Growth inhibitory effects of gossypol and related compounds on fungal cotton root pathogens.

UNLABELLED: The effect of the terpenoids gossypol, 6-methoxygossypol, 6,6'-dimethoxygossypol, gossypolone and apogossypolone on growth of fungal soil pathogens was investigated. The compounds were tested at a concentration of 100 µg ml(-1) in a Czapek Dox agar medium at 25°C. Gossypol, gossypolone and apogossypolone demonstrated strong growth inhibitory activity (≥90%) against Pythium irregulare, Pythium ultimum and Fusarium oxysporum. These same terpenoids provided good growth inhibition against most Rhizoctonia solani isolates. Methylated gossypol derivatives generally yielded reduced growth inhibition against the tested fungi compared with gossypol. Dose-response effects of gossypol, gossypolone and apogossypolone were determined over a concentration range of 5-100 µg ml(-1) against P. irregulare CR1, P. ultimum ATCC 56081 and R. solani CR15. At lower concentrations, gossypol proved to be a more potent growth inhibitor of P. irregulare (ED50  = 4 µg ml(-1) ) and P. ultimum (ED50  = 13·2 µg ml(-1) ) than the other tested compounds. Rhizoctonia solani CR15 was more resistant to growth inhibitory effects of all tested terpenoids (ED50  = 35-43 µg ml(-1) ). SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates that gossypol is an effective natural antimicrobial agent against a wide range of potential fungal pathogens of cotton. Relative to gossypol, methylated gossypol derivatives that are also found naturally in root tissue were less effective at inhibiting the growth of soil fungal pathogens. However, by virtue of their significant concentration in root tissue, they still may contribute to cotton defence.

Effects of temperature and medium composition on inhibitory activities of gossypol-related compounds against aflatoxigenic fungi.

AIMS: To investigate the effects of temperature and medium composition on growth/aflatoxin inhibitory activities of terpenoids gossypol, gossypolone and apogossypolone against Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: The compounds were tested at a concentration of 100 µg ml(-1) in a Czapek Dox (Czapek) agar medium at 25, 31 and 37°C. Increased incubation temperature marginally increased growth inhibition caused by these compounds, but reduced the aflatoxin inhibition effected by gossypol. Gossypolone and apogossypolone retained good aflatoxin inhibitory activity against A. flavus and A. parasiticus at higher incubation temperatures. However, increased temperature also significantly reduced aflatoxin production in control cultures. The effects of the terpenoids on fungal growth and aflatoxin production against the same fungi were also determined in Czapek, Czapek with a protein/amino acid addendum and yeast extract sucrose (YES) media. Growth of these fungi in the protein-supplemented Czapek medium or in the YES medium greatly reduced the growth inhibition effects of the terpenoids. Apogossypolone displayed strong anti-aflatoxigenic activity in the Czapek medium, but this activity was significantly reduced in the protein-amended Czapek and YES media. Gossypol, which displayed little to no aflatoxin inhibitory activity in the Czapek medium, did yield significant anti-aflatoxigenic activity in the YES medium. CONCLUSIONS: Incubation temperature and media composition are important parameters involved in the regulation of aflatoxin production in A. flavus and A. parasiticus. These parameters also affect the potency of growth and aflatoxin inhibitory activities of these gossypol-related compounds against aflatoxigenic fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies utilizing gossypol-related compounds as inhibitory agents of biological activities should be interpreted with caution due to compound interaction with multiple components of the test system, especially serum proteins.

Inhibitory effects of gossypol-related compounds on growth of Aspergillus flavus.

