Changes in fungi and mycotoxins in pearl millet under controlled storage conditions

Pearl millet is increasingly being grown as a premium-value grain for the recreational wildlife and poultry industries in the southern US. We conducted three experiments to assess grain mold development in storage conditions typically encountered in the region of production. Variables included production year, temperature, relative humidity, atmosphere, and grain moisture content. In the first experiment, grain was stored for 9 weeks at 20 or 25°C and maintained at 86% or 91% relative humidity (r.h.). In the second experiment, grain was stored for 9 weeks at 20 or 25°C in either air (aerobic) or N₂ (anaerobic), and maintained at 100% r.h. In the third experiment, high-moisture grain was stored for 3 weeks at 20 or 25°C and maintained at 100% r.h. Grain was sampled at weekly intervals and plated to determine changes in fungal frequency. Fungi isolated included Fusarium chlamydosporum (19% of grain), Curvularia spp. (14%), F. semitectum (16%), Alternaria spp. (9%), Aspergillus flavus (8%), “Helminthosporium”-type spp. (6%), and F. moniliforme sensu lato (3%). Year of grain production significantly affected isolation frequency of fungi. Isolation frequencies from low-moisture grain were rarely affected by temperature, relative humidity, or atmosphere treatments, but was affected by storage duration for some fungi. Changes in isolation of toxigenic fungi occurred in high-moisture grain. Isolation frequency of F. chlamydosporum increased in grain stored at 86% and 91% r.h. Incidence of A. flavus increased in high-moisture grain treatments, particularly at 25°C. Incidence of deoxynivalenol was not affected by storage treatment. Low concentrations of nivalenol were detected in most grain incubated at 100% r.h. Zearalenone was detected only when grain moisture content was 20–22%. Aflatoxin contamination averaged 174 ng g⁻¹ over all treatments, and increased up to 798 ng g⁻¹ in high-moisture grain at stored at 25°C.     

Cytotoxicity of Fusarium species mycotoxins and culture filtrates of Fusarium species isolated from the medicinal plant Tribulus terrestris to mammalian cells

Ayurvedic medicine, which uses decoctions made of medicinal plants, is used to cure diseases in many Asian countries including Sri Lanka. Although proper storage facilities for medicinal plants are unavailable in Sri Lanka, neither the potential for growth of toxigenic fungi nor their ability to produce mycotoxins in stored medicinal plants has been investigated. We isolated three Fusarium species, F. culmorum, F. acuminatum and F. graminearum from the medicinal plant Tribulus terrestris. Culture extracts of the 3 Fusarium spp. were cytotoxic to mammalian cell lines BHK-21 and HEP-2. Three toxic metabolites produced by Fusarium spp; T-2 toxin, zearalenone, and diacetoxyscirpenol were also cytotoxic to the same mammalian cell lines. The 3 Fusarium spp. grown on rice media produced zearalenone. Plant material destined for medicinal use should be stored under suitable conditions to prevent growth of naturally occurring toxigenic fungi prior to its use.     

Long-term storage of Pennisetum glaucum (L.) R. Br. pollen

Summary Pearl millet [Pennisetum glaucum (L.) R. Br.] pollen has been successfully stored for 2,615 and 2,911 days at -18° and -73 °C, respectively, and continues to be viable. Viability of pollen stored at -73 °C appears to be little affected either by pollen storage moisture contents below 7.2% or by storage in glass vial or zip-lock plastic bag containers. Pollen moisture content appears to be more critical for maintaining viability at -18°C than at -73°C. Glass vials appear to be more desirable for longer term (>3 years) storage at -18°C.     

