liti3l5, This is to certify that the dissertation entitled DESIGN AND FUNCTION OF A MODIFIED ATMOSPHERE PACKAGE FOR PRECOOLED TULIP BULBS presented by Timothy A. Prince has been accepted towards fulfillment of the requirements for Ph.D. degmin Horticulture MGM ‘ Major professor Date July 29, 1983 M5u;.,...ur .> i t ,1 :nr . , . . 0.12771 bVlES‘.) RETURNING MATERIALS: Place in book drop to LIBRAfiJES remove this checkout from _:—. your record. FINES will be charged if book is returned after the date stamped below. -, «- nlJO H12543 ‘ (”Meme 090 stse am 29 '88 iii . 5 1hr: DESIGN AND FUNCTION OF A MODIFIED ATMOSPHERE PACKAGE FOR PRECOOLED TULIP BULBS By Timothy A. Prince A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1983 ABSTRACT DESIGN AND FUNCTION OF A MODIFIED ATMOSPHERE PACKAGE FOR PRECOOLED TULIP BULBS By Timothy A. Prince A prediction method was demonstrated as an aid in selection of a polymeric film for modified atmosphere (MA) packaging of precooled tulip bulbs (Tulipa gesneriana L. 'Kees Nelis'). A small consumer 2 package of 5 precooled (5°C) bulbs sealed in ca. 800 cm of LDF-301 low density polyethylene film obtained equilibrium levels of ca. 5% 0 4% C02, and 0.1 ul/liter ethylene at 20°. This package 29 maintained excellent bulb flowering ability through 4 wks of storage while non-packaged bulbs flowered poorly. Fusarium oxysporum infec- tion of bulbs of other cultivars yielded package ethylene levels of 2-47 ul/liter, which reduced subsequent flowering. Penicillium was shown to infect the root plate of the packaged 'Kees Nelis' tulip bulbs. Infection was associated with increased package ethylene and 602 as well as reduced 02 levels, and led to reduced rooting and increased floral abortion during subsequent forcing of the bulbs. Prochloraz and vanguard pretreatment of the bulbs prior to packaging controlled Penicillium growth. Benomyl, captan, and chlorine dip pretreatments did not control infection. Three pathogenic isolates Timothy A. Prince of Penicillium corymbiferum and one B. rugulosum isolate were obtained from the tulip bulbs. One 3. corymbiferum isolate displayed benomyl resistance and produced 1.5 ul/cmzohr of ethylene when grown on PDA. Vanguard and prochloraz controlled growth of this isolate on PDA. Packages of 'Kees Nelis' bulbs stored at 20° for 1 wk followed by 3 wks of temperature fluctuation between 15 and 25° displayed little change in package C02 and 02 levels. The temperature adaptability appeared due to both changing bulb respiration rates and changing film permeabilities to C02 and 02. Non-packaged 'Kees Nelis' bulbs at 20° lost 45%/35% of scale fresh/dry weight and 38%[20% of floral shoot fresh/dry weight during 4 storage wks. The daughter bulbs within these non-packaged bulbs increased 7-fold in fresh and dry weight. Bulbs in packages yielded little change in fresh or dry weight of any bulb organs. ACKNOWLEDGEMENTS I acknowledge the guidance of my advisor, Dr. R. C. Herner, and that of the other members of my committee: Drs. C. T. Stephens, J. Lee, D. R. Dilley, and R. D. Heins. The critical review of this manuscript by Dr. A. C. Cameron also was appreciated. Special thanks is given to Tom Stebbins, Denise Cerny, and Mollie Stark for their many hours of technical assistance. Finally I thank the Netherlands Flower Bulb Institute, New York, N. Y., for their financial support; and Dow Chemical, U.S.A., for supplying various polymeric films. ii Guidance Committee: The paper format was adopted for this dissertation in accordance with departmental and university regulations. Section I, Section II, and Section III are to be submitted to the Journal of the American Society for Horticultural Science; Section IV is to be submitted to Phytopatholggy_. TABLE OF CONTENTS Page LIST OF TABLES .......................... vi LIST OF FIGURES ......................... viii INTRODUCTION ........................... 1 LITERATURE REVIEW ........................ 2 SECTION I: DESIGN OF A MODIFIED ATMOSPHERE PACKAGE FOR MARKETING 0F PRECOOLED TULIP BULBS ................... 34 Abstract .......................... 35 Introduction ........................ 36 Materials and Methods ................... 37 Results and Discussion ................... 45 Literature Cited ...................... 60 SECTION II: CONTROL OF INFECTION BY PENICILLIUM SPP. OF PRECOOLED TULIP BULBS IN A MODIFIED ATMOSPHERE PACKAGE ......... 63 Abstract .......................... 64 Introduction ........................ 64 Materials and Methods ................... 66 Results and Discussion ................... 70 Literature Cited ...................... 94 SECTION III: CULTIVAR RESPONSE, TEMPERATURE FLUCTUATION EFFECTS, AND BULB ORGAN FRESH AND DRY MATTER DISTRIBUTION IN A MODIFIED ATMOSPHERE PACKAGE 0F PRECOOLED TULIP BULBS . . . . . . . . . . 96 Abstract . . - ........................ 97 Introduction ........................ 98 Materials and Methods ................... 99 Results and Discussion ................... 103 Literature Cited ...................... 129 iv Page SECTION IV: PATHOGENICITY, FUNGICIDE RESISTANCE, AND ETHYLENE PRODUCTION OF PENICILLIUM SPP. ISOLATED FROM TULIP BULBS . . . 132 Abstract .......................... 133 Introduction ........................ 134 Materials and Methods ................... 135 Results and Discussion ................... 140 Literature Cited ...................... 154 Table LIST OF TABLES Section I Permeabilities to C02, 02, and ethylene of 3 low density polyethylene films utilized for packaging precooled tulip bulbs ....................... Flowering obtained from packaged and non-packaged post precooled 'Kees Nelis' tulip bulbs stored at 20° and 25°C for 2 and 3 wks ..................... Forcing characteristics of packaged and non-packaged post precooled 'Kees Nelis' tulip bulbs stored at 20° and 25°C for 2 and 3 wks ..................... Section II Infection by Penicillium spp. of tulip bulb root plates after prepackaging dips or dusts and subsequent storage for 2 or 3 wks at 20°C in LDF—301 film packages or non-packaged ....................... Flowering of precooled 'Kees Nelis' tulip bulbs after prepackaging treatment and subsequent storage for 2 wks at 20°C in LDF-3OI film packages or non-packaged ..... Flowering of precooled 'Kees Nelis' tulip bulbs after prepackaging treatment and subsequent storage for 3 wks at 20°C in LDF-301 film packages or non-packaged ..... Infection by Penicillium spp. of inoculated and non- inoculated precooled 'Kees Nelis' tulip bulbs after fungi- cide or sterilant prepackaging treatments and subsequent storage for 3 and 4 wks at 20°C in LDF-301 film packages or non-packaged . . .* .................. Flowering of inoculated and non-inoculated precooled 'Kees Nelis' tulip bulbs after fungicide or sterilant prepackaging treatments and subsequent storage for 3 wks at 20°C in LDF-301 film packages or non-packaged ..... vi Page 72 79 80 89 Table Flowering of inoculated and non-inoculated precooled 'Kees Nelis' tulip bulbs after fungicide or sterilant prepackaging treatments and subsequent storage for 4 wks at 20°C in LDF-301 film packages or non-packaged ...... Section III Flowering of 12 cultivars of precooled tulip bulbs after storage for 3 wks at 20°C in LDF-301 film packages or non-packaged ........................ Flowering of 12 cultivars of precooled tulip bulbs after storage for 4 wks at 20°C in LDF-301 film packages or non-packaged ........................ Ethylene levels after 5, 17, and 27 days, and Fusarium oxysporum presence, on 12 cultivars of tulip bulbs in LDF-301 film packages during 4 wks of storage at 20°C Permeabilities to C02 and 02 of LDF-301 low density polyethylene film at 15, 20, and 25°C ........... Flowering of precooled 'Kees Nelis' tulip bulbs after storage in LDF-301 film packages or non-packaged for 1 wk at 20°C and an additional wk at 6 temperature regimes Flowering of precooled 'Kees Nelis' tulip bulbs after storage in LDF-301 film packages or non-packaged for 1 wk at 20°C and an additional 2 wks at6 temperature regimes . . . Flowering of precooled 'Kees Nelis' tulip bulbs after storage in LDF-30l film packages or non-packaged for 1 wk at 20°C and an additional 3 wks at6 temperature regimes . . . Section IV Pathogenicity and virulence of 4 Penicillium spp. isolates on excised root plates of precooled 'Kees Nelis' tulip bulbs . . Growth ratings of 4 Penicillium isolates on PDA containing 4 fungicides after 4 days at 23-26°C . ........... Ethylene levels accumulated in 473 ml jars containing cultures of Penicillium spp. during 6 days at 20°C ..... Infection of excised tulip bulb root plates pretreated with benomyl, vanguard, or water, and subsequently inoculated with a spore suspension of benomyl resistant, non-resistant, or mixed isolates of Penicillium corymbiferum, or of non- inoculated controls .................... Page 90 106 108 109 112 119 120 122 141 143 145 152 LIST OF FIGURES Figure Page Section I 1. C02 and 02 levels in jars containing 1 or 2 precooled tulip bulbs and sealed with four different film types . . . 46 2. C02 and 02 levels in 3 low density polyethylene film packages of precooled 'Kees Nelis' tulip bulbs during 24 days of storage at 20° and 25°C ............. 50 3. Ethylene levels in 3 low density polyethylene film packages of precooled 'Kees Nelis' tulip bulbs during 24 days of storage at 20° and 25°C .................. 52 4. Representative pots of forced 'Kees Nelis' tulip bulbs after 3 wks of storage at 20°C ............... 55 Section II 1. Infection by Penicillium spp. of precooled 'Kees Nelis' tulip bulb root plates pretreated with benomyl at 2000 pg a.i.lml compared to non-infected bulbs treated with vanguard at 240 pg a.i.lml. All were packaged in LDF-301 film for 3 wks at 20°C ........................ 71 2. Effect of prepackaging treatment on 02 levels in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C . . . .......... . ......... 74 3. Effect of prepackaging treatment on C02 levels in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C .......... . ............ 75 4. Effect of prepackaging treatment on ethylene levels in LDF—301 film packages of precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C ................ 77 5. Correlations of fresh root weight with percentage of root plate diseased and floral ratings of precooled 'Kees Nelis' tulip bulbs after prepackaging treatments and subsequent storage for 2 wks at 20°C in LDF-301 film packages ...... 82 viii Figure Page 6. Correlations of fresh root weight with percentage of root plate diseased and floral ratings of precooled 'Kees Nelis' tulip bulbs after prepackaging treatments and subsequent storage for 3 wks at 20°C in LDF-301 film packages ....................... 83 7. Effect of prepackaging treatment on 02 levels in LDF-301 film packages of inoculated and non-inoculated precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C ....... 86 8. Effect of prepackaging treatment on C02 levels in LDF-301 film packages of inoculated and non-inoculated precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C . . . . . . . 87 9. Effect of prepackaging treatment on ethylene levels in LDF-301 film packages of inoculated and non-inoculated precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C . . 88 10. Representation of 0-4 root rating. Flowering of non- inoculated 'Kees Nelis' tulip bulbs after vanguard pretreatment and 4 wks of storage at 20°C in LDF-301 film packages or non-packaged . . . ............... 92 Section III 1. C02 levels in LDF-301 film packages of 'Prominence','Abra', and 'Oskar' precooled tulip bulbs through 4 wks at 20°C . . . 104 2. 02 levels in LDF-301ifilnipackages of 'Prominence', 'Abra', and 'Oskar' precooled tulip bulbs through4wks at 20°C . . . 105 3. Arrhenius plots of the permeability constants of LDF-301 film to 002 and 02 permeation . .............. 113 4. C02 levels and 02 levels in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs during storage for 1 wk at 20°C followed by 3 wks at 15, 20, or 25°C ........ 114 5. 002 levels and 02 levels in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs during storage for 1 wk at 20°C followed by 3 wks at 15° for 2 days/20° for 5 days; 25° for 2 days/20° for 5 days; or continuous 20° storage . . 116 6. C02 levels and 0% levels in LDF-301 film packages of precooled 'Kees elis' tulip bulbs during storage for 1 wk at 20°C followed by 3 wks at 25° for 2 days/15° for 2 days/ 20° for 3 days; or continuous 20° storage ......... 118 ix Figure Page 7. Dry weights, fresh weights, and fresh/dry weight ratios of various organs of precooled 'Kees Nelis' tulip bulbs stored in the open or in LDF-301 film packages through 4 wks at 20°C ....................... 124 Section IV 1. Ethylene levels in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C after tunic removal and root plate inoculation with a spore suspension of an ethylene producing or a non-ethylene producing Penicillium corymbiferum ............. 148 2. 02 levels in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C after tunic removal and root plate inoculation with a spore sus- pension of an ethylene producing or a non-ethylene producing Penicillium corymbiferum . . . . . . . ...... 150 3. C0 levels in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C after tunic removal and root plate inoculation with a spore sus- pension of an ethylene producing or a non-ethylene producing Penicillium corymbiferum ............. 151 INTRODUCTION Successful marketing of precooled (5°C) tulip bulbs in consumer packages has not been reported to date. These prepared bulbs could be marketed through mail-order or other retail channels for mid- winter indoor forcing or spring planting in the Northern United States or winter outdoor planting in the South. A marketing period exceeding the 8 days now recommended and maintenance of excellent bulb flowering ability without a refrigeration requirement would be necessary. Maintenance of flowering ability in these post precooled bulbs has been shown to be enhanced by low 02 atmospheres. However, normal methods of controlled atmosphere (CA) maintenance would be impractical and expensive during marketing of these bulbs. Since many partially successful modified atmosphere (MA) packages of various commodities have been reported, the objective of this research was to develop and evaluate an MA marketing package for tulip bulbs utilizing a semi- permeable polymeric film for atmosphere maintenance. A simple prediction method for package parameter optimization was utilized in package development. Disease control, ethylene accumulation, bulb organ fresh and dry matter distribution, and effects of temperature fluctuation, as well as maintenance of bulb flowering ability, were considerations in package evaluation. LITERATURE REVIEW This review comprises two sections. The first outlines storage of various commodities in polymeric film packages. Included in this section are prediction methods for package parameter optimization, effects of temperature fluctuation on the package environment, and the development of diseases in packages. The second section outlines tulip bulb morphology and physiology as they relate to bulb storage in various atmospheres. I. Commodities in Polymeric Films Polymeric film packaging is used for a wide variety of fruits, vegetables, and ornamentals. Controversy exists as to whether the polymeric films can be used most effectively for perforated packages to aid only in moisture retention of a product, or for sealed packages to create modified atmosphere (MA) conditions. In early research, Scott and Tewfik (94) found that deleterious high C02 and low 02 levels occurred in sealed cellophane (CP) and pliofilm (PF) packages of tomatoes, snap beans, sweet corn, and apples. All stored for one week or less with poor results. They concluded that different film wraps would be required for each commodity to avoid deleterious effects of gaseous modification unless the consumer packages were perforated. They also reported that existing consumer packages were not airtight, so that deleterious effects were not 3 occurring in the marketplace. Film perforation was supported by Allen and Allen (4) who found that all films in use in 1950 were not permeable enough to 02 to be used for sealed market packages. Upon testing 40 films for produce packaging, Schomer (88) found that refrigeration was more important than film type, since all films required perforation to avoid anaerobic effects. One exception noted was cherries, which could be sealed in PF-type FM1-80 without ventilation (32). This film was found to be permeable enough to prevent fogging and deleterious atmosphere modification during a handling and marketing period. Sommer and Luvisi (101) noted the occasional success of a sealed package in delaying fruit ripening, but still recommended perforated packages. In a review of the subject, Hardenburg (37) concluded that, in general, perforation of packages was necessary to eliminate anaerobic respira- tion, development of off flavors, and swelling due to C02 accumulation in the package. Citrus in particular appears to require ventilation in a polymeric film package. As few as 4 to 8 (107) and as many as 72 I" holes (45) per 5 lb polyethylene (PE) bag of oranges have been recommended. Reduc- tions have been observed in weight loss, peel color changes, stem-end rot, and deformation of grapefruit in individual vented PE bags as compared to non-bagged storage (62). Similar wrapping of grapefruit reduced the required relative humidity range during shipment without increasing weight loss (63). This kept fiberboard boxes drier and stronger, which minimized box distortion during handling. In surrmary, the role of perforated polymeric film packaging in reducing trans- pirational loss of produce appears well documented. 