Controlled Environment Room Research Articles

The potential of entomopathogenic nematodes as biocontrol agents for clover root weevil (<i>Sitona lepidus</i>)

The efficacy of the entomopathogenic nematodes Heterorhabditis zealandica H bacteriophora Steinernema carpocapsae and S feltiae against clover root weevil (CRW) larvae and pupae was determined in petri dish and pot experiments Field collected third instar (L3)pupal CRW immature stages were used and experiments were conducted in a controlled environment room at 18C Weevil survival and development was assessed 7 and 10 days after nematode inoculation for petri and pot experiments respectively All nematodes significantly reduced CRW survival and all except S feltiae prevented adult development Under the conditions used in this study Heterorhabditis spp were generally more effective than Steinernema spp Results are discussed in terms of the potential use of entomopathogenic nematodes as biocontrol agents for this clover pest

Determining uptake of ‘non‐labile’ soil cadmium by Thlaspi caerulescens using isotopic dilution techniques

We assessed the ability of several populations of the metal‐hyperaccumulator species, Thlaspi caerulescens, to mobilize non‐labile cadmium in soils historically contaminated by Pb/Zn mine spoil or sewage sludge. Radio‐ labile Cd was determined chemically as an ‘E‐value’, [CdE], and biologically as an ‘L‐value’, [CdL]. For comparison, chloride‐extractable Cd, [Cdchlor], was also determined using 1 M CaCl2 as a single‐step soil extractant. Values of [CdL] were measured for six populations of T. caerulescens that varied substantially in their ability to assimilate soil Cd, and a non‐accumulator species with a similar growth habit, Lepidium heterophyllum. Seeds were sown in soil spiked with 109Cd and grown for 9–12 wk in a controlled environment room. Values of [CdL] were determined from the specific activity of 109Cd and concentration of Cd in the plant leaves. For the six soils studied, [CdE] ranged from 4.9 to 49% of total soil Cd [CdT]. Values of [CdL] were, in general, in close agreement with both [CdE] and [Cdchlor] and substantially less than [CdT]. However, [CdL] showed no correlation with the concentration of Cd in plant tissue, [Cdshoot]. This suggests that, in the soils studied, T. caerulescens did not mobilize non‐labile soil Cd by producing root exudates or altering rhizosphere pH. The results imply that there may be significant restrictions to metal bioavailability, even to hyperaccumulator species, in heavily contaminated soils in which a large proportion of the metal may be present in ‘non‐labile’ forms.

Laboratory rearing of lesser appleworm (Lepidoptera: Tortricidae).

The lesser appleworm, Cydia prunivora (Walsh), was reared successfully in the laboratory. Larvae of various instars were collected in the field from hawthorn fruit, Crataegus spp. Initially, immature apples served as the food source for the larvae in the laboratory. Rearing was conducted in a greenhouse and later in combination with a controlled environment room at 25 degrees C, 50-60% RH, and a photoperiod of 18:6 (L:D) h. Under these conditions, a generation required approximately 30 d. Fifty-six adult lesser appleworm moths emerged from the original field collected hawthorn fruits. After a decline in the number of the F1 generation to 39 moths, the colony on mature apples, increased to in excess of 10,000 moths by the fifth generation with a mean survival rate to adult of 68.0%. When production on immature apples was compared with that on four artificial diets, the most promising of the artificial diets was the lima bean-based diet currently used to rear the oriental fruit moth, Cydia molesta (Busck), with a mean survival rate of 46.4%. The other bean-based diets tested were not as satisfactory. Pear foliage was the preferred oviposition substrate of those tested, including apple and hawthorn foliage. No eggs were deposited on plain waxed paper or glass microscope slides; however, large numbers of eggs were deposited on waxed paper treated with a water extract of pear foliage and immature apples.

Population differences in Trifolium repens L. response to ultraviolet-B radiation: foliar chemistry and consequences for two lepidopteran herbivores.