Effect of temperature and moisture content on Egyptian peanut seed-borne fungi

In peanut seeds adjusted to various levels of moisture content (8.5 %, 13,5 %, 17.5 % and 21 % on a dry-weight basis) and stored for six months at 5°, 15°, 28° and 45 °C, seed-borne fungi were monthly identified and counted using the dilution plate method, and the germinability of seeds was tested. The total count of seed-borne fungi (recovered at 28 °C) increased regularly and the germinability declined with the rise moisture content and with the lengthening of the storage period. At 5°, 15° and 28 °C,A. fumigatus was almost the most dominant fungal species followed by several fungi such asAspergillus flavus, A. niger, A. terreus, Penicillium funiculosum, P. notatum, Pyrenochaeta decipians andScopulariopsis brevicaulis. The degree of dominance of each fungal species depended upon the conditions of storage and the length of storage period.A. flavus gained its highest count in seeds adjusted to 13.5 % stored at 15 °C for 1 month.Penicillium predominated at 17.5 % and 21 %, and at 13.5 % and 17.5 % when the seeds were stored for long periods at 5° and 15 °C respectively.The total count of thermophilic fungi (recovered at 45 °C) significantly increased with the rise of moisture content and with the lengthening of the storage period between 1 and 2 months and regularly declined after 4 months.A. fumigatus was so extremely dominant that it was the main component of the total fungal flora. Several fungi truly thermophilic were isolated also. They were,Mucor pusillus, Humicola grisea var.thermoidae, H. insolens, H. lanuginosa (Thermomyces lanuginosus) andSporotrichum thermophile.     

Cold storage ofPhytoseiulus persimilis (Phytoseiidae)

Phytoseiulus persimilis is commerelally mass-reared for use as a biological control agent for spider mites, primarilyTetranychus urticae, and cold storage is a potentially valuable aspect of mass-production. Cold storage ofP. persimilis in empty containers was found to be unsatisfactory, but provision of moisture during cold storage greatly increased survival. Provision of food further increased survival even though the mites were stored at temperatures below their threshold for development of 11°C. When food was provided, survival at 7.5°C was 97% after 4 weeks and 80% after 6 weeks. Subsequent longevity and fecundity of mites that survived 8 weeks at 7.5°C was comparable to mites taken directly from mass-rearing cultures. Survival of mites packaged in bran or vermiculite and held at 6°C for 10 days ranged from 75% to 85% and was not decreased by agitating the containers to simulate shipping. However, survival of mites held in bran or vermiculite at 5°C or 8°C for 4 weeks was poor, ranging from 0–19%, due to growth of mould in the media.Phytoseiulus persimilis can be successfully stored for 4 to 6 weeks in containers provisioned only with food and moisture; granular media used for distribution of the mites should be added just prior to shipping.     

Invasion of sorghum seed by storage fungi at moisture contents of 13.5–15 % and condition of samples from commercial bins

In sorghum seed stored at 22–25° C for 535 days, invasion by storage fungi and loss of germinability increased greatly with small increases in moisture content between 13.5 and 15.5 %. Seeds dried for 18 hours at 70° C, then exposed to a relative humidity of 75 %, had a lower equilibrium moisture content, but were more heavily invaded by storage fungi and lost germinability fsater, than those that had been conditioned to 20 % moisture before storage at 75 % relative humidity. The 10 samples of Grade No. 2 sorghum examined averaged about 13.0 % moisture, germinated an average of 59 %, yieldedAlternaria from 75 % of the surface-disinfected kernels andAspergillus glaucus from 4 %; judged by these criteria, the lots from which the samples came were in good condition for continued storage.     