4 However, other findings suggest that it is possible to extend applications of polymeric film packaging into the MA realm. These studies involved specific commodities and specific films. One example is the extensive work reported on the development of MA shipping and marketing containers for bananas (65, 89-92, 111, 112). Scott and Roberts (92) first reported an increase of 6 days in maintenance of the keeping quality of bananas in large PE bags at 20°. Ripening appeared to be significantly delayed by the gaseous atmosphere modification. In further work (91), they found that KMn04-saturated vermiculite reduced the ethylene levels in the bags and yielded increased firmness of the fruit. The authors suggested that the treated vermiculite absorbed ethylene produced by a few early ripening fruits and thereby inhibited premature ripening. Even though the packaging was successful, large variabilty in C02 (1.3-20.7%) and 02 levels (1.2- 16.3%) occurred, possibly indicating leakage problems. Noodruff (112) demonstrated that 1 ppm ethylene was the threshold level leading to increased banana respiration in PE packages. Accordingly, addition of the ethylene scrubber Purafil to PE bags of bananas was reported to absorb ethylene and extend storage life (65). Oxygen levels in these packages, near 1% by the 8th storage day, were generally lower than measured by Scott and coworkers (91, 92). No anaerobic effects were reported, although the ratio of film surface area to banana weight appeared different from that used by Scott and coworkers, which may have led to lower 02 levels. However, Scott and Gandanegara (90) reported that PE bagged bananas had increased storage life over a wide range of shipping temperatures, while again reporting a variable gaseous environment. They concluded that a careful 5 consideration of film surface area to banana weight ratio was not important precisely because bananas respond to a wide range of C02 and 02 levels. This generalization about the commodity/film ratio may not hold for other commodities if a narrower range of MA conditions is necessary for proper storage and/or subsequent ripening. Packaging of bananas in consumer-size sealed packages also has been investigated (16). Four types of polyvinylchloride (PVC) films were used to overwrap bananas on trays. The most permeable film could extend the shelf-life of bananas to 30 days at 15° by maintaining ca. 3% CO2 and 02 in the trays. Consumer packaging of tomatoes also has been investigated. Tomatoes sealed in many different films showed varied weight losses during storage (7). Cellulose acetate (CLA) was found to yield the best quality tomatoes presumably due to its high permeability to C02 and 02. Off flavors, condensation, and rot occurred in some of the packages, apparently due to anaerobic conditions, although atmospheric levels were not reported. Tomatoes packed in PVC or Haiesu-film (HF) bags kept well for 5 days at 20-24° (78), but subsequently softened, changed color, and produced a fermentation odor. Fruit packaged in PE displayed delayed ripening while the climacteric C02 production was reduced by 40% (77). The most successful results with tomatoes were obtained in PVC and PE packages by Saguy and Mannheim (86), who reported satisfactory quality after 21 days at 25° compared to a shelf-life of less than 7 days for controls held in air. Limited packaging studies with lettuce have been undertaken. Detrimental effects were observed when heads were packaged in PE and 6 then put under controlled atmosphere (CA) conditions (2.5% CO and 02) 2 (96). It is possible that anaerobic or high CO2 effects were present although no internal atmospheric levels were reported. The CA and CA plus packaging conditions did yield higher retention of starch and total sugars than cold storage alone (97). Lettuce in sealed PE bags with and without an initial N2 flush gave better keeping quality than lettuce stored in air at 1° (1). The NZ flush gave better results presumably because the 02 level was reduced faster. Atmospheres reached 2-3% C02 and 12-13% 02 in the bags. The C02 appeared to damage crisphead lettuce but not Romaine. Shredded lettuce also has been packaged in sealed film (80). Control PE bags began with air only while others had injectionsix>30% 02. The 02 level fell to 1-2% and CO2 reached 10% after 1 wk at 2.5°. It appeared that 02 injection did not greatly affect the time to equilibrium or the ultimate 02 level obtained. Other vegetables have been stored in MA packages. Pea pods, kidney beans, lettuce, and bell peppers were found to store better in PE and polystyrene (PS) bags than in other films tested (50). Again, N2 flushing led to faster atmospheric equilibrium and extended the keeping quality. Carrots stored in sealed PE bags (81) attained tat10°. 0f additional interest was levels of ca. 3% CO and 17% 0 2 2 the low phenolic increase in carrots in bags, presumably due to the CO2 levels and not the relatively high 02 levels. Similar atmospheric levels were obtained in trays of bell peppers overwrapped with PVC film (11). However, the attributes of this package seemed to be due mainly to reduction of transpiration. 7 Research in the shipment of pallet loads of strawberries covered with PE film has been reported by Harvey and coworkers (39-41). Addi- tions of C02 gas or dry ice in the pallet before shipment were successful with dry ice addition being optimal. The CD2 suppressed fungus growth and slowed ripening of the berries. There has been some interest in the use of sealed film packages to reduce chilling injury in citrus. Chilling injury of grapefruit has been reported to be at least partially controllable with high CO2 levels (106), but less so by low 02 levels (34). Chilling injury was prevented for 1 month at 4.5° by sealing fruits in containers with PVC and cast vinyl (CV) films (109). It was presumed that the CO2 levels reduced the symptoms although the increased RH levels could have aided also. No attempt was made in this research to develop a practical package for chilling injury reduction in citrus. Polymeric film packaging of cut flowers has been investigated as well. Hauge and coworkers (42) used heat sealed CP packages for rose storage at 4-7° for 5 days. Packaged roses had better keeping qualities than non-packaged controls presumably due to C02 accumu- lation (5%) and 02 depletion (15%). Carnations in the same packages for 8 days lasted 2-3 times longer than controls. Results with pompon Chrysanthemums were less successful due to a larger headspace in the package which slowed the CO2 accumulation. Other studies utilizing 7 different film types for packaging various cut flowers have been reported (43). Roses stored at 10° and below for 5 days were in good condition in all films except 300 LSAT film (Dupont) which yielded 02 levels less than 1%. Chrysanthemums 8 packaged in 300 PMBS film (Sylvania) were stored for periods up to 28 days at 0-10°. The CO2 levels ranged from 1 to 11% with higher temper- atures and longer durations yielding the highest levels. High CO2 levels were associated with abnormal flowers. Carnations sealed in 300 MSAT film (Dupont) showed little 02 depletion (17%) or C02 accumu- lation (2%) at 0-10° for 10 days. However, carnation respiration rate was found to increase in the package, most likely due to ethylene accumulation (not reported). Von Oppenfeld et al. (108) sealed cut tulips in consumer units with PE, saran, CP, and plastic-coated CP. Flower quality was acceptable after storage at 0° for all films except the plastic- coated CP, which yielded CO2 levels up to 21%. CP film wrapped packages of cut tulips at 5° gave mixed results (76). However, cut tulips kept well for 10 days in laminated PP-CP bags inflated with N2 and air (5). No package atmospheres were reported, however. PE film packaging of cut roses with initial N2 flushing kept blooms in the tight bud stage for 40 hrs at 15° (105). The 02 levels rose from near 0 to 2.5% during the 40 hours. Attempts at packaging entire plants also have been reported. Foliage, flowering, and bedding plants were packed in 3 mil coextruded PE-PP packs which were sealed and injected with air to form pressurized containers (35). These were held in the dark for 2 days at 16°, then fbr 30 days under low light. Only purple passion plant (Gynura sarmentosa) stored successfully. The most frequent problem was that plants totally collapsed upon removal from the bags. Since no atmos- pheric levels were reported, the cause could not be determined. Possibly ethylene, high C02, or low 02 levels were responsible. 9 Further work with potted foliage plants yielded a good marketable product with a few species after 60 days without a watering require- ment (36). Plant collapse was still seen occasionally. Again no package atmospheric data were provided. Work by French researchers (31, 67) has described the use of silicone rubber (SR) membranes to create controlled atmospheres in storage units of apples and vegetables. Specific PE bags with varied amounts of SR membranes were designed for specific amounts of commodities. Chosen levels of CO2 and 0 were obtained within pallet- 2 ized loads. The advantages of this system were that storage rooms were accessible at any time and that possible losses were reduced to indi- vidual bags instead of entire storage rooms. Cost was also determined to be 25% cheaper than standard CA storage units. The SR membrane was also used in a circulating system to maintain CA conditions in entire storage rooms by utilizing varied exposures of the membrane. Recent work has centered upon individual wrapping of fruits and vegetables. Avocados packed individually in PE bags had increased storage life while KMnO4 addition further delayed fruit softening (79). The KMnO4 addition was found to have little effect upon 02 and ethylene levels, but did appear to decrease CO2 in the packs (13). Avocados appeared to respond to wide ranges of CO2 and 02, a response similar to that noted earlier for bananas. Citrus fruit individually wrapped in high density polyethylene (HOPE) had twice the storage duration and a 5-fold reduction in weight loss compared to open controls (9). Respiratory activity and ethylene production both were reduced by wrapping, although no differences from controls in internal CO2 and 02 levels were noted after 1 month. 10 Storage of kiwifruit (Actinidia chinensis) in individual PE bags resulted in variable CO2 levels as well as variable results in flesh softening data (70). Generally, bagged storage yielded less flesh softening than air storage. Kawada (61) reported that individual vacuum film packaging of tomatoes, persimmons, and grapefruit was more effective than non-vacuum packaging in extending storage life. However, the author cautioned that this system was only a supplement to proper refrigeration. In summary, many perforated polymeric film packages have been shown to function well by reducing transpirational water loss from a commodity. In addition, examples of at least partially successful MA packages have been reported. However, much of this work has been performed with somewhat arbitrarily chosen parameters (i.e. film type, package size, amount of commodity, etc.). It is impossible to estimate how poor choices of packaging parameters have influenced the results of MA packaging efforts. It does appear that definitive and faster results could be obtained if package parameters were optimized before testing. This aspect of packaging deserves futher discussion. Optimizing Package Parameters. Henig (46) described a produce packaging system as a dynamic one where respiration and permeation are occurring simultaneously. Therefore factors affecting either respira- tion or permeation rate or both must be considered when designing a package. He identified commodity weight, stage of maturity, membrane permeability, temperature, 02 and C02 partial pressures, ethylene level, light, and possibly other factors as affectors of produce respiration rate. In addition, variables affecting gas permeation 11 through a film were identified as structure of the film, thickness, area, temperature, and 02 and CO2 concentrations. Henig recommended that the package design be directed towards achieving the optimal gaseous composition ina package. He also noted that perforation destroys the semipermeable nature of a film, making gas exchange independent of the chemical nature of the gas or its interaction with a polymer. This results in less control over the package gaseous environment by the package designer. Tomkins (104) studied the dynamics of a polymeric film package and found that after a period of adjustment, equilibrium levels of CO2 and 02 were obtained. At this point, the respiration rate (CO2 production or 02 consumption) was assumed to be equal to the perme- ation rate. If the package was poorly designed, anaerobic conditions were established before equilibrium was obtained. It was concluded that to produce specific package conditions, the correct package size and permeability had to be chosen, and the temperature must be held within fairly narrow limits. Jurin and Karel (55) studied the package permeation/respiration rate interaction in apple and banana packages. They devised a graphcial solution to predict equilibrium package conditions. The method involved plotting the rate of respiration and permeation at different 02 concen- trations on the same set of coordinates. The equilibrium 02 and C02 concentrations were indicated by the intersection of the curves. These calculations assumed that CO2 accumulation did not affect the respir- ation rate and that the R0 was equal to 1. The experimental equilibrium values were in good agreement with the predicted values (ca. 9% O2 and 3% C02). 12 A detailed computer prediction method for CO2 and 02 levels in produce packages has been published (44, 47). To demonstrate this method, tomatoes were enclosed in chambers with known headspaces and film areas. Samples were withdrawn and analyzed periodically for 02 and CO2 levels. These values were used to develop regression equa- tions for prediction of 02 consumption and 002 production rates under different 02 and CO2 atmospheric concentrations. Two first-order differential equations were developed and solved. This computer- aided iteration technique provided the 02 and C02 concentrations in a package at 1 hour intervals until equilibrium conditions were obtained. Good agreement was found between the experimental and computer calcu- lated results. They also reported that an increase in package headspace only lengthened the time to obtain equilibrium, but did not affect the levels obtained. From these examples it is apparent that much research time can be saved if some method of predicting the permeability requirements of a film is utilized before any extensive trials are begun. These methods should indicate a range of acceptable film permeabilties for a specific commodity that will yield an acceptable MA package. They may preclude further tests by indicating that with existing films no reasonable film surface area/commodity ratio exists. In this case, prediction methods would at least quantitate the required permeability, with the hope of future film development. Even with the use of a prediction method, the problems of disease control and temperature fluctuation during marketing and handling remain to be solved for a successful MA packaging system. 13 Temperature Fluctuation. Tomkins (104) reported that increases in temperature of storage increased the equilibrium CO2 concentrations within sealed packages. He also noted that temperature decreases could lead to condensation inside the package, which could leach solutes from the produce and support the growth of bacteria and molds. Fisher (29) reported that CP packaging of carnations and pompon Chrysanthemums gave good results after 1 month at 0°, but that 7° storage resulted in worthless carnations due to mold development. No package atmospheres were reported in this study, however. In his review of packaging, Hardenburg (37) displayed data from packages of green beans and the effects of temperature upon the internal package atmosphere. He concluded that temperature had profound effects on package C02 and 02 levels, yielding little hope for practical MA packages. However, it appears that his conclusions were faulty since the packages were not optimized and eventually all would have become anaerobic. Apparent was the effect of temperature upon the rate of obtaining atmospheric equilibrium and not upon the ultimate equilibrium levels. Other studies have shown varied effects of temperature fluctu- ation upon MA packages. Roses packaged in various film kept well for 5 days at 0-10° but not at higher temperatures (43). No great atmos- peric differences were found between individually sealed PE bags of avocados at 20 or 30° (13). Consumer PVC packages extended the shelf- life of bananas at 15° while odor and taste development was abnormal at 22° (16). The abnormal ripening process appeared to be due to increased CO2 levels, since no significant 02 level differences were found. PE bags were found to increase the shipping life of bananas 14 at 13—37°, although wide ranges of C02 and 02 were reported (90). Henig and Gilbert (47) found that final 02 and CO2 levels were nearly the same at 15° and 23° for PVC type RMF-61 and VF-7I film packages of tomatoes. They suggested that temperature changes affected both respiratory activity and film permeability to the same degree with their packages. However, other work with sealed PE bags of head lettuce demonstrated atmospheric changes from 2% C02 and 12% 02 at 1° to 4% CO2 and 7% 02 at 20° after only 2 days (1). The varied effects of temperature upon the success of MA packaging can be explained by the dynamics ofaipackage system itself (46). Tem- perature change affects both commodity respiration and film permeability. The effect on film permeability is entirely a physical one. Karel (58) has shown by Arrhenius plots that film permeability varies log-linearly with the reciprocal of the absolute temperature and that breaks, or changes of the slope of the line, are rare for films. The effects of temperature on respiration, however, can vary between commodities (30), and depends upon the range of temperatures considered. I have already noted differences in the commodity quality response to various CO2 and O2 regimes. It seems apparent, then, that the parameters of film permeability, commodity respiration, and comnodity quality response to various atmospheres interact to determine the effect of temperature change upon the success of an MA package. Thus, the effects of temperature change must be evaluated individually for each package commodity system. Diseases 19 MA Packages. Various packaging techniques have been shown to decrease/increase disease growth on stored commodities. 