Spectral filters for the control of Botrytis cinerea

Experiments performed in vitro examined the sporulation of Botrytis cinerea (grey mould) under different spectral distributions. Eighty‐three isolates, taken from plants of primula (Primula vulgaris) at different locations throughout the UK, were incubated in the dark, with visible light only and visible plus near‐ultraviolet (nUV) light. On average, compared to isolates not exposed to nUV, sporulation was increased 54‐fold following illumination with nUV light. No isolates showed complete insensitivity to near ultraviolet. New polyethylene materials with different optical properties were then tested on two typical isolates. A film which removed nUV up to 405 nm, compared to a film with nUV absorption up to 384 nm, resulted in the lowest production of conidia (by 5‐fold). The former film was used to clad horticultural polyethylene tunnels in which crops of P. vulgaris and strawberry were grown for two seasons and the incidence of B. cinerea assessed throughout the growth of the crops. The incidence of infection on the P. vulgaris and strawberries was reduced by c. 50% and c. 26% respectively with the nUV blocking film compared to a standard film. The results are discussed in terms of the potential of spectral filters as a novel means of grey mould control in greenhouse‐produced crops.     

Ability of the antagonistic yeast Cryptococcus albidus to control Botrytis cinerea in strawberry

The yeast Cryptococcus albidus, originally isolated from mature strawberry fruits, was tested for antagonistic activity against Botrytis cinerea, the causal agent of grey mould in strawberries. Conidial germination and germ tube growth of conidia of B. cinerea were inhibited by a cell suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) after 6 and 24 hours of incubation. Application of a cell suspension (1 × 10⁶ cells/ml) on detached strawberry leaf disks incubated at 10°C reduced incidence and conidiophore density of B. cinerea by 86 and 99%, respectively, but effectiveness was reduced at higher temperatures. Treatments with C. albidus during bloom of strawberries reduced incidence of grey mould on ripe strawberry fruits after harvest by 33, 28 and 21% in three years of field trials. The effectiveness of the yeast was increased when formulation substances (alginate, xanthan and cellulose) were added to the cell suspension.     

Mycoflora of tuber surface of white yam (Dioscorea rotundata poir) and postharvest control of pathogens with Bacillus subtilis

Bacillus subtilis (Enrenberg) Cohn was investigated for its antagonistic properties against surface mycoflora of yam (Dioscorea rotundata Poir) tubers in storage. Yam tubers inoculated with a spore suspension of B. subtilis in potato dextrose broth using a knapsack sprayer showed a drastic reduction in the range and number of mycoflora, including pathogens of the tuber surface in contrast to the control tubers, during the five-month storage period in a traditional yam barn. However, B. subtilis maintained a high frequency of occurrence during the same period. Botryodiploidia theobromae Pat, Fusarium moniliforme Wollen and Reink., Penicillium sclerotigenum Yamamoto, and Rhizoctonia sp. were displaced completely on the treated tubers. The antagonism of B. subtilis was so effective that the normal tuber surface mycoflora was greatly reduced throughout the storage period of five months by a simple initial application of the antagonist.This revised version was published online in October 2005 with corrections to the Cover Date.     

Preliminary phenotypic characterization of newly isolated halophilic microorganisms by footprinting: a rapid metabolome analysis

The emerging need for rapid screening and identification methods for microbiological purposes necessitates the combined uses of high-tech instruments. In this work, electrospray ionization mass spectrometry was used to visualize the relation of ten newly isolated moderately halophilic microorganisms, to Halomonas salina DSMZ 5928 and Halomonas halophila DSMZ 4770. The method was based on the global analysis of the metabolites in culture media and is termed as metabolic footprinting. Since it was not possible to gain insight into the similarities solely based on the visual inspection of the chromatograms, principal component (PC) analysis was applied on the data. Three PCs alone were able to explain 99% of the information in the data set. The score plots revealed the relation of the new isolates to the two type strains whereas the loading plots gave important clues on the significant ions responsible for the observed clustering. Loading plots also indicated inversely correlated ions that give clues on differing metabolic pathways. The work described here offers a potentially useful way for preliminary rapid phenotypic characterization of new and closely related isolates and a method for screening of similar microorganisms for different and valuable secondary metabolites.     