White clover growing in New Zealand is experiencing increasing levels of ultraviolet-B (UV-B) radiation as a result of ozone depletion. We evaluated the effects of UV-B radiation on the foliar chemistry of two populations of white clover (Trifolium repens L.), 'Huia' and 'Tienshan,' and the consequences for the performance of armyworms (Spodoptera litura) and cutworms (Graphania mutans). Plants were grown in controlled environment rooms with and without supplemental UV-B radiation at a dose of 13.3 kJ m-2 day-1, corresponding to a 25% mid-summer ozone depletion above Palmerston North, New Zealand. In both white clover populations, UV-B radiation elicited changes in foliar chemistry, including slight increases in nitrogen concentrations and decreases in carbohydrate concentrations. In addition, the 'Huia' population showed decreases in fiber concentrations and marked increases in cyanogenic activity. No change in UV-absorbing compounds was detected in either population. Long- and short-term feeding trials were conducted to assess dietary effects on insect growth, consumption, and food utilization. Changes in the performance of both insect species were generally small. The most pronounced effect was a 36% reduction in weight of S. litura after 2 weeks of feeding on Huia grown at high UV, but larval development times were only slightly prolonged and pupal weights were unaffected. S. litura short-term performance was affected by differences in white clover population. The long-term performance of G. mutans was not affected and its short-term performance (stadium duration and consumption rate) was only marginally affected by the high-UV treatment. We conclude that the effects of elevated UV-B radiation on white clover plant chemistry can be specific to certain plant populations. The differences in sensitivity of the two generalist insect species suggest that effects may also be specific to certain plant-herbivore associations. These results indicate that future UV-B herbivory studies should examine genotypic effects in both plants and animals.

Water Relations of Cut Roses as Influenced by Vapor Pressure Deficits and Temperatures.

Freshly harvested 'Bridal Pink' roses (Rosa hybrida L.), with their stem bases in test tubes containing deionized water, were placed in a glass tank and held in a controlled environment room at 14, 20, or 30°C. The vapor pressure deficit (VPD) in the tank was maintained at nearly 0 kPa (no VPD : NVPD) or 0.9 kPa (intermediate VPD : IVPD). At all temperatures and VPDs, the fresh weight of cut roses increased initially and then decreased ; the decrease occurred earlier in IVPD than in NVPD and at higher temperatures. Necks of all flowers placed in IVPD became bent within 48, 144, and 312 hr of postharvest at 30, 20, and 14°C, respectively ; whereas, bent neck did not develop in NVPD. Irrespective of temperature, transpiration and water uptake rates of the roses placed in IVPD were markedly higher than those in NVPD. In IVPD, these rates increased initially, but decreased after 48, 72, and 96 hr at 30, 20, and 14°C, respectively. Petal water potential gradually decreased during the first 36 hr at 30°C in IVPD, but did not change at 14°C. The osmotic potential increased with time and was higher at 30°C than at 14°C. Fructose, glucose, and sucrose were the major sugars in petals. Concentrations of these sugars decreased during the first 36 hr, the decrease being greater at 30°C than at 14°C. The contribution of these sugars to the petal osmotic potentials was only 10%. These data indicate that the water relations of cut roses, immediately after harvest, was greatly influenced by high VPD by hastening the transpiration rate, and subsequently by increasing temperature through rise in the osmotic potential which was partly attributable to the consumption of respiratory substrate.

Stock-type patterns of phosphorus uptake, retranslocation, net photosynthesis and morphological development in interior spruce seedlings

One-year-old interior spruce (Picea glauca (Moench) Voss × Picea engelmannii Parry) spring-stock and summer-stock were grown under two phosphorus (P) fertility regimes, with (+P) or without (−P), followed by a simulated winter, and a second growing period under an adequate fertility regime in a controlled environment room. The two stock-types differed in their response to low P availability. For spring-stock, morphological development, phosphorus-use efficiency (PUE) and P specific absorption rate (SAR) were similar between −P and +P seedlings. For summer-stock, −P seedlings compared to +P seedlings had lower (p ≤ 0.05) morphological development, but greater PUE and SAR. For both stock-types, P content increased in +P seedlings, remained low in −P seedlings, and P concentration decreased in nursery-needles (i.e., formed in the nursery) of −P seedlings. The difference in stock-type response to low phosphorus availability (−P) was attributed to internal supply of P and it's retranslocation. Assimilation (A) of CO2 in nursery-needles was similar between −P and +P seedlings for both stock-types. For spring-stock, +P seedlings had greater A in new-needles (i.e., needles formed during the trial) than −P seedlings. It was recommended that the spring-stock be selected over summer-stock for sites low in P availability.

