Immunochemical analysis of trichothecenes produced by various fusaria

The production of type A trichothecene mycotoxins by 19 Fusaria, including 12Fusarium sporotrichioides, 4F. chlamydosporum and 3F. graminearum at 15°C and 25°C over a 35-day period was analyzed by ELISA using antibodies cross-reactive with most type A trichothecenes after conversion to T-2 tetraol tetraacetate. The toxin production peaked at 20–25 days of incubation with maximum yield between 4–6 mg type A trichothecene/ml of culture medium for 5F. sporotrichioides cultures and between 1 to 2 mg/ml for 6F. sporotrichioides cultures. OneF. sporotrichioides produced 700 µg type A trichothecenes/ml of culture medium. Detectable type A trichothecene was also found in the culture extracts ofF. chlamydosporum andF. graminearum, but the yield was very low (less than 100 µg/ml). Quantitative determination of individual trichothecenes was achieved by separation of different toxin in HPLC and followed by ELISA analysis. Eight to 10 immunoreactive peaks, corresponding to various type A trichothecenes, were detected in all the fungal extracts. T-2 tetraol (T-2-4ol), 4-acetyl-T-2 tetraol (4-Ac-T-2-4ol), neosolaniol (NEOS), diacetoxyscirpenol (DAS), HT-2 and T-2 toxin accounted for more than 85% of the total toxins. In general, low temperature was preferred for total type A trichothecene production. More T-2-4ol, 4-Ac-T-2-4ol, HT-2 and DAS were produced at 25°C. In contrast, more T-2 toxin and NEOS were produced at 15°C. Transformation of T-2 toxin and NEOS to polar metabolites such as T-2-4ol, 4-acetyl-T-2-4ol and HT-2 by various strains were observed at both temperatures after 25 days incubation.     

Production of type A trichothecenes and enniatin B byFusarium sambucinum Fuckel sensu lato

Twenty-nineFusarium isolates, representing three new taxa originated by Nirenberg fromF. sambucinum Fuckel sensu lato, namely:F. sambucinum Fuckel sensu stricto,F. venenotum Nirenb., andF. torulosum (Berk. & Curt.) Nirenb., were tested for in vitro production of toxic secondary metabolites on autoclaved corn kernels.F. sambucinum sensu stricto was able to produce type A trichothecenes and enniatin B (EB). In particular, amongst the 14 isolates tested, 5 produced only diacetoxyscirpenol (DAS) (up to 700 µg/g); 1 produced only neosolaniol (NEOS) (250 µg/g); 2 produced T-2 toxin (T-2) + NEOS (up to 175 and 150 µg/g, respectively); 1 produced NEOS + DAS (300 and 100 µg/g, respectively); and 5 produced DAS + EB (up to 500 and 140 µg/g, respectively). All six isolates ofF. venenotum were able to produce only DAS (up to 100 µg/g).F. torulosum produced no trichothecenes, but four out of nine tested isolates were able to produce EB (up to 140 µg/g). Zearalenones and type B trichothecenes were not found. The toxicity of the culture extracts towardsArtemia salina L. was correlated in general with the occurrence of the above toxins, except for someF. torulosum strains. However, the lack of correlation between the amounts of toxins recovered and toxic activity observed in theGeotrichum candidum Link ex Pers. andA. salina assays suggested the presence of unknown toxic compounds.     

Effect of<Emphasis Type="Italic">Trichoderma</Emphasis> species on growth of Fusarium<Emphasis Type="Italic">proliferatiom</Emphasis> and production of fumonisins,fusaproliferin and beauvericin