Salinicoccus salsiraiae sp. nov.: a new moderately halophilic gram-positive bacterium isolated from salted skate

Two moderately halophilic low G + C Gram-positive bacteria were isolated from a sample of salted skate (Class Chondrychthyes, Genus Raja). Phylogenetic analysis of the 16S rRNA gene sequence of strains RH1^T and RH4 showed that these organisms represented a novel species of the genus Salinicoccus. The new isolates formed pink–red colonies and flocculated in liquid media, with optimum growth in media containing 4% NaCl and pH of about 8.0. These organisms are aerobic but reduce nitrate to nitrite under anaerobic conditions. Acid is produced from several carbohydrates. Oxidase and catalase were detected. Menaquinone 6 was the major respiratory quinone. The major fatty acids of strains RH1^T and RH4 were 15:0 anteiso and 15:0 iso. The G + C contents of DNA were 46.2 and 46.0 mol%, respectively. The peptidoglycan was of A3alpha L-Lys-Gly_5–6 type. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we suggest that strain RH1^T (=LMG 22840 = CIP 108576) represents a new species of the genus Salinicoccus, for which we propose the name Salinicoccus salsiraiae.     

Salinicoccus salitudinis sp. nov., a new moderately halophilic bacterium isolated from a saline soil sample

A novel pale-yellow-pigmented, moderately halophilic, facultatively alkaliphilic, non-motile, non-spore-forming, catalase- and oxidase-positive, obligately aerobic Gram-positive coccus, strain YIM-C678^T was isolated from a saline soil sample collected from a hypersaline habitat in the Qaidam basin, northwest China. The organism grew at 4–37°C and pH 6.0–11.0, with optimum growth at 25°C and pH 8.0. Strain YIM-C678^T grew optimally in the presence of 10–12% (w/v) NaCl and growth was observed in 1–25% (w/v) NaCl. The cell wall murein type was l-Lys-Gly₅. Major cellular fatty acids were anteiso-C_15:0, iso-C_15:0, iso-C_16:0 and C_16:0. Menaquinone 6 (MK-6) was the major respiratory quinone. The DNA G + C content was 46.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain YIM-C678^T belonged to the family Staphylococcaceae and was most closely related to the eight described species of the genus Salinicoccus with sequence similarities from 92.2 (S. luteus YIM 70202^T) to 97.5% (S. kunmingensis YIM Y15^T). The DNA–DNA relatedness between strain YIM-C678^T and S. kunmingensis YIM Y15^T was 35.4%. Chemotaxonomic data and 16S rRNA gene sequence analysis supported the affiliation of strain YIM-C678^T with the genus Salinicoccus. The combination of phylogenetic analysis, phenotypic characteristics, chemotaxonomic differences and DNA–DNA hybridization data supported the view that the bacterium represents a novel species of the genus Salinicoccus, for which the name Salinicoccus salitudinis sp. nov. is proposed, with YIM-C678^T (=DSM 17846 = CGMCC 1.6299) as the type strain.     

Biological control of Botrytis cinerea on tomato using naturally occurring fungal antagonists

Abstract

In order to evaluate the potential of naturally occurring filamentous fungi having potential as biocontrol agents effective against grey mould and post-harvest fruit rot caused by Botrytis cinerea on tomato, fungal saprophytes were isolated. They were obtained from leaves, fruits and flowers belonging to different species of cultivated and spontaneous Solanaceous plants collected at the horticultural area of La Plata, Argentina. Of 300 isolates screened for inhibition of B. cinerea using the dual culture technique on agar plate, 12 strains inhibited strongly mycelial growth of the pathogen. Among the antagonists one isolate of Epicoccun nigrum (126), four of Trichoderma harzianum (110, 118, 248 and 252) and four isolates of Fusarium spp. decreased the spore germination of B. cinerea between 30 and 70%. These isolates were probed on tomato fruits to evaluate their biocontrol activity against post-harvest grey mould. In growth chamber tests, E. nigrum (27), F. equiseti (22, 105) and T. harzianum (118, 252) reduced the diameter of fruit lesions by 50 – 90% and were selected for further biocontrol assays of tomato plants in the greenhouse. Although there were not significant differences between the treatments and the control, F. equiseti (105), E. nigrum (27) and T. harzianum (118) reduced by 20, 22 and 22 respectively the disease on whole plants. The targeted application of isolates of E. nigrum, T. harzianum and F. equiseti provides a promising alternative to the use of fungicide spray to control B. cinerea on tomatoes.     