15 Decreases of disease have been mainly due to prevention of cross inocu- lation by individual wrapping of commodities, while increased disease has generally resulted from the high relative humidity in the package. Grapefruit that were individually wrapped in non-sealed PE had less stem-end rot than those stored in film-lined boxes (62). It was noted that more calyxes remained green in PE and were thus less likely to become infected. Individual vacuum packaging of other fruits was found to prevent decay organisms from cross-infecting other fruit (61). When adequate fungicides were used, decay also was believed to be low because there was no condensation between the fruit and the tightly wrapped film. Karel (58) has outlined the barrier properties of polymeric films to passage of spores from disease organisms that appear to have functioned in the above studies. Overwrapping pallet loads of strawberries with PE films and purging with C02 gas (IO-13%) has led to less Botrytis incidence during transport (40). Transport temperature was ca. 5° and the diff- erence in disease incidence was evident after a subsequent 2 days at 15.5°. Sommer et al. (100) had previously demonstrated the effective- ness of high CO2 (5-15%) in suppressing strawberry gray mold at 5° or above. However, the common CA conditions of 2-3% 02 and 5% CO2 have shown only moderate suppression of most commodity disease organisms (28). Therefore, it is not surprising that in many MA packages, increases of disease incidence have been shown to occur. Spore germination and invasion of a commodity occurs easily in the near 100% RH of a film package. Therefore, the use of low storage temperatures and wrapping films of high water vapor permeability have been recommended (4). Wounds have been suggested as the main avenue 16 of disease entrance within packaged commodities (101). Humid atmos- pheres, while reducing commodity shrinkage, also make an excellent environment for colonization of wounds by pathogens. Fungicide treatment has controlled disease infection in certain instances. Okubo and Maezawa (78) reported that mold development was sometimes a problem with tomatoes stored for 5 days in PVC and HF bags. Similar mold growth on tomatoes packaged in several PVC and PE packages at 25° was reduced by the use of the most permeable films and by pretreatment of the fruit with 25 ppm chlorine, 1000 ppm Nipagin-M, or 1000 ppm Nipacide (86). Best results were noted with fruit treated with 25 ppm chlorine and wrapped in VF-71 and TPM-87 PVC films. Decay was 0-5% after 21 days at 25°, while control deterioration was 40%. Disease growth has been a problem in perforated as well as sealed packages. Grierson (33) compared disease growth in mesh vs. perforated PE bags of tangerines and oranges. Results of 35% vs. 55% spoiled packages for tangerines and 3% vs. 10% for oranges were obtained. The use of diphenyl or 2-aminobutane reduced decay in the packages. Scott and Roberts (93) reported that thiabendazole could control the black-end storage rot of bananas during shipment. However, it was found that the handling method determined the severity of the disease in PE packaged bananas (89). Benomyl or thiabendazole was recommended if the fruits were packaged in PE bags as hands or as single fruits. If they were shipped as a bunch, fungicide treatment was not recom- mended. Disease entrance through cut tissue of hands or single fruits was involved in the disease severity difference. Storage rots were also found to be a problem with a small per- centage of avocados held in individual PE bags at ambient temperatures 17 (20-30°) for ca. 10 days. Anthracnose rot occurred in benlate dipped avocados after 40-50 days at 10° in individual PE bags (79). Storage was terminated even though fruits stored with KMnO4 were still firm. Infection was observed mostly at the stem end where the pedicel dried during storage. Erwinia carotovora was the predominant cause of decay in packaged bell peppers (11). The bacterial soft rot was more severe in PE film packs than in PVC. Overall, however, very little decay occurred at 7, 12, and 25° in the packs. Mold growth has been noted as a problem in packages of tulips (76, 108), carnations (29), and Chrysanthemums (29) in various films. Species of bacteria, as well as Botrytis, Fusarium, and Pythium species, were isolated from flowering pot plants and bedding plants stored in coextruded PE-PP bags (35). It was not determined whether they were pathogenic or secondary invaders, but packaging led to decayed flowers and foliage, making the product unmarketable. In addition to the commodity destruction and cosmetic damage caused by disease growth in a package, ethylene production by the host and/or the pathogen during disease development can be detrimental. In a study of 228 species of fungi examined in pure culture, about 26% produced ethylene as a metabolic product (52). The host tissue itself has also been shown to produce ethylene upon infection. Williamson (110) was the first to clearly recognize that typical disease symptoms were due to an increase in ethylene production by infected tissues. He reported that high ethylene levels were associated with blackspot of roses (Diplocarpon rosae), Chrysanthemum flowers infected with Ascochyta chrysanthemi, Septoria leaf spot of Chrysanthemums, and Alternaria leaf 18 spot of carnations. Cut carnations infected with Botrytis also dis- played a marked surge of ethylene production at the onset of fungal attack (98). Detailed studies demonstrating ethylene production by potato tubers infected with the black rot fungus Ceratocystis fimbriata (12, 53, 102) and tomatoes infected with Fusarium (27) have been published. The soft rot bacterium, Erwinia carotovora also has been shown to induce host ethylene production (66). All of these studies indicate that commodity infection by disease organisms could lead to ethylene accumulation in an MA package. The level obtained would ultimately depend upon the production rate and the permeation rate through the specific polymeric film being utilized. II. Tulip Bulb Storage Studies This section discusses the limited studies of storage of tulip bulbs under various atmospheres. To provide a framework for this discussion, an outline of the tulip bulb life cycle and factors affecting bulb respiration rate and ethylene production rate is first presented. Tulip Bulb Life Cycle. The tulip bulb life cycle has been out- lined by Rees (84). The tulip bulb is composed of concentric scales separated by short internodes. The outer scale, or tunic, is brown and papery. The scales are joined at the basal plate from which emerge the flowering shoot and the roots. Daughter bulbs are initiated in the axils of the scales. Each mother bulb planted in the autumn dies upon flowering and is replaced by daughter bulbs. The initial starch content of the mother bulb scales has been shown to account for only 19 17% of the starch found in matured daughter bulbs. Thus about 83% of the starch in matured daughter bulbs is derived from photosynthesis before leaf senescence (6). Accordingly, the total dry weight of the daughter bulbs increases rapidly during the spring, with the increase peaking at the time of leaf senescence. The apices within the daughter bulbs are still in the vegetative state at lifting time in July in the Netherlands. Flower initiation occurs when the bulbs are in storage or during shipment. Differen- tiation progresses from the tepals to the anthers, being completed with formation of the tri-lobed gynoecium. With the completion of the differentiation process, the bulbs are said to have reached stage G (8). Low Temperature Rquirement. After stage G is attained, a period of low temperature is necessary for normal flower stalk elongation during the spring under natural conditions or during forcing (38, 84). Special precooling (or 5° storage) has been described as storage of tulip bulbs at 5° in a dry, unplanted state to satisfy completely the low temperature requirement before planting (17). This method is in contrast to more standard methods whereby all or part of the cold treatment (generally 9°) is applied to planted bulbs. The rate of shoot growth and number of bulbs flowering has been shown to increase with duration of 5° storage, with 12 to 14 wks duration appearing optimal (74). DeHertogh (17) recommends this duration to forcers in the United States. However, Kawata (60) found that a treatment of 17° for 2 wks plus 5° for 9 wks, or the standard Japanses treatment of 15° for 2 wks plus 2° for 7-8 wks, gave early and high quality 20 flowers. More recently it has been shown that some -1° treatment may be advantageous (75), although treatment below 5° has sometimes resulted in more blasted flowers upon forcing (60). Additionally, Hoogeterp (51) has suggested a 2° treatment for late forcing only. Thus it appears that the optimal combination of temperature and duration has yet to be established. Interruption of the precooling after 6 wks by a period of warm storage did not nullify the cold treatment effects for tulip bulbs (85) as has been reported for lily bulbs (69). Such an interruption hastened rather than delayed flowering of tulip bulbs except at 30°, where all of the flowers aborted. This finding indi- cates that warm temperatures during a post-precooling marketing period before planting may not erase the effects of a previous precooling period. Bulb Respiration Rate. Studies of bulb respiration rate before, during, and following the cold treatment have been reported. Algera (2, 3) found that after a minimum was reached in mid-August, CO2 production by tulip bulbs slowly increased. Low temperatures were found to reduce respiration while a return to 20° increased it. Removal of the tunic increased respiration two- to three-fold. Rees (84) found that after lifting, 0 consumption was maximal 2 and then decreased to a steady state through October. Bulbs held at 17° through the winter months had low CO2 production rates until January when they increased (83). This same study showed that bulbs precooled at 5° for 12 wks or longer had higher respiration rates than bulbs precooled less than 12 wks. Since some have considered 21 this duration optimum, the post-precooling respiratory increase may be associated with completion of the cooling requirement. Studies of mitochondria isolated from tulip bulb scales sub- jected to different cold treatments also have been reported. Cooled bulbs showed more active mitochondria than uncooled bulbs when measured as ability to oxidize succinate, malate, or 2-oxoglutarate (49). Arrhenius plots of mitochondrial oxidation in uncooled bulbs showed a single transition point, but bulbs cooled for 8 wks or longer at 2° showed indications of two discontinuities in the diagrams (48). The author suggested that tulip bulbs are chilling sensitive with a phase change occurring in the mitochondrial lipid bilayer during cooling. In summary, bulb cooling has been shown to increase subsequent isolated mitochondrial as well as whole bulb respiratory activity. This increased activity may be involved in the subsequent shoot elongation and flowering in the tulip. Ethylene Effects. A review by Kamerbeek and DeMunk (57) lists the major effects of ethylene on the tulip as gummosis of the bulb scales, bud necrosis, flower bud blasting, and morphological changes resembling those caused by unfavorable temperatures. Fusarium oxysporum tulipae, which may infect young growing bulbs just prior to harvest, has been found to produce ethylene abun- dantly in gitrg (103). This ethylene in turn is involved in bud necrosis, a storage disorder of tulip bulbs, related to bulb mite infestation (20-23, 25). DeMunk (24) also has shown that ethylene exposure during storage before planting resulted directly in flower 22 bud blasting. The blasting increased with the period before exposure, the storage temperature, the concentration of ethylene, and the exposure period. However, DeHertogh et al. (19) found that 10 of 27 cultivars tested were resistant to the blasting effect of ethylene. Bulbs infected with Fusarium also can cause problems in the greenhouse after precooling and planting. Symptoms include growth retardation and yellowing of the leaves, and death of the plant before flowering (87). Ethylene up to 10 ppm has been measured in the soil atmosphere sur- rounding bulbs infected with Fusarium (87). The production of ethylene by healthy, uncooled tulip bulbs was reported as too low for detection in samples of surrounding air by DeMunk (22). However, Prince et al. (83) reported that ethylene 1 1 production of bulbs kept at 17° fluctuated between 0 and 1 ml-kg' -day’ until January, after which it increased and became very variable. Moe et al. (71)were the first to report on the ethylene production of precooled bulbs. Production reached a peak 3-4 days after removal of the bulbs from 5° to 21°. One wk later, a second peak related to flower blasting was observed. Increasing the duration of 5° storage increased the ethylene production. Ethylene emanation during 5° precooling also has been reported (83). An initial peak of ethylene production occurred during the second wk of cooling, followed by a major increase after 12 wks. This latter increase may have been related to completion of the cooling requirement. Storage Studies. The use of low pressure storage of non-cooled tulip bulbs, and the storage of precooled tulip bulbs in various packages, closed systems, and low 02 ventilated systems has been noted. 23 Storage of non-cooled tulip bulbs for 14 days in August at pres- sures of 76 or 150 mm Hg suppressed leaf growth and floral development (18). The treatments delayed flowering after subsequent forcing of most cultivars. The authors concluded that low pressure storage offered no advantage over ventilated temperature-controlled units now being used for shipping. Application of low pressure during other phases of the tulip bulb forcing season has not been investigated. Various storage methods for bulbs removed from 5° treatment have been studied. Precooled bulbs held in poorly ventilated cardboard boxes for more than 4-8 days at warm temperatures formed many deformed, blasted, or poor quality flowers upon forcing (73). These floral dis- orders differ from blindness, which results when no floral organs form within the bulb before precooling. Symptoms of floral bud injury were found within bulbs 8 days after removal from 5° to 21°, and increased with temperature, ethylene level, and duration of storage (71, 72). However, bulbs kept in static chambers where atmospheres approached 1% 02 and 16% C02 at 15 or 21° did not show injury. Since a ventilated system with 4.5% CO2 did not prevent the injury, it appeared that the effect was due to low 02. From these studies, practical recommen- dations were made that special precooled bulbs be held at 15° or lower; that maximum ventilation be provided to avoid ethylene damage; and that the shipping period not exceed 8 days. Prince et al. (82) studied further the effects of low 02 levels on precooled bulbs. Flowering of 'Kees Nelis' bulbs was not impaired after 4 wks of storage at 17° in either 3 or 5% O2 in a ventilated system. 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Yang. 1982. Effects and fate of carbon monoxide in shredded lettuce, p. 267-277. In: D. G. Richardson and M. Meheriuk (eds.). Controlled atmospheres for storage and transport of perishable agricultural commodities. Oregon State Univ. School of Agr. Symp. Series. 1. Phan, C. T. 1974. Use of plastic films in the storage ofcarrots. Acta Hort. 38:277-290. Prince, T. A., R. C. Herner, and A. A. DeHertogh. 1981. Low oxygen storage of special precooled 'Kees Nelis' and 'Prominence' tulip bulbs. J. Amer. Soc. Hort. Sci. 106(6):747-751. , and . 1982. Increases in ethylene and carbon diOXide prodUction by Tulipa gesneriana L. 'Prominence' after completion of the cold requirement. Sci. Hortic. 16:77-83. Rees, A. R. 1972. The growth of bulbs. Academic Press, London. 311 p. 1973. Effects on tulip bulbs of warm storage following low temperature treatment. J. Amer. Soc. Hort. Sci. 48:149-154. Saguy, I. and C. H. Mannheim. 1975. The effects of selected plastic films and chemical dips on the shelf life of Marmande tomatoes. J. Food Tech. 10:547-556. Schenk, P. K. and B. H. H. Bergman. 1969. Uncommon disease symptoms caused by Fusarium oxysporum in tulips forced in the glasshouse after precooling at 5°C. Neth. J. Plant Path. 75: 100-104. Schomer, H. A. 1953. Films for produce. Mod. Pack. 26(8): 191-196. Scott, K. J. 1975. The use of polyethylene bags to extend the life of bananas after harvest. Food Tech. in Australia 27: 481-482. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 32 , and S. Gandanegara. 1974. Effect of temperature on the storage life of bananas held in polyethylene bags with ethylene absorbent. Trop. Agric. 51(1):23-26. , W. B. McGlasson, and E. A. Roberts. 1970. Potassium permanganate as an ethylene absorbent in polyethylene bags to delay ripening of bananas during storage. Austral. J. Expt. Agr. & Anim. Husb. 10:237-240. , and E. A. Roberts. 1966. Polyethylene bags to delay ripening of bananas during transport and storage. Austral. J. Expt. Agr. & Anim. Husb. 10:237-240. , and . 1967. Control in bananas of black-end rot caused by Gloeosporium musarum. Austral. J. Expt. Agr. & Anim. Husb. 7:283-286. Scott, L. E. and S. Tewfik. 1947. Atmospheric changes occurring in film-wrapped packages of vegetables and fruits. Proc. Amer. Soc. Hort. Sci. 49:130-136. Singh, B., N. A. Littlefield, and D. K. Salunkhe. 1970. Effect of CA storage on amino acids, organic acids, sugar and rate of respiration of 'Lambert' sweet cherry fruit. J. Amer. Soc. Hort. Sci. 95:458. , D. J. Wang, and D. K. Salunkhe. 1972. Controlled atmosphere storage of lettuce, I. Effects on quality and the respiration rate of lettuce heads. J. Food Sci. 37:48-51. , and . 1972. Controlled atmosphere storage of lettuce, 11. Effects on biochemichal com- position of the leaves. J. Food Sci. 37:52-55. Smith, W. H., D. F. Meigh, and J. C. Parker. 1964. Effect of damage and fungal infection on the production of ethylene by carnations. Nature 204:92-93. Smock, R. M. 1979. Controlled atmosphere storage of fruits. Hort. Rev. 1:301-336. Sommer, N. F., R. J. Fortlage, F. G. Mitchell, and E. C. Maxie. 1973. Reduction of postharvest losses of strawberry fruits from gray mold. J. Amer. Soc. Hort. Sci. 98:285-288. , and D. A. Luvisi. 1960. Choosing the right package for fFesh fruit. Pack. Eng. 5(12):37-43. Stahman, M. A., B. G. Clare, and W. Woodbury. 1966. Increased disease resistance and enzyme activity induced by ethylene and ethylene production by black rot infected sweet potato tissue. Plant Physiol. 41:1505-1512. — — - ‘ —-—— 4 A ’ e._a.~‘_" .m-v—v , 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 33 Swart, A. and G. A. Kamerbeek. 1977. Ethylene production and mycelium growth of the tulip strain of Fusarium oxysporum as influenced by shaking of and oxygen supply to the culturenmdium. Physiol. Plant 39:38-44. Tomkins, R. G. 1962. The conditions produced in film packages by fresh fruits and vegetables and the effect of these condi- tions on storage life. J. Appl. Bact. 25(2):290-307. Uota, M. 1969. Commodity requirements and recommendations for flowers and nursery stock, p. 109-112. In: D. H. Dewey, R. C. Herner, and D. R. Dilley (eds.). Controlled atmospheres for the storage and transport of horticultural crops. Mich. State Univ. Hort. Rpt. 9. Vakis, N., W. Grierson, and J. Soule. 1970. Chilling injury in tropical and subtropical fruits, III. The role of C02 in suppres- sing chilling injury of grapefruit and avocados. Proc. Trop. Region Amer. Soc. Hort. Sci. 14:89-100. Vines, H. M. and M. F. Oberbacher. 1961. Changes in carbon dioxide concentrations within fruit and containers during stor- age. Proc. Fla. State Hort. Soc. 74:243-246. Von Oppenfeld, H., R. S. Lindstrom, D. H. Dewey, and J. W. Goff. 1955. Cold storage of field-grown cut tulips. Quart. Bull. Mich. Agr. Expt. Sta. 38:273-278. Wardowski, W. F., W. Grierson, and G. J. Edwards. 1973. Chilling injury of stored Times and grapefruit as affected by differentially permeable packaging films. Hortscience 8(3):173-175. Williamson, C. E. 1950. Ethylene, a metabolic product of diseased or injured plants. Phytopath. 40:205-208. Wilson, L. G. 1976. Handling of postharvest tropical fruit crops. Hortscience 11(2):120-121. Woodruff, R. E. 1969. Modified atmosphere storage of bananas, p. 80-94. In: D. H. Dewey, R. C. Herner, and D. R. Dilley (eds.). Controlled atmospheres for the storage and transport of horticul- tural crops. Mich State Univ. Hort. Rpt. 9. SECTION I DESIGN OF A MODIFIED ATMOSPHERE PACKAGE FOR MARKETING OF PRECOOLED TULIP BULBS 34 Abstract. A simple prediction method utilizing mathematical descriptions of bulb respiration and film permeation was demonstrated as an aid in selection of a polymeric film for modified atmosphere (MA) packaging of precooled (5°C) tulip bulbs (Tulipa gesneriana L. 