Trichoderma species has been suggested as potential biocontrol agent forFusarium verticillioides on maize. In this cereal,F. verticillioides and F. proliferatum contributed to fumonisin accumulation. In addition,F. proliferatum could produce beauvericin and fusaproliferin. The aim of this work was to evaluate the effect ofTrichoderma spp. on growth and fumonisin B¹ fusaproliferin and beauvericin production byF. proliferatum. Dual cultures of F.proliferatum andT. harzianum ITEM 3636 andT. longibrachiatum ITEM 3635 on maize meal agar at 0.995 aw were done. The effect ofTrichoderma spp. on the lineal growth ofF. proliferatum was determined. The effect ofTrichoderma species on fumonisin B¹, fusaproliferin and beauvericin production byF. proliferatum was determined on co-inoculated maize kernels by HPLC.T. harzianum suppressedF. proliferatum growth once contact between the colonies occurred.T. longibrachiatum showed a less antagonistic effect againstF. proliferatum. A reduction on fumonisin B¹ production of 98% and 88% was observed in the co-incubation ofF. proliferatum withT. harzianum andT. longibrachiatum, respectively. The decrease of FB¹ production was significant even in maize kernels on whichF. proliferatum had been growing 7 days prior to the addition ofTrichoderma spp. The concentration of beauvericin and fusaproliferin produced during 30 days coincubation ofF. proliferatum with bothTrichoderma spp. did not differ to those produced byF. proliferatum alone. These mycotoxins might enter the food chain causing so far unknown consequences to the health of domestic animals and humans. For this reason it is important, when a potential biocontrol agent is under study, to test the effect on the fungal growth and on the putative mycotoxin produced. Part of the information was presented at the Mycotoxin Prevention Cluster Dissemination Day and Mycoglobe Launch Conference, Brussels, Belgium, Oct 20–21, 2004 Financial support: Agenda Córdoba Ciencia, grant N^o 0279–000431/00     

Protection of maize seedlings byFusarium moniliforme against infection byFusarium graminearum in the soil

The incidence ofFusarium moniliforme in surface-sterilized kernels in two commercial South African white maize cultivars was 64% and 6%, respectively. Heat treatment completely eliminated seedborneF. moniliforme from kernels of both cultivars. Heat treated, uncontaminated maize germlings were pre-inoculated with different isolates ofF. moniliforme and planted in steam-treated soil containing inoculum of different isolates ofF. graminearum Group 1 and Group 2. Seedling weights of germlings pre-inoculated with some isolates ofF. moniliforme were significantly higher than those of controls when exposed to some isolates ofF. graminearum in the soil. The protective effect of pre-inoculation withF. moniliforme was particularly evident in maize seedlings exposed to inoculum of an aggressive isolate ofF. graminearum Group 1. This is the first report of the protection of maize seedlings byF. moniliforme against infection byF. graminearum in the soil.     

Occurrence and toxicity ofFusarium subglutinans from Peruvian maize

Twenty-five samples of maize kernels collected at harvest time from geographically different corn fields in Peru, were examined for the occurrence of toxigenicFusarium species. The most frequently recovered species wereF. subglutinans (48%),F. moniliforme (46%), andF. equiseti (5%). OtherFusarium species isolated (up to 1%) includedF. graminearum, F. acuminatum, F. solani, F. oxysporum, andF. culmorum. Assays ofFusarium culture extracts usingArtemia salina larvae, showedF. subglutinans as one of the most toxigenic species, and its toxicity was mostly correlated to the capability to produce beauvericin (BEA). All eight tested isolates ofF. subglutinans grown on autoclaved corn kernels produced BEA (from 50 to 250 mg/Kg) as well as moniliformin (M) (from 70 to 270 mg/Kg). This is the first report on BEA and M production by maize isolates ofF. subglutinans from South America.     

Production of zearalenone,nivalenol, moniliformin,and wortmannin from toxigenic cultures ofFusarium obtained from pasture soil samples collected in New Zealand

One culture ofF avenaceum, 4 cultures ofF oxysporum, and 11 cultures of Fsambucinum were isolated from soil samples of pasture in New Zealand in 1987. All cultures, when grown on rice media and fed to rats caused a weight loss in rats as well as toxic signs including hemorrhaging and congestion, uterine enlargement, and hematuria. 6 out of 16 cultures caused death in rat feeding tests.F oxysporum #1 killed rats (feeding test) within 5-12hrs. 10 cultures produced zearalenone (19 to 8,849 ppm), 8 cultures produced nivalenol (32 to 117 ppm), 1 culture,F sambucinum #8, produced wortmannin (40 ppm), and 5 cultures produced moniliformin (19 to 9,000ppm). We report for the first time the co-occurrence of zearalenone, nivalenol, and moniliformin produced byF sambucinum #3 in culture.F avenaceum #1 andF oxysporum cultures (nos 1, 2, and 3) produced moniliformin alone.F oxysporum #4 produced zearalenone alone as well.F sambucinum #5 caused erythema in the small intestine of rats and 100% mortality and did not produce any known toxin(s). Nivalenol when administered to the stomach of rats orally at levels 10, 20, and 40mg/kg body weight caused inflammation in the intestines, coma, and death. The mycotoxins T-2 toxin, HT-2 toxin, T-2 tetraol, diacetoxyscirpenol (DAS), monoacetoxyscirpenol (MAS), deoxynivalenol (DON), 3-acetyl-and 15-acetyldeoxynivalenol, depoxynivalenol, fusarenon-X, alpha-and beta-zearalenone, and fusarochromanone (TDP-1) were not detected in the extracts of these cultures.     

