An ABC Transporter and a Cytochrome P450 of Nectria haematococca MPVI Are Virulence Factors on Pea and Are the Major Tolerance Mechanisms to the Phytoalexin Pisatin

The fungal plant pathogen Nectria haematococca MPVI produces a cytochrome P450 that is responsible for detoxifying the phytoalexin pisatin, produced as a defense mechanism by its host, garden pea. In this study, we demonstrate that this fungus also produces a specific ATP-binding cassette (ABC) transporter, NhABC1, that enhances its tolerance to pisatin. In addition, although both mechanisms individually contribute to the tolerance of pisatin and act as host-specific virulence factors, mutations in both genes render the fungus even more sensitive to pisatin and essentially nonpathogenic on pea. NhABC1 is rapidly induced after treatment with pisatin in vitro and during infection of pea plants. Furthermore, NhABC1 was able to confer tolerance to the phytoalexin rishitin, produced by potato. NhABC1 appears to be orthologous to GpABC1 of the potato pathogen Gibberella pulicaris and, along with MoABC1 from Magnaporthe oryzae, resides in a phylogenetically related clade enriched with ABC transorters involved in virulence. We propose that NhABC1 and the cytochrome P450 may function in a sequential manner in which the energy expense from pisatin efflux by NhABC1 releases the repression of the cytochrome P450, ultimately allowing pisatin tolerance by two mechanisms. These results demonstrate that a successful pathogen has evolved multiple mechanisms to overcome these plant antimicrobial compounds.

Potentially dangerous fusarioid microorganisms associated with rot of hops (Humulus lupulus L.) plants in field culture

Several fusarioid microorganisms were isolated as potential pathogens of hop (<i>Humulus lupulus</I> L.) but their virulence was not proved in inoculation trials in field conditions. Molecular search for other possible pathogens was then performed. Using terminal restriction fragment length polymorphism (TRFLP), <i>Gibberella pulicaris</I> (anamorph: <i>Fusarium sambucinum</I>) was identified as a probable cause of the hop wilting. The primary cause of the disease is wounding of hop crowns by feeding of rosy rustic moth (<i>Hydraecia micacea</I>) caterpillars or by defect pruning and other unfavourable circumstances. The specific primer HLf1 was designed that can be used to detect the pathogen in soil and in damaged plant tissues.

Tri16 Is Required for Esterification of Position C-8 during Trichothecene Mycotoxin Production by Fusarium sporotrichioides

ABSTRACT We previously characterized Tri1, a gene required for hydroxylation of the C-8 position during trichothecene mycotoxin biosynthesis in Fusarium sporotrichioides NRRL 3299. Sequence analysis of the region surrounding Tri1 revealed a gene, named Tri16, which could encode an acyltransferase. Unlike the wild-type parent strain NRRL 3299, which accumulates primarily T-2 toxin along with low levels of diacetoxyscirpenol (DAS) and neosolaniol (NEO) and trace amounts of 8-propionyl-neosolaniol (P-NEO) and 8-isobutyryl-neosolaniol (B-NEO), mutants containing a disruption of Tri16 were blocked in the production of the three C-8 esterified compounds T-2 toxin, P-NEO, and B-NEO and accumulated the C-8-hydroxylated compound NEO along with secondary levels of DAS. These data indicate that Tri16 encodes an acyltransferase that catalyzes the formation of ester side groups at C-8 during trichothecene biosynthesis. We also report the presence of a Tri16 ortholog in Gibberella pulicaris R-6380 that is likely linked to a presumably inactive ortholog for Tri1.

An ATP-binding Cassette Multidrug-Resistance Transporter Is Necessary for Tolerance of Gibberella pulicaris to Phytoalexins and Virulence on Potato Tubers

The necrotrophic pathogen Gibberella pulicaris infects potato tubers through wounds that contain fungitoxic secondary metabolites such as the phytoalexins rishitin and lubimin. In order to colonize tuber tissue, the fungus must possess a mechanism to tolerate potato defense compounds. In this paper, we show that a gene, Gpabc1, that codes an ATP-binding cassette (ABC) transporter is required for tolerance to these phytoalexins and for virulence on potato. The Gpabc1 gene, isolated in the course of a differential cDNA screen, shares high sequence homology with the ABC1 gene of Magnaporthe grisea. G. pulicaris mutants deficient in Gpabc1 were still able to metabolize rishitin but lost their tolerance to this phytoalexin as well as their virulence on potato. These results strongly suggest that the Gpabc1-encoded ABC transporter is necessary for tolerance of G. pulicaris to rishitin and that this tolerance is required for virulence on potato.

Metabolism of the Phytoalexin Rishitin by Gibberella pulicaris Is Highly Reduced in Liquid Culture

Gibberella pulicaris is a causal agent of potato dry rot. The fungus is able to metabolize the potato phytoalexin rishitin, a trait which is possibly associated with virulence against potato tubers. Metabolism of the plant defence compound on agar medium is completed within 24 h. In contrast, incubations in various liquid media and buffers highly reduced degradation of rishitin with a maximal reduction of substrate down to 30% of the initial concentration within five days. The structurally related sesquiterpene lubimin was degraded completely within 12 hr in all tested liquid media. Our data suggest that rishitin metabolism is under an unusual genetic control requiring growth on a solid surface for efficient metabolism.

Effects of Antagonist Cell Concentration and Two-Strain Mixtures on Biological Control of Fusarium Dry Rot of Potatoes

Eighteen bacterial strains were individually assayed against Gibberella pulicaris (5 × 105 conidia per ml) by coinoculating antagonist and pathogen in wounds in cv. Russet Burbank potatoes. All antagonist concentrations (106