'Kees Nelis'). The method predicted that a small consumer package of 5 precooled bulbs sealed in 2 of LDF-301 low density polyethylene film (Dow ca. 800 cm Chemical) would obtain atmospheric equilibrium at 3-5% CO2 and 02 while maintaining normal bulb flowering ability. Two other films, LDF-550 and PSO-599, were predicted as unsuccessful for a package. Packaging trials verified the predictions. Bulbs packaged in LDF-301 film for 3 wks at 20° yielded 70% normal flowers upon forcing. Bulbs in the other films and non- packaged bulbs flowered poorly. Penicillium infection of the bulbs increased C02 and decreased 02 levels in the package, and also decreased the flowering percentage. The package atmosphere appeared to protect the bulbs from package ethylene accumulations to 0.5 ul/liter. Tepal length, plant height, and bottom and top internode lengths upon flowering of bulbs held in the LDF-301 film packages for up to 3 wks at 20° were similar to those from bulbs planted immediately after precooling. 35 36 The marketing of precooled bulbs through mail-order or other retail channels for mid-winter indoor forcing in the northern United States or outdoor planting in the South has not been investigated. DeHertogh (5) describes 5° precooling as storage of tulip bulbs in open tray cases at 5° to satisfy all of the bulb cold requirement (8, 24) before planting. These bulbs could offer consumer conven- ience since no cold treatment would be required for flowering. Current recommendations suggest that these bulbs be held at 15° or lower, that maximum ventilation be provided, and that a shipping period not exceed 8 days (16). Some storage at warmer temperatures has been attempted. Moe and Hagness (18) showed that precooled bulbs held in poorly ventilated cardboard boxes for more than 4 to 8 days formed many deformed, blasted, or poor quality flowers upon forcing. Moe et al. (16, 17) found symptoms of floral bud injury within tulip bulbs 8 days after removal from 5° to 15° or 21° environments. The symptoms increased with temperature, ethylene concentration, and duration of storage. Addition of 4.5% CO2 in a ventilated system did not prevent injury, but storage in static chambers where the 02 content reached 1% did prevent injury. Subsequently, Prince and coworkers (22) found flowering of 'Kees Nelis' bulbs to be unimpaired through 4 wks of storage at 17° in either 3 or 5% O2 in ventilated systems, while air storage yielded poor flowering. Low 02 storage also was found to reduce ethylene- induced floral abortion and to reduce the post-cooling respiratory rise compared to that of bulbs stored in air. Since low 02 appeared to offer advantages, a practical method for obtaining this condition during shipping and marketing needed to 37 be investigated. One such method is the use of sealed polymeric film packages. Beneficial effects from this method have been reported for many commodities (4, 7, 9, 15, 20, 27). Henig (11) has described a produce packaging system as a dynamic one where respiration and permeation are occurring simultaneously. Various methods of predicting the optimal film parameters have been published. Jurin and Karel (13) devised a graphical solution to predict equilibrium package conditions. A more complex computer aided method (10, 12) has utilized differential equations that were developed and solved to predict package atmospheric levels. The objective of this research was to develop and evaluate a consumer size polymeric film package for marketing precooled tulip bulbs under low oxygen regimes without a refrigeration requirement. The application of a simple prediction method for package parameter optimization is demonstrated. Subsequently, the effects of specific polymeric films upon package gaseous atmospheres and the resultant maintenance of bulb flowering ability are outlined. Materials and Methods This research was performed during the 1979-80 and 1980-81 forcing seasons. Tulip bulbs (12-14 cm in circum.) were shipped to East Lansing, Michigan, from the Netherlands in open tray cases in temperature controlled containers at 17-20°. The shipping/arrival dates were Aug. 17/Sept. 7, 1979, and Sept. 10/26, 1980. All bulbs had reached stage G upon arrival and were stored at 13° until the 5° precooling period began. 38 Package Parameter Optimization. A simple method of package parameter optimization (5. Gyesly, personal communication) was used during the first season. 'Kees Nelis' bulbs were precooled in open tray cases at 5° (i 0.5°) from Nov. 11 to Feb. 7 (13 wks). At the end of the precooling period, one or two bulbs were placed in 473 ml canning jars and sealed with ca. 40 cm2 of one of each of four selected poly- meric films. The films were sealed on the jars with stopcock grease, rubber O-rings, and the jar bands. Three jars (reps) were used for each film/bulb number treatment combination. Internal headspaces were ca. 400 ml (1 bulb) or ca. 320 ml (2 bulbs). The jars were placed randomly in a 20° room. The CO2 and 02 levels were monitored over a 15 day period by withdrawing a 2 ml sample through ports of silicone rubber caulking (Dow Chemical) applied to the film surface. Gas chromatography analysis was on a Carle GC-87OO equipped with a thermal conductivity detector. Four types of polymeric films with widely varied permeabilities were obtained from the School of Packaging at Michigan State University for sealing the jars. The films' permeabilities were measured with the Oxtran 100 (02) and Permatran (C02) devices (Modern Controls, U.S.A.) 2 l'day_1 in liter-m7 -atm' at 20-23°. The films and their 02/C02 permeabilities (i 10%) respectively were: Mylar (0.08/0.20); polypropylene (2.8/3.3); polyethylene (6.5/31.0); and pliofilm (28.0/102.0). All were 0.025 mm thick except the Mylar, which was 0.013 mm thick. The changes in jar headspace CO2 and O2 quantities between each sampling were calculated. These changes were assumed to be simul- taneously a function of bulb respiratory activity and C02 and 02 39 permeation through the films. Therefore, the following steady-state equations were used to estimate the C02 emanation and 02 consumption of the bulbs between each sampling: Acog "ZE‘ = [5(002) " P(COZHPCOZD’ (1) where ACO: change in headspace C02 between samplings in ml-bulb'l, -1 -1 E(coz) bulb co2 emanation in ml-bulb oday P(C02) CO2 permeation through each selected film in ml atm 1 day 1, pCOZ = average CO2 partial pressure (atm) in headspace during interval t, At interval in days, and H A02 “ 4sz ,[o 21 - p01 - c(0 ,1, (2) where A0? = change in headspace 02 between samplings in ml-bulb'l, C = bulb 0 consumption in ml-bulb'loday'l, P(02) = 02 permeation through each selected film in ml-atm'l-day'l, p02 = average 02 partial pressure (atm) in headspace during interval t, At = interval in days. P(C02 ) and P(02 ) were calculated from the known permeabilities of the four2 films and the film surface area available for permeation (40 cm 2). The ambient conditions were measured and found to be ca. 0.21 atm O2 and 0.0 atm C02. 40 These two equations were solved for E(CO ) and C(0 ) yielding: 2 2 Acog E(C02) = At + P(COZ)[pC02], and (3) A0; C(02) = P<02)[0.21 - p02] - Kt‘ . (4) The E and C of the bulbs were thus calculated between each (c021 (oz) sampling period. Since the average CO2 and O2 partial pressures external to the bulbs for each sampling interval were also calculated, regression equations relating the E(CO ) and C(0 ) of the bulbs to 2 2 levels of C02 and 02 external to the bulbs were developed, such that: E(C02) = f[C02][02], and (5) c( f'ICOZJIOZJ. (6) 2) where [C02] = 100(pC02), [02] = 100(p02), and f and f' yielded units of ml-bulb-l-day-l. The polynomial functions f and f' were fitted using the stepwise multiple regression program of the STAT IV statistical package. Each variable up to the third order was added if it was significant at the 10% level. For simplicity, the independent variables were entered as 100 times the partial pressures (e.g. 0.10 atm was entered as 10). These equations then allowed the calculation of the desired C02 and O2 permeation for an optimized package. It has been demonstrated that at package equilibrium, the E(C0 ) and C(02) of a commodity are 2 equal to their corresponding film permeation rates (26). 41 Therefore, two equations described the relationship between E(CO ) or 2 C(0 ) and film permeation at equilibrium of an optimized package: 2 _ X E(C02) - P(C02)[pC02], and (7) _ X _ C(02) P(02)[0.21 p02], (8) where E(C02) or C(O bulb CO2 emanation or 02 consumption 1 ) - 2 at package equilibrium in ml-bulb'l-day , Px or Px (C02) (02) required CO2 or 02 permeation of package at equilibrium in ml-bulb'l-day'loatm'l,and pCO2 or p02 = desired package C02 or 02 partial pressure (atm) at equilibrium. Equations 7 and 8 applied only if C02 and 02 partial pressures outside of the package were equal to ca. 0.0 and 0.21 atm respectively. Algebraic substitution then yielded: _ X f[C02][02] - P(c02)[pC02], and (9) . x f [c02][02] P(02)[O.21 - p02]. (10) Desired C02 and 02 levels for an optimized package were then substituted into both sides of equations 9 and 10, which then were solved for PICO ) and PIO ). The number of bulbs per package, as well as a practical 2 packagezsize (film surface area), were decided upon before film selec- tion. This allowed calculation of the required film permeability for an optimized package as follows: X Y P(c02)[B] P(c02) =T , and (11) X P [B] 0 ( 2) (12) v P =-——————-, (02) SA 42 Y Y . . . . where P(C02) or P(0 = requ1red f1lm C02 or 02 permeab1l1ty 1 2 2) _1 _ -day -m , in mloatm' required CD2 or 02 permeation of package 1-atm'1, P1602) or P?02) at equilibrium inml ~bulb'1-day' B = desired number of bulbs in package, and SA = package film surface area (m2). Films with specific manufacturer's numbers were selected fortflmipackaging trials so as to insure repeatability. Bulb Packagigg. Packaging trials were performed during the second forcing season using three films chosen according to the method 2 of outlined above. Films were selected for a package of ca. 0.08 m film surface area (20 x 20 cm, top and bottom) to contain 5 bulbs. This package size was selected to allow for insertion of the bulbs into a prototype plastic holding device before sealing, for subsequent marketing studies. This device is part of a hydroponic forcing pot for tulip bulbs designed for home forcing of bulbs (Netherlands Patent 14.28.24). The permeabilities of the films were measured with a custom-made stainles steel permeability cell to yield more precise measurements than those obtained from perviously-noted industrial equipment. This permeability cell was divided into 2 sections by ca. 44 cm2 of the selected film and sealed with stopcock grease and 2 rubber O-rings which were tightened above and below the plane of the film. This created 2 sections of ca. 60 cm3, separated by the film. Each section was equipped with inlet, outlet, and sampling ports. Through the top section pure C02, pure 02, or 7000 ppm ethylene in air was passed to 43 yield at least 1 air exchange/min. Pure N2 at 8-15 ml/min was passed through the bottom section, which was then sampled for C02, 02, or ethylene analysis until a constant value was obtained. Gas analysis was performed with a Carle GC-87OO (CO2 and 02) and a Varian 1700 (ethylene) gas chromatograph equipped with thermal conductivity and flame ionization detector respectively. The permeability of the films to each gas then was calculated. Three separate determinations were made for each film at 20°. 'Kees Nelis' bulbs were precooled at 5° and 80-90% RH from Oct. 21, 1980, to Jan. 23, 1981 (13.5 wks) for the packaging trial. At the end of the precooling period, 5 bulbs were heat sealed inside each polymeric film package. The resultant package headspace was determined to be ca. 500 ml. Any bulbs with desiccated root plates, a disorder that occurs with a small number of precooled bulbs, were not used in the packages. Four packages (reps) of each film/duration treatment combination were placed randomly into each of two rooms at 20° (35-55% RH) and 25° (20-35% RH). Non-packaged control bulbs were also stored in the open at each temperature. Four replicates of initial post precooled control bulbs were also planted at the start of the experiment to determine if any disorders existed in the bulbs prior to packaging. The C02, 02, and ethylene levels in the 4 wk duration packages were monitored during the 24 days by withdrawing a 2 ml (CO2 and 02) or 1 ml (ethylene) sample through ports of applied silicone rubber for gas chromatography as previously described. At the end of the 2 and 3 wk storage periods, bulbs were removed from the packages for forcing in the greenhouse. 44 Upon opening the packages, the tunics were removed from the bulbs to facilitate rooting (5). The bulbs were planted 5/pot in 15 cm pots containing 20% vermiculite, 20% perlite, and 60% peat (VSP-Mix, Michigan Peat Company). After planting, each pot was drenched with 0.2% benomyl and placed randomly on the greenhouse bench with a minimum night temperature of 16-17°. The plants were fertilized once with 20N-8.6P-16.6K at 200 ppm N when the roots first reached the bottom of the pots. When two-thirds of the flower bud developed color, a plant was considered to have flowered. The days to flower, tepal size, plant height from nose of bulb to top of flower, as well as the bottom and top internode lengths of each normal flower were recorded. The average observation from the normal flowers as well as the percent normal flowers then were calculated for each pot (rep). Statistical Analysis. Stepwise multiple regression was used in the prediction experiment as previously described. Analysis of vari- ance was performed on the package trial data where possible. No statistical comparison was valid between the two temperatures, nor between the two storage durations, due to possible confounding by changing daytime greenhouse conditions during subsequent forcing of the bulbs. The percent normal flower data were based on a small sample (5 bulbs) and did not fit the assumptions of the analysis of variance. For this reason, the Kruskal-Wallis non-parametric statistic was used for testing overall treatment significance while the Mann- Whitney statistic was used for more specific mean separation (14). These tests are based on ranks and have fewer distribution assumptions. 45 Results and Discussion The results of the package optimization study are shown in Figure 1. The trends in the CO2 and 02 levels in the jars over the 15 days reflected the relative permeabilities of the 4 films utilized. Jars containing 2 bulbs obtained higher CO2 and lower 02 levels than those containing 1 bulb, due to the greater amount of respiring tissue. The various film/bulb number combinations resulted in the bulbs being exposed to varied levels of CO2 and 02. This was desirable for developing the prediction equations. The pliofilm sealed over 2 bulbs stretched during the experiment. This altered the film permeation during the study and led to an 02 increase at day 15. These data were excluded from the regression analysis. The two equations obtained from the multiple stepwise regression were: f[C02][02] = 9.1 - 0.18[C02] + 0.059[C02][02] - O.OO8[C02]2[02] with R2 = 0.81, and f'[C02][02] = 15.1 - 2.89[C02] + 1.01[02] + 0.176[c02]2 - (14) 2 = 0.88. 0.003[c02]3 - 0.003[02]3 with R These equations did not elucidate any biological relationship between CO2 or 02 levels external to the bulbs and bulb respiration, but defined the best fit expressions for prediction purposes only. These2 functions were substituted into equations 9 and 10 to allow calculation of PICOZ) and pl02)° A range of 3 to 5% O2 and CO2 was selected as desirable for the package since earlier research had indicated that these ranges could maintain bulb flowering ability (21, 22). Substitution of this range into equations 9 and 10 yielded P§C02) and P%02) ranges of 200-330, and 42-81 ml-bulb"1-atm"1-day'1 respectively. These 46 Figure 1. C02 and 02 levels in jars containing 1 or 2 precooled tulip bulbs and sealed with four different film types. (Values are means of 3 replications.) 47 30 ' Mylar Polypropylene 25 20 0/015 25 , Polyethylene Pliofilm 48 values and the desired package surface area (0.08 m2) to contain 5 bulbs then were substituted into equations 13 and 14 to yield P(CO ) and P102). Package headspace was not considered a critical parameteF for the prediction since studies have indicated that headspace affects only the time to obtain equilibrium, but not the ultimate equilibrium levels (12). Substitution yielded final predicted permeability ranges of 12-26 and 2.6-5.1 liters-m'z-atm'loday'1 for CO2 and 02 respectively. Dow Chemical supplied 3 films with permeabilities near these requirements. All were types of low density polyethylene with good heat-sealable properties. The film permeabilities are listed in Table 1. The permeabilities to CO2 and 02 of the LDF-301 film appeared to be near the middle of the predicted required ranges. It appeared that the LDF-550 film would be marginally effective due to an O2 permeability near the acceptible low limit and a C02 permeability below the limit. The PSD-599 film had permeabilities too low for both gases. While no prediction for an ethylene permeability range was made, the levels were measured and found to be similar to 02 permeability for the 3 films. Packagjng Experiment. The C02 and 02 levels through 24 storage days at 20° and 25° are depicted in Figure 2. Ambient conditions in the storage rooms during the experiment were ca. 0.15% CO2 and 20.5% 02. Equilibrium 02 levels in the 2-3% range were obtained in the LDF-550 and PSD-599 packages at both temperatures, with 25° yielding a faster decrease to equilibrium. These levels were lower than the predicted desired range. The LDF-301 packages at 20° appeared to level off at 5% 02, but subsequently O2 fell to less than 3% sometime 49 Table I. Permeabilities to C02, 02, and ethylene of3low density poly- ethylene films utilized for packaging precooled tulip bulbs.z Permeability (liter-atm'loday'lom'z)y Film Thickness type (mm) C02 02 CZH4 LDF-301 0.051 16.43 (0.53) 4.17 (0.12) 5.21 (0.03) LDF-550 0.076 7.30 (0.12) 2.21 (0.09) 2.55 (0.08) PSD-599 0.152 4.30 (0.07) 1.43 (0.05) 1.09 (0.06) zFilms supplied by Dow Chemical, U.S.A. yValues are means (i 1 SD) of three determinations at 20°C. 50 16 14 P80 599 12 10 « LDF 550 8 o~ s b a? a? LDF 301 veco, 15 DAYS Figure 2. C02 and 02 levels in 3 low density polyethylene packages of precooled 'Kees Nelis' tulip bulbs during 24 days of storage at 20° and 25°C. (Values are means of 4 replications. Mean separation within sampling date by Duncan's multiple range test, 5% level. Absence of letters indicates no significant differences on that sampling date.) 51 between day 17 and day 24. This decrease, as well as decreased 02 levels in other packages between day 17 and day 24, was likely due to increased respiratory activity in the packages due to infection of the bulb root plates by Penicillium. This decrease was eliminated when Penicilliumwas controlledin later packaging studies (Section II). The equilibrium 02 levels in LDF-301 packages at 25° were near 6%. This was most likely due to increased film permeation at 25°. The effects of temperature change upon the function of the package were subsequently investigated in more detail (Section III). Levels of C02 in the 4-5% range were obtained in the LDF-301 packages at 20° with an increase at day 24, again probably due to Penicillium infection of the bulbs. Both LDF-550 and PSD-599 packages resulted in CO2 levels above the 5% level. These high levels have previously been associated with "stem topple“, a disorder where the tulip stem collapses just at flowering (21). Storage at 25° appeared to result in higher CO2 levels than 20° storage for LDF-550 and PSD-599 packages, with little difference occurring for the LDF-301 packages. Ethylene levels in the packages (Figure 3) were variable and were apparently the result of production by the bulbs in response to precooling (15, 23) and as a result of Penicillium infection (Sections 11 and IV). Ambient ethylene levels were ca. 10 nl/l during the experiment. The bulb flowering percentages, after storage and forcing, are shown in Table 2. Non-normal flowers displayed varied degrees of floral abortion and shoot growth retardation. These disorders were quantified in later studies (Sections 11 and 111). After 2 wks at 20° 52 LDF301 DF550 P80599T_ 14 21 7 DAYS .5[ 200 11. LDF301 Z? 3- E: E! 0 ER 2- C) .1. DAYS Figure 3. Ethylene levels in 3 low density polyethylene film packages of precooled 'Kees Nelis' tulip bulbs during 24 days of storage at 20° and 25°C. (No significant differences within sampling dates.) 