Diurnal variations in hydraulic conductivity and root pressure can be correlated with the expression of putative aquaporins in the roots of lotus japonicus

The hydraulic conductivity of excised roots (Lp(r)) of the legume Lotus japonicus (Regel) K. Larsen grown in mist (aeroponic) and sand cultures, was found to vary over a 5-fold range during a day/night cycle. This behaviour was seen when Lp(r) was measured in roots exuding, either under root pressure (osmotic driving force), or under an applied hydrostatic pressure of 0.4 MPa which produced a rate of water flow similar to that in a transpiring plant. A similar daily pattern of variation was seen in plants grown in natural daylight or in controlled-environment rooms, in plants transpiring at ambient rates or at greatly reduced rates, and in plants grown in either aeroponic or sand culture. When detached root systems were connected to a root pressure probe, a marked diurnal variation was seen in the root pressure generated. After excision, this circadian rhythm continued for some days. The hydraulic conductivity of the plasma membrane of individual root cells was measured during the diurnal cycle using a cell pressure probe. Measurements were made on the first four cell layers of the cortex, but no evidence of any diurnal fluctuation could be found. It was concluded that the conductance of membranes of endodermal and stelar cells may be responsible for the observed diurnal rhythm in root Lp(r). When mRNAs from roots were probed with cDNA from the Arabidopsis aquaporin AthPIP1a gene, an abundant transcript was found to vary in abundance diurnally under high-stringency conditions. The pattern of fluctuations resembled closely the diurnal pattern of variation in root Lp(r). The plasma membranes of root cells were found to contain an abundant hydrophobic protein with a molecular weight of about 31 kDa which cross-reacted strongly to an antibody raised against the evolutionarily conserved N-terminal amino acid sequence of AthPIP1a.

A thermal time model of post‐initiation flower development in the shade plant, cineraria

SummaryEffects of temperature on flower development in cineraria cv. Cindy Blue were studied in controlled environment rooms and in glasshouses. The base, optimum and maximum temperatures respectively for progress to macroscopic flower appearance after flower initiation respectively were 1.6°C, 19.3°C and 39.8°C. From these cardinal temperatures, a thermal time requirement for flower appearance after flower initiation was calculated to be 130°Cd. The base, optimum and maximum temperatures for progress to anthesis after flower initiation were respectively 1.7°C, 22.3°C and 37.1°C and from these values, the thermal time required to reach anthesis after flower initiation was calculated to be 555°Cd. No significant difference was demonstrated between thermal times for flower development in plants grown in controlled environment growth rooms or under glasshouse conditions where irradiance and photoperiod varied markedly.

Toxicity of Ethylene Glycol Vapors to Cucumbers

Vapors of ethylene glycol, 1,2 ethanediol, were found to cause serious injury to cucumbers in controlled-environment rooms in which this chemical was utilized for heat transfer. A chlorotic injury, which later became necrotic, occurred at the leaf apices and progressed along the margins to cause stunting and cupping of the leaves. High concentrations caused cessation of shoot growth and produced florets with short petals and stunted sepals. The source of the contaminating vapors appeared to be residual ethylene glycol from leaks over the years that had impregnated the floors or walls of the rooms. Activated charcoal or potassium permanganate filters installed in the circulating air stream of the rooms effectively prevented injury.

DISTRIBUTED LONWORKS CONTROL AND MONITORING SYSTEM FOR ENVIRONMENT AND SECURITY

An integrated monitoring and control system was needed to monitor and control over 100 items of research,security, and safety equipment in 17 buildings at the Southeast Poultry Research Laboratory. A LonWorks-based systemwas chosen to provide maximum interconnectability between three existing systems, to provide a variety of wired orwireless connection options, and unlimited expansion capabilities. The resulting network has 36 nodes includingcontrollers, network access cards and routers connected by a twisted pair of wires, and it monitors equipment such asincubators, hatching cabinets, freezer and walk-in cooler temperatures, controlled environment rooms, static pressures,fire alarm, door access, and sewage tank cooking parameters in 17 buildings. It also controls two sewage cooking tanksand access for three doors and two gates. Local and remote access to the system is possible through any one of threedesktop computers; two of which are accessed using PCanywhere (Symantec Corp., Cupertino, Calif.), a remote controlprogram for PCs, and one of which is accessed by telephone using Win911 (Specter Instruments, Austin, Tex.), a programfor remote telephone access of the system and alphanumeric alarm paging. Alarms are annunciated locally and throughdigital and alphanumeric pagers. The system has been in use for approximately 24 months and overall it has performedvery reliably. Most failures have been caused by insufficient suppression devices to protect against high voltage transientsduring thunderstorms, but new suppressors have corrected this problem. The generic nature of the system and easyexpansion lends itself to many applications.