Prevalence and development of storage fungi in peanut Arachis hypogaea seed

Summary A batch of peanutArachis hypogaea seed were divided into 4 2-kilogram portions and stored 6 months at 22–28° C. One portion was stored with the moisture content slowly increasing from 4 to 28 %; the other portions were maintained at approximately 4.5, 8.9 and 11.5 %. After 1, 2, 3, 4, and 6 months storage, 100 seed of each treatment were cultured on malt-10 % NaCl agar and the fungi isolated identified. The storage fungi present after intervals of storage were compared with those initially present. In seed stored at 4.4 % moisture,Aspergillus species remained about the same andPenicillium decreased. At 8.9 % moistureA. flavus, A. repens andPenicillium were reduced, andA. amstelodami andA. ruber increased. At 11.5 % moisture, 100% of the seed yieldedA. ruber after 4 months storage and the other storage fungi decreased to 0–2 %. The moisture content of seed stored at 99 % R. H. increased slowly to 28 % and resulted in monthly changes in the dominant fungi. Under such a circumstance with competition between these storage fungi,A. ruber grew best at 12–15 %,A. repens at 18.5 %,A. flavus andPenicillium at 20 %, andA. amstelodami between 20–28 %.Fusarium increased very rapidly as moisture content increased above 18 %.This investigation was supported by Agricultural Research Service, U.S. Department of Agriculture, Contract No. 12-14-100-8179 (34), administered by the Crops Research Division, Beltsville, Maryland.Former Research Associate, North Carolina State University, Raleigh, N. C., currently Research Plant Pathologist, Market Quality Research Division, Agricultural Research Service, United States Department of Agriculture; and Professor, North Carolina State University, Raleigh, N.C.Paper number 2489 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina.     

Influence of Different Storage Conditions on the Mycotoxin Production and Quality of Fusarium-Infected Wheat Grain

Wheat seed samples with different initial infection levels of Fusarium culmorum were kept under different storage conditions for 36 weeks. Samples for analysis were drawn before storage and at intervals of 6‐8 weeks to determine the mycotoxin contents, seed health and seed quality. Zearalcnone (ZEA) accumulated to higher kernel contents towards the end of storage, when the seed was stored under warm and humid conditions [25°C/90% relative humidity (RH)], whereas the deoxynivalenol (DON) content of severely infected kernel samples (> 50%) remained unchanged under any of the conditions. On the other hand, DON contents increased in samples with a slight (4%) or moderate (15%) Fusarium infection level. when the seed was stored under Warm and humid conditions. Nivalenol (NIV) was not found in any samples immediately after harvest but later on in storage, and only under cool or warm but very humid conditions (15°C/84% RH and 25°C/90% RH). During storage, the mycotoxin contents of the samples did not reflect the percentage of Fusarium infected kernels. Under warm but dry conditions (25°C/62% RH) the seed germination rate showed a slight increase or remained nearly constant; at the same time the Fusarium infection level of the kernels decreased fairly fast. Cool and dry conditions (15°C/56% RH) maintained good seed quality but the Fusarium infection level of the kernels remained largely the same. Warm and humid conditions are not appropriate to maintaining quality of both seed and grain product.     

Effects of Moisture Content on Germination of Seeds of Hancornia speciosa Gom. (Apocynaceae)

Seeds of Hancornia speciosa germinated best at a temperatureof 20–30 °C. The viability of the seeds during storagewas short and the best storage conditions for viability entailedkeeping the seeds in polyethylene bags. Seed viability was maintainedonly when the seeds were stored at a moisture content above30%; storage conditions which allowed dehydration resulted ina rapid loss of viability (the seeds showed recalcitrant behaviour). Low temperature during storage did not improve longevity. Arelationship between germination and moisture content was established,but when the moisture content fell below 25% there was a drasticreduction of germination. After 9 weeks of storage, even athigh moisture content, seeds lost viability. Loss of seed viability during seed dehydration was associatedwith increased leakage of electrolytes and organic solutes,and reduced tetrazolium staining during subsequent imbibition. Hancornia speciosa, germination, recalcitrant seeds, storage, moisture     

Effect of incubation temperature on zearalenone production by strains of Fusarium crookwellense

After 6 weeks incubation on rice 2 strains of Fusarium crookwellense produced more zearalenone (6060–5010 mg/kg dry wt of culture) at ambient temperature (16–29°C) in daylight than at ambient temperature (18–23 °C) in darkness or at controlled temperatures of 11 °C, 20 °C or 25 °C in darkness. Yields at 25 °C were low. Incubation at 11 °C during the second 3 weeks incubation increased yields only when preliminary incubation had been at 25 °C. After 6 weeks incubation at controlled temperatures in darkness, 4 strains produced most zearalenone at 20 °C (2460-21 360 mg/kg), 1 strain at 11 °C (6570 mg/kg). Yields at a temperature oscillating daily from 10–20 °C were less than at 15 °C. One of the 5 strains produced appreciable amounts of a-zearalenol (1645 mg/kg at 20°C) and 2 of nivalenol (340 and 499 mg/kg at 20 °C).     