Use of some essential oils as post-harvest botanical fungicides in the management of grey mould of grapes caused by Botrytis cinerea

During screening of twenty six essential oils against Botrytis cinerea, the essential oils of the ten plants viz. Chenopodium ambrosioides, Eucalyptus citriodora, Eupatorium cannabinum, Lawsonia inermis, Ocimum canum, O. gratissimum, O. sanctum, Prunus persica, Zingiber cassumunar and Z. officinale were found to exhibit absolute fungitoxic activity (100% growth inhibition). The essential oils of O. sanctum, P. persica and Z. officinale were selected for further investigation because these oils showed lower Minimum Inhibitory Concentration (MIC) as compared to the other fungitoxic oils. The selected oils were subsequently standardized through physico-chemical and fungitoxic properties. The MIC values of O. sanctum, P. persica and Z. officinale were found to be 200, 100 and 100 ppm (mg/l) respectively. The oils showed fungistatic nature at their respective MIC. The oils were thermostable, and exhibited a wide range of fungitoxicity against 15 other post-harvest fungal pathogens. The oils had the potency to withstand high inoculum density. The antifungal potency of oils was found to be greater in comparison to some prevalent synthetic fungicides. Practical applicability of the essential oils was observed in control of grey mould of grapes caused by B. cinerea during storage. The O. sanctum- and P. persica-oil-treated grapes showed enhancement of storage life up to 5 and 4 days respectively. The storage life of Z. officinale-oil-treated grapes was found to be enhanced up to 6 days. The oils did not exhibit any phytotoxic effect on the fruit peel. Therefore, the oils could be recommended as a potential source of ecofriendly botanical fungicide, after long term and wide ranging trials.     

Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide-producing species of Halomonas

Some members of the moderately halophilic genus Halomonas, such as H. eurihalina, H. maura, H. ventosae and H. anticariensis, produce exopolysaccharides with applications in many industrial fields. We report here that these four species also produce autoinducer molecules that are involved in the cell-to-cell signaling process known as quorum sensing. By using the N-acyl homoserine lactone (AHL) indicator strains Agrobacterium tumefaciens NTL4 (pZRL4) and Chromobacterium violaceum CV026, we discovered that all the Halomonas strains examined synthesize detectable AHL signal molecules. The synthesis of these compounds was growth-phase dependent and maximal activity was reached during the late exponential to stationary phases. One of these AHLs seems to be synthesized only in the stationary phase. Some of the AHLs produced by H. anticariens FP35^T were identified by gas chromatography/mass spectrometry and electrospray ionization tandem mass spectrometry as N-butanoyl homoserine lactone (C₄-HL), N-hexanoyl homoserine lactone (C₆-HL), N-octanoyl homoserine lactone (C₈-HL) and N-dodecanoyl homoserine lactone (C₁₂-HL). This study suggests that quorum sensing may also play an important role in extreme environments.     

Biological control of bacterial wilt caused by Pseudomonas solanacearum in India with antagonistic bacteria

From among 125 strains of fluorescent and 52 strains of nonfluorescent bacteria initially screened in the laboratory for their antibiosis towards the bacterial wilt pathogen, Pseudomonas solanacearum, strain Pfcp of Pseudomonas fluorescens and strains B33 and B36 of Bacillus spp., were chosen and evaluated further in greenhouse and field tests. Pfcp treated banana (Musa balbisiana), eggplant and tomato plants were protected from wilt upto 50, 61 and 95% in greenhouse and upto 50, 49 and 36% respectively in field. Protection afforded by the Bacillus strains was lower. In bacteria-treated plants which were subsequently inoculated with P. solanacearum plant height and biomass values increased and were close to those of nontreated and noninoculated control plants.     

