Depression of the minimum concentration of methyl bromide effective against diapausing warehouse moth larvae at 15°c by prior exposure at a moderate concentration

Warehouse moth (Ephestia elutella) larvae in diapause were exposed at 15°C to methyl bromide at 8 mg litre^?1 for 14.5 h and then immediately exposed at a lower concentration. The exposure at 8 mg litre^?1 killed 44–69% of the larvae treated. Subsequent concentrations down to 1.1 mg litre^?1 obeyed Haber's rule (concentration × time= k, a constant for mortality), but a higher concentration-time product (ct) was required for over 90% kill at 0.8 mg litre^?1. Only concentrations down to 1.9 mg litre^?1 obey Haber's rule if there is no prior exposure at a higher concentration. Although minimum effective concentrations are lower at 15°C than at 25°C, exposure at a higher concentration depresses the subsequent level to a similar extent at each temperature. The contribution to the efficacy of a treatment, of low concentrations persisting at the end of fumigation, is thus likely to be even greater at moderate to low temperatures than at 25°C. The implications for the development of resistance to methyl bromide are discussed.     

Effect of temperature on the toxicity of low concentrations of methyl bromide to diapausing larvae of the warehouse moth Ephestia elutella (Hübner)

The performance of low concentrations of methyl bromide against diapausing larvae of Ephestia elutella at 15 and 25°C was assessed in extended exposure periods. At concentrations of 1.9 mg litre^?1 and below, test batches required higher concentration-time (ct) products for 100% kill at 25°C than at 15°C. The minimum concentration at which the concentration: time relationship still applied was between 1.3 and 1.9 mg litre^?1 at 15°C, whereas at 25°C it was between 2.7 and 4.0 mg litre^?1. For many individuals within each population sample, however, lower concentrations at moderate dosage levels remained lethal. At 25°C, a ct product of about 90 mg litre^?1 h gave between 53 and 77% kill at 6.1, 4.0, 2.7 and 1.9 mg litre^?1. The trends observed suggest that the most tolerant members of the population have an enhanced ability to detoxify methyl bromide at the higher temperature. The implications of the results for the build-up of resistance and for practical control measures are discussed.     

Electrophysiological identification of site-insensitive mechanisms in knockdown-resistant strains (kdr,super-kdr) of the housefly larva (Musca domestica)

The effects of pyrethroids were studied upon isolated segmental nerves and neuromuscular junctions in both susceptible (Cooper) and knockdown-resistant (kdr; super-kdr) strains of housefly larvae (Musca domestica L.). Isolated segmental nerves contained neither cell bodies nor synaptic contacts; thus, any effects of pyrethroids were attributed solely to their actions upon voltage-dependent Na⁺ channels. Threshold concentrations of the type II pyrethroid, deltamethrin, required to elevate the spontaneous firing rate of these nerves were determined. Both resistant strains were about ten times less sensitive to deltamethrin than the susceptible strain, but insensitivity of super-kdr nerves was no greater than in the less resistant kdr strain. At neuromuscular junctions, the minimum concentrations of pyrethroids needed to trigger massive increases in the frequency of miniature excitatory postsynaptic potentials (mEPSPs) were determined for deltamethrin and the type I pyrethroid, fenfluthrin. With fenfluthrin there was no detectable difference between the junctions of kdr and super-kdr strains, which were both about ten-fold less sensitive than Cooper junctions. With deltamethrin, kdr junctions were about 30 times less sensitive than those of Cooper; super-kdr junctions were dramatically insensitive to deltamethrin, being some 10000- and 300-fold less sensitive than those of Cooper and kdr respectively. Thus, in the synaptic assay, super-kdr conferred an extension in resistance over kdr only against the type II pyrethroid, it being ineffective against fenfluthrin. We suggest that kdr resistance comprises at least two site-insensitive areas within the nervous system. One involves insensitivity of the Na⁺ channel and has similar efficacy in both kdr and super-kdr strains against type I and II pyrethroids; the other is associated with the presynaptic terminal and is particularly effective in super-kdr resistance against type II pyrethroids. The latter could be associated with Ca²⁺-activated phosphorylation of proteins involved with neurotransmitter release. Such phosphorylation reactions are known to be perturbed by pyrethroids, especially by type II compounds.     