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

The role of root-associatedKlebsiella pneumoniae in the nitrogen nutrition ofPoa pratensis andTriticum aestivum as estimated by the method of15N isotope dilution

Summary The technique of¹⁵N isotope dilution was used to verify that nitrogen was fixed and transferred to the plant byKlebsiella pneumoniae strain Pp in association withPoa pratensis orTriticum aestivum. Surface sterilized, sprouting seeds were inoculated withK. pneumoniae and grown in sand in modified Leonard jars. Potassium nitrate enriched with¹⁵N was used to provide N concentrations ranging from 10–40 mg Nl^–1 nutrient solution. After 10–18 weeks the shoots and roots were analyzed separately for dry matter, N content, total N, and atom %¹⁵N excess. The acetylene reduction technique was used to test for the presence of N₂-fixing organisms on the roots. The data from¹⁵N isotope dilution demonstrated that up to 33.8% of N in the shoots ofP. pratensis and 15.9% in those ofT. aestivum were derived from associative N₂ fixation byK. pneumoniae. In most experiments the dry matter yield, N content, and total N yield of the shoots ofP. pratensis were increased byK. pneumoniae inoculation, whereas inoculation had no significant effect on the dry matter yield, N content or total N of the shoots ofT. aestivum.     

Effects of beauvericin to mammalian tissue and its production by Austrian isolates ofFusarium proliferatum and Fusarium subglutinans

Austrian isolates ofFusarium subglutinans andFusarium proliferatum were studied for their ability to produce beauvericin, moniliformin and fumonisin B₁ and B₂ under laboratory conditions. Analytical methodology for beauvericin was specially adapted for this task. Our analyses showed that the strains produced beauvericin up to 687 mg /kg maize and moniliformin up to 70 mg/kg. The culture ofF. proliferatum in addition produced fumonisin B₁ and B₂ at levels of 106 and 61 mg/kg,respectively. The preliminary toxicity experiments performed in this study clearly indicated a toxic effect of beauvericin on the contractility of mammalian smooth muscle and thus on mammalian cells.     

Anatoxin-a concentration inAnabaena andAphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxicAnabaena-strains — a laboratory study

Anatoxin-a-concentration in cells ofAnabaena- andAphanizomenon-strains and in their growth media were studied in the laboratory in batch cultures at different temperatures, light fluxes, orthophosphate and nitrate concentrations and with different nitrogen sources for growth. Toxin concentrations were detected by HPLC. Also, the growth of the toxicAnabaena-strains was compared to that of a non-toxic one. The non-toxicAnabaena was never found to produce anatoxin-a. The amount of toxin in the cells of the toxic strains was high, often exceeding 1% of their dry weight. High temperature decreased the amount of the toxin regardless of growth. Growth limiting low and growth inhibiting high light decreased the amount of the toxin in the cells ofAnabaena-strains. The highest light flux studied did not limit the growth or decrease the level of the toxin in the cells ofAphanizomenon. Growth in N-free medium (i.e. N₂ fixation) showed that the cells contained more toxin than growth in N-rich medium. Orthophosphate concentration had no effect on toxin levels, although the lowest concentrations limited the growth of all strains studied. The toxic strains tolerated higher temperatures than the non-toxic one, but the non-toxic strain seemed to be more adjustable to high irradiance than the toxic ones. The yields (dry weight) of non-toxic and toxic strains differed significantly in different phosphate concentrations.     

