desirable cultivar because of its susceptibility to soft rot. Experiment No. 3, 1983 This experiment was designed to determine if the various between row and within row (between plants) spac- ings on raised beds would significantly affect the phyllosphere environment and subsequently the incidence of bacterial soft rot. Daily phyllosphere air temperatures were recorded with a portable thermograph during the last two weeks before harvest. 'Tropical Pride', a susceptible cultivar, was direct seeded on raised beds on June 17 and harvested from September 3 to September 15. The between and within row spacings in raised bed culture had no significant effects on rot incidence or the number of heads harvested for the cultivar 'Tropical Pride' (Table 10). The between X within row interaction also did not effect any of the parameters measured. Headweight was significantly affected by within row spacing (p=.05) (Table 10). As the within row Spacing increased, the headweight significantly increased, 52 .mCOmflHMQEOO SEDHOU Gflflflflké HON mun. GDHMNV OmnH Hm. Amo.v omq os.ms «.mm mH.Hm p.mm em.H me.m Hm Hm.mv m.wm ma.em a.Hm eH.H em.~ we mm.qm a.mm ms.mfi o.oH mo.H mm.m om Amx mafim onmc Awe Amx mqflm one. Ame Imxv I.nHI lace ocflommm umm>nmm w pom w unmflmz comm 30m canons .mphaflnmomxums can .pon .uanoB new: no .mcflum HMOAQCHB. Hm>flpaso wmmnnmo mmwcflzo mo mcflommm Aucmam coonmnV 30H cflnuflz mo pommmw one .OH mange 53 suggesting that competition for nutrients, water, space, and light between plants decreased as within row spacing increased. Experiment No. 4, 1984 The purpose of this experiment was to determine the efficacy of the use of raised bed culture and various between and within row spacing combinations in reducing the incidence of soft rot and in maximizing summer pro- ductivity. Three cultivars, 'Tropical Pride', 'Asveg #1', and 'Nagaoka No. 2' were used. Seed were sown on June 17 on raised beds and in flat culture and harvested from August 30 to September 17. There were no main effects of cultivar or treat- ment on soft rot, however, the incidence of soft rot was significantly affected by a culture type x cultivar inter- action (p=.001) (Table 11). 'Tropical Pride' showed a significantly higher incidence of soft rot in flat culture than when grown on raised beds. 'Asveg #1' also had more soft rot in the flat culture than the raised beds while 'Nagaoka No. 2' had less soft rot in the flat culture when compared to raised beds (p=.05). Tanaka and Kikumoto (1976) also reported that Chinese cabbage leaves with high water potential were correlated with early soft rot symptoms of the heads. Since tropical cultivars require a shorter maturity period than the spring and fall 54 Table 11. The effect of cultivar and culture type on the incidence of soft rot on three Chinese cabbage cultivars. Values in columns are arc sine /§ and %, respectively. Cultivar Type of Tropical Nagaoka Asveg Culture PrI e No. 2 #1 ($5) ($5) (%) Raised Beds 6.65 ( 1.34) 18.55 (10.12) 3.00 (0.27) Flat Culture 25.55 (18.60) 12.12 ( 4.41) 11.07 (3.69) LSD (.001)=9.52 LSD (.05 )=5.56 LSD values are for arc sine /%comparisons within columns. cultivars ('Nagaoka No. 2), 'TrOpical Pride' may be more succulent and have a higher incidence of soft rot than 'Nagaoka No. 2' due to its rapid growth, turgid cells, and a thin cuticle. The turgid cells and thin cuticle may increase the incidence of petiole cracking which promotes insect damage and provides infection sites for Erwinia. Leaves with low water potentials failed to develop lesions. The rate of growth of 'Nagaoka No. 2' in flat cul- ture may have been decreased due to greater soil moisture and lack of oxygen than on raised beds. The root system of 'Tropical Pride' may have been more shallow than 'Nagaoka No. 2' allowing for sufficient available oxygen to reach the root system and prevent any appreciable 55 decrease in growth rate. Since the correlation of the Erwinia population in the soil with the physiological age of the plant is important for infection (Togashi, 1972), any disruption in the relationship will decrease the incidence of soft rot in 'Nagaoka No. 2'. Upon initial examination of the differences in rot incidence, it appeared that the use of raised beds reduced the temperature in the immediate phyllosphere to create a less conducive environment for the incidence of soft rot. Ten daily afternoon air temperature readings recorded from heading until harvest showed differences between the raised beds and flat culture (Table 12). Erwinia carotovora ssp. carotovora growth is promoted in high humidity (95%) as well as high temperatures. According to Togashi (1972), as the plant grows, evaporation of water from the surface of the soil in the phyllosphere is inhibited by the older leaves which cover the phyllosphere resulting in a humid microclimate. It is possible that increased aeration in the phyllosphere on the raised beds reduced the air temperature and humidity and reduced the incidence of soft rot in the field. The low incidence of soft rot on the raised beds may also be due to the location of the lower leaves in the phyllosphere. The lower leaves are more exposed than the same leaves of plants grown in flat culture which may allow for prOper insecticide coverage. 