53 Table 2. Flowering obtained from packaged and non- packaged post precooled 'Kees Nelis' tglip bulbs stored at 20° and 25°C for2 and 3 wks. % normal flowers)"x Film 20° 25° type 2 wks 3 wks 2 wks 3 wks LDF-301 75a 70b 85b 45b LDF-550 808 5a 20ab Oa PSD-599 55a Oa Ga Ga Non-packaged 708 0a 35ab 0a ZInitial post precooled bulbs yielded 95% normal flowers. yMeans of 4 replications of 5 bulbs xMean separation within columns by the Mann-Whitney non-parametric test, 5% level. 54 no significant differences in flowering were visible between treatments. All yielded between 55 and 80% normal flowers, including the open controls. This level of flowering from non-packaged bulbs was better than levels previously reported from bulbs stored in poorly ventilated cardboard boxes (18). The ventilation of endogenous ethylene by open storage alone appeared to lessen floral abortion. Cultivar response differences also could have been involved here (6). The LDF-301 packages maintained a high level of flowering after 3 wks at 20°, compared to negligible flowering for all other treatments. Penicillium infection of the bulbs was observed in all packages and was believed responsible for reduced flowering from the bulbs in LDF- 301 compared to initial post precooled controls. Representative pots of forced bulbs from these treatments are shown in Figure 4. Flowering of bulbs after 2 wks in LDF-301 packages at 25° was 85%, while large variability in the flowering response was observed from LDF-550 packages and non-packaged storage at the same temperature and duration. The LDF-301 packaging for 3 wks at 25° maintained some degree of flowering, but was less successful than at 20°. However, while high temperatures may limit the package success, continuous 25° in a marketing environment is unlikely. The LDF-301 package has subse- quently been demonstrated to be adaptable to ambient temperature fluctuation between 15° and 25° with only small changes occurring in the package environment, and with good maintenance of flowering ability (Section III). The poor performance of bulbs from LDF-550 and PSD-599 Packages after 2 wks at 20°, and after 2 or 3 wks at 25°, was likely due to low 02 and/or high CO‘2 effects. Because of these poor results, GMT); LDF-301 film was used in further studies. 55 Figure 4. Representative pots of forced 'Kees Nelis' tulip bulbs after 3 wks of storage at 20°C. (Left to right: non-packaged, in LDF-301, LDF-550, and PSD-599 packages.) 56 The forcing characteristics of the normal flowers obtained from packaged and non-packaged bulbs are shown in Table 3. The LDF-301 packages maintained most characteristics at similar levels to initial post precooled bulbs through 3 wks. After 2 wks at 20°, non-packaged bulbs yielded lower values than bulbs from LDF-301 packages for total plant height and for bottom and top internode lengths. This indicated some degeneration of the non-packaged bulbs after 2 wks that was not apparent from the percent normal flowering data. Studies have shown that both the leaves and the floral organs provide auxin-like sub- stances which control the elongation of the floral shoot (25), with the gynoecium exerting the greatest control over the top internode (19). Endogenous bulb ethylene levels may have inhibited polar auxin trans- port (2) in the floral shoot yielding reduced elongation from open stored bulbs. The LDF-301 package may have preserved the normal hormonal status of the shoot by the antagonistic effects on ethylene action of reduced 02 and elevated CO2 levels (1, 3), even though some package ethylene accumulation was evident. Previous research has demonstrated that the bulbs could tolerate these ethylene levels if exposed to a 3-5% 02 atmosphere, without exhibiting a great degree of floral bud blasting upon forcing (22). Bulbs subjected to any post precooling storage flowered in less days than initial post precooled control bulbs, with open stored bulbs flowering in less days than any 0f the packaged bulbs after 2 wks at 20°. This indicated some shoot development in open stored bulbs that was slowed by the package atmosphere. 57 Am.Hv m.mH Am.HV ¢.oH Am.ov m.m A~.~v ¢.om A~.ov m.¢ Homing; 3.x.mm\axz m Ao.ov m.mH Am.ov m.NH Ao.ov m.m Am.HV o.om flfi.ov m.¢ Honing; 3.x.om\axz m ae.m~ a~.m a~.m am.- aam.a eamaxuaa-eee um.o~ no.HH no.m amm.¢m m¢.¢ mmmtomm unN.mH n~.oH am.m m¢.mm new.e ommiu34 no.mH nm.- ao.m am.om no.m Homtmob 11111111111111111111 mfifi§M11ii:111111111111111 cmzopc AEuV Asov Asuv Asuv max“ on muoccwucw muoccmucw mNVm m~_m EFPJ mxmo mop souuom vamp; Foam» N.mxz m eea N Lee 8mm nee com “a eaeepa 882:8 aw_=u .mmez mmmx_ empooomca umoa ummmxumaico: use vmmmxuma mo muwpmwcmuumcmgu m:_ucom .m msamh 58 .Aom H my memos mcm mama: .cowpmuwpamc cmaoca cow mcmzopm Peace: 3mm oou umcpmw» mpocucou :mao use .mmmiomm .ommimobx ._m>mp gm .pmwp mace; mpneupze m.:mu::o xa mcszfiou segue; cowumcmamm cams» .xpco mcmzopw FmELoc soc» muwumwcmpumcugu PP<~ 3.8 8.3 3.3 3: :.8 ca 3.3 8.8 :.8 9m 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 memaemauaigm 88.3 11111111111111111 cmzopm Asov Azuv Asuv Aeov mama op mcoccmpcw mooccmucm m~wm mNPm sped mxdo new sopuom acmpa —mamh .A.u.u:ouv m m_amh 59 This study has demonstrated the application of a simple prediction method as an aid in the choice of a polymeric film for MA packaging. The method determined a range of required film permeation for each unit of a commodity to be packaged to yield a chosen atmosphere. While a very specific package was developed and tested, further package parameter changes could be made, provided that the permeation to commodity ratio is maintained. The sealed package of LDF-301 film developed here maintained flowering ability of precooled tulip bulbs. Actual marketing studies with this package will be performed in the future. It is hoped that this method will aid others in modified atmosphere package development. LITERATURE CITED 10. 11. LITERATURE CITED Beijer, E. M. Jr. 1979. Effect of silver ion, carbon dioxide, and oxygen on ethylene action and metabolism. Plant Physiol. 63:169-173. Burg, s. P. and E. A. Burg. 1967. Inhibition of polar auxin L. transport by ethylene. Plant Physiol. 42:1224-1228. - , and . 1967. Molecular requirements for the biological activity of ethylene. Plant Physiol. 42: 144-152. 17 Daun, H. K., S. G. Gilbert, Y. Ashkenazi, and Y. Henig. 1973. Storage quality of bananas packaged in selected permeability films. J. Food Sci. 38:1247-1250. DeHertogh, A. A. 1977. Holland bulb forcer's guide. Neth. Flower Bulb Inst., New York, NY. , D. R. Dilley, and N. Blakely. 1980. Response variation of tulip cultivars to exogenous ethylene. Acta Hort. 109:205-210. Harbaugh, B. K., G. J. Wilfret, and F. J. Marousky. 1978. Use of sealed polyethylene packages for marketing potted plants. Hortscience 13:669-670. Hartsema, A. M. 1961. Influence of temperature on flower for- mation and flowering of bulbous and tuberous plants. Encyl. Plant Physiol. 16:123-167. Hauge, A., W. Bryant, and A. Laurie. 1947. Prepackaging of cut flowers. Proc. Amer. Soc. Hort. Sci. 49:427-432. Hayakawa, K. I., Y. S. Henig, and S. G. Gilbert. 1975. Formulae for predicting gas exchange of fresh produce in polymeric film packages. J. Food Sci. 40:186-191. Henig, Y. S. 1975. Storage stability and quality of produce packaged in polymeric films, p. 144-152. In: N. F. Haard and D. K. Salunkhe (eds.). Postharvest biology and handling of fruits and vegetables. A. V. I. Westport, CN. 60 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 61 , and S. G. Gilbert. 1975. Computer analysis of the variables affecting respiration and quality of produce packaged in polymeric films. J. Food Sci. 40:1033-1035. Jurin, V. and M. Karel. 1963. Studies on control of respira- tion of McIntosh apples by packaging methods. Food Tech. 17(6): 104-108. Meddis, R. 1975. Statistical handbook for non-statisticians. McGraw-Hill, London. Mitchell, F. G., M. L. Arpaia, and G. Mayer. 1982. Modified atmosphere storage of kiwifruits (Actinidia chinensis), p. 235- 238. In: D. G. Richardson and M. Meheriuk (edST). Controlled atmospheres for the storage and transport of perishable agri- cultural commodities. Oregon State Univ. School of Agr. Symp. Series. 1. Moe, R., A. A. DeHertogh, and D. R. Dilley. 1978. Influence of post special precooling treatments on flower development and ethylene evolution from water and growth regulator injected tulip bulbs. Sci. Rpt. Agr. Univ. Norway 57(46):1-18. , and . 1978. Influence on growth and flowering of special precooled tulips of growth regulators and sucrose injections, and of temperature and ethylene exposures under ventilated and non-ventilated conditions. Sci. Rpt. Agr. Univ. Norway 57(45):1-19. , and A. Hagness. 1975. The influence of storage temperature and 2-chloroethylphosphoric acid (ethephon) on shoot elongation and flowering in tulips. Acta Hort. 47:307-318. Op den Kelder, P., M. Benschop, and A. A. DeHertogh. 1971. Factors affecting floral stalk elongation of flowering tulips. J. Amer. Soc. Hort. Sci. 96(5):603-605. Oudit, D. D. and K. J. Scott. 1973. Storage of 'Hass' avocados in polyethylene bags. Trop. Agric. 50(3): 241- 243. Prince, T. A. 1980. Controlled atmosphere storage of special precooled tulip bulbs (Tulipa gesneriana L., 'Kees Nelis' and 'Prominence'). M.S. Thesis, Michigan State Univ., East Lansing,MI. R. C. Herner, and A. A. DeHertogh. 1981. Low oxygen storage of special precooled 'Kees Nelis' and 'Prominence' tulip bulbs. J. Amer. Soc. Hort. Sci. 106:747-751. , and . Increases in ethylene and carbon diOX1de production by TUlipa gesneriana L. 'Prominence' after completion of the cold requirement. Sci. Hortic. 16:77-83. 24. 25. 26. 27. 62 Rees, A. R. 1972. The growth of bulbs. Academic Press. 311 p. Saniewski, M. and W. J. DeMunk. 1981. Hormonal control of shoot elongation in tulips. Sci. Hortic. 15:363-372. Tomkins, R. G. 1962. The conditions produced in film packages by fresh fruits and vegetables and the effect of these conditions on storage life. J. Appl. Bact. 25(2):290-307. Von Oppenfeld, H., R. S. Lindstrom, D. H. Dewey, and J. W. Goff. 1955. Cold storage of field-grown cut tulips. Quart. Bull. Mich. Agr. Expt. Sta. 38:273-278. SECTION II CONTROL OF INFECTION BY PENICILLIUM SPP. 0F PRECOOLED TULIP BULBS IN A MODIFIED ATMOSPHERE PACKAGE 63 Abstract. Root plates of precooled tulip bulbs (Tulipa _gesneriana L. 'Kees Nelis') maintained in sealed modified atmosphere packages of low density polyethylene film for 4 wks became infected with species of Penicillium. Infec- tion led to increased ethylene and C02 as well as reduced 02 levels in the packages. Infection also led to reduced rooting and increased floral abortion during subsequent forcing of the bulbs. Prochloraz and vanguard pretreat- ment of the bulbs prior to packaging controlled the damage caused by Penicillium spp. through 4 wks of storage in the packages. Control of infection resulted in equilibrium levels of ca. 5% 0 4% C02, and 0.1 ul/liter ethylene in 2, the packages. Pretreated bulbs also retained excellent bulb flowering ability through 4 wks of storage while non- packaged bulbs flowered poorly. Benomyl, captan, and chlorine dip pretreatments did not control root plate infection in the packages. The design and initial testing of a modified atmosphere (MA) package for marketing of precooled tulip bulbs has been outlined (Section I). This system, which consists of a consumer-size package of 5 bulbs sealed in a low density polyethylene film (LDF—301, Dow Chemical), was partially successful in maintaining flowering ability of the bulbs through 3 wks at 20°C. However, infection of the bulb 64 65 root plates by Penicillium spp. during storage appeared responsible for unfavorable package atmospheres and for the prevention of normal flowering upon forcing of the bulbs in the greenhouse. These effects of infection by Penicillium spp. could limit the future commercial success of the package. MA packaging has been shown to increase the shelflife of various commodities by reducing transpirational water loss, product respiration, and the detrimental effects of ethylene exposure (3, 11, 14, 15). However, the high relative humidities in MA packages have been shown to create excellent environments for commodity infection by both fungi and bacteria. It appears that the comnon controlled atmosphere (CA) conditions of 2-3% 02 and 5% CO2 suppress only moderately most commo- dity disease organisms (4). Disease causing fungal organisms were observed to be detrimental to cut tulips (10, 20), carnations, and Chrysanthemums (5) stored in various film packages, while the bacteria Erwinia carotovora caused decay of bell peppers packaged in poly- ethylene (PE) and polyvinylchloride (PVC) films (2). Anthracnose was seen to invade the stem end of avocados in individual PE bags after 40-50 days at 10°, even though the fruit were benomyl dipped prior to packaging (11). Various bacteria and fungi were isolated from flowering pot plants and bedding plants sealed in coextruded PE-polypropylene (PP) film packages (6). Although identification and pathogenicity studies of the isolates were not performed, decayed flowers and foliage made the plants unmarketable. Fungicide or sterilant pretreatment of commodities has been instru- mental to the success of some sealed packages. A chlorine dip controlled mold development on tomatoes in PVC and PE packages (14) which led to a 66 fruit shelflife of 21 days at 25°. Benomyl or thiabendazole controlled occasional infections by Gloeosporium musarum in bananas in large sealed PE shipping containers (15, 16). This MA packaging technique is now used commercially for distant market shipment of bananas (21). Thus, while disease growth is common in sealed commodity packages, fungicide pretreatment has led to control in some instances. The control of infection by Penicillium spp. of precooled tulip bulbs in an MA package could make the marketing of these bulbs a commercial reality. Therefore, the purpose of this study was to determine the effectiveness of various fungicide pretreatments in controlling Penicillium root plate infection of the packaged bulbs. The nature of the detrimental effects of Penicillium growth upon both the package gaseous atmosphere and the subsequent flowering ability of the bulbs also was investigated. Materials and Methods This research was performed during the 1981-82 and 1982-83 bulb forcing seasons. Tulip bulbs (12-14 cm in circum.) were shipped to East Lansing, Michigan, from the Netherlands in open tray cases. Tem- peratures during shipment were 13-17° (1981-82) and 17-20° for 10 days followed by 4 days at 7-15° (1982-83). The shipping/arrival dates were Sept. 11/Oct. 6, 1981; and Aug. 16/30, 1982. All bulbs had reached stage G upon arrival and were stored at 13° (1981-82) and 17-20° (1982- 83) until the 5° precooling period began. Expt. 1. 4(1981-82). 'Kees Nelis' bulbs were precooled at 5° and 80-90% RH from Oct. 8 to Dec. 30 (12 wks). At the end of the precooling period, the bulbs were treated with various fungicides 67 prior to packaging in LDF-301 film. The following fungicides were applied: benomyl (Benlate 50 WP) [methyl-I-(butylcarbamoyl) benzi- midazole-Z yl carbamate]; vanguard (CGA-64251 10 W6) [1-[[2-(2,4- dichlorophenyl)-4-ethyl, 1,3-dioxolan-2-yl]methyl]-1 H-1,2,4-triazole]; prochloraz (BTS-4O 542 40 EC) [1-(N-propyl-N-(2-(2,4,6-trichlorophenoxy) ethyl)carbamoyl)imidazole]; and captan 50 WP [N-trichloromethyl- mercapto-4-cyclohexene-1,2-dicarboximide]. All fungicides were applied by dipping the bulbs for 20 min in well-agitated water suspensions in 4 liter containers at 21°. Captan was applied both as a suspension and as a dust. Two rates of each fungicide were used. These are shown in Table 1 in pg of active ingredient per ml. The low rate of captan dust was prepared from captan 50 WP and talc. Bulbs dipped in water without fungicide and non-dipped bulbs were utilized as controls. All dipped bulbs were thoroughly dried under flowing air from a small fan before packaging. Five bulbs from each treatment then were sealed in each LDF-301 film package and placed randomly in a 20° room (40-50% RH) for dura- tions of 2 and 3 wks. Two cm lengths of adhesive tape (Scotch® Patch and Repair Tape) were utilized as gas sampling ports on the film surface of the 3 wk packages. The top length of tape was used to seal previous sampling holes on the bottom length. At the end of each storage period the bulbs were removed from the packages, and the bulb tunics were removed. The percentage of the surface area of each bulb root plate infected with Penicillium spp. was estimated and recorded. The package parameters and atmosphere monitoring, the use of initial post precooled controls, and the forcing of the bulbs were identical to those reported earlier (Section 1), except that each pot 68 of 5 bulbs from every treatment was drenched with 0.2% benomyl and 0.4% ethazol after planting. Upon forcing, the percent normal flowers obtained from each pot was recorded. In addition, the abnormal plants were rated for floral and shoot abnormalities. The 0-4 point rating system utilized was: 0 - no shoot elongation above the bulb; 1 - some shoot elongation, but flower unemerged from leaves; 2 - tepals visible, but dried and yellow; 3 - tepals turgid, but no color development; and 4 - abnormal or mis- shapen tepals. The ratings from each abnormal plant were averaged to yield an abnormality rating for each pot. For the correlation analysis all plants were rated, with normal flowers additionally rated as 5 points. These averages for each pot were designated as floral ratings. After flowering, all bulbs were removed from the pots and the soil was carefully washed from the roots. They were blotted dry and cut from the bulbs, and an average bulb fresh root weight was determined for each pot. Expt. 2. (1982-83). 