The influence of spray droplet characteristics on the efficacy of permethrin against the diamondback moth Plutella xylostella: The effect of drop size and concentration on the potency of ulv- and ec-based residual deposits

The effect of droplet diameter (36-274 μm) and concentration (10-400 g litre^?1) of permethrin on the knockdown and mortality of 2nd-instar Plutella xylostella larvae was investigated. Larvae were placed on brussels sprout leaf discs treated with residual deposits of permethrin applied as ULV or emulsion formulations. The LD₅₀ of permethrin decreased with droplet size at all concentrations tested. This effect could not be accounted for solely by increased drop numbers per unit area of leaf, suggesting that for both formulations transfer of permethrin to larvae is more efficient for deposits composed of small drops. The concentration of permethrin in the ULV formulation could be reduced to approach a minimum quantity of toxicant per unit area to maintain 50 per cent mortality. The approach to the minimum dose varied with drop size and drop number per unit leaf area. The ULV formulation was approximately 2.7 times more effective at killing larvae than the emulsion, presumably as a result of greater availability of toxicant and transfer to larvae.     

Metabolism of amitrole in apple: III. Model systems

Excised shoots from apple trees and cell suspension cultures were used as model systems to study the metabolism of [3,5-¹⁴C]amitrole in Malus domestica Borkh. Significant differences in the metabolism of the compound applied were observed with excised shoots, cultured cells and whole apple trees. The major metabolite in excised shoots was aminotriazolylalanine which occurred both in the free form and as conjugates. The major metabolite from whole plants. triazolylalanine, was detected in shoots in minor amounts only. In cell suspension cultures, the type of metabolism strongly depended on the concentration of amitrole when initially applied. At 10 ^?3 m or lower, mainly aminotriazolylalanine was formed. Depending on the concentration of the active ingredient, this metabolite predominantly occurred in free form or as glycosides. At concentrations above 5 × 10^?4 M a new metabolite, 3,5-dihydroxytriazole, was detected which was the only metabolite found at 5 × 10^?3M. Significant amounts of nonmetabolized amitrole remained in the medium.     

Increased activity and reduced sensitivity of glutamine synthetase in glufosinate‐resistant vetiver (Vetiveria zizanioides Nash) cells

Vetiver (Vetiveria zizanioides Nash) cells derived from an inflorescence were cultured in a modified N6 liquid medium supplemented with 10 µm 2,4‐D and 10 mm proline. Exponentially growing cell suspensions were subcultured with a selection medium containing glufosinate (ammonium dl ‐homoalanin‐4‐yl(methyl)phosphinate). The glufosinate‐resistant cells which can grow in a medium containing 5 × 10^?5 M glufosinate was selected by a stepwise selection, and its I₅₀ value was determined to be 4.2 × 10^?5 M. The growth of susceptible cells was inhibited by lower concentrations of glufosinate and its I₅₀ value was 2.5 × 10^?7 M. This indicated that the selected cells were 170‐fold resistant compared with the susceptible cells. Glutamine synthetase (GS) activity of the resistant cells was twice as high as that of the susceptible cells. The I₅₀ values of glufosinate were 3.2 × 10^?5 M and 9.0 × 10^?7 M for GS from the resistant and susceptible cells, respectively. The accumulation of ammonia caused by GS inhibition was higher in the susceptible cells. Absorption of [3,4–¹⁴C]glufosinate was not significantly different between the resistant and susceptible cells. Both cell types did not metabolize glufosinate. These results suggest that the resistance of the selected vetiver cell suspension to glufosinate is mainly due to increased GS activity and its decreased sensitivity to the herbicide.     

Temperature adaptation in isolates of Sclerotinia sclerotiorum affects their ability to infect Brassica carinata