Biological control of Phytophthora blight in red pepper (Capsicum annuum L.) using Bacillus subtilis

Phytophthora blight is one of the most important devastating diseases of red pepper plants. Forty-one bacterial isolates were obtained from rhizosphere soil and subsequently tested for antagonistic activity under in vitro and in vivo conditions. Among the 41 isolates tested, 12 exhibited a maximum antagonistic activity in dual culture assay. These 12 isolates were further screened for disease suppression on red pepper plants in both natural and greenhouse conditions. All the antagonists showed varying levels of antagonism, whereas the isolates R33 and R13 exhibited the maximum (86.8 and 71%) ability to reduce the disease severity in in vivo conditions. Based on the 16S rDNA sequencing, the most effective isolates were identified as Bacillus subtilis. In addition, the isolates were also screened for siderophores, hydrogen cyanide and hydrolytic enzymes. Further, the isolates increased the root and shoot length of the red pepper, which is an added advantage of the isolates while performing the desired function.     

Biological control of alligatorweed (Alternanthera philoxeroides) with a Fusarium sp.

Alligatorweed (Alternanthera philoxeroidesG.) has become a serious weed in different crops in China. A fungal pathogen was found in Chongqing and Sichuan Provinces and was identified as a species in the Fusarium genus. The fungus produced macroconidia and chlamydospores abundantly on potato sucrose agar (PSA) plates. The bestconidial production and germination and colonygrowth of Fusarium sp. were at 23–31°C and pH 6.7–7.0. Light period and flooding did not affect fungal growth and conidium formation. The herbicides, glyphosate and paraquat, inhibited the fungal development in vitro. The fungus did not affect seed germination and seedling growth of paddy rice, wheat, maize, oilseed rape and broad bean inlaboratory or greenhouse trials. Inoculum density and wetness duration influenced the efficiency of Fusarium sp. to control alligatorweed; a concentration of 1.0 × 10⁵ spores^–1 ml and 12 h of high humidity duration after inoculation produced goodinfections on the weed at 23°C in the laboratory. When the fungus was applied to alligatorweed grown in greenhouse and in the field, good biocontrol efficiency was obtained: the plants started to wilt after four to five (greenhouse) or six days (field), and were killed 9–10 (greenhouse) or 13–14 (field) days after spraying the fungal inoculum. This was similar to the control efficiency resulting from glyphosate treatment. Therefore, this Fusarium sp. appeared to be a good candidatefor further studies and a promising biocontrol agent to manage alligatorweed in some terrestrial and aquatic crops.     

Isolation of mannan-utilizing bacteria and the culture conditions for mannanase production

A locally isolated strain, Bacillus subtilis NM-39, was selected as an active mannan-utilizing bacterium based on high saccharifying activities on coconut residue and locust bean gum galactomannan. The optimal pH and temperature ranges for activity of the crude enzyme were 5.0 to 6.0 and 50 to 60°C, respectively. The organism gave maximum mannanase activity when grown in liquid mineral salts medium containing 1% (w/v) each of coconut residue and soybean flour, as carbon and nitrogen sources, respectively, at pH 7.0 and in aerobic growth for 28 h at 37°C. High saccharifying activity on coconut mannan was also observed.The authors are with the Industrial Technology Development Institute, Department of Science and Technology, Manila, Philippines. M. Arai and T. Kawaguchi are also currently with the Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan; T. Yoshida is also with the Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan.     

Evaluation of formulations of Bacillus licheniformis for the biological control of tomato gray mold caused by Botrytis cinerea

Bacillus licheniformis N1, which has previously exhibited potential as a biological control agent, was investigated to develop a biofungicide to control the gray mold of tomato caused by Botrytis cinerea. Various formulations of B. licheniformis N1 were developed using fermentation cultures of the bacteria in Biji medium, and their ability to control gray mold on tomato plants was evaluated. The results of pot experiments led to the selection of the wettable powder formulation N1E, based on corn starch and olive oil, for evaluation of the disease control activity of this bacterium after both artificial infection of the pathogen and natural disease occurrence under production conditions. In plastic-house artificial infection experiments, a 100-fold diluted N1E treatment was found to be the optimum biofungicide spray formulation. This treatment resulted in the significant reduction of symptom development when N1E was applied before Bo. cinerea infection, but not after the infection. Both artificial infection experiments in a plastic house and natural infection experiments under production conditions revealed that the N1E significantly reduced disease severity on tomato plants and flowers. The disease control value of N1E on tomato plants was 90.5% under production conditions, as compared to the 77% conferred by a chemical fungicide, the mixture of carbendazim and diethofencarb (1:1). The prevention of flower infection by N1E resulted in increased numbers of tomato fruits on each plant. N1E treatment also had growth promotion activity, which showed the increased number of tomato fruits compared to fungicide treatment and non-treated control and the increased fruit size compared the non-treated control under production conditions. This study suggests that the corn starch-based formulation of B. licheniformis developed using liquid fermentation will be an effective tool in the biological control of tomato gray mold.     