Wheat regenerated from scutellum callus as a source of material for transformation

Reports of wheat transformation efficiencies vary from less than 1% to more than 5% for individual experiments. Rarely are negative experiments reported though we estimate that between one in two and one in three of all experiments fail to produce transformed plants. Consequently if transformation efficiencies were calculated from the total number of scutellum bombarded rather than from only those experiments which produced transformed plants there would be a significant fall in reported efficiencies. The use of scutellum-derived material from plants regenerated from scutellum callus and grown in a controlled environment room significantly reduced the number of experiments failing to produce plants. Though there is a small but significant increase in transformation efficiencies for individual experiments, the recovery of plants, as a direct consequence of the reduction in the number of failed experiments, increases nearly 350%, from 4.8 plants/1000 scutella from seed gown plants bombarded to 17 plants/1000 embryos bombarded from tissue culture regenerated plants. There appears to be no additional gains to be made from plants which are cycled through tissue culture more than one time.

Requirements of Gap-Demanding Species in Chalk Grassland: Reduction of Root Competition Versus Nutrient-Enrichment by Animals

Summary 1 Arenaria serpyllifolia and Cerastium fontanum (two non-mycorrhizal species of small gaps in chalk grassland) were grown from seed on topsoil (0-5cm) and subsoil (712cm) from a chalk grassland for 100-105 days in a controlled environment room with and without root competition from Bromus erectus (a matrix-forming perennial grass) and with and without addition of nitrate and phosphate. 2 Neither species grew appreciably without addition of P; there was no interaction of P and N. In the presence of P or P + N, both species showed strong inhibition by root competition from Bromus. 3 In a second experiment the Arenaria and the Cerastium were grown for 64 days on four different chalk grassland (CG) soils (pH 6.8-7.6), and on a soil from a ploughed chalk heath grassland (CH; pH 6.4), with and without addition of P and P + N. Both species failed to grow on two of the CG soils, and grew only a little on the other two; both species grew faster with addition of P, but showed no extra effect of P + N. Both species grew rapidly on the CH soil, on which both occur spontaneously in turf 1 cm tall. 4 The mean dry mass of an Arenaria plant in the CH turf was strongly negatively related to the dry mass of grass shoots in a circle of 2.5 cm radius around it. 5 It is concluded that, on most chalk grassland soils, Arenaria and Cerastium are not prevented from invading short turf simply by root competition, but require addition of P via faeces. On soils of lower pH, in which greater quantities of phosphate ions are available, the studied species can grow without nutrient-enrichment by animal activity, but are very sensitive to root competition.

Effect of high ambient temperature on feed digestibility in broilers

The effect of chronic heat exposure on feed digestibility of broilers was investigated. Eighty 4-wk-old male chickens were brooded in individual battery cages in two controlled-environment rooms at a constant ambient temperature (22 or 32 C) until 6 wk of age. They were equally distributed into three treatments: 22 C, ad libitum feed consumption (22AL); 32 C, ad libitum feed consumption (32AL), and 22 C, pair-feeding on the daily feed intake of heat-exposed chickens (22PF). Broilers were fed either a standard corn-soybean meal diet (control diet) or a practical seasonal diet containing several ingredients including wheat, spring pea, and animal fat (summer diet). Digestibility of energy, dry matter, protein, fat, starch, and nitrogen, and total mineral balances were measured between 38 and 42 d of age. Apparent metabolizable energy content of summer diet was significantly decreased in 32AL compared to 22AL, whereas AME of the control diet did not change. Nitrogen retention was significantly reduced in 32AL birds compared to 22AL and 22PF birds, irrespective of the diet. Taking into account these differences in nitrogen balance, AMEn was reduced under hot exposure: -72 and -155 kcal for control and summer diets respectively, in 32AL compared to 22PF chickens. This reduction could be explained by a significant decrease of nutrient digestibility:protein: -4.2 percentage units irrespective of the diet, fat: -1.7 and -5.2 percentage units for control and summer diets respectively, and starch: -4.2 percentage units for summer diet. It thus appears worthwhile to take into account such reduction in digestibility to formulate practical diets for brooding under hot conditions. High quality oil and protein sources should also be used instead of low quality feedstuffs, like animal sources, in such conditions.