Sclerotinia stem rot (Sclerotinia sclerotiorum) is a serious disease in oilseed Brassica crops worldwide. In this study, temperature adaptation in isolates of S. sclerotiorum collected from differing climatic zones is reported for the first time on any crop. Sclerotinia sclerotiorum isolates from oilseed rape (Brassica napus) crops in warmer northern agricultural regions of Western Australia (WW3, UWA 7S3) differed in their reaction to temperature from those from cooler southern regions (MBRS‐1, UWA 10S2) in virulence on Brassica carinata, growth on agar, and oxalic acid production. Increasing temperature from 22/18°C (day/night) to 28/24°C increased lesion diameter on cotyledons of B. carinataBC054113 more than tenfold for warmer region isolates, but did not affect lesion size for cooler region isolates. Mean lesion length averaged across two B. carinata genotypes (resistant and susceptible) fell from 4·6 to 2·4 mm for MBRS‐1 when temperature increased from 25/21°C to 28/24°C but rose for WW3 (2·35 and 3·21 mm, respectively). WW3, usually designated as low in virulence, caused as much disease on stems at 28/24°C as MBRS‐1, historically designated as highly virulent. Isolates collected from cooler areas grew better at low temperatures on agar. While all grew on potato dextrose agar between 5 and 30°C, with maximum growth at 20–25°C, growth was severely restricted above 32°C, and only UWA 7S3 grew at 35°C. Oxalate production increased as temperature increased from 10 to 25°C for isolates MBRS‐1, WW3 and UWA 7S3, but declined from a maximum level of 101 mg g^?1 mycelium at 20°C to 24 mg g^?1 mycelium at 25°C for UWA 10S2.     

CNS insensitivity to pyrethroids in the resistant kdr strain of house flies

Solutions of tetramethrin, RU 11679, or cismethrin caused uncoupled convulsions in 30–40 min in exposed thoracic ganglia from S_NAIDM house flies at concentrations down to 10^?10M: whereas these same compounds at 10^?6M concentrations failed to produce poisoning symptoms when perfused onto the exposed ganglia of the kdr strain of house fly. The pyrethroid analogs examined had a negative temperature coefficient of action on the exposed thoracic ganglia from S_NAIDM flies. DDT and GH-74 possessed positive temperature coefficients of action on the exposed thoracic ganglion of susceptible house flies. It is concluded that the central nervous system of the kdr strain of house fly is resistant to pyrethroid action; furthermore, the resistance appears to be widespread throughout the house fly nervous system, involving sensory, motor, and central neural elements.     

Pyrethroid resistance in a strain of Spodoptera littoralis is correlated with decreased sensitivity of the CNS in vitro

The effects of permethrin and cypermethrin on the isolated abdominal nerve cord of insecticide-resistant [R] and -susceptible [S] strains of Spodoptera littoralis larvae have been studied. Above ca. 19°C, permethrin at 10^?7M caused a prolonged spike train to follow electrical stimulation of the nerve cord. The time of onset of this repetitive firing was significantly greater for the [R] strain. Moreover, cypermethrin, to which this strain shows negligible resistance, did not cause such repetitive discharges. Thus, resistance to permethrin but not to cypermethrin appears to be based on a qualitative difference between the pyrethroids. Nerve blockage by the two pyrethroids was also investigated, with particular reference to temperature. Once again, differences were apparent: when considered relative to untreated controls, permethrin caused quicker nerve blockage as temperature was reduced whereas the blocking action of cypermethrin was not affected by temperature. However, the times taken to cause nerve blockage by permethrin in [R] and [S] larvae were not significantly different, making it unlikely that nerve blockage plays a major role in this resistance. Two methods were employed to reduce the resistance factor in vitro. The synergist dodecyl imidazole failed to significantly reduce the time taken for permethrin to cause either repetitive firing or nerve blockage. However, reducing the calcium concentration in the saline did significantly reduce the latency of repetitive firing caused by permethrin in [R] larvae, thus increasing the nerve sensitivity to approximately the same level as normal calcium, [S] insects.     

The action of insecticides on neurosecretory neurons in the stick insect, Carausius morosus

The action of insecticides on the spontaneous electrical activity of neurohemal tissue in the stick insect, Carausius morosus, has been studied using extracellular electrodes. The pyrethroid, permethrin, causes a massive increase in the frequency of the spontaneously generated action potentials at concentrations between 5 × 10^?5 and 5 × 10^?8M. Concentrations as low as 5 × 10^?11M are still effective in producing bursting activity.DDT, at concentrations between 5 × 10^?5M and 5 × 10^?6M, produces an overall increase in activity although the bursting activity is less violent than that shown with permethrin. DDT, 5 × 10^?7M, is ineffective at altering the resting pattern.Carbaryl and coroxon cause a transitory increase in electrical activity at 1 × 10^?4M, but are ineffective at 1 × 10^?5M.It is concluded that insecticides could have a direct effect upon the neurohormonal balance in insects.     