'Kees Nelis' bulbs were precooled at 5° and 80-90% RH from Sept. 17 to Dec. 10 (12 wks). Some Penicillium growth was observed on the bulb tunics during the precooling period. To avoid development of high inoculum levels, the bulbs were dusted with plain talc on Nov. 23 to lower the available surface moisture for pathogen growth while avoiding fungicide use. At the end of the precooling period, one half of the bulbs had inoculum applied to them in addition to that naturally present. Application was by dipping the base of the bulbs in a spore suspension for a few seconds. The remaining bulbs had no additional inoculum applied to them. The mixed spore suspension 69 of ca. 107 spores/ml was prepared from cultures of various isolates of Penicillium spp. These isolates were collected during the I981-82 season from infected bulbs and were maintained in culture on potato- dextrose agar. A few drops of Tween-20 surfactant were added to the suspension to facilitate spore wetting. Investigations of the patho- genicity, fungicide resistance, and other properties of the individual Penicillium isolates are reported elsewhere (Section IV). Fungicide or sterilant treatments used before bulb packaging were: vanguard (240 pg a.i./ml), prochloraz (600 pg a.i./ml), and bleach (6000 ppm available chlorine, pH 7.6). In addition, water- dipped and non-dipped control bulbs were packaged. Both inoculated and non-inoculated, non-packaged control bulbs were utilized and initial post precooled bulbs were planted at the start of the experi- ment. The inoculation/fungicide/storage duration treatment combinations were arranged in a 2 x 6 x 2 factorial with 4 replications (packages) of each combination in a completely randomized design. The fungicide application, packaging, storage, disease evalu- ation, planting, forcing, and flowering evaluation were the same as utilized in Expt. 1 except for the following changes. Bulbs were stored for 3 and 4 wks. The planting mix was 50% muck peat, 25% perlite, and 25% vermiculite. An additional fertilization of Ca(N03)2 at 2.4 g/liter was applied one week after the first fertilization. At flowering, the fresh root weight was not determined. Instead, a 0-4 root rating was recorded for each bulb. A photograph of this rating scale was maintained to assure repeatability (Figure 10). An average root rating then was calculated for each pot. 70 Statistical Analysis. Analysis of variance was performed where possible. Data transformations were used where noted in the tables and figures. When the data could not be transformed to meet the assumptions of the analysis of variance, the Kruskal-Wallis non- parametric statistic was used for testing overall treatment signifi- cance, while the Mann-Whitney statistic was used for more specific mean separation. Results and Discussion Expt. 1. (1981-82). The root plate of the precooled tulip bulbs provided an excellent site for Penicillium infection. This was likely due to the slight emergence of the root initials from the root plate during the precooling and storage in LDF-301 film packages. This emergence is visible on the non-infected root plates shown in Figure 1. Infection also was seen on any occasional wounds on the outer fleshy scale of the bulbs. Saturated relative humidity conditions in the package were indicated by the soft and watersoaked tunics of bulbs removed from the packages as compared to the dry tunics of the bulbs at the time of packaging. This condition was ideal for infection since both free water and nutrients that may leach from the tissue have been demonstrated to be necessary for Penicillium spore germi- nation (7, 12). The infection of root plates after bulb prepackaging treatments and storage in LDF-301 film packages is shown in Table 1. The water dip pretreatment apparently spread spores to the root plates yielding greater infection than on non-dipped bulbs. In addition, uptake of water through the root plate during the 20 min treatment may have led 71 Figure 1. Infection by Penicillium spp. of precooled 'Kees Nelis' tulip bulb root plates pretreated with benomyl at 2000 pg a.i./ml (top) compared to non-infected bulbs treated with vanguard at 240 ug a.i./ml (bottom . All were packaged in LDF-301 film for 3 wks at 20°C (1981-82 . 72 Table 1. Infection by Penicillium spp. of tulip bulb root plates after prepackaging dips or dusts and subsequent storage for 2 or 3 wks at 20°C in LDF-301 film packages or non-packaged (1981-82). % of root plate infectedZ Prepackaging Ratey treatment (pg a.i./ml) 2 wks 3 wks ”2° _3_4_ f: No dip ---- 0 10 Benomyl 1000 47 ns 88 2000 54 ns 97 Prochloraz 300 O 2 600 0 1 Vanguard 120 0 0 240 O 1 Captan 1200 18 ns 48 ns 2400 10 ns 47 ns Captan dust 10% 1 4 50% 0 3 Non-packaged H20 ---- 1 2 No dip ---- 0 1 zMeans within columns different from H20 dip controls at 5% level by Mann-Whitney nonparametric statistic, except those marked nonsignifi- cant ns . yResponse from two application rates was not significantly different for any fungicide. 73 to deep spore penetration and enhanced infection. Non-dipped bulbs in the packages displayed a range of 0-80% infection of each bulb root plate after 3 wks although the average infection was only 10%. This large variation likely reflected differences in natural inoculum on the bulbs and/or bulb susceptibility differences. Benomyl treatment yielded more infection than that observed on water dipped bulbs (Table 1 and Figure 1). This was most likely due L to uninhibited growth of a benomyl-resistant isolate of_P. corymbiferum that was later obtained in pure culture from the bulbs (Section IV). Both prochloraz (19) and vanguard (17), two new unregistered chemicals, yielded excellent control of infection. These compounds, at the rates i utilized here, have been shown to be active against benomyl resistant P. expansum isolates on stored apples (1). Apparently, they are active against the resistant E. corymbiferum as well. No phytotoxic effects from these two chemicals were visible on the bulbs after removal from the packages or upon forcing in the greenhouse. Captan treatment was unsuccessful as a dip application, but appeared to control infection when applied as a dust. Decreased bulb surface moisture and the lack of an inoculum spreading during dipping were likely explanations. The dust was unsightly and considered unacceptable from a consumer viewpoint. However, these results indi- date that enclosure of a packeted desiccant in the package could possibly control the infection without the use of a fungicide, if bulb desiccation did not occur. This possibility deserves investigation in the future. The effects of prepackaging treatment on package 02 and CO2 levels are shown in Figures 2 and 3 respectively. The treatments that 74 vanguard ‘: control (nodip) ' captan dust prochloraz captan ‘ benlate control 111,01 WEEKS Figure 2. Effect of prepackaging treatment on 02 levels in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C (1981-82). (Mean separation within sampling datetnrDuncan's multiple rante test, 5% level. Absence oflettersindicates no sig- nificant differences on that date.) 75 b captan benlate 9.002 b control (H20) prochloraz - captan dust a. vanguard WEEKS Figure 3. Effect of prepackaging treatment on CO levels in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C (1981-82). (Mean separation within sampling date by Duncan's multiple range test, 5% level. Absence of letters indicates no sig- nificant differences on that date.) 76 yielded the most severe Penicillium infection (benomyl and water dips) resulted in the lowest package 02 levels and the highest CO2 levels. Ambient levels during storage were ca. 0.10% C02 and 20.2% 02. Increased bulb respiration in response to infection and the additional respiration of the fungus itself were likely causes for the differences in package atmospheres. When Penicillium spp. were controlled, package equilibrium was obtained at ca. 5% 02 and 4% C02. The packages of non-dipped bulbs did not show 02 declines below 5% as was observed in earlier studies with bulbs that were not pretreated with fungicide (Section I). Those bulbs were packaged on Jan. 23 while bulbs for this study were packaged on Dec. 30. Bulb root plates have been observed to become more protruded as the period of storage after harvest but before planting lengthens (8). Greater root plate protrusion on bulbs utilized in the earlier trials could have increased susceptibility to infection and ultimately decreased the package 02 levels. Differences in natural inoculum levels could have been involved also. Package ethylene levels are shown in Figure 4. Tulip bulbs them- selves have been demonstrated to produce ethylene in response to precooling (9, 13). The ethylene levels in packages of vanguard treated bulbs, where little Penicillium spp. growth occurred, were further evidence for this production. The subsequent forcing of the bulbs indicated that these ethylene levels in the package atmos- phere were not detrimental to flowering. Increased package ethylene levels resulted from pretreatments that poorly controlled infection by Penicillium spp. Ambient levels during storage were ca. 50 nl/l. The benomyl resistant P. corymbiferum isolated from the bulbs has been shown to produce ethylene in pure culture. This isolate appeared 77 captan benlate In control (Hp) bc' captan dust 02H4 ( ppm) b prochloraz I: control (nodip . o vanguard WEEKS Figure 4. Effect of prepackaging treatment on ethylene levelsirlLDF-BOI film packages of precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C (1981-82). (Mean separation within sampling date by Duncan's multiple range test, 5% level. Absence of letters indicates no sig- nificant differences on that date. Data analyzed on log transformed sca e. 78 responsible for at least some of the ethylene accumulation, especially within packages of benomyl treated bulbs. However, increased bulb ethylene production in response to infection as well as pathogen ethylene production could have occurred in the packages (Section IV). The cause of the ethylene accumulation in packages of bulbs pretreated with captan dust was unclear. The results of subsequent forcing of the bulbs after 2 and 3 wks of storage at 20° are shown in Tables 2 and 3 respectively. All treatments yielded nearly perfect flowering after 2 wks except for packages of benomyl and water dipped bulbs. The ratings indicated that the average abnormal flower from these two treatments had dried and yellow tepals. These two treatments also yielded the poorest root growth during forcing. Prochloraz and vanguard dipped and captan dusted bulbs in packages yielded nearly perfect flowering after 3 wks of storage (Table 3). However, captan, water, and benomyl treated bulbs in packages flowered very poorly. The floral abnormalities and reduction of root growth observed during forcing of benomyl treated bulbs were even more severe after 3 wks than after 2 wks of storage. The non-pretreated packaged bulbs did flower acceptably after 3 wks. This indicated that natural inoculum and/or bulb susceptibility to infection in this experiment may have both been low enough to allow flowering without fungicide pre- treatment. Non-packaged control bulbs yielded only 40-60% normal flowers. Those non-packaged bulbs that were dipped in water had lower abnormality ratings and reduced root growth compared to their non- dipped counterparts. While little disease was evident on any non- packaged bulbs at the end of storage, some infection may have 79 Table 2. Flowering of precooled 'Kees Nelis' tulip bulbs after pre- packaging treatment and subsequent storage for 2 wks at 20°C in LDF-301 film packages or non-packaged (1981-82).Z Prepackaging Rate % normal Abnormality Root fresh treatment (ug a.i./ml) flowersy ratingx wt. (gm)w H20 ---- _gg;_ 2.5 2.8ab No dip ---- 95 --- 7.1cde Benomyl 1000 *75 ns 2.0 3.7abc 2000 45 ns 2.3 1.9a Prochloraz 300 90 3.5 6.9cde 600 100 --- 7.4cde Vanguard 120 100 --- 7.8de 240 100 --- 8.8e Captan _ 1200 80 --- 5.1a-d 2400 100 --- 5.5a-e Captan dust 10% 95 --- 6.7b-e 50% 100 --- 8.6d-e Non-packaged H20 ---- 100 --- 6.Ia-e No dip ---- 95 --- 6.3b-e 2Initial post precooled controls yielded 100% normal flowers and 7.4 gm fresh root wt. yMeans different from H20 dip controls at 5% level by Mann-Whitney nonparametric statistic except those marked nonsignificant (ns). Difference between fungicide rates at 5% level indicated by (*). xNo si nificant differences. Ratings shown only when at least 2 reps (pots? contained abnormal flowers. Each abnormal plant rated from 0-4 according to symptoms of shoot and floral abnormality (see text). wMean separation within columns by Duncan's multiple range test, 5% level. 80 Table 3. Flowering of precooled 'Kees Nelis' tulip bulbs after pre- packaging treatment and subsequent storage for 3 wks at 20°C in LDF-301 film packages or non-packaged (1981-82).z Prepackaging Rate % normal Abnormality Root fresh treatment (ug a.i./ml) flowersy ratingx’v wt.(gm)WsV H20 ---- _1§__ 1.8cd 0.4abc No dip ---- 87 --- 3.3efg Benomyl 1000 5 ns 1.1abc O.Iab 2000 5 ns 0.4a 0.1a Prochloraz 300 95 --- 3.8fg 600 95 --- 3.8fg Vanguard 120 100 --- 5.09 240 100 --- 4.2fg Captan 1200 50 1.7bcd 1.5cde 2400 45 2.5de 1.4b-e Captan dust 10% 90 3.0e 4.4fg 50% 95 --- 4.6fg Non-packaged H20 ---- 40 0.8ab 0.98-d No dip ---- 60 2.3de 2.3def 2Initial post precooled controls yielded 100% normal flowers and 7.4 gm fresh root wt. yMeans different from H20 dip controls at 5% level by Mann-Whitney nonparametric statistic except those marked nonsignificant (ns). There were no differences between fungicide rates. xRatings indicated only when at least 2 reps (pots) contained abnormal flowers. Each abnormal plant rated from 0-4 according to symptoms of shoot and floral abnormality (see text). wData were analyzed on log (X + 1) transformed scale. vMean separation within columns by Duncan's multiple range test, 5% level. 81 developed during forcing yielding the more severe symptoms of the dipped bulbs. A negative correlation was evident between infection of the bulb root plate by Penicillium spp. and subsequent root growth after 2 and 3 wks of storage in packages (Figures 5 and 6). The root growth also was positively correlated with the floral rating. This indi- cated that floral abortion was possibly induced by a reduction in root growth during forcing. This reduction likely resulted from infection of the root plates in the packages. The increased package ethylene levels which resulted from infection also could have induced floral abortion. Expt. 2. (1982-83). In spite of the application of spore suspen- sion to the bulb bases, disease severity was similar to that on bulbs with only naturally occurring inoculum. Only prepackaging treatment and duration of storage led to significant differences in disease severity for this experiment (Table 4). It was unlikely that poor contacttyfthe spores with the bulb root plate occurred during inocula- tion. The tunics of the bulbs were cracked near the protruding root plates, which allowed suspension contact with the root plates to be easily achieved. However, bulbs dipped in water and stored for 3 wks displayed less severe infection than similarly treated bulbs during 1981-82 (Table 1). This suggested that bulbs used in Expt. 2 were less susceptible to infection. Possibly infection sites on the root plate, rather than inoculum supply, were limiting to infection by Penicillium spp. The lack of increased disease incidence following 82 100 5 I 80 4 I c2” 3', 60 3 1': <3; <1: 8 «x . ‘I 5 40 >3) 2 3:. , ’15: (I \0 . 6.. 0 0 20 611,-}; 1 d 1 * ‘27 l o 2.0 2.5 3.0 LOG ROOT WT. (MG) Figure 5. Correlations of fresh root weight with percentage of root plate diseased and floral ratings of precooled 'Kees Nelis' tulip bulbs after prepackaging treatments and subsequent storage for 2 wks at 20°C in LDF-301 film packages (1981-82). (Plotted are the means of 4 replications.) 83 100 1 .. 1 (25 H 60 E g a: 5 4O 23:" 5 C3 5 20 1 17'. 1 2 3 4 LOG ROOT WT. (MG) Figure 6. Correlations of fresh root weight with percentage of root plate diseased and floral ratings of precooled 'Kees Nelis' tulip bulbs after prepackaging treatments and subsequent storage for 3 wks at 20°C in LDF-301 film packages (1981-82). (Plotted are the means of 4 replications.) 84 Table 4. Infection by Penicillium spp. of precooled 'Kees Nelis' tulip bulbs after fungicide or steri- lant prepackaging treatments and subsequent storage for 3 and 4 wks at 20°C in LDF-301 film packages or non-packaged (1982-83).Z % of root plate infectedy Prepackaging treatment 3 wks 4 wks H20 26de 36ef No dip 9c 26de Chlorine 12cd 53f (6000 ppm) Vanguard 0a lab (240 pg a.i./ml) Prochloraz 2ab 3b (600 pg a.i./ml) Non-packaged lab 3b zTreatments applied to inoculated and non-inoculated bulbs. Inoculated bulbs dipped briefly in mixed spore suspension (107 spores/ml). Non-inoculated bulbs infected only by naturally-occurring inoculum. There were no significant infection level differ- ences within any treatment between the inoculated and non-inoculated bulbs. Data shown are means of inoculated and non-inoculated bulbs. yData were analyzed on log (X + 1) transformed scale. Treatment x wks interaction significant at 5% level. Separation of any means by Duncan's multiple range test, 5% level. 85 application of inoculum in addition to that present on the bulbs was evidence for infection site limitations. Vanguard and prochloraz again were most effective in controlling infection through 4 wks of storage (Table 4). Chlorine pretreatment was not successful. Apparently, not all spores were killed by the sterilant. Similar control failure has been observed with citrus inoculated with Penicillium and treated with chlorine (18). Van der Plank (18) suggested that oxidizable substances in the fruit peel reduced the active chlorine before it could penetrate to the pathogen deep within a wound. This may have occurred on the bulb root plates. Possibly, the solution did not even contact spores deeply imbedded in the root plates. Additionally, the lowering of the population of competetive microorganisms on the surface of the root plate may have ultimately increased the spread of infection during the duration of storage. Package 02 levels were again reduced when Penicillium infection was poorly controlled (Figure 7). However, little difference in package CO2 levels was observed (Figure 8). Package ethylene levels also paralleled disease infection although the levels were ca. 50% of those seen in Expt. 1 (Figure 9). This could have been due to less severe infection, to less production by the bulbs, or to a different population mix of ethylene and non-ethylene producing Penicillium spp. on the root plates. Ambient storage conditions were ca. 0.22% 002, 20.6% 02, and 20 nl/l ethylene. Non-inoculated bulbs pretreated with vanguard or prochloraz before packaging yielded excellent flowering after 3 wks (Table 5) and 4 wks of storage (Table 6). While inoculation previously was 86 1 o—-—oNO DIP CONTROL o—o H20 CONTROL ‘2 r—d BLEACH b—dVANOUARO .——-O PROCH LORAZ 1 2 3 14 WEEKS Figure 7. Effect of prepackaging treatment on 02 levels in LDF-301 film packages of inoculated and non-inoculated precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C (1982-83). (Inoculation effect was nonsignificant. Data are means from packages of inoculated and non-inoculated bulbs. Mean separation within sampling date by Duncan's multiple range test, 5% level. Absence of letters indicates no significant differences on that date.) 87 o—-o NO DIP CONTROL o—o H20 CONTROL I-—I BLEACH L————‘ VANGUARD o—o PROCHLORAZ 96 (:(32 4 _ l 2 3 4 WEEKS Figure 8. Effect of prepackaging treatment on 002 levels inLDF-301 film packages of inoculated and non-inoculated precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C (1982-83). (Inoculation effect was nonsignificant. Data are means from packages of inocu- lated and non-inoculated bulbs. Mean separation within sampling date by Duncan's multiple range test, 5% level. Absence of letters indicates no significant differences on that date.) 88 0.4 d o—-—o NO DIP CONTROL .