AIMS: The objective of this study was to test a series of gossypol-related compounds for growth inhibition against Aspergillus flavus. METHODS AND RESULTS: A series of chiral and achiral gossypol derivatives, some natural products of the cotton plant and others prepared by synthesis from gossypol, were incorporated into agar plates to follow the rate of A. flavus isolate AF13 colony growth. All tested compounds exhibited some growth inhibition against this organism. The synthetic compounds, gossypolone and apogossypolone, exhibited greater activity than either racemic or chiral gossypol. Methylated derivatives (i.e. 6-methoxy and 6,6'-dimethoxy derivatives) generally exhibited less activity than the nonmethylated parent compounds. The (-)-optical form of gossypol was found to be slightly more active than the (+)-optical form, and this trend was observed regardless of the presence of methoxy groups at the 6-position. Growth inhibition of gossypolone and apogossypolone was concentration dependent. For gossypolone, the 50% effective dose was 90 µg ml⁻¹ of medium (165 µmol l⁻¹). For apogossypolone, the most active compound in the study, the 50% effective dose was 19 µg ml⁻¹ (38·7 µmol l⁻¹). The presence of gossypol-related terpenoids appeared to stimulate production of A. flavus sclerotia, although replicate variability was so large that it was not possible to determine a significant correlation between the mass of sclerotia formed and compound growth inhibition. CONCLUSIONS: The quinone derivatives of gossypol, gossypolone and apogossypolone demonstrated significant fungal growth inhibitory activity against A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: These gossypol derivatives may provide a new class of fungicide for use against the mycotoxigenic fungus A. flavus.

Ecology of aflatoxin producing fungi and biocontrol of aflatoxin contamination.

Aflatoxins, highly toxic and carcinogenic compounds that frequently contaminate foods and feeds, are produced by several genera in the genusAspergillus. Aspergillus flavus, the most common species causing crop contamination, is a common inhabitant of the Sonoran desert of North America where it resides in complex communities composed of diverse individuals. This diversity reflects divergent adaptation to various ecological niches. SomeA. flavus isolates that are well adapted to plant associated niches do not produce aflatoxins yet have the capacity to competitively exclude aflatoxin producers. These atoxigenic strains can serve as biological control agents for management of aflatoxins in crops. Detailed knowledge of the ecology of aflatoxin-producing fungi may lead to novel practical methods for limiting contamination.

Influence of lipids with and without other cottonseed reserve materials on aflatoxin B(1) production by Aspergillus flavus.

Cottonseed storage lipids (primarily triglycerides), in either crude or refined form, were found to support growth and aflatoxin B(1) production by Aspergillus flavus. When lipids were removed from ground whole cottonseed by petroleum ether extraction, aflatoxin production dropped by more than 800-fold. Reconstitution of the lipid-extracted ground whole seed with a crude preparation of cottonseed lipids restored aflatoxin production to the previous levels. Fungal utilization of the three major cottonseed reserve materials, raffinose, triglycerides (refined cottonseed oil), and cottonseed storage protein, was monitored in vitro over a 7 day fermentation period. The fermentation medium contained the reserve compounds in proportions approximating those found in mature cottonseed. A. flavus rapidly converted raffinose to fructose and melibiose, presumably by action of invertase, and then hydrolyzed the melibiose. These simple sugars apparently supported initial growth and aflatoxin B(1) production. Raffinose and the resulting melibiose were nearly exhausted by day 2. Fungal hydrolysis of triglycerides began as exhaustion of carbohydrate approached. After day 2, rapid catabolism of the released fatty acids began and coincided with glucose regeneration through gluconeogenesis, which peaked on day 6. The fungus did not preferentially utilize specific fatty acids. A. flavus also produced a number of storage metabolites, including arabitol, erythritol, mannitol, and trehalose. Mannitol was produced in much higher concentrations than the other storage metabolites. Selective use of simple carbohydrates by A. flavus to drive aflatoxin production may suggest strategies for reducing vulnerability of cottonseed to aflatoxin contamination.

Demonstration of aflatoxin inhibitory activity in a cotton seed coat xylan.