Multifactorial resistance to transpermethrin in field-collected strains of the tobacco budworm Heliothis virescens F.

The penetration, degradation and excretion of [³H]transpermethrin were examined in susceptible and field-collected pyrethroid-resistant strains of the tobacco budworm Heliothis virescens. No consistent differences in labelled materials excreted or recovered in cuticle rinses were found between the resistant (R) and susceptible (S) larvae. Considerably lower levels of the parent compound were present internally in R compared with S larvae after 24h (P <0.01), clearly identifying a metabolic resistance mechanism in Meloland and Westmorland larvae. Moderate levels of absorbed permethrin accompanied by an absence of poisoning symptoms were observed in certain individuals of both R strains, suggesting a second resistance mechanism. Neurobioassays of R larvae showed a consistently lower sensitivity of the neuromuscular system to pyrethroids when compared with the S larvae, thus confirming the indication from metabolic studies of the additional (site-insensitive) mechanism. Toxicity values suggest a cross-resistance to other pyrethroids.     

Breakdown rates of dimethoate in fruit and vegetable dips

In order to determine the effect of pH and temperature on post-harvest dip solutions of dimethoate (500 mg litre^?1), the half-lives and pseudo first-order rate constants were calculated from measurements at pH 4, 6, 8, 10, 11.5, and at two temperatures 25 and 52°C. The half-lives ranged from 206 days to 39.3 min at 25°C, and from 5.6 days to 205s at 52°C; the rate constants ranged from 3.9 × 10^?8 s^?1 to 2.9 × 10^?4 s^?1 at 25°C, and from 1.4 × 10^?6 s^?1 to 3.4 × 10^?3 s^?1 at 52°C. The results show that the water used in dips should have a pH≤7. The addition of benomyl to the dip solutions at two concentrations (0.5 and 1.0 g litre^?1) had no effect on the half-lives and rate constants. The use of hard and salted waters in dips also showed no major effect. A formula was developed that gives the half-life of the dimethoate as a function of the pH and temperature.     

Evaluation of field resistance in field-collected mosquito Culex quinquefasciatus Say through quantification of ULV permethrin/PBO formulation in field bioassays

BACKGROUND

Pyrethroids are among the most applied adulticides worldwide to control mosquito vectors for prevention of arboviral diseases transmission. However, pesticide resistance development in a mosquito population could lead to decreased control efficacy. While most studies investigate the resistant genotype (i.e. kdr, CYP450, etc.) as explanatory variables, few field efficacy studies have measured pesticide quantities deposited at different distances from the sprayer in association with observed mosquito mortality. The current study determined field delivered amounts of an applied ULV permethrin/PBO formulation (31% permethrin + 66% piperonyl butoxide) by GC/MS and estimated practical resistance ratios using caged mosquito females.

RESULTS

For field samples, the extraction method recovered 78 ± 3.92–108 ± 8.97% of the permethrin/PBO formulation when utilizing the peaks of PBO from GC/MS to estimate the concentrations of adulticide deposited near the mosquito cages. The field bioassay showed that the spatial distribution of permethrin/PBO formulation was heterogeneous among three pseudo-replicates within the same distance. Within the quantifiable permethrin/PBO range of 15.7–51.4 ng/cm², field-collected mosquito mortalities started at 64% and linearly increased reaching 100% only in two areas, while all Sebring susceptible mosquitoes died. The field LC₉₅ resistance ratio (RR) of F₀ Cx. quinquefasciatus ranged from 2.65–3.51, falling within the 95% CI of RR₉₅ estimated by laboratory vial assays. Tests with and without PBO indicated P450's enzymes contributed to field resistance.

CONCLUSION

Results showed the suitability of the collection and quantification method to estimate the field resistance ratio at the applied pesticide rate. Pesticide quantification would also allow the association of the known frequencies of resistance mechanisms (e.g. kdr, CYP450) with field mortalities to estimate the resistance level conferred by such mechanisms. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Genetic and biochemical studies of resistance to permethrin in a pyrethroid-resistant strain of the housefly (Musca domestica L.)