——o 11,0 cowrnm. o-——I BLEACH b 03 H VANGUARD ' r——e PROCHLORAZ C2H4 (pl/l) P N 0.1 Figure 9. Effect of prepackaging treatment on ethylene levels in LDF-301 packages of inoculated and non-inoculated precooled 'Kees Nelis' tulip bulbs through 3 wks at 20°C (1982-83). (Data were analyzed on log transformed scale. Inoculation effect was non- significant. Data are means from packages of inoculated and non-inoculated bulbs. Mean separation within sampling date by Duncan's multiple range test, 5% level. Absence of letters indicates no significant differences on that date.) 89 Table 5. Flowering of inoculated and non-inoculated precooled 'Kees Nelis' tulip bulbs after fungicide or sterilant prepackaging treat- ments and subsequent storage for 3 wks at 20°C in LDF-301 film packages or non-packaged (1982-83).z Prepackaging Inoculation % nonnal Abnormality Root treatment (i) flowersy ratingx ratingw H20 - _§9_ 2.8 3.4abc + 70 2.1 3.08 No dip - 55 3.0 3.5abc + 60 2.9 3.7bc Chlorine - 85 3.0 3.1a 65 3.1 3.3ab Vanguard - *100* --- 3.9c + 60 3.2 3.9c Prochloraz - 80 3.0 3.8bc + 95 --- 3.8bc Non-packaged - 60 3.1 3.9c + 60 2.9 3.8bc 2Initial post precooled control bulbs yeilded 95% normal flowers and a root rating of 3.9. y(*) to the right of means within column indicates difference at 5% level from H 0 by Mann-Whitney nonparametric statistic. (*) between means indicages significant inoculation effect by same statistic. xRatings indicated only when at least 2 reps (pots) contained abnormal flowers. Each abnormal plant rated from 0-4 according to symptoms of shoot and floral abnormality (see text). There were no significant differences. wRoot growth from each bulb rated from 0-4 (see Figure 10). Mean separation by Duncan's multiple range test, 5% level. 90 Table 6. Flowering of inoculated and non-inoculated precooled 'Kees Nelis' tulip bulbs after fungicide or sterilant prepackaging treat- ments and subsequent storage for 4 wks at 20°C in LDF-301 film packages or non—packaged (1982-83).z Prepackaging Inoculation % normal Abnormality Root treatment (i) flowers ratingx ratingw H20 - _475_ 1.2 2.9bc + 70 2.3 3.00 No dip - 80 3.1 2.9bc + 90 1.5 3.4cd Chlorine - 40* 1.7 1.6a + 45 1.9 1.9ab Vanguard - 95* --- 3.9d 95* --- 3.9d Prochloraz - 100* --- 3.8d + 100* --- 3.8d Non-packaged - 5* 1.2 3.7d + 0* 1.7 3.7d 2Initial post precooled control bulbs yielded 95% normal flowers and a root rating of 3.9. y(*) to the right of means within column indicates difference at 5% level from H20 by Mann-Whitney nonparametric statistic. xRatings indicated only when at least 2 reps (pots) contained abnormal flowers. Each abnormal plant rated from 0-4 according to symptoms of shoot and floral abnormality (see text). differences. wRoot growth from each bulb rated from 0-4 (see Figure 10). Mean separation by Duncan's multiple range test, 5% level. There were no significant 91 shown not to increase disease in the package during 3 wks of storage, it did yield reduced flowering with vanguard treated bulbs. Even though good root development was obtained from these inoculated bulbs, the roots did exhibit a brown discoloration which may have indicated disease development. This discoloration was similar to that seen during pathogenicity trials with inoculated excised root plates (Section IV). While a benomyl and ethazol post planting drench was used as a standard treatment for prevention of Pythium and Rhizoctonia root rots, it appeared not to prevent this effect. When the roots emerged from the bulbs after planting, they may have been exposed to a large spore population remaining on the bulb surface that induced the discoloration. Neither this effect nor reduction of flowering from inoculated vanguard treated bulbs was seen after 4 wks of storage. Possibly some greenhouse environmental factor during the first week of forcing enhanced disease development on the 3 wk bulbs. Additional vanguard application at planting or an increased pretreatment application rate may prevent this occurrence. Poor flowering and poor root growth resulted with chlorine pretreated bulbs after 4 wks of storage due to poor control of infec- tion by Penicillium spp. in the packages (Table 6). The benefit of the LDF-301 film package was highly apparent after 4 wks of storage, with vanguard and prochloraz pretreated bulbs yielding nearly perfect flowering while non-packaged bulbs flowered negligibly (Table 6 and Figure 10). However, good root growth was obtained from the non- packaged bulbs. In conclusion, infection of the root plate of precooled tulip bulbs by Penicillium spp. has been shown to be detrimental to the 92 Figure 10. Representation of 0—4 root rating (top). Flowering of non-inoculated 'Kees Nelis' tulip bulbs (bottom) after vanguard pretreatment and 4 wks of storage at 20°C in LDF—30l film packages (left 3 pots) or non-packaged (right 3 pots). 93 function of the LDF-301 film packages. Infection increased C02 and ethylene levels and reduced 02 levels in the packages. Decreased root growth and increased floral abortion during forcing were the end results of infection. Moderate amounts of infection by Penicillium spp. occurred on non-pretreated packaged bulbs in these studies. However, temperature fluctuation under actual marketing conditions could lead to condensation in the packages (Section III) which could increase fungus growth. Variability in natural inoculum levels and/or bulb susceptibility to infection also could exist. Therefore, fungicide pretreatment seems warranted. Benomyl pretreatment was unsuccessful due to the presence of a tolerant isolate of E. corymbiferum on the bulbs. However, prochloraz and vanguard, two unregistered fungicides, controlled infection in the packages. LITERATURE CITED 10. LITERATURE CITED Burton, C. L. and D. H. Dewey. 1981. New fungicides to control benomyl-resistant Penicillium expansum in apples. Plant Dis. 65:881-883. Bussel, J. and Z. Kenigsberger. 1975. Packaging green bell r peppers in selected permeability films. J. Food. Sci. 40: 1300-1303. Daun, H. K., S. 0. Gilbert, Y. Ashkenazi, and Y. Henig. 1973. Storage quality of bananas packaged in selected permeability : films. J. Food Sci. 38:1247-1250. 5 El-Goorani, M. A. and N. F. Sommer. 1981. Effects of modified atmospheres on postharvest pathogens of fruits and vegetables. Hort. Rev. 3:412-461. Fischer, C. W. 1953. Long-term holding of cut flowers. Proc. Amer. Soc. Hort. Sci. 61:585-592. Harbaugh, B. K., G. J. Wilfret, A. W. Engelhard, W. E. Waters, and F. J. Marousky. 1976. Evaluation of 40 ornamental plants for a mass marketing system utilizing sealed polyethylene pack- ages. Proc. Fla. State Hort. Soc. 89:320-323. Kavanagh, J. A. and R. K. Wood. 1967. The role of wounds in the infection of oranges by Penicillium digitatum Sacc. Ann. Applied Biol. 60:375-383. LeNard, M. 1983. Physiology and storage of bulbs: Concepts and nature of dormancy in bulbs, p. 191-230. In: M. Lieberman (ed.). Post-harvest physiology and crop improvement. Plenum Press, New York, NY. Moe, R., A. A. DeHertogh, and D. R. Dilley. 1978. Influence of post special precooling treatments on flower development and ethylene evolution from water and growth re ulator injected tulip bulbs. Sci. Rpt. Agr. Univ. Norway 57(46):1-18. New, E. H. 1964. Lasting qualities of selected clones of field-grown cut tulips following cold storage. Proc. Amer. Soc. Hort. Sci. 85:647-656. 94 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 95 Oudit, D. D. and K. J. Scott. 1973. Storage of 'Hass' avocados in polyethylene bags. Trop. Agric. 50:241-243. Pelser, P. T. and J. W. Eckert. 1977. Constituents of orange juice that stimulate the germination of conidia of Penicillium digitatum. Phytopath. 67:747-754. Prince, T. A., R. C. Herner, and A. A. DeHertogh. 1982. Increases in ethylene and carbon dioxide production by Tulipa gesneriana L. 'Prominence' after completion of the cold require- ment. Sci. Hortic. 16:77-83. Saguy, I. and C. H. Mannheim. 1975. The effect of selected plastic films and chemical dips on the shelflife on 'Marmande' tomatoes. J. Food Tech. 10:547-556. r Scott, K. J. 1975. The use of polyethylene bags to extend the life of bananas after harvest. Food Tech. in Australia 27: 481-482. , and E. A. Roberts. 1967. Control in bananas of Tu. black-end rot caused by Gloeosporium musarum. Aust. Journ. Expt. Agr. & Anim. Husb. 7:283-286. Staub, T., F. Schwinn, and P. Urech. 1979. CGA-64251, a new broad spectrum fungicide. Phytopath. 69:1046. Van der Plank, J. E. 1945. The use of hypochlorous acid and its salts in citrus packhouses for bleaching sooty blotch and as disinfectants against mould. Union 5. Africa Dept. Agr. Bull. 241:1-60. Van Gestel, J., J. Heeres, M. Janssen, and G. Van Reet. 1980. Synthesis and screening of a new group of fungicides: 1-(2 phenyl- 1,3 dioxolan-2 ylmethyl)-1,2,4-triazoles. Pestic. Sci. 11: 95-99. Von Oppenfeld, H., R. S. Lindstrom, D. H. Dewey, and J. W. Goff. 1955. Cold storage of field-grown cut tulips. Quart Bull. Mich. Agr. Expt. Sta. 38:273-278. Wilson, L. G. 1976. Handling of postharvest tropical fruit crops. Hortscience 11:120-121. SECTION III CULTIVAR RESPONSE, TEMPERATURE FLUCTUATION EFFECTS AND BULB ORGAN FRESH AND DRY MATTER DISTRIBUTION IN A MODIFIED ATMOSPHERE PACKAGE 0F PRECOOLED TULIP BULBS 96 Abstract. 'Abra', 'Bing Crosby', 'Favourite', and 'Parade' tulip bulbs (Tulipa gesneriana L.) yielded 87-93% normal flowers after 3 wks in low density polyethylene film packages at 20°C whereas non-packaged bulbs displayed aborted flowers. Storage of the same cultivars for 4 wks in the packages was . less successful. Infection of some bulbs by Fusarium oxysporum caused package ethylene levels to rise to 2-47 ul/liter which reduced subsequent flowering. Similar packages of 'Kees Nelis' i bulbs stored at 20° for 2wks followed by 3 wks of temperature fluctuation between 15 and 25° displayed little change in package CO2 and 02 levels. The temperature adaptability appeared due to both changing bulb respiration rates and changing film permeabilities to CO and 02. Packaged bulbs at any of the 2 temperatures used yielded 80-100% normal flowers after 3 total storage wks with slightly reduced flowering after 4 wks. Non- packaged 'Kees Nelis' bulbs at 20° and 40-50% RH lost 30% of bulb fresh weight (FW) and 25% of bulb dry weight (DW) during 4 wks of storage. The scales of these bulbs lost 45% of FW and 35% of DW; the floral shoot lost 38% of FW and 20% of DW; while the root plate displayed a 37% loss in tissue hydration level. The daughter bulbs within these non-packaged bulbs displayed 7-fold increases in FW and DW during 4 wks. Bulbs in packages yielded little change in FW or DW of any bulb organs. 97 98 Control of infection of precooled tulip bulbs by Penicillium spp. in a modified atmosphere (MA) package has led to maintenance of bulb flowering ability for as long as 4 wks at a constant 20°C (Section II). However, temperature fluctuation can be expected under marketing conditions. Any resultant change in the package atmosphere must not be detrimental to subsequent flowering of the bulbs if success of the package is to be achieved in the marketplace. There is conflicting information in the literature on temperature fluctuation effects upon sealed packages. Tomkins (27) reported that CO2 increased with temperature in sealed packages of various commo- dities. He also observed that condensation could occur in response to temperature decline, leading to increased mold infestation. Consumer-size polyvinylchloride (PVC) packages have been reported to extend the shelflife of bananas at 15°, while odor and abnormal taste development was apparent at 22° (4). Roses packaged in various films kept well for 5 days at 0-10°, but not at higher temperatures (12). In his review of packaging, Hardenburg (11) displayed data from packages of green beans and the effects of temperature upon the internal package atmosphere. He concluded that temperature had profound effects upon package CO2 and 02 levels yielding little hope for practical MA packages. However, a reexamination of his data suggests that his packages were not properly optimized so that all eventually would have become anaerobic. Apparent in his data is the effect of temperature upon the rate of 02 decline and not upon an ultimate equilibrium level. No great atmospheric differences were found between individually sealed polyethylene bags of avocados at 20 or 30° (3). Henig and 99 Gilbert (13) found that final C02 and 02 levels were nearly the same at 15° and 23° for PVC packages of tomatoes. They suggested that temper- ature changes affected both respiratory activity and film permeability to the same degree with their packages. Karel (16) demonstrated the temperature dependence of film permeability and found the level of dependence to vary with film type. The possibility that changes in film permeability to CO2 and 02 could allow an MA package of precooled tulip bulbs to adapt to a reasonable marketplace temperature fluctu- ation was investigated in this study. Prince and coworkers (22) have demonstrated that storage of precooled bulbs in a 3-5% 02 atmosphere leads to reduction in both bulb respiration rate and floral abortion due to ethylene exposure. These reductions may allow the maintenance of bulb flowering ability in the MA package. Ethylene induced shoot abortion is apparently prevented while the reduced respiration maintains high bulb carbo- hydrate levels for subsequent shoot elongation. However, LeNard (18) has recently suggested that when the planting or rooting 0f tulip bulbs is delayed after the precooling period, it is the predominance of daughter bulb enlargement that leads to floral shoot abortion. These studies report the effects of MA packaging upon daughter bulb enlargement and other bulb organ changes that occur during 20° storage. The varied response of 12 tulip cultivars to the MA packaging also is reported. Materials and Methods The study of fresh and dry matter distribution among the bulb organs was performed during the 1981-82 forcing season while the 100 cultivar evaluation and temperature fluctuation studies were performed during the 1982-83 season. Tulip bulbs (12-14 cm in circum.) were shipped to East Lansing, Michigan, from the Netherlands in open tray cases. Temperatures during shipment were 13-17° (1981-82) and 17-20° for 10 days followed by 4 days at 7-15° (1982-83). The shipping/ arrival dates were Sept. 11/Oct. 6, 1981, and Aug. 16/30, 1982. All bulbs had reached stage G upon arrival. They were stored at 13° (1981-82) or 17-20° (1982-83) prior to the precooling except for the ., bulbs utilized in the temperature study, which were moved to 13° on Oct. 20 for storage prior to precooling. All bulbs utilized in the 1982-83 season had talc applied on Nov. 23 to lower the available surface moisture to reduce growth 0f Penicillium spp. during the precooling. All bulbs from both seasons were dipped in vanguard solution (240 pg a.i./ml) at the end of the precooling period as previously described for control of Penicillium spp. (Section II). Cultivar Evaluation. Bulbs of 12 cultivars were precooled at 5° and 80-90% RH from Sept. 20 to Dec. 22 (13 wks). Following the pre- cooling and fungicide treatment, one half of the bulbs were sealed in LDF-301 film packages (5 bulbs/package) and the remainder were not packaged. Three packaged and non-packaged replicates of each cultivar were placed randomly in a 20° storage room (40-50% RH) for each 3 or 4 wk duration. All other materials and methods utilized were identical to those in Expt. 2 of Section 11, except that no bulbs were inocu- lated and only 2 pots of each cultivar were planted as initial post precooled controls. 101 Upon forcing, the percent normal flowers obtained from each pot was recorded. In addition, the abnormal plants were rated for floral and shoot abnormalities. The 0-4 point rating system utilized was: 0 - no shoot elongation above the bulb; 1 - some shoot elongation, but flower unemerged from leaves; 2 - tepals visible, but dried and yellow; 3 - tepals turgid, but no color development; and 4 - abnormal or mis- shapen tepals. The ratings from each abnormal plant were averaged to yield an abnormality rating for each pot. At flowering, a 0-4 root r rating was recorded for each bulb, with 0 indicating no root growth and 4 indicating excellent growth. A photograph of this rating scale 'FYI'E 'I- . " is shown in Section II. An average root rating was then calculated for each pot. Temperature Fluctuation Study. 'Kees Nelis' bulbs were precooled at 5° and 80-90% RH from Nov. 2, 1982, to Jan. 27, 1983 (12 wks). Following the precooling and fungicide treatment, one half of the bulbs were sealed in LDF-301 film packages (5 bulbs/package) and the remainder were not packaged. All were placed at 20° (40-50% RH) for 1 wk to allow package atmosphere equilibrium to be obtained. Four packaged and non-packaged replicates then were randomly assigned to each of 6 temperature regimes for an additional 1, 2, or 3 wks of storage. The temperature regimes utilized were: constant 20°; constant 25°; constant 15°; 25° for 2 days/20° for 5 days; 15° for 2 days/20° for 5 days; and 25° for 2 days/15° for 2 days/20° for 3 days. The fluctuating temperatures were repeated each week. The ambient RH was 20-25% at 25° and 25-35% at 15°. Four packaged and non-packaged replicates of each treatment were removed at the end of 102 2, 3, and 4 total storage wks for forcing evaluation. All other materials and methods including the floral and root ratings were as described above for the cultivar evaluation except that 4 pots of initial post precooled control bulbs were planted at the start of the experiment. The permeability of the LDF-301 film at 15, 20, and 25° was determined with a custom-made permeability cell as previously described (Section 1). Bulb Organ Study. 'Kees Nelis' bulbs were precooled at 5° and 80-90% RH from Dec. 7, 1981, to March 9, 1982 (13 wks). Following precooling and fungicide treatment, one half of the bulbs were sealed in LDF-301 film packages (5 bulbs/package) and the remainder were not packaged. All were randomly placed at 20° (40-50% RH). At the start of the experiment and at weekly intervals through 4 wks, 3 replicates of packaged and non-packaged bulbs were removed from storage. Two bulbs from each replicate were randomly selected for dissection into shoot, inner daughter bulbs, outer daughter bulb, scales, and root plate. The separate organs from the 2 bulbs were pooled and the fresh and dry matter were determined. The outer daughter bulb, which underlies the papery tunic, was analyzed separately from the other daughter bulbs due to previously-observed size variability and occasional absence due to bulb handling. Statistical Analysis. The analysis of variance or the Mann- Whitney nonparametric statistic was used. Statistical comparison of forcing results between temperature regimes was not utilized since the temperature rooms were not actually replicated. However, com- parison of forcing results between packaged and non-packaged bulbs 103 was valid. The validity of the statistical comparison between package atmospheres at different temperatures was limited due to the replication problem. Trend analysis was utilized for the bulb organ study to elucidate duration and treatment effects. Separate analyses were performed for the fresh weight, dry weight, and fresh/dry weight ratio trends for each bulb organ. Results and Discussion Cultivar Evaluation. The package CO2 and 02 levels during 4 wks of storage are shown in Figures 1 and 2, respectively, for 3 of the cultivars utilized. 