Dry Matter Yield, Leaf Growth and Population Dynamics in Lolium perenne/ Trifolium repens-Dominated Pasture Turves Exposed to Two Levels of Elevated CO 2 </latex&#62

1. Dry matter yield, leaf growth and population dynamics of turves taken from an old Lolium perenne/Trifolium repens-dominated pasture were studied in controlled environment rooms at CO 2 concentrations of 350 μmol mol -1 , 525 μmol mol -1 and 700 μmol mol -1 . Starting with September data the turves were subjected sequentially to the mean monthly temperature and photoperiod taken from long-term climatic data for the area of New Zealand where the pasture was located. Each temperature and photoperiod regime was applied for 21 days to provide 12 different simulated 'months' of environmental conditions. The experiment ran for 14 simulated months, with September and October conditions being repeated at the end of the first simulated 'year'. Mean photon flux density throughout was 500 μE m -2 s -1 . 2. The total quantity of herbage harvested was increased by 7% and 14% at 525 μmol mol -1 and 700 μmol mol -1 CO 2 , respectively. The increase in the amount of T. repens harvested by the end of the experiment was 63% at 525 μmol mol -1 CO 2 and 48% at 700 μmol mol -1 CO 2 . In contrast, neither the yield of C3 grasses nor the yield of the only C4 grass present, Paspalum dilatatum, was significantly affected by CO 2 concentration. The implications of this increase in the proportion of T. repens in temperate pastures at elevated CO 2 is discussed briefly. 3. With the exception of a small increase in the specific leaf area of T. repens, detailed measurements of leaf growth on marked tillers (L. perenne and P. dilatatum) and growing points (T. repens) showed no consistent effects of exposure to elevated CO 2 concentrations. 4. Differences in yield between CO 2 concentrations were mainly attributable to changes in the number and balance of population units. By the middle of the 'winter' conditions T. repens growing point densities at 700 μmol mol -1 CO 2 were more than double those found at 350 μmol mol -1 but total grass tiller densities were unchanged. Growing point densities were also more than doubled at 525 μmol mol -1 CO 2 compared with 350 μmol mol -1 but grass tiller densities were reduced significantly below those recorded in the other two treatments. The relationship between the stability of herbage production and population densities is discussed and the potential interaction between population density, elevated CO 2 and grazing considered. 5. Although exposure to elevated levels of CO 2 did result in large changes in population numbers, this did not happen immediately and so the yield response of this particular commuyity to CO 2 varied with time. The average yield increases recorded here at elevated CO 2 may therefore tend to underestimate those likely to be shown by communities that, at the population level, have become fully adapted to growth in a CO 2 -enriched atmosphere.

Fine root electrolyte leakage and moisture content: indices of Sitka spruce and Douglas-fir seedling performance after desiccation

In spring 1992, 2-year-old bare-rooted seedlings of Sitka spruce (Picea sitchensis (Bong.) Carr.) and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) were exposed for 0, 0.5, 1.0, 1.5, or 3 h in a controlled environment room at 20°C. Plants were desiccated in batches during February and March; 100 seedlings of each species were planted in 5 20-plant plots at each of 6 planting sites using the normal planting times and methods of each area. The effect of exposure was assessed within 24 h of desiccation on a subsample of plants by measuring fine root moisture content and electrolyte leakage and, in a limited number of cases, needle water potential. These measures were related to survival and growth after the first and second growing seasons.

Chronic Heat Exposure Enhances Fat Deposition and Modifies Muscle and Fat Partition in Broiler Carcasses

The effect of chronic heat exposure on carcass quality of broilers: proportion of lean and fat tissues, fat content, and fatty acid composition, was investigated. One hundred and eight 4-wk-old male chickens were brooded in individual battery cages in two controlled-environment rooms at constant ambient temperature (22 or 32 C) until 7 wk of age. They were equally distributed into three treatments: 22 C, ad libitum feeding (22AL); 32 C, ad libitum feeding (32AL); and 22 C, pair-feeding on the daily feed intake of heat-exposed chickens (22PF).At 7 wk of age, heat-exposed chickens (32AL) had a lower body weight gain than the other birds: −47% compared to 22AL and −31% compared to 22PF. At 32 C, broilers exhibited a lower breast to body weight proportion: 11.9 vs 13.4% for 22AL. Abdominal, subcutaneous, and intermuscular fat deposits were enhanced in hot conditions, respectively, 15, 21, and 22% compared to 22AL and 58, 64, and 33% compared to 22PF. However, lipid contents of abdominal, subcutaneous, intermuscular, and intramuscular tissues were not affected by heat exposure but were significantly reduced in the 22PF birds. In heat-exposed birds, although saturated fatty acid proportions, particularly palmitic acid (C16:0), were increased, unsaturated fatty acids as a percentage of total fatty acids were decreased, especially oleic (C18:1) and linoleic (C18:2) acids in fat tissues. Consequently, under ad libitum feeding conditions, heat exposure significantly decreased the unsaturated to saturated fatty acid ratio in the abdominal and subcutaneous fat tissues, but not in intermuscular and intramuscular fats.