An inhibitor of aflatoxin biosynthesis localized in the seed coats of developing cotton was partially purified and characterized. Aqueous extracts from 25-day postanthesis seed coat tissue inhibited aflatoxin (B(inf1)) production in liquid cultures of Aspergillus flavus AF13. Inhibition was concentration dependent, with a 50% effective dose of 173 (mu)g of crude extract per ml of medium. The inhibitor was neutral in charge. Two active fractions were obtained from crude preparations by gel filtration chromatography (BioGel P-100). The purest fraction eluted in the void volume. Carbohydrate composition analysis of this void volume inhibitor indicated a composition of xylose (>90%) and mannose. Aflatoxin production in vitro was inversely related to inhibitor concentration in the fermentation medium (log of aflatoxin versus log of [inhibitor]; r(sup2) = 0.82; P < 0.002). The void volume inhibitor had a 50% effective dose of 6.2 (mu)g/ml, a 28-fold purification of the inhibitor material. These data support the hypothesis that seed coat inhibitory activity is associated with a cottonseed-specific xylan.

Expression of elastinolytic activity among isolates in Aspergillus section flavi.

A survey of the distribution of elastinolytic potential among 32 culture collection isolates of Aspergillus flavus. A. oryzae, A. parasiticus, A. sojae, A. nomius, and A. tamarii revealed this character to be highly conserved within Aspergillus Section Flavi. Furthermore, 144 isolates of A. flavus from environmental samples from six separate regions of the United States produced elastase on solid medium. Most previously described polymorphisms in elastinolytic potential were attributed to the toxicity of borate buffers. Replacement of borate with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) resulted in detection of elastase production on solid medium by all tested fungal isolates except two that had been in culture over 50 years. In liquid culture, only isolates of A. flavus, A. tamarii, and A. oryzae accumulated elastase activity. Although isoelectric focusing revealed only one isoform (pI 9.0) of elastase in these culture filtrates, elastinolytic activity in filtrates was partially inhibited by both 1,10-phenanthrolene (2 mM) and phenylmethylsulfonylfluoride (2 mM), suggesting the presence of both metallo and serine elastinolytic proteinases.

Purification and Characterization of Isoperoxidases Elicited by Aspergillus flavus in Cotton Ovule Cultures.

Two anionic isoperoxidases were isolated from media of Aspergillus flavus-inoculated cotton (Gossypium hirsutum L.) ovule cultures and purified about 150-fold to apparent homogeneity by treatment with Cell Debris Remover and ion exchange chromatography on Accell QMA medium. These isoperoxidases were present in noninoculated cotton ovule cultures at low levels. The major activity peak (B) represented 90% of the recovered peroxidase activity and was electrophoretically homogeneous. The minor activity peak (A) was about 95% pure. Isoelectric focusing analysis showed that B was greater than 95% pure with respect to other peroxidase isozymes, while the enzyme in A was about 90% isozymically pure. Each isoperoxidase displayed a molecular mass of 56 kilodaltons by interpolation from denaturing gel electrophoresis. The B isozyme displayed a molecular mass of 55 kilodaltons by gel filtration chromatography. The pH optima for the cotton ovule isoperoxidases were similar, 5.0 for isozyme A and 6.0 for isozyme B. The isoelectric points for isozymes A and B were 4.2 and 4.4, respectively. Eugenol, guaiacol, and 3,3',5,5'-tetramethylbenzidine were good electron donor substrates, whereas 4-aminoantipyrine was a poor substrate. The absorption spectrum of the material in B revealed a major peak at 400 nanometers and a minor peak at 280 nanometers. The molar extinction coefficient at 400 nanometers (pH 7.0) was calculated to be 1.07 x 10(5) per square centimeter per mole. Amino acid analysis of isozyme B confirmed the acidic nature of this protein and identified a number of similarities to the anionic peroxidases from tobacco and potato. This glycoprotein was found to contain 12 to 14% sugar (by weight), mainly in the form of galactose and mannose.

Variation in polygalacturonase production among Aspergillus flavus isolates.

Pectinase production by Aspergillus flavus was determined by measuring clear zones formed around colonies stained with ruthenium red. Several isolates produced red zones instead of clear zones. Red zones were reproduced with pectinesterase and correlated with absence of specific polygalacturonases. Of 87 isolates tested, 15 produced red zones.