56 Table 12. Mean of ten daily afternoon air temperatures (°C) within the phyllosphere of culture types and ambient air temperature during the last two weeks prior to harvest of Chinese cabbage. Raised Beds Flat Culture Air August 15—21 29.0 31.0 28.0 August 22-30 28.3 30.0 28.3 Between and within row spacing treatment combina- tions did not significantly effect the incidence of soft rot. The tropical cultivars, 'Tropical Pride' and 'Asveg #1', produced visible flower stalks prior to harvest. 'Nagaoka No. 2', a spring/summer cultivar, produced signifi- cantly fewer heads with flower stalks when compared to the tropical cultivars (p=.001) (Table 13). The response to cool summer temperatures by the trOpical cultivars sug- gests that cultivars of tropical origin are more sensitive to vernalization than the spring cultivars. Temperature readings for the growing season show that minimum air temperatures during the second and third week after planting were low enough to vernalize the seedlings (Table 14). Generally, it only takes two weeks' exposure to minimum daily temperatures of 13°C or lower to initiate the development of a flower stalk within the plant. Since 'Nagaoka No. 2' was developed as a spring/summer cultivar, it has more resistance to bolting than the tropical 57 Table 13. Percentage of bolters produced by three culti- vars of Chinese cabbage under summer growing conditions. Values in column are arc sine /§ and %, respectively. Cultivar % Bolt (%) Tropical Pride 15.25 ( 6.90) Nagaoka No. 2 1.47 ( 0.06) Asveg #1 31.94 (27.98) LSD (.001) 6.75 LSD ( .01) 5.23 LSD ( .05) 3.95 Table 14. Mean maximum/minimum daily air temperatures (°C) for the 1984 growing season. Date Maximum Minimum June 17-24 27.6 16.1 June 25-July 2 24.5 12.1 July 3—10 26.7 13.6 July 11-18 28.3 16.6 July 19-26 28.5 15.3 July 27-August 3 26.9 12.3 August 4-11 29.6 19.1 August 12-19 28.0 14.7 August 20-30 28.9 14.7 58 cultivars. Guttormsen and Moe (1985) found that two tropical cultivars, 'Saladeer' and 'Nagaoka Tropicana' had less resistance to bolting than a spring cultivar, 'Nagaoka 60'. It is also possible that the spring/summer culti- var, 'Nagaoka No. 2' is better devernalized by the higher air temperatures reached during the day than the tropical cultivars. Elers and Wiebe (1984) reported that there is a devernalization (antivernalization) effect as temperatures increase in Chinese cabbage. A rise in temperature from 18 to 26°C delayed bolting. Since soft rot incidence of the tropical cultivars were higher in flat culture than raised beds, the number of heads harvested was also effected by a culture type X cultivar interaction (p=.01) (Table 15). Both 'Tropical Pride' and 'Asveg #1' showed a significantly lower per- centage of harvested plants in flat culture when compared to raised beds. Between and within row spacing treatment combina- tions did not significantly affect the percentage of marketable heads. Headweight was significantly affected by a culture type X cultivar interaction. The headweights of 'Nagaoka No. 2' and 'Asveg #l' were significantly lower in flat culture when compared to the raised beds (p=.001) (Table 16). 'Tropical Pride' however produced heads 59 Table 15. The effect of culture type on the percentage of marketable heads of three Chinese cabbage cultivars. Values in columns are arc sinc /§ and %, respectively. Cultivar EZIEuig Tropical Pride Nagaoka No. 2 Asveg #1 (%) (%) (%) Raised Beds 70.37 (88.70) 65.40 (82.60) 62.88 (79.20) Flat Culture 49.71 (58.20) 57.92 (71.80) 48.65 (56.40) LSD (.01)==10.84 LSD (.05)== 7.66 LSD values represent arc sine /% comparisons within columns. Table 16. The effect of culture type on headweights of three Chinese cabbage cultivars. Culture Tropical Pride Nagaoka No. 2 Asveg #1 Type (kg) (kg) (kg) Raised 3.29 (1.49) 6.24 (2.83) 3.18 (1.44) Beds Flat . . . . . . 4 Culture 3 16 (1 43) 4 88 (2 21) 2 30 (l 0 ) LSD (.001) = .67 LSD (0.05) = .39 LSD values represent lb. comparisons within columns. 