Mycotoxin formation in moist wheat under controlled temperatures

One-kilogram parcels of wheat with 20.5% moisture content were maintained at 15° and 22 °C for 10 weeks to study quality changes. Temperature, moisture, oxygen and carbon dioxide levels, microfloral incidence and abundance, seed germination, fat acidity values, aflatoxins, sterigmatocystin, ochratoxin A, penicillic acid, citrinin and zearalenone were monitored. By two weeks, trace levels of ochratoxin had formed at both temperatures. By 10 weeks, the wheat contained at least three times more ochratoxin A at 22 °C than at 15 °C. Strains of Penicillium verrucosum var. cyclopium were associated with ochratoxin A production. No other mycotoxins were detected. The effect of temperature was significant for all variables (greater effect at 22 °C) except A. glaucus gr. and Penicillium (P<.01). The effect of time was significant for all variables except bacteria (P<.01). The shape of the response was fully characterized by the linear and quadratic terms, except for % moisture which was linear only, and for bacteria for which time was not significant. The interaction between time and temperature was significant (P<.01) for total fungal propagule count, % moisture, and Aspergillus versicolor, indicative of the steeper rise in slope for 22 °C.     

Effects of long-term storage on Fusarium toxin concentrations in wheat - sources of error of the analytical results

The concentrations of the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) of a heavily contaminated wheat grain batch were followed over a period of 1 year by taking samples 15 times every 28 days. The air temperature and relative humidity at the top of the wheat batch ranged between 7 and 22°C and 44 and 55%, respectively, and corresponded to a variation in the moisture content of the wheat grain between 11.5 and 12.3%. None of these fluctuations were related to ZON and DON concentrations, which varied between 0.46 and 0.66 and 15.0 and 19.5 mg/kg DM. Therefore, the data were used to analyse the error sources for the analytical results. It was found that the variance proportions due to sampling and sample preparation plus analysis were not similar for DON and ZON. The variance proportion due to sampling was found to be 0.62 for ZON, which corresponded to a variance proportion of 0.38 due to sample preparation plus analysis. In contrast, the latter variance proportion for DON was estimated to be 1.0 and consequently completely superimposed the sampling error. It is concluded that long-term storage of contaminated wheat grain does not affect the concentrations of DON and ZON considering the measured fluctuations in ambient temperature, relative humidity and moisture content of the grain. Therefore, no degradation of DON and ZON occurred during the storage of wheat for a period of one year under ambient conditions. The effects of sampling and sample preparation plus analysis on the final analytical results are different for DON and ZON and require further consideration.     

Studies on the fungus flora of three grains in Egypt

In wheat, corn and sorghum grains twenty-six genera and seventy-seven species including sixteen ofAspergillus and twenty-one ofPenicillium were identified.In grain samples adjusted to various moisture levels (up to 18.8 % on a dry-weight basis) and stored for 4 months at 8, 20 and 30 C seed-borne fungi were monthly identified and counted, and the germinability of the grains was tested. In the control samples (moisture content 7.1–8.2 %) temperature had no effect on the fungal counts and on the precentage germination. With the rise of temperature and moisture content the fungal counts markedly increased and the germinability declined.The list of fungi dominant in one or more of the experiments, included eight species ofAspergillus, six ofPenicillium, two ofFusarium and one each ofAlternaria andCurvularia. The order of dominance of these fungi varied according to the type of grain, the length of storage and the treatment.A. niger was the dominant organism in the control samples of the three grains. It could grow successfully at high moisture contents but above 15 %, it was usually overgrown by other fungi. In wheatP. citrinum andA. sydowii, in cornP. citrinum andA. terreus, and in sorghumA. terreus andA. niger were the dominant fungi at moisture contents above 15 % after four months storage at 30 C. When stored at 8 CPenicillium species tended to predominate over other fungi in grains with high moisture contents.In surface-sterilized grains adjusted to 15 % moisture content, inoculated with six dominant fungi separately and incubated at 30 C,A. niger, P. citrinum andP. variabile caused severe deterioration in the three grains;A. ochraceus in wheat only andF. moniliforme in wheat and corn;C. spicifer was slightly injurious to the three grains.     