Formulation of the Biocontrol FungusCladorrhinum foecundissimumto Reduce Damping-Off Diseases Caused byRhizoctonia solaniandPythium ultimum

Five isolates ofCladorrhinum foecundissimum, added to soilless mix as 10-day-old fresh bran preparations (1.0% w/w), significantly reduced (P≤ 0.05) damping-off of eggplant and pepper caused byRhizoctonia solanistrain R-23. After 4 weeks of growth, plant stands in the biocontrol-amended, pathogen-infested treatments (>80%) were comparable to those in the noninfested controls. Since plant stands were similar at 2 and 4 weeks, most of the disease was preemergence damping-off. The bran preparations also reduced saprophytic growth of the pathogen, and there was an inverse correlation (r²= −0.94) between saprophytic growth and eggplant stand. Added to soilless mix at a rate of 2.0% (w/w), alginate prill containing 20% fermentor-produced biomass of six biocontrol isolates ofC. foecundissimumreduced (P≤ 0.05) damping-off of eggplant caused byR. solani, but only the prill with biomass of isolates Cf-1 or Cf-2 yielded plant stands (>80%) comparable to that in the noninfested control. As with the bran preparations, there was also an inverse correlation (r²= −0.80) between saprophytic growth of R-23 and eggplant stand with the alginate prills. Alginate prill with biomass of Cf-1 or Cf-2 also reduced (P≤ 0.05) damping-off of eggplant and pepper caused by other isolates (195, NG-2, DPR-1) ofR. solani, but only the stands (>80%) of pepper were similar to that in the noninfested control. Alginate prill formulations ofC. foecundissimum(Cf-1, Cf-2, and Cf-3) also reduced (P≤ 0.05) populations of the pathogen and damping-off of eggplant and pepper caused byPythium ultimum(PuZ3). However, although the plant stands in the treatments were not as high as those in the noninfested controls, they were higher than those in the pathogen-infested controls. The treatments also reduced populations ofP. ultimumin the soilless mix so that there were inverse correlations between the pathogen population and eggplant stand (r²= −0.81) and pepper stand (r²= −0.78). Extruded flour/clay granules containing 5.0% biomass of Cf-1 and Cf-2, added toR. solani-infested soilless mix (2.0%), reduced (P≤ 0.05) damping-off of eggplant and pepper. However, only the Cf-2 treatments resulted in stands (>80%) equal to those in the noninfested controls for the crops after 4 weeks of growth. The influence of bran and alginate prill of Cf-1 or Cf-2 on the spatial spread ofR. solaniand its ability to incite damping-off of eggplant showed that prill with Cf-1 or Cf-2 and bran with Cf-2 were equally effective in reducing the spread of the pathogen from the point source of the inoculum to the center of the flats.     

Genetic and mutational tools for investigating the genetics and molecular biology of trichothecene production inGibberella pulicaris (Fusarium sambucinum)

Gibberella pulicaris (Fusarium sambucinum) is a promising organism for studying the genetics and regulation of trichothecene biosynthesis; conditions for obtaining fertile crosses have been defined (Desjardins & Beremand, 1987) and crosses between natural variants have provided some information about the number, location, arrangement, and role of genes which determine trichothecene production (Desjardins & Beremand, 1987; Beremand & Desjardins, 1988). The development of some additional experimental tools and methodologies required for the further genetic analysis of trichothecene production inG. pulicaris are described in the present study. A highly fertile, isogenic line was constructed forG. pulicaris strain R-6380. The ability to readily generate mutants in this strain was also demonstrated. Both biochemical and morphological mutants were obtained following UV-mutagenesis. The inheritance of some of these mutations through meiosis indicated that they will be useful genetic markers for crosses and mapping studies. Since strain R-6380 is also transformable (Salch & Beremand, 1988), it is an excellent choice for transmission and molecular genetic studies involving trichothecene production.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Biological factors affecting leafhopper transmission of purified maize chlorotic dwarf machlovirus