60 which were similar in weight in both culture types. It is possible that the soil moisture content in flat bed cul- ture was higher than on raised beds. The decrease in headweight of the cultivars in flat culture may be due to the lack of adequate oxygen in the root zone for good root develOpment. Koda et a1. (1974) found that there can be significant differences in size and depth of root zones between cultivars. In flat culture production, cultivars with shallow root zones may be more severely stunted than cultivars with deep root zones. It is interesting to note that 'Tropical Pride' is a cultivar that does well in both culture systems. The cultivars, 'Nagaoka No. 2' and 'Asveg #1' may have more shallow root zones than 'Tropical Pride', however, evaluation of the root systems was not conducted. Calcium Effects Foliar Calcium Application: Field The objective of this study was to determine the effect of foliar applications (of CaCl2 on petiole calcium level and to determine the effect of petiole calcium levels on the incidence of soft rot. Two surfactants, crOp oil concentrate (COC) and X—77 (Ortho) were added to the CaCl solutions to determine if calcium absorption were 2 enhanced. The seedlings of the cultivar 'Tropical Pride' 61 received a total of three weekly foliar applications and the last treatment was applied prior to transplant- ing. The use of X—77 surfactant (.5%) with the CaCl2 solution, severely damaged the seedlings. The leaves of the transplants were extensively burned and the plants were stunted (Fig. 3). The damage was consistent in all treatments containing X-77 so the X—77 treatments were eliminated from the next foliar experiment. At harvest, 6.45 cm2 samples from the third and ninth developed petioles were inoculated with Rifampin (antibiotic) resistant Erwinia carotovora ssp. carotovora. Dead or senescent petioles at the base of the plant were not counted when the index petioles were sampled. Portions of these petioles were also freeze dried for calcium analysis. The total calcium concentration of the petioles was determined by atomic absorption spectrophotometry. The use of COC did not enhance calcium absorption by the outer or inner petioles when compared to foliar applications without COC. Foliar application (7gm/1) significantly increased the calcium content in the inner petioles when compared to the control (p=.05) (Table 17). Higher application rates (14 and 22gm/1) did not result in significantly different petiole calcium contents between the two treatments (p=.05) Foliar applications of CaCl2 62 .Anhlx msaflauucmfin .nnlxub o mvcflacomm omnnnwo omocwau co DGMDUMMHSm nhlx mo uomwmomoww .m musmflm 64 Table 17. The effects of foliar applied calcium chloride on inner petiole calcium content (% dry weight). gms/l (CaClZ) 0 7 14 21 1.9 2.4 2.9 2.5 LSD (.05) = .45 to the outer petioles did not significantly increase the petiole calcium content over the control. The apparent increase in calcium absorption from the foliar treatments may be due to the absence of a more developed cuticle in the inner petioles than the outer petioles, resulting in a more reduced physical barrier for calcium absorption. Inoculation of petiole sections with Erwinia carotovora ssp. carotovora did not show any differences in the incidence and progression of soft rot between the inner and outer petioles. Evaluation of soft rot inci- dence and progression 36 hours after inoculation was con- ducted with a symptom rating scale from 1 to 5 (Fig. 1) (1=no rot symptoms, 5-completely rotted). The results suggest that the age of plant tissue did not effect petiole susceptibility to soft rot. However, Kikumoto (1984) reported that petiole susceptibility was reduced as plant age increased. The absence of differences in 65 rot progression may have been due to the small increase in calcium level in the petioles (Fig. 4). Since the plants received only three foliar treatments, an addi— tional experiment was conducted to determine if more frequent applications would have a significant effect on calcium absorption. Fractionation of freeze dried petiole tissue into calcium pectate and calcium oxalate was attempted to determine if a specific calcium fraction had an effect on the incidence and progression of soft rot. The procedure, described by Ferguson and Turner (1980), resulted in variable fraction values from identical tissue subsamples. A modification in the procedure may be necessary to obtain accuracy and consistency in calcium fractionation when working with Chinese cabbage tissue samples. Foliar Calcium Application: Greenhouse The purpose of this experiment was to determine if frequent applications of CaCl2 would significantly increase calcium content in petioles of Chinese cabbage. Periodic samplings of the cultivar 'Tropical Pride' were also taken to study the effect of plant age on the incidence and pro— gression of soft rot in inoculated petioles. To facilitate the determination of calcium effects on the incidence and progression of soft rot in Chinese 66 .wucflom memo mo mummfi co comma mum mafia :oflmmmnmmu may psonc mpamb mocmcflmcoo “maoz .Amo.nmv ommnnmo omwcflcu mo Esfloamo Damoumm oHOADom Hoccfl co coaumuucmocoo Edfloamo mmumm unflaom mo uoommm one .v onsmflm 67 on A :m v zo: mmjocmd mmzz_ mo1v:> . 1 are 08 Honma ESQ moMU owv onoeuom nouno no noeuonnnoonoo Enfloamo noennaom unoenunn mo noommo one .m onnmem 82 Sec 2295 20:38 w m I. n N _ —-r~ lfi '1‘1 [VII U ‘ Ul'jjlt UT‘UT'UU - — p — p — p — P — p — E — p F p — - Beam”. xnoo...xm¢m.+-.uu> m p F . P p p P 'd' t’) (w “P %) wnmvo t0 are 08 50:5 E50 mo F j N I n (mm 9: 33m) 103 30 333330 P p n F n Améo 08 50:5 ESQ Z