Cercosporin is a light-activated, non-host-selective toxin produced by many Cercospora fungal species. In this study, a polyketide synthase gene (CTB1) was functionally identified and molecularly characterized to play a key role in cercosporin biosynthesis by Cercospora nicotianae. We also provide conclusive evidence to confirm the crucial role of cercosporin in fungal pathogenesis. CTB1 encoded a polypeptide with a deduced length of 2,196 amino acids containing a keto synthase (KS), an acyltransferase (AT), a thioesterase/claisen cyclase (TE/CYC), and two acyl carrier protein (ACP) domains, and had high levels of similarity to many fungal type I polyketide synthases. Expression of a 6.8-kb CTB1 transcript was highly regulated by light and medium composition, consistent with the conditions required for cercosporin biosynthesis in cultures. Targeted disruption of CTB1 resulted in the loss of both CTB1 transcript and cercosporin biosynthesis in C. nicotianae. The ctb1-null mutants incited fewer necrotic lesions on inoculated tobacco leaves compared with the wild type. Complementation of ctb1-null mutants with a full-length CTB1 clone restored wild-type levels of cercosporin production as well as the ability to induce lesions on tobacco. Thus, we have demonstrated conclusively that cercosporin is synthesized via a polyketide pathway, and cercosporin is an important virulence factor in C. nicotianae. The results also suggest that strategies that avoid the toxicity of cercosporin will be useful in reduction of disease incidence caused by Cercospora spp.

Colletotrichum acutatum infects citrus petals and induces premature fruit drop and the formation of persistent calyces. The accumulation of hormones and other growth regulators, and differential gene expression in affected flowers and young fruit, was examined following fungal infection. Ethylene evolution increased threefold and indole-3-acetic acid (IAA) accumulation was as much as 140 times. Abscisic acid (ABA) levels showed no significant response. After infection, both trans- and cis-12-oxo-phytodienoic acid increased 8- to 10-fold. No significant difference of transjasmonic acid (JA) was observed in citrus flower petals or pistils. However, a fivefold increase of cis-JA was detected. The amount of salicylic acid (SA) was elevated twofold in affected petals, but not in pistils. Northern blot analyses revealed that the genes encoding ACC oxidase or ACC synthase, and 12-oxo-phytodienoic acid (12-oxo-PDA) reductase, were highly expressed in affected flowers. The genes encoding auxin-related proteins also were upregulated. Application of 2-(4-chlorophenoxy)-2-methyl-propionic acid (clofibrate; a putative auxin inhibitor), 2,3,5-triiodobenzolic acid (an auxin transport inhibitor), or SA after inoculation significantly decreased the accumulation of the gene transcripts of auxin-responsive, GH3-like protein and 12-oxo-PDA reductase, but resulted in higher percentages of young fruit retention. The results indicate that imbalance of IAA, ethylene, and JA in C. acutatum-infected flowers may be involved in symptom development and young fruit drop.

Postbloom fruit drop (PFD) of citrus and Key lime anthracnose (KLA) are caused by Colletotrichum acutatum. Both fungal isolates can infect flower petals, induce young fruit abscission and result in severe yield loss on many citrus cultivars. Previous studies revealed that infection of citrus flowers by C. acutatum caused higher levels of indole-3-acetic acid (IAA), which could be synthesized from the host plant and/or the fungal pathogen. The ability for IAA production by C. acutatum isolates was investigated. Similar to many microorganisms, the production of indole compounds in the medium by C. acutatum was dependent solely on the presence of tryptophan (Trp). In total, 14 PFD and KLA fungal isolates were tested, and revealed that they all were capable of utilizing Trp as a precursor to synthesize IAA and other indole derivatives. High-performance liquid chromatography analysis and chromogenic stains after a fluorescence thin-layer chromatography separation unambiguously identified IAA, tryptophol (TOL), indole-acetaldehyde, indoleacetamide (IAM), indole-pyruvic acid, and indole-lactic acid (ILA) from cultures supplemented with Trp. The data suggest that C. acutatum may synthesize IAA using various pathways. Interestingly, increasing Trp concentrations drastically increased the levels of TOL and ILA, but not IAA and IAM. The ability of C. acutatum to produce IAA and related indole compounds may in part contribute to the increased IAA levels in citrus flowers after infection. ß

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