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.

Characterization of the Aspergillus ochraceoroseus aflatoxin/sterigmatocystin biosynthetic gene cluster.

Production of carcinogenic aflatoxins has been reported from members of Aspergillus section Flavi, Aspergillus section Nidulantes and a newly proposed Aspergillus section Ochraceorosei that consists of Aspergillus ochraceoroseus and A. rambellii. Unlike members of section Flavi, A. ochraceoroseus and A. rambellii have been shown to accumulate both aflatoxin (AF) and the aflatoxin precursor sterigmatocystin (ST). Alhough morphologically distinct from A. nidulans, molecular characterization of A. ochraceoroseus AF/ST genes and physiological characteristics of AF/ST production indicated that A. ochraceoroseus is more closely related to A. nidulans than to A. flavus. Knowing that the A. nidulans ST gene cluster is organized differently from the A. flavus AF gene cluster, we determined the genetic organization of the AF/ST biosynthetic cluster in A. ochraceoroseus. Sequencing of overlapping lambda clones and genomic PCR fragments obtained by gene-walking techniques demonstrated that the A. ochraceoroseus AF/ST gene cluster is organized much like the A. nidulans ST gene cluster except that the region from aflN to aflW is located directly upstream of aflC and in reverse orientation such that aflW represents the distal end and aflY the proximal end of the cluster. The A. ochraceoroseus cluster genes demonstrated 62-76% nucleotide identity to their A. nidulans ST cluster gene homologs. Transformation of an A. nidulans aflR mutant with the A. ochraceoroseus aflR restored ST production in A. nidulans transformants. PCR amplification of A. rambellii genomic DNA demonstrated that the AF/ST gene cluster is organized in the same manner as that of A. ochraceoroseus.

Characterization of aflatoxin-producing fungi outside of Aspergillus section Flavi.

Most aspergilli that produce aflatoxin are members of Aspergillus section Flavi, however isolates of several Aspergillus species not closely related to section Flavi also have been found to produce aflatoxin. Two of the species, Aspergillus ochraceoroseus and an undescribed Aspergillus species SRRC 1468, are morphologically similar to members of Aspergillus section Circumdati. The other species have Emericella teleomorphs (Em. astellata and an undescribed Emericella species SRRC 2520) and are morphologically distinctive in having ascospores with large flanges. All these aflatoxin-producing isolates were from tropical zones near oceans, and none of them grew on artificial media at 37 C. Aflatoxins and sterigmatocystin production were quantified by high-pressure liquid chromatography (HPLC) and confirmed by HPLC-mass spectrometry (LC-MS) detection. Phylogenetic analyses were conducted on these four species using A. parasiticus and Em. nidulans, (which produce aflatoxin and the aflatoxin precursor sterigmatocystin, respectively) for comparison. Two aflatoxin/sterigmatocystin biosynthesis genes and the beta tubulin gene were used in the analyses. Results showed that of the new aflatoxin-producers, Aspergillus SRRC 1468 forms a strongly supported clade with A. ochraceoroseus as does Emericella SRRC 2520 with Em. astellata SRRC 503 and 512.

Phylogenetic and morphological analysis of Aspergillus ochraceoroseus.

Aspergillus ochraceoroseus produces the yellow-gold conidia and other characteristics of Aspergillus subgenus Circumdati section Circumdati. However, this species produces aflatoxin, a secondary metabolite characteristic of some members of subgenus Circumdati section Flavi and sterigmatocystin, a related secondary metabolite usually associated with subgenus Nidulantes sections Nidulantes and Versicolores, as well as members of several other genera. Our morphological data support the placement of A. ochraceoroseus in subgenus Circumdati. Sequence data from A. ochraceoroseus aflatoxin and sterigmatocystin genes aflR and nor-1/stcE, as well as 5.8S ITS and beta tubulin genes, were compared to those of aspergilli in sections Circumdati, Flavi, Nidulantes and Versicolores. In the sequence comparisons, A. ochraceoroseus was related more closely to the species in subgenus Nidulantes than to species from subgenus Circumdati.