A helper component from maize chlorotic dwarf machlovirus (MCDV)-infected plants was necessary for insect transmission of purified MCDV. Purified MCDV, WS strain, when acquired by membrane feeding, was transmitted byGraminella nigrifrons (Forbes) (Homoptera: Cicadellidae) which fed first on MCDV (M1 strain)-infected maize. Conversely, feeding on MCDV-WS-infected maize allowed transmission of purified MCDV-M1, indicating that the helper component was not strain specific. ViruliferousG. nigrifrons lost the ability to transmit MCDV-M1 after feeding for 24 h on healthy maize, but retained the ability to acquire and transmit purified MCDV-WS for up to 36 h. Wheng. nigrifrons fed initially on MCDV-infected plants and then on a second strain of purified MCDV, the second strain of MCDV could be transmitted alone, suggesting that the helper component from MCDV-infected plants was something other than the virion itself. Lengthening acquisition feeding ofG. nigrifrons on MCDV-M1-infected maize or purified MCDV-WS did not significantly change the transmission frequency of MCDV-WS.Amblysellus grex (Oman), an experimental vector of MCDV, also transmitted purified MCDV-WS after an initial acquisition feeding on MCDV-M1-infected maize. This suggests that the helper component is not vector species specific.     

Studies on metal resistance system inKluyveromyces marxianus

Through preliminary plate tests,Kluyveromyces marxianus was found to be much more resistant to toxic heavy metals compared to aCUP1 ^R strain ofSaccharomyces cerevisiae. Specific growth rate and maximum dry weights affected by increasing metal concentrations were determined to obtain precise patterns of resistance. Metal biosorption was also monitored during the course of growth in synthetic media containing respective metals at 0.5 mM final concentration. Although Zn- and Co-binding was negligible, as much as 90% of silver, 60% of copper, and 65% of cadmium were found to be absorbed by the end of active growth. Analysis of the protein profiles ofS. cerevisiae andK. marxianus on metal exposure suggested constitutive production of metallothionein inK. marxianus. Furthermore, a smaller protein synthesized byK. marxianus on induction by silver or cadmium accounts for the high resistance of the organism to these metals.     

Screening fusarium strains isolated from overwintered Canadian grains for trichothecenes

A survey of 38 samples of Canadian overwintered grains showed that 14 (37 %) contained viableFusarium. Of a total of 38Fusarium isolates, cultured on autoclaved corn, 20 (from 7 grain samples) showed toxicity to brine shrimp larvae and 12 (from 5 samples) produced levels of trichothecenes detectable by thin layer chromatography. The principal trichothecene found was T-2 toxin, produced by 10 strains and accompanied in half of these by neosolaniol; some of these strains were identified asF. sporotrichioides Sherbakoff. Two strains ofF. poae (Peck) Wollenw. formed small amounts of diacetoxyscirpenol. T-2 toxin was the most toxic of 8 trichothecenes tested on brine shrimp larvae; the wide range of toxicities limits the usefulness of this bioassay as a general screening method for trichothecenes.     

Competitive growth of slow growingRhizobium japonicum against fast growingEnterobacter andPseudomonas species at low concentrations of succinate and other substrates in dialysis culture

A cultivation system with simultaneous growth of six bacterial cultures in separate bags in dialysis culture was developed. In a medium with no added carbon source (one half concentrated Hoagland solution, water deionized and distilled), cell number ofRhizobium japonicum increased during a 7 day period by a factor of 35, whereas the number ofEnterobacter aerogenes cells decreased to one half. With a concentration of 100 nM succinate as an additional carbon source in the inflow,Rhizobium japonicum 61-A-101 cell number increased by a factor of 50 during an 8 day period, whereas cell number ofEnterobacter cloacae NCTC 10005 only doubled and ofEnterobacter aerogenes NCTC 10006 decreased. At 10 mM concentration of succinate in the inflow, doubling time the twoEnterobacter strains was about 12 h, compared to about 24 h for theRhizobium japonicum strain. Varying the succinate concentration from 10 mM to 100 nM in the inflow,Rhizobium japonicum 61-A-101 surpassed theEnterobacter aerogenes strains in the growth rate between 1 mM and 100 M succinate in the inflowing medium. Three otherRhizobium japonicum strains (fix⁺ and fix^-) did grow with a similar rate as strain 61-A-101 at very low concentrations of substrate. Growth rates for the strains were confirmed by protein data per culture. Growing in competition with twoPseudomonas strains,Rhizobium japonicum RH 31 Marburg (fix^-) did overgrow alsoPseudomonas fluorescens, was however outgrown byPseudomonas putida. In utilizing low concentrations of a¹⁴C labelled organic acid (malonate), three strains ofRhizobium japonicum left 2–4 times smaller amounts of¹⁴C in the medium than two species ofPseudomonas and two species ofArthrobacter.On sabbatical leave at ANU     