Some characteristics of peroxidase secreted by cotton ovule cultures.

Cotton (Gossypium hirsutum L.) ovule cultures secreted a soluble peroxidase into the surrounding medium, resulting in a 200-fold increase in this activity during the 30-day growth period. The peroxidase activity was thermostable from 4°C to 60°C and displayed a pH optimum of 5.5 to 6.0. The ovule peroxidase was susceptible to periodate treatment and very resistant to protease digestion. The data suggest that the peroxidase activity is a glycoprotein. Interpretation of peroxidase data may be complicated by the presence of phenol oxidase activity in the same preparations. The presence of phenol oxidases was ruled out by the inaction of a tyrosinase-specific inhibitor, tropolone.

Interaction of a hydroxyproline-rich glycoprotein from tobacco callus with potential pathogens.

A hydroxyproline-rich glycoprotein was isolated from tobacco (Nicotiana tabacum L.) callus tissue cultures by an acidic-ethanol extraction procedure and purified to about 95% homogeneity by ion exchange chromatography on carboxymethyl cellulose. This glycoprotein agglutinated cells of an avirulent strain (B-1) of the bacterial pathogen Pseudomonas solanacearum but not its parental, virulent isolate (K-60). Bacterial lipopolysaccharide (from K-60 strain) inhibited this agglutination. The tobacco glycoprotein also agglutinated zoospores of both compatible and incompatible races of Phytophthora parasitica var. nicotianae. Although 34 potential haptens were tested, no low-molecular-weight carbohydrate that inhibited bacterial or fungal agglutination was found. The agglutination activity of the tobacco glycoprotein was sensitive to pronase and sodium periodate. The apparent molecular weight of the glycoprotein was 120,000. The protein moiety was basic (12% lysine and 5% histidine) and contained 38% hydroxyproline. The carbohydrate moiety comprised 26% (by weight) of the glycoprotein, and contained 87% arabinose, 8% galactose, and 5% glucose. The glycoprotein labeled with fluorescein isothiocyanate bound significantly better to the avirulent isolate (B-1) of P. solanacearum than to the virulent strain (K-60). Binding to the avirulent cells was inhibited by incubation in a higher ionic strength medium (e.g. 0.2 m NaCl). The labeled glycoprotein also bound to cystospores and mycelia of both races of P. parasitica var. nicotianae. This fungal-glycoprotein interaction was inhibited by the lipopolysaccharide from strain K-60 and by higher ionic strength conditions.

Diterpene biosynthesis in maize seedlings in response to fungal infection.

A cell-free system which catalyzes the biosynthesis of terpene hydrocarbons when supplemented with mevalonate, Mn(2+), and ATP was prepared from the scutellum-embryonic axis region of maize seedlings. The capacity of this system for the production of terpene hydrocarbons was enhanced 50- to 100-fold when the seedlings were exposed for 48 hours to the fungus Rhizopus stolonifer prior to tissue homogenization. The fungi Aspergillus niger, Fusarium moniliforme, and Verticillium albo-atrum also elicited this biosynthetic enhancement. The terpene hydrocarbon products were separable into six fractions by argentation thin layer chromatography. Radioactivity was contributed to five of these fractions when either geranylgeranyl pyrophosphate or copalyl pyrophosphate was supplied as substrate, suggesting that polycyclic diterpenoid hydrocarbons were the main products. Large scale biosynthetic reactions led to the acquisition of about 1 milligram of terpene hydrocarbon products plus some more polar terpenoid products. Analysis of the hydrocarbon products by gas chromatography and mass spectrometry led to the separation of six distinct diterpene hydrocarbons plus a fraction with a molecular weight of about 550. Three of the diterpene hydrocarbons were identified as kaur-16-ene, kaur-15-ene (isokaurene), and pimara-8(14),15-diene. None of the terpene hydrocarbon fractions tested displayed antifungal activity in the Cladosporium cucumerinum thin layer plate assay.