Operational and storage stability of neutral β-mannanase from Bacillus licheniformis

The stability of neutral -mannanase from Bacillus licheniformis during operation and storage was investigated. The enzyme activity decreased by 70% with a hydrolysate of glucomannan at 20 g l^–1 over 30 min at 25 °C. In an enzymatic membrane reactor operated at 50 °C after 24 h, the loss of enzyme activities were 23% and 9% in the absence/presence of the substrate. The residual activities of the enzyme were 21% and 90%, respectively, when stored in 30% (v/v) glycerol solution and in solid state at 4 °C after one year.     

Effects of Chilling Temperatures on Ethylene Binding by Banana Fruit

Banana fruit are highly susceptible to chilling injury during low temperature storage. Experiments were conducted to compare ethylene binding during storage at chilling (3 and 8 °C) versus optimum (13 °C) temperatures. The skins of fruit stored at 3 and 8 °C gradually darkened as storage duration increased. This chilling effect was reflected in increasing membrane permeability as shown by increased relative electrolyte leakage from skin tissue. In contrast, banana fruit stored for 8 days at 13 °C showed no chilling injury symptoms. Exposure of banana fruit to the ethylene binding inhibitor 1-methylcyclopropene (1 l l^-1 1-MCP) prevented ripening. However, this treatment also enhanced the chilling injury accelerated the occurrence of chilling injury-associated increased membrane permeability. ¹⁴C-ethylene release assay showed that ethylene binding by banana fruit stored at low temperature decreased with reduced storage temperature and/or prolonged storage time. Fruit exposed to 1-MCP for 12 h and then stored at 3 or 8 °C exhibited lower ethylene binding than those stored at 13 °C. Thus, chilling injury of banana fruit stored at low temperature is associated with a decrease in ethylene binding. The ability of tissue to respond to ethylene is evidently reduced, thereby resulting in failure to ripen.     

Seed germination of Lasia spinosa as a function of temperature,light, desiccation,and storage

Lasia spinosa seeds were not dormant at maturity in early spring. The most favorable temperatures for germination were between 25 and 30 °C, and final percentage and rate of germination decreased with an increase or decrease in temperature. When L. spinosa seeds were transferred to 25 °C, after 60 days at 10 °C (where none of the seeds germinated), final germination increased from 0% to 78%. Seeds germinated to high percentage both in light and in dark, although dark germination took more than twice as long as in the light. During desiccation of seeds at 15 °C and 45% relatively humidity, moisture loss decreased exponentially from 2.02 to 0.13 g H₂O g⁻¹ dry wt within 16 days, and only a few seeds (12%) survived 0.13 g H₂O g⁻¹ dry wt moisture content. Seeds stored at 0.58 g H₂O g⁻¹ dry wt moisture content at four constant temperatures (4, 10, 15, and −18 °C) for up to 6 months exhibited a well-defined trend of decreasing viability with decreasing temperature. Thus, we concluded that freshly harvested L. spinosa seeds are non-dormant and recalcitrant. Also, the seeds with 0.58 g H₂O g⁻¹ dry wt moisture content could be effectively stored for a few months between 10 and 15 °C although the most appropriate temperature for wet storage appears to be 10 °C, as it is close to the minimum temperature for germination and so there will be less pre-sprouting compared to 15 °C.     

Characterization of a combined DNA initiation and cell division mutant of Bacillus subtilis.