One or more weak factors of resistance on autosome 2, and barely detectable resistance on autosome 3, confer moderate resistance to several pyrethroids (5–13-fold) in the field-collected Ipswich strain of houseflies. In these flies, which unlike other pyrethroid-resistant strains lack kdr or super-kdr, pyrethroid resistance probably developed in response to prolonged treatment of buildings for animals with pyrethrins synergised with piperonyl butoxide. Substrains, isolated genetically from Ipswich flies and with resistance only on autosome 2, degraded permethrin more rapidly than susceptible flies and produced larger amounts of very polar metabolites. In this, they differed from flies with kdr or super-kdr which resembled susceptible flies in their metabolism of permethrin. NIA 16388 (propyl prop-2-ynyl phenylphosphonate) was a better synergist and reduced the metabolism of permethrin more than piperonyl butoxide in both the susceptible and resistant insects. The slight increase in synergism and minimal decrease in metabolism when piperonyl butoxide was applied with NIA 16388 indicated that the latter also inhibited detoxication that was sensitive to piperonyl butoxide.     

Ecology of Xiphinema vuittenezi and Xiphinema pachtaicum in vineyards of north-east Croatia1

During a 3-year field study on two vineyards of north-eastern Croatia, the qualitative and quantitative composition as well as the vertical dynamics of Xiphinema spp. were determined each month. The greatest number of fertile X. vuittenezi females was noted in August-September at a soil moisture of 18–20%. The greatest number of larvae of this species was determined in September-October in a temperature range of 14–18°C and soil moisture of 18–22%. The development cycle of X. vuittenezi lasts about 24–33 months under natural conditions and that of its larval stages 3–8 months. The nematodes of this species are susceptible to high temperatures (above 20°C) and drought (under 13%). The greatest number of fertile females of X. pachtaicum was determined in July at a soil temperature of 20–24°C, absolute soil moisture of 16–20%. The greatest number of larvae was noted in September-October at a soil temperature of 16–21°C and soil moisture of 13–23%. The development cycle of X. pachtaicum in field conditions lasts about 12–13 months and that of the larval stages 2–3 months. This species demonstrated reduced activity at soil temperatures under 10°C and at soil moisture under 13%; larvae were less active than females at temperatures over 20°C. On the basis of the results obtained, it is suggested that sampling of vineyards to determine the distribution and population density of the two Xiphinema spp. should be performed at depths down to 50 cm in spring and autumn, which are also the most favourable times for nematicide application.     

Insecticide resistance in house flies from caged‐layer poultry facilities

The frequency of resistance of eight strains of house flies, Musca domestica L., collected from caged‐layer poultry facilities across New York state, to nine insecticides (dimethoate, tetrachlorvinphos, permethrin, cyfluthrin, pyrethrins, methomyl, fipronil, spinosad and cyromazine) was measured relative to a laboratory susceptible strain. Percentage survival was evaluated at five diagnostic concentrations: susceptible strain LC₉₉, 3 × LC₉₉, 10 × LC₉₉, 30 × LC₉₉ and 100 × LC₉₉. The highest levels of resistance were noted for tetrachlorvinphos, permethrin and cyfluthrin. There was substantial variation in the levels of resistance to the different insecticides from one facility to another, independent of their geographical location. There was very little cross‐resistance detected in these populations to either fipronil or spinosad. Overall, there was a good correlation between insecticide use histories and the levels of resistance. The apparent isolation of fly populations within poultry facilities suggests that there are good opportunities for the implementation of successful resistance management strategies at these facilities. Differences between these results and those of a resistance survey on New York dairy farms in 1987 are discussed. © 2000 Society of Chemical Industry     

Interpretation of the EC method for the detection of latent Corynebacterium sepedonicum infections in potato1

The EC method for the detection of latent ring-rot infections (Corynebacterium sepedonicum) consists essentially of an indirect immunofluorescence test and a pathogenicity test on eggplant (EP). When interpreting the results of this method, care should be taken that: (a) eggplants are grown at 21°C. At 28–29°C the detection level of the EP was increased 10² to 10³-fold to 10⁵ to 10⁶ cells ml^-1 and latent infections (which may go unnoticed) occurred with 10⁴ to 10⁵ cells ml^-1; (b) reisolations are made from symptomless eggplants grown at the optimum temperature (21°C) as latent infections can also occur in these plants at concentrations of 10² to 10³ cells ml^-1; (c) potato tuber extracts are tested without freezing or stored freeze-dried or in a proper protective agent. Freezing at -20°C in maceration buffer of cells of C. sepedonicum and Pseudomonas solanacearum (the latter was included for comparison) reduced their numbers by 90–98% after one day. This could easily cause viable cell numbers to drop below the detection level of the pathogenicity test.     

