Peter J. Cotty

Genomic Islands in the Pathogenic Filamentous Fungus Aspergillus fumigatus

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated “gene dumps” and, perhaps, simultaneously, as “gene factories”.

Outbreak of an Acute Aflatoxicosis in Kenya in 2004: Identification of the Causal Agent

ABSTRACT Maize contaminated with aflatoxins has been implicated in deadly epidemics in Kenya three times since 1981, but the fungi contaminating the maize with aflatoxins have not been characterized. Here we associate the S strain of Aspergillus flavus with lethal aflatoxicoses that took more than 125 lives in 2004.

Agriculture, Aflatoxins and Aspergillus

Human activities affect both the size and structure of fungal populations. Construction, war, recreation, and agriculture disrupt large expanses of vegetation and soil; disruption causes redistribution of fungal propagules and makes nutrients available to fungi. Many fungi, including the aspergilli, exploit these human engineered resources. This results in the association of large fungal populations with various human activities, especially agriculture. When crops are grown or animals raised, fungi are also grown. From a human perspective, most fungi associated with cultivation increase inadvertently. Human activity, however, partly dictates which and how many fungi occur and the fungi, both directly and through fungal products, influence human activities, domestic animals, and even humans themselves.

Aflatoxin-producing potential of communities of Aspergillus section Flavi from cotton producing areas in the United States

Communities of Aspergillus section Flavi resident in soils planted with cotton were compared among several areas in the southern United States. Incidence of A. flavus and A. tamarii differed among areas. A. flavus incidence increased with temperature and decreased with latitude. Less than 1% of isolates were A. nomius or A. parasiticus. A. flavus isolates were assigned to either the S or L strains on the basis of sclerotial morphology. S strain isolates produced numerous small ( A. flavus was found in all areas. Aflatoxin-producing potential of A. flavus differed among areas and was correlated with S strain incidence. L strain isolates produced only 33% as much aflatoxin B 1 as S strain isolates. No S strain isolate produced both aflatoxin B 1 and aflatoxin G 1 . Correlations indicated that L strain toxigenicity but not S strain toxigenicity varied geographically. While toxigenicities of most isolates were stable through single conidial transfer, 28% of isolates expressed altered levels of toxigenicity after transfer. The observed differences among communities may reflect geographic isolation and/or adaptation, and may cause different vulnerabilities to aflatoxin contamination among crops planted in diverse locations.

Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation

ABSTRACT Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.

Reduction in Aflatoxin Content of Maize by Atoxigenic Strains of Aspergillus flavus

In field plot experiments, an atoxigenic strain of Aspergillus flavus interfered with preharvest aflatoxin contamination of corn when applied either simultaneously with or one day prior to a toxigenic strain. The atoxigenic strain reduced preharvest aflatoxin contamination 80 to 95%. The atoxigenic strain was also effective in reducing postharvest aflatoxin contamination caused by both an introduced toxigenic strain and by strains resident on the kernels. The results suggest that atoxigenic strains of A. flavus may have potential use as biological control agents directed at reducing both preharvest and postharvest aflatoxin contamination of corn.

Distribution and toxigenicity of Aspergillus species isolated from maize kernels from three agro-ecological zones in Nigeria

Maize samples were collected during a survey in three agro-ecological zones in Nigeria to determine the distribution and aflatoxin-producing potential of members of Aspergillus section Flavi. The three agro-ecological zones were, Derived Savannah (DS) and Southern Guinea Savannah (SGS) in the humid south and North Guinea Savannah (NGS) in the drier north. Across agro-ecological zones, Aspergillus was the most predominant fungal genera identified followed by Fusarium with mean incidences of 70 and 24%, respectively. Among Aspergillus, A. flavus was the most predominant and L-strains constituted >90% of the species identified, while the frequency of the unnamed taxon S(BG) was <3%. The incidence of atoxigenic strains of A. flavus was higher in all the districts surveyed except in the Ogbomosho and Mokwa districts in DS and SGS zones, respectively, where frequency of toxigenic strains were significantly (P<0.05) higher than that of atoxigenic strains. The highest and lowest incidence of aflatoxin positive samples was recorded in the SGS (72%) and NGS (20%), respectively. Aflatoxin contamination in grain also followed a similar trend and the highest mean levels of B-aflatoxins were detected in maize samples obtained from Bida (612 ng g(-1)) and Mokwa (169 ng g(-1)) districts, respectively, in the SGS. Similarly, the highest concentrations of G-aflatoxins were detected in samples from Akwanga district in the SGS with a mean of 193 and 60 ng g(-1), respectively. When agro-ecological zones were compared, B-aflatoxins were significantly (P<0.05) higher in SGS than in NGS, and intermediate in maize samples from the DS agro-ecological zone.

An isolate of Aspergillus flavus used to reduce aflatoxin contamination in cottonseed has a defective polyketide synthase gene

Contamination of certain foods and feeds with the highly toxic and carcinogenic family of Aspergillus mycotoxins, the aflatoxins, can place a severe economic burden on farmers. As one strategy to reduce aflatoxin contamination, the non-aflatoxin-producing A. flavus isolate AF36 is currently being applied to agricultural fields to competitively exclude aflatoxin-producing Aspergillus species. We now show that the polyketide synthase gene (pksA) required for aflatoxin biosynthesis in AF36, and in other members of the same vegetative compatibility group, possesses a nucleotide polymorphism near the beginning of the coding sequence. This nucleotide change introduces a premature stop codon into the coding sequence, thereby preventing enzyme production and aflatoxin accumulation.

Aflatoxin biosynthesis gene clusters and flanking regions

Aims:  To compare the biosynthetic gene cluster sequences of the main aflatoxin (AF)‐producing Aspergillus species.

Evaluation of atoxigenic isolates of Aspergillus flavus as potential biocontrol agents for aflatoxin in maize

Aflatoxin contamination resulting from maize infection by Aspergillus flavus is both an economic and a public health concern. Therefore, strategies for controlling aflatoxin contamination in maize are being investigated. The abilities of eleven naturally occurring atoxigenic isolates in Nigeria to reduce aflatoxin contamination in maize were evaluated in grain competition experiments and in field studies during the 2005 and 2006 growing seasons. Treatments consisted of inoculation of either grains in vials or ears at mid-silking stage in field plots, with the toxigenic isolate (La3228) or atoxigenic isolate alone and co-inoculation of each atoxigenic isolate and La3328. Aflatoxin B1 + B2 concentrations were significantly (p < 0.05) lower in the co-inoculation treatments compared with the treatment in which the aflatoxin-producing isolate La3228 was inoculated alone. Relative levels of aflatoxin B1 + B2 reduction ranged from 70.1% to 99.9%. Among the atoxigenics, two isolates from Lafia, La3279 and La3303, were most effective at reducing aflatoxin B1 + B2 concentrations in both laboratory and field trials. These two isolates have potential value as agents for the biocontrol of aflatoxin contamination in maize. Because these isolates are endemic to West Africa, they are both more likely than introduced isolates to be well adapted to West African environments and to meet regulatory concerns over their use throughout that region.