Procedures for Mass Rearing the Buffalo Fly, Haematobia irritans exigua De Meijere (Diptera: Muscidae)

ABSTRACTProcedures are described for mass rearing the buffalo fly, Haematobia irritans exigua. Adult flies are fed on bulls in a controlled environment room. The eggs are laid on the dung which is incubated for 2 d and then reconstituted into 2–3 L pads in a room at 30°C 80% R.H. Average daily production was 3,700 pupae (maximum 17,000) for each bull over a 6–month period, while over 7 years the number of flies on each bull averaged 1,000–2,000 with the introduction of 200 puparia/bull/day.

キュウリの初期生長と無機栄養に及ぼす高気温と高地温の影響

Relative contributions of supraoptimal air and root-zone temperatures on the growth inhibition of cucumber (Cucumis sativus L.) during hot seasons were evaluated. Seedlings of a Chinese cultivar, Sangoh (Ji huang gua 3) and a Japanese cultivar, Sharp I were grown in water culture for 8 days in controlled environment rooms. The environmental conditions were : 26/26 °C, 26/37 °C, 37/26 °C or 37/37 °C daytime air/root-zone temperature regimes (air temperature in the night was 5 °C lower than that in the day), 80/90 % relative air humidity, and a 15-hr photoperiod of ca. 480 μmol•m-2•s-1 PPFD.Temperature of leaves was approximately equal to air temperature during the daytime. Although plants thrived best at 26/26 °C and worst at 37/37 °C combinations, when growth at 26/37 °C and 37/26 °C regimes was compared, high air temperature was more inhibitory than high root-zone temperature. Root growth, leaf expansion rate, leaf water content, photosynthetic rate and leaf mineral concentrations were reduced more severely by high root-zone temperature. Seedlings of 'Sangoh' were significantly more tolerant to high root-zone temperature than were those of 'Sharp I'. Possible causes of growth inhibition by high air temperature compared to that by high root-zone temperature and the mechanisms of the cultivarietal difference in thermotolerance of cucumber plants are discussed.

Effect of soil water content and temperature on the transmission of Pseudomonas syringae pv. pisi from pea seed (Pisum sativum) to seedling

The effect of soil water content on the transmission of Pseudomonas syringae pv. pisi from pea seed (cv. Buckley) to seedling in field soil was investigated in controlled environment rooms maintained at 5°C and 15°C. Artificially inoculated seed was sown in soils of various water contents, and the seedlings tested for the presence of P. syringae pv. pisi using an ELISA based test. Soil water content had a significant effect on the transmission of the bacterium from seed to seedling. Transmission was reduced, but not prevented, at low soil water contents. The effect of temperature on transmission was marginally non-significant (P=0.056). Since transmission off! syringae pv. pisi still occurred at low soil water content it is unlikely that sowing into a dry seed bed can be used to prevent transmission of P. syringae pv. pisi from seed to seedlings.

Frost tolerance ofTrifoliumspecies

Abstract Thirteen Trifolium species were artificially frosted at ‐4, ‐8, ‐12, and ‐16°C in controlled environment rooms. This was carried out in such a way that soil freezing was avoided and only shoots were frosted. Frost tolerance was primarily assessed as the percentage of dead to total leaf dry weight present 1 week after frosting. Trifolium arvense, T. dubium, and T. hybridum were the most frost‐tolerant, whereas T. ambiguum, T. subterraneum, T. glomeratum, T. vesiculosum, and T. cernuum were the most frost‐sensitive, based on the temperature required to kill 50% of leaves. Trifolium ambiguum may have been particularly sensitive to frosting because of its poor vigour (size).