Biological control of crown rot of bananas with Pichia anomala strain K and Candida oleophila strain O

The antagonistic activity of two yeast strains (Pichia anomala (E.C. Hansen) Kurtzman, strain K and Candida oleophila Montrocher, strain O) against the parasitic complex responsible for banana crown rot was evaluated. The strains were applied at three different concentrations (10⁶, 10⁷, 10⁸ cfu/ml) and their efficacy tested in vivo on three separate fungi (Colletotrichum musae (Berk. & Curt.) Arx, Fusarium moniliforme Sheldon, and Cephalosporium sp.) and on a parasitic complex formed by association of these three fungi. At the concentrations used C. musae appeared to be the most pathogenic. The complex showed intermediate aggressiveness between C. musae and both other fungi.Statistically significant antagonistic effects were observed on C. musae, F. moniliforme, and the fungal complex. The highest protection level (54.4%) was observed with strain O added at 10⁸ cfu/ml on crowns previously inoculated with the fungal complex. The level was lower when the fungi were inoculated separately.Furthermore, the antagonistic effect was strongly reinforced when strain O at 10⁸ cfu/ml was applied 24 h before fungal complex inoculation (59.9%), as compared to its application 15 min (24.3%) or 3 h (27.3%) after fungal complex inoculation. Bananas showed increased susceptibility to the fungal complex from March to June, and this influenced the level of protection by yeast, which decreased over the same period. A strict negative correlation (R² = 0.83) was highlighted between susceptibility of banana to crown rot and protection provided by yeast.     

<Emphasis Type="Italic">Halobacillus naozhouensis</Emphasis> sp. nov., a moderately halophilic bacterium isolated from a sea anemone

A moderately halophilic, Gram-positive, catalase- and oxidase-positive, rod-shaped, aerobic bacterium, designated strain JSM 071068^T, was isolated from a sea anemone (Anthopleura xanthogrammica) collected from the Naozhou Island on the Leizhou Bay in the South China Sea. Cells were motile by means of peritrichous flagella and formed ellipsoidal endospores lying in subterminal swollen sporangia. Strain JSM 071068^T was able to grow with 1–20% (w/v) total salts (optimum, 6–9%), at pH values of 6.0–10.0 (optimum, pH 7.5) and a temperature range of 10–35°C (optimum, 25°C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The predominant menaquinone was MK-7 and the major cellular fatty acids were anteiso-C_15:0, anteiso-C_17:0 and iso-C_15:0. The genomic DNA G + C content was 42.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 071068^T belonged to the genus Halobacillus. The 16S rRNA gene sequence similarities between strain JSM 071068^T and the type strains of the recognized Halobacillus species ranged from 97.9% (with Halobacillus alkaliphilus) to 95.3% (with Halobacillus kuroshimensis). The levels of DNA–DNA relatedness between the new isolate and the type strains of H. alkaliphilus, Halobacillus campisalis, Halobacillus halophilus and Halobacillus seohaensis were 25.6, 22.1, 10.8 and 13.2%, respectively. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 071068^T represents a new species of the genus Halobacillus, for which the name Halobacillus naozhouensis sp. nov. is proposed, with JSM 071068^T (=DSM 21183^T =KCTC 13234^T) as the type strain. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 071068^T is EU925615.     