N-Nitrosodimethylamine content of aqueous dimet hylamine and phenoxy herbicide/dimethylamine formulations during storage

N-Nitrosodimethylamine (NDMA) levels in samples of 600 g litre^?1 aqueous dimethylamine (DMA) solution stored at 4, 25 and 40°C, in formulations of the DMA salt of 2, 4-dichlorophenoxyacetic acid (2, 4-D) and of the DMA salt of 4-chloro-2-methylphenoxyacetic acid (MCPA) at 40°C were monitored using a gas-liquid chromatograph coupled with a thermal energy analyser (g.l.c./t.e.a.). The NDMA content of aqueous DMA at 4°C increased from 0.32μgg^?1 to 8.6μgg^?1 in 156 days. At 25°C it increased from 0.20μgg^?1 to 10.5 μgg^?1 in 156 days and at 40°C from 0.76μgg^?1 to 7.12μgg^?1 in 54 h. At 40°C, the NDMA in the 2, 4-D/DMAsalt increased from 0–53μgg^?1 to 2.79μgg^?1 in 96 days and in the MCPA/DMA salt from 0.48μgg^?1 to 5.51 μgg^?1 in 21 days.     

Fate and short-term persistence of permethrin insecticide injected in a northern Ontario (Canada) headwater stream

1·275 dm³ of a l·3g dm^?3 aqueous emulsion of permethrin (i.e. 1·658 g permethrin) was applied to the surface of a fast-flowing stream by manual injection. Water samples were collected from the top 1-cm layer at different intervals of time at various distances downstream from the site of application. Caged samples of stream detritus, crayfish, brook trout and stonefly nymphs, bottled sediment and potted aquatic plants were placed 280 m from the treatment site, and collected afterwards for residue analysis. The downstream movement of the chemical was not uniform with peak concentrations of about 12 ng ml^?1 at 30 m and 0·1 ng ml^?1 at 730 m from the treatment site. The peak concentrations (ng g^?1) in potted aquatic plants, caged detritus, crayfish and brook trout were about 18, 39, 14 and 11 respectively. The residues were higher than the ambient water concentration (1·67 ng ml^?1) and dissipated slowly. The peak concentration in the bottled sediment was 2·75 ng g^?1, and the residues were lost rapidly. In contrast, the naturally occurring sediment in a beaver pond showed high residues that persisted for a longer period of time. Permethrin concentrations in invertebrate drift ranged from 9 to 357 ng g^?1, depending on the distance from the application site. The study demonstrated that aquatic invertebrates, plants and stream detritus acted as the major sink for the chemical.     

Combination of hot water, <Emphasis Type="Italic">Bacillus subtilis</Emphasis> CPA-8 and sodium bicarbonate treatments to control postharvest brown rot on peaches and nectarines

The aim of this study was to evaluate the effect of hot water (HW), antagonists and sodium bicarbonate (SBC) treatments applied separately or in combination to control Monilinia spp. during the postharvest storage of stone fruit. Firstly, we investigated the effect of HW temperatures (55–70°C) and exposure times (20–60 s), seven antagonists at two concentrations (10⁷ or 10⁸ cfu ml⁻¹) and four SBC concentrations (1–4%). The selected treatments for brown rot control without affecting fruit quality were HW at 60°C for 40 s, SBC at 2% for 40 s and the antagonist CPA-8 (Bacillus subtilis species complex) at 10⁷ cfu ml⁻¹. The combinations of these treatments were evaluated in three varieties of peaches and nectarines artificially inoculated with M. laxa. When fruit were incubated for 5 d at 20°C, a significant additional effect to control M. laxa was detected with the combination of HW followed by antagonist CPA-8. Only 8% of the fruit treated with this combination were infected, compared to 84%, 52% or 24% among the control, CPA-8, and HW treatments, respectively. However, the other combinations tested did not show a significant improvement in effectiveness to control brown rot in comparison with applying the treatments separately. When fruit were incubated for 21 d at 0°C plus 5 d at 20°C, the significant differences between separated or combined treatments were reduced and generally the incidence of brown rot was higher than when fruit were incubated for 5 d at 20°C. Similar results were observed testing fruit with natural inoculum.