Summary The temperature-sensitive mutation in Bacillus subtilis 168-134ts, a conditional lethal DNA initiation mutant, was transferred to the minicell producing strain, CU 403 div IV-B1, to study he relationship of DNA synthesis to cell division. Markers in the combined mutant were verified by transduction. DNA replication kinetics, genome location by autoradiography, and clonal analysis of cell division patterns during spore outgrowths were investigated. Growth of the double mutant at the restrictive temperature results in an impressive reduction of the percentage cell length covered by DNA grain clusters (60.2% at 30° C compared to 8.6% after 2 h at 45° C). The probability of a minicell producing division in double mutant clones is essentially the same at 30° C and during the initial 2–3 h growth at 45° C at which time lysis begins. Residual division at 45° C is attributable to processes initiated at 30° C. The CU 403 div IV-B1, 134ts, double mutant divides about 25% as frequently relative to growth as do wild type CU 403 clones when incubated at permissive temperature. This is approximately 15% greater division suppression than previously found in the CU 403 div IV-B1 mutant strain, and is presumably due to interactions of the mutant gene products both of which affect DNA.     

Screening of zearalenone-producing Fusarium species in Egypt and chemically defined medium for production of the toxin

296 isolates of Fusarium spp. from 100 samples of cereale grains were examined for their ability to produce zearalenone on liquid culture medium. Thin layer Chromatographic analysis revealed that the mycotoxin was detected from 45 isolates, (F. oxysporum, 36; F. moniliforme, 8; and F. equiseti, one isolate). A suitable liquid medium and some optimal conditions for the biosynthesis of zearalenone were reported. Glutamine and riboflavin stimulated the production of the toxin. The maximum amount of zearalenone appeared at pH 7, after 12 days of incubation at 20 °C.     

Effects of temperature on acaricidal and insecticidal activities of the benzoylureas flucycloxuron and diflubenzuron

Effects of temperature on the activity of flucycloxuron on larval stages of Panonychus ulmi (Koch), based on LC₅₀ values, were highly significant (P < 0.001) with temperature coefficients of-1.7 in both the ranges of 15° to 25°C and 20° to 30°C. The slopes of probit regression lines at 15° and 20°C were significantly steeper than those at 25° and 30°C. As a consequence the temperature coefficients based on LC₉₀ values were-4.4 and-2.2, for the 2 temperature ranges. The ovicidal activity of flucycloxuron on P. ulmi was low and was only statistically detectable at 20°C (LC₉₀ of 84 mg a.i./l). In studies with larvae of Aedes aegypti (Linnaeus), Leptinotarsa decemlineata (Say), Plutella xylostella (Linnaeus), Spodeptera exigua (Hübner) and Spodoptera littoralis (Boisduval) probit regression lines were parallel over temperature. The activity of flucycloxuron on these five insect species was not affected by temperature. Based on LC₅₀ values, diflubenzuron showed positive temperature coefficients on P. xylostella of + 2.1 at 15° to 25°C and + 2.5 at 20° to 30°C. For S. littoralis the temperature coefficient was positive (+ 2.4) at 15° to 25°C but negative (-1.9) at the 20° to 30°C range. Temperature coefficients of diflubenzuron were neutral for A. aegypti, L. decemlineata and S. exigua. In the design and analysis of these studies special allowance was made for date effects and variation in natural mortality over temperature.     

Effect of water activity and temperature on Tenuazonic acid production byAlternaria alternata on sunflower seeds

Alternaria alternata is isolated in high frequency in sunflower seeds both in the field and storage. This species produces several toxic metabolites among them alternariol, alternariol monomethyl ether and tenuazonic acid. The accumulation of mycotoxins is regulated by physical, chemical and biological factors and for their production in many commodities nothing is known with regards to these conditions. In sunflower seeds the optimal conditions of temperature and water activity for tenuazonic acid production are unknown. The aim of this work was to investigate the effect of temperature and water activity on tenuazonic acid production byAlternaria alternata ITEM 539 in sunflower seeds. The temperature conditions evaluated were: 20, 25, and 30 °C and the water activities were 0.97, 0.90, 0.87, 0.80. The tenuazonic acid determinations were carried out during the incubation period at intervals of 7 days. Under conditions of constant temperature 25 °C) and variable water activities, 0.90 was the optimal value for tenuazonic acid production. At this water activity of the optimal temperature for tenuazonic acid production was 25 °C.