Biological treatments to control bacterial canker of greenhouse tomatoes

Experiments were conducted to determine the effects of treatments on Clavibacter michiganensis subsp. michiganensis in vitro and on young seedlingsinoculated with the pathogen under greenhouseconditions. Lysozyme was bactericidal at 10 g/l concentration in vitro. Tomato plantstreated with lysozyme at 10 g/l and 100 g/lshowed significantly higher plant heightcompared with the inoculated control plants,and plants in these treatments were as tall asthose observed in untreated uninoculatedcontrol plants. Treatments with B. subtilis (Quadra 136) and Trichoderma harzianum (RootShield®), lysozyme,vermicompostea, Rhodosporidium diobovatum(S33), B. subtilis (Quadra 137) appliedas a spray at 0.3 g/l, 0.6 g/l, 10 g/l,concentrated, 1 × 10⁹ CFU/ml, and 0.5 g/l,respectively, have the ability to prevent theincidence of bacterial canker of tomato plantscaused by C. michiganensis subsp.michiganensis under greenhouse conditions.     

Osmotic stress adaptation, compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat

Fusarium head blight (FHB) caused by Gibberella zeae (anamorph = Fusarium graminearum) is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. Biological control has been demonstrated to be effective under laboratory conditions but a few biocontrol products have been effective under field conditions. The improvement in the physiological quality of biocontrol agents may improve survival under field conditions, and therefore, enhance biocontrol activity. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were isolated from wheat anthers and showed significant effect on control of FHB under greenhouse assays. This study showed the effect of water availability measured as water activity (a_W) using a growth medium modified with NaCl, glycerol and glucose on: (i) osmotic stress tolerance, (ii) viability in modified liquid medium, (iii) quantitative intracellular accumulation of betaine and ectoine and (iv) the biocontrol efficacy of the physiologically improved agents. Viability of B. subtilis RC 218 in NaCl modified media was similar to the control. Brevibacillus sp. RC 263 showed a limited adaptation to growth in osmotic stress. Betaine was detected in high levels in modified cells but ectoine accumulation was similar to the control cells. Biocontrol activity was studied in greenhouse assays on wheat inoculated at anthesis period with F. graminearum RC 276. Treatments with modified bacteria reduced disease severity from 60% for the control to below 20%. The physiological improvement of biocontrol agents could be an effective strategy to enhance stress tolerance and biocontrol activity under fluctuating environmental conditions.     

Preventive and Curative Biological Treatments for Control of Botrytis cinerea Stem Canker of Greenhouse Tomatoes

The effects of biological treatments with PlantShield^®, Prestop^®, Quadra 136, RootShield^®, and S33 (Rhodosporidium diobovatum) and chemical treatment with Decree^® applied as a preventive or curative sprays on stem canker caused by Botrytis cinerea on tomato plants grown in sawdust were studied under near-commercial greenhouse conditions. Prestop^® and Decree^®, applied as preventive or curative sprays, PlantShield^® applied as curative spray, and S33 and Q-136 applied as preventive or preventive plus one spray to wounded surface provided season-long protection from B. cinerea stem canker. These treatments also increased fruit yield and decreased the number of dead plants compared with the inoculated control.     

Dynamics of heat destruction of spores: A new view

Summary Discrepancies between actual, viable spore populations and those predicted by a classical model during heat sterilization of food and pharmaceutical products have long concerned food engineers and scientists as they pursue new sterilization techniques, including ultra-high temperature processes. Among potential causes of those discrepancies, activation of dormant spores is significant, and models addressing that factor were developed recently. This paper reviews historic and current views on the biology and models of microbial spore populations during heat sterilization. Activation and inactivation of viable spores are emphasized, with each viewed as a first-order reaction. Rate constants of those reactions may differ significantly, inactivation rates of dormant and activated spores may differ, and variations of all rate constants with temperature appear to be well described by Arrhenius equations. Model-based analyses show how categories of survivor response curves observed during isothermal heat treatments can arise from simultaneous activation and inactivation of spores in an overall population. Effects of different distributions of initial subpopulations, different distributions of rate constants, and heat shock for homogenizing an indicator population are shown. The complexity of new, multiple process models has not increased greatly, but the potential for accurate, dynamic prediction of product safety after prescribed sterilization has. The relevant biology is understood and accounted for more thoroughly, and it is anticipated that the new models will aid design and evaluation of new and improved sterilization processes for food and pharmaceuticals.Mention of brand or firm names does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.