'Prominence' bulbs had the lowest mass of the cultivars utilized; 'Abra' bulbs were intermediate; and 'Oskar' bulbs had the highest mass. The small differences in package atmospheres observed were likely due to the different masses of tissue respiring within the packages. The other cultivars all yielded package atmos- pheres within the same range. Thus it appears that different film surface areas need not be utilized for packaging different cultivars to achieve a desirable atmosphere. 'Abra', 'Bing Crosby', 'Favourite',and 'Parade' bulbs all flowered acceptably (87-93% normal flowers) after 3 wks in LDF-301 film packages at 20° (Table 1). The other cultivars did not flower acceptably. It appeared that some of the cultivars did not respond well to the precooling technique itself as indicated by the flowering of initial post precooled control bulbs. All non-packaged bulbs flowered poorly after 3 storage wks and had abnormalities that ranged from no shoot growth to dried and yellow tepals. Except for 'Apeldorn', 'Golden Oxford', and 'Oxford', the abnormalities displayed by the packaged bulbs were less severe than from non-packaged bulbs. 104 8 o-—-o PROMINENCE - 25g o————o ABBA-339 7 o___o OSKAR-44g 1 2 3 4 WEEKS Figure 1. CO levels in LDF-301 film packages of 'Prominence', 'Abra', and 'Oskar' precooled tulip bulbs through 4 wks at 20°C. (Mean separation within sampling date by Duncan's multiple range test, 5% level. No letters indicates no significant differences on that date. None of the other cultivars displayed significantly higher or lower values than the range shown on any date. Also shown are the average per bulb fresh weights for the 3 cultivars.) 105 14 F b 0—0 PROMINENCE-ZSg -———c ABBA-339 12 ‘ab A—A OSKAR-44g 1 2 3 4 WEEKS Figure 2. 0 levels in LDF-301 film packages of 'Prominence', Abrau and 'Oskar' precooled tulip bulbs through 4 wks at 20°C. (Mean separation within sampling date by Duncan's multiple range test, 5% level. No letters indicates no significant differences on that date. Noneof the other cultivars displayed significantly higher or lower values than the range shown on any date. Also shown are the average per bulb fresh weights for the 3 cultivars.) 106 Table 1. Flowering of 12 cultivars of precooled tulip bulbs after storage for 3 wks at 20°C in LDF-301 film packages or non-packaged.z % normal Abnormalit Root flowersy ratingsxa ratings",v non- LDF non- LDF non- LDF Cultivar packaged 301 packaged 301 packaged 301 Abra 0 * 93 0.8a-e -- 1.9c-e 3.7gh Apeldorn 0 * 73 1.6c-g 2.59 2.9e-9 3.8gh Bing Crosby * 93 0.38b -- 3.1f-h 3.9h Favourite 13 * 87 0.58-c -- 1.18-c 3.9h Golden Melody 0 27 0.08 1.3c-f 0.28 2.2d-f Golden Oxford 7 60 2.0fg 2.0fg 3.7gh 3.9h Monte Carlo 0 27 0.08 1.7d-g 3.58b 3.38b Oskar 0 * 47 0.6a-d 2.4fg 1.7bd 3.4gh Oxford 7 * 67 1.9e-g 2.69 3.7gh 3.9h Parade 13 * 93 1.9e-9 -- 3.59h 4.0h Paul Richter O * 47 0.38b 2.4fg 0.6a 3.59h Prominence O * 67 0.18 2.0fg 0.8ab 3.59h zInitial post precooled control bulbs yielded 100% normal flowers except Golden Oxford and Bing Crosby--80%; Oskar and Parade--70%; and Oxford and Monte Carl0--60%. All yielded root ratings of 3.6-4.0. y(*) indicates significant difference between packaged and non-packaged bulbs within cultivar at 5% level by Mann-Whitney nonparametric stat- istic. xRatings indicated only when at least 2 reps (pots) contained abnormal flowers. Each abnormal plant rated 0-4 according to symptoms ofshoot and floral abnormality (see text). wRoot growth from each bulb rated 0-4 (see text). vMean separation between rows and columns by Duncan's multiple range test, 5% level. 107 The root ratings of packaged bulbs after 3 storage wks were in the 3.3-3.9 range for all cultivars except 'Golden Melody'. This suggested that rooting differences were likely not responsible for the flowering differences observed between packaged bulbs of the different cultivars. The root rating of non-packaged bulbs of 'Golden Oxford', 'Monte Carlo', 'Oxford', and 'Parade' were in the 3.5-3.7 range. This indicated that poor rooting of non-packaged bulbs was not the limiting factor for all cultivars but may have been for some of them after 3 wks of storage. None of the cultivars evaluated appeared to respond to the packaging as well as 'Kees Nelis', the cultivar utilized in other studies (Section 11), since none of the tested cultivars flowered acceptably after packaging for 4 wks (Table 2). However, some did flower significantly better than their non-packaged counterparts. The floral abnormalities from packaged and non-packaged bulbs after 4 wks appeared more severe than after 3 wks of storage. Root growth was poor from most non-packaged cultivars. Rooting may have become an increasingly limiting factor for non-packaged bulbs between the 3 and 4 wk durations due to root plate desiccation (see below). 'Abra', 'Golden Oxford', 'Oxford', and 'Parade' packaged bulbs rooted well despite the poor flowering obtained. Infection by Fusarium oxysporum f. sp. tulipae appeared to limit packaging success with many of the cultivars (Table 3). 'Abra', 'Apeldorn', 'Favourite', 'Golden Melody', 'Oskar', 'Paul Richter', and 'Prominence' bulbs all displayed some infection at the end of 4 wks of storage. Infection occurred at the root plate and was quite variable. Infection created extremely variable package ethylene levels, depending 108 Table 2. Flowering of 12 cultivars of precooled tulip bulbs after storage for 4 wks at 20°C in LDF-301 film packages or non-packaged.z % normal Abnormalit Root flowersy ratingsx’ ratings"”v non- LDF non- LDF non- LDF Cultivar packaged 301 packaged 301 packaged 301 Abra 0 * 33 0.3a-c 2.2e-g 1.28-c 3.7gh Apeldorn O 0 1.08-d 0.9a-d 1.5b-d 2.3c-e Bing Crosby 0 20 0.3a-c 3.09 1.9c-e 2.6d-9 5 Favourite 0 40 0.38-c 0.9a-d 0.68b 2.5d-f Golden Melody 0 0 0.08 0.28-c O.la 0.68b Golden Oxford 0 * 47 0.3a-c 2.69 1.18-c 3.5f-h , Monte Carlo 0 0 0.0a 1.2de 2.6e-g 2.7e-g A Oskar 0 7 O.Iab 2.3fg 0.68b 2.5d-f Oxford 0 * 53 0.5a-d 3.09 1.9c-e 3.7gh Parade 0 7 1.4d-f 2.2e-g 3.0e-h 3.9h PaUl Richter O 20 O.Iab 1.1b-d O.Ia 2.5d-f Prominence 0 0 0.08 0.9a-d 0.28 2.lc-e 2Initial post precooled control bulbs yielded 100% normal flowers except Golden Oxford and Bing Crosby--80%; Oskar and Parade--70%; and Oxford and Monte Carlo--60%. All yielded root ratings of 3.6-4.0. y(*) indicates significant difference between packaged and non-packaged bulbs within cultivar at 5% level by Mann-Whitney nonparametric stat- istic. xRatings indicated only when at least 2 reps (pots) contained abnormal flowers. Each abnormal plant rated 0-4 according to symptoms of shoot and floral abnormality (see text). wRoot growth from each bulb rated 0-4 (see text). vMean separation between rows and columns by Duncan's multiple range test, 5% level. Table 3. 109 Ethylene levels after 5, 17, and 27 days, and Fusarium oxysporum presence, on 12 cultivars of tulip bulbs in LDF-301 fi m packages during 4 wks of storage at 20°C. C2H4 Range (pl/liter) FusariumZ Cultivar Day 5 Day 17 Day 27 'EFEEEHEE Abra 0.05 0.08 0.33 - 0.81 0.10 - 2.06 + Apeldorn 0.04 0.06 0.07 - 5.38 0.29 - 46.9 + Bing Crosby 0.03 0.04 0.05 - 0.23 0.07 - 0.17 0 Favourite 0.06 0.15 0.35 - 0.68 0.89 - 14.3 + Golden Melody 0.05 3.28 0.27 - 15.3 7.27 - 29.8 + Golden Oxford 0.03 0.05 0.05 - 0.21 0.07 - 0.19 0 Monte Carlo 0.06 0.07 0.35 - 0.87 0.35 - 1.70 0 Oskar 0.03 0.16 0.16 - 1.29 0.17 - 8.00 + Oxford 0.03 0.14 0.06 - 0.07 0.06 - 0.18 0 Parade 0.02 0.04 0.12 - 0.21 0.05 - 0.50 0 Paul Richter 0.15 0.21 0.24 - 10.2 6.28 - 37.3 + Prominence 0.05 0.22 0.42 - 3.51 2.44 - 17.7 + 2(0) indicates no Fusarium observed; (+) indicates Fusarium observed on 1 or more bulbs in at least 1 package. 110 upon whether an individual package contained an infected bulb (Table 3). The ethylene production of Fusarium oxysporum f. sp. tulipae has been documented (26). Infection led to package ethylene levels of 2-47 ul/ liter, while packages without infected bulbs contained less than 2 pl/liter. The floral abortion response of bulbs to ethylene exposure has been shown to vary with cultivar (6). While a 3-5% 02 exposure caused less floral abortion of 'Kees Nelis' bulbs (as compared to air storage) when exposed to 10 ul/liter of ethylene (22), it cannot be assumed that the package atmosphere could protect the bulbs from the levels reported here. The infected bulbs also could have continued to damage other bulbs once planted, since ethylene up to 10 pl/liter has been measured in the soil atmosphere surrounding bulbs infected with Fusarium oxysporum f. sp. tulipae (25). Ethylene exposure also could have caused the reduced rooting observed (15, 19). DeMunk has demonstrated susceptibility differences among different tulip cultivars to infection by Fusarium oxysporum f. sp. tulipae (7). These differences appeared to have affected this experiment. However, different inoculation levels in the bulb production fields in the Netherlands may also have been involved. Vanguard treatment of all the bulbs prior to packaging did not prevent the development of Fusarium infection. Damage from the disease has typically been minimized by removal of infected bulbs (5). It appeared that infection by Fusarium oxysporum f. sp. tulipae was the primary limiting factor in the packaging of certain cultivars. However, the poor flowering of 'Golden Oxford', 'Oxford', and 'Parade' after 4 wks in the package, despite good root growth and the lack of infection, indicated other possible cultivar limiting factors. 111 Therefore, selection of bulbs free of infection and pr0per choice of cultivar will both be necessary for successful packaging. Temperature Fluctuation Study. The permeabilities to CO2 and 02 of LDF-301 film at 3 temperatures are shown in Table 4. A tem- perature increase from 15 to 25° resulted in a 63% increase in C02 permeability and an 82% increase in 02 permeability. Arrhenius plots of the permeability constants further elucidated the tempera- ture response and allowed calculation of activation energies (E8) of the permeation process (Figure 3). Karel (16) has indicated that discontinuities in the slope of the Arrhenius plots, which would indicate changing Ea over temperature, are very rare for polymeric films. The higher calculated activation energy for 02 than for C02 permeation indicated that 02 permeation would change more than the CO2 permeation for any given temperature change. The respiratory rate change of the bulbs in response to tempera- ture change also affected the package atmosphere. Figure 4 depicts the CO2 and 02 levels in packages during 1 wk at 20° followed by 3 wks at a constant 15, 20, or 25°. Package 02 level was increased by a temperature drop to 15° while an increase to 25° resulted in little 02 level change compared to a constant 20°. Bulb Ozconsump- tion likely increased in response to temperature increase. While a Q10 of respiration of precooled tulip bulbs has not been published, an assumption near the 2-3 range would be reasonable (9). The lack of significant package 02 decline at 25° was likely due to the increased film permeation. However, temperature decline to 15° apparently lowered bulb O2 consumption more than it decreased film 112 Table 4. Permeabilities to 002 and 02 of LDF-301 low density polyethylene film at 15, 20, and 25°C.2 Permeability (liter-atm'loday'lom'z)y Temp. (°C) CO2 02 15 12.38 (0.37) 2.82 (0.17) 20 16.43 (0.53) 4.17 (0.12) 25 20.22 (0.62) 5.14 (0.21) zFilm supplied by Dow Chemical, U.S.A. Film thickness was 0.051 mm. yValues are means (i 1 SD) of three determinations. 113 LDF 301 3.0 I'm-0.9977 (COZ) log Fan-1.84%) :28 1011.18 7 E581I8.4|!!EL. mole 11- 2.6 J o? 8 2" L 7" rat-0.91" (02) log Pct @3513) 23 + 10. o E. I 10.3—1468' mole 3.35 3.40 3.45 3.50 [1h <°K>l “0 3 Figure 3. Arrhenius plots of the permeability constants (Pc = ml-mm- atm771oday71-m7:) of LDF-301 film to CO2 and 02 penneation. (Slope of lines (x 10 3) = -Ea/2. 3R. Values plotted are means of three determinations. (**) indicates significant r at 1% level.) 114 Figure 4. C02 levels (top) and 02 levels (bottom) in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs during storage for 1 wk at 20°C followed by 3 wks at 15, 20, or 25°C. [(*) indicates significant difference from 20° mean within sampling date by Dunnett's procedure, 5% level.] 115 permeation, leading to increased package 02. The difference in package response between the two temperature changes of equal increment appeared due to the logarithmic nature of the film permeability temperature response. A temperature change from 20 to 15° decreased permeation by 32% while a change from 20 to 25° increased permeation by only 23% (Table 4). In addition, the tulip bulbs possibly changed 010 of respiration within the 15-25° interval. The package CO2 levels responded in a manner opposite to 02 (Figure 4). A change from 20 to 15° decreased while 20 to 25° increased the package CO2 levels, although the latter increase was minor. The demonstrated lowerlevel of temperature responsiveness of film CO2 permeation apparently caused bulb CO2 production change to be a more important determinant of package C02 level change than was film permeation change. All package CO2 and 02 levels displayed greater variability during the final storage wk. During that same period, the 20° control packages tended toward slightly decreased 02 and increased CO2 levels. Package CO2 and 02 levels displayed similar responses to 2 day fluctuations to 15 or to 25° during the second storage wk (Figure 5). Both levels returned to the levels of the 20° continuous controls upon return to 20°. During the last 2 storage wks, only package C02 appeared to respond to the temperature decline to 15° while 02 levels remained unchanged. This suggested that after 2 wks of storage, the 02 consumption of the bulbs became less responsive to temperature fluctuation while CO2 production continued to respond. A change in the R0 of the bulbs possibly had occurred in response to continued exposure of the bulbs to the MA conditions. This effect was further 116 23°-2 days - 23°- 0 2| -1dlye| adaysu 3" l, 52‘” A O tflzaayso 1’-3dav" Iiizdayss Figure 5. 002 levels (top) and 02 levels (bottom) in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs during storage for 1 wk at 20°C followed by 3 wks at 15° for 2 days/20° for 5 days; 25° for 2 days/20° for 5 days; or continuous 20° storage. [(*) indicates significant difference from continuous 20° mean within sampling date by Dunnett's procedure, 5% level.] 117 elucidated by the 2 days at 25° followed by 2 days at 15° treatment (Figure 6). No significant effect upon package 02 level was observed at any time while C02 responded as before. Apparently, the 25° exposure before the 15° one yielded enough exposure of the bulbs to the MA conditions to induce the change in RQ and eliminate the 02 increase at 15° even during the second wk. An additional effect of 15° exposure was the condensation observed on the inside of the LDF-301 film packages. This disappeared upon return of the packages to 20°. Some root emergence did occur from the bulbs in response to the condensate. However, this emergence did not hinder subsequent rooting or flowering, since damage or desic- cation of the roots was avoided during planting. While package atmospheres did display some statistically sig- nificant changes in response to temperature fluctuation, these differences had little practical effect upon subsequent flowering of the bulbs. All packaged and non-packaged bulbs flowered and rooted well after a total of 2 wks at any temperature (Table 5). The LDF-301 film packaging for 3 total wks of storage was successful at all tempera- tures. At a constant 15° exposure, there was excellent flowering from packaged and non-packaged bulbs alike (Table 6). This indicated that at temperatures near 15°, the package may possibly maintain flowering for periods longer than 4 wks. Rooting was excellent from all packaged bulbs. The average abnormal flower obtained from the non-packaged bulbs had dried and yellow tepals. There were significantly lower root ratings from the non-packaged bulbs than from the packaged bulbs for all but 2 of the temperature regimes. 118 2070 0 ° c s .. as -2dayou ”'3 933'3' ”-2 days- N O U o é~4,_ . A 3 dan- 2 days. 157 Zdayso 1 1 J 157-3 daysl ° ns-z days. 0 lS-ZGOYSI )- 25° 4 2 days. 23°-2 days. A . f 25-2 days- I 1 1 __1 WEEKS Figure 6. C02 levels (top) and 02 levels (bottom) in LDF-301 film packages of precooled 'Kees Nelis' tulip bulbs during storage for 1 wk at 20°C followed by 3 wks at 25° for 2 days/15° for 2 days/20° for 3 days; or continuous 20° storage. [(*)indicates significant difference from continuous 20° mean within sampling date by Dunnett's procedure, 5% level.] 119 Table 5. Flowering of precooled 'Kees Nelis' tulip bulbs after storage in LDF-301 film packages or non-packaged for 1 wk at 20°C and an additional wk at 6 temperature regimes.z % normal Abnormality Root flowersX ratingsw ratingsv Temperature non- LDF non- LDF non- LDF regimey packaged 301 packaged 301 packaged 301 20° - constant 95 100 --- --- 3.8 3.8 | 25° - constant 85 95 2.0 --- 3.6 3.9 15° - constant 95 95 --- --- 3.9 3.9 . 25° - 2 days/wk 95 100 --- --- 3.6 * 4.0 3 15° - 2 days/wk 95 95 --- --- 3.7 3.9 5 25° - 2 days/wk, E. 15° - 2 days/wk 95 95 --- --- 3.8 3.9 2Initial post precooled control bulbs yielded 100% normal flowers and a root rating of 3.9. yBulbs under non-constant temperatures returned to 20°C for the remainder of each wk of storage. xNo significant differences between packaged and non-packaged bulbs within temperature regime by Mann-Whitney nonparametric statistic. wRatings indicated only when at least 2 reps (pots) contained abnormal flowers. Each abnormal plant rated 0-4 according to symptoms ofshoot and floral abnormality (see text). vRoot growth from each bulb rated 0-4. (*) indicates difference between packaged and non-packaged bulbs within temperature regime by LSD test at 5% level. (See text for root rating scheme.) 120 Table 6. Flowering of precooled 'Kees Nelis' tulip bulbs after storage in LDF-301 film packages or non-packaged for 1 wk at 20°C and an additional 2 wks at 6 temperature regimes.Z % nonnal Abnormality Root flowersx ratingsw ratingsv Temperature non- LDF non- LDF non- LDF regimey packaged 301 packaged 301 packaged 301 20° - constant 5 * 95 1.8 --- 3.0 * 3.9 25° - constant 0 * 100 1.7 --- 2.4 * 3.9 15° - constant 95 90 --- --- 3.6 3.9 25° - 2 days/wk 15 * 90 1.9 2.0 2.9 * 3.8 15° - 2 days/wk 35 * 95 2.0 --- 3.2 * 3.9 25° - 2 days/wk, 15° - 2 days/wk 25 * 80 2.4 2.6 3.6 4.0 ZInitial post precooled control bulbs yielded 100% normal flowers and a root rating of 3.9. y Bulbs under non-constant temperatures returned to 20°C for the remainder of each wk of storage. x(*) indicates significant difference between packaged and non-packaged bulbs within temperature regime at 5% level by Mann-Whitney nonpara- metric statistic. ' wRatings indicated only when at least 2 reps (pots) contained abnormal flowers. Each abnormal plant rated 0-4 according to symptoms