Fusarium toxin contents of maize and maize products purchased in the years 2000 and 2001 in Germany

Samples (n=106) of maize and maize products were analysed for 13 trichothecene toxins and zearalenone (ZON). All 14 toxins examined were detected, although with varying frequency. Cooccurrence of two or more toxins was observed in 96% of samples. The toxins of the scirpenol group scirpentriol, 15-monoacetoxyscirpenol and diacetoxyscirpenol were detected in 14, 27 and 3% of the samples analysed, the toxins of the T-2 group T-2 toxin, HT-2 toxin, T-2 triol und T-2 tetraol were found in 33, 66, 2 and 7%. Toxin content was higher in feeds than in foods (semolina and flour). In food samples, the German regulatory level for DON (500 μg/kg) was not exceeded, three samples of maize flour contained ZON above the regulatory level (50 μg/kg). Presented at the 26^th Mykotoxin-Workshop in Herrching, Germany, May17–19, 2004     

Production of beauvericin byFusarium proliferatum from maize in Italy

Beauvericin (BEA), a toxic cyclodepsipeptide, was purified from corn kernel cultures of a toxigenic strain ofFusarium proliferatum, Isolated from corn ear rot in northern Italy (45 mg/kg dry culture). The strain, designed ITEM-1503, also produced fumonisin B, (2,250 mg/kg dry culture), and moniliformin (150 mg/kg dry culture). Thin-layer chromatography, high-performance thin layer chromatography, high-performance liquid chromatography, low-resolution electronic impact mass spectrometry and 1H, 13C nuclear magnetic resonance spectroscopy were used for BEA Isolation and confirmation. This is the first report on the production of BEA byF. proliferatum.     

Growth and development of Bacillus thuringiensis Cry1Ab-susceptible and Cry1Ab-resistant sugarcane borer on diet and conventional maize plants

The sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), is a dominant maize borer pest and a major target of Bacillus thuringiensis (Bt)‐maize in Louisiana and the Gulf Coast area of Texas (USA). Growth and development of D. saccharalis on non‐toxic diet, diet treated with three low concentrations (0.01, 0.05, and 0.1 μg g^?1) of Cry1Ab toxin, and on non‐Bt maize plants were compared for five insect genotypes: a Bt‐susceptible strain (BT‐SS), a Cry1Ab‐resistant strain (BT‐RR), a back‐crossed and re‐selected resistant strain (BT‐R’R’), and two F₁ progeny of the BT‐SS and BT‐R’R’ strains. Fitness of the five genotypes was examined by infesting neonates on diet with/without Cry1Ab toxin in the laboratory and on intact non‐Bt maize plants in the greenhouse. Biological parameters measured were neonate‐to‐pupa development time and pupation rate, larval survival, larval and pupal weight, and sex ratio. Larvae of BT‐SS and BT‐R’R’ on non‐toxic diet and non‐Bt maize plants grew normally and there were no significant differences between the two strains in all measured parameters, suggesting a lack‐of‐fitness cost of the Cry1Ab resistance in D. saccharalis. Except for the development time on non‐Bt diet, all other parameters on both non‐Bt diet and non‐Bt maize plants were similar among the five genotypes. Larval development of BT‐SS was significantly affected on diet treated with Cry1Ab toxin at 0.05 and 0.1 μg g^?1, whereas the effect to BT‐RR and BT‐R’R’ was not significant. Pupal weight and sex ratio reared on Cry1Ab‐diet were similar and there were no significant differences among the five genotypes. Neonate‐to‐pupation rate decreased as Cry1Ab concentrations increased but the decrease was more significant for BT‐SS than for the other four genotypes. The lack‐of‐fitness costs of Bt resistance in D. saccharalis imply a greater challenge in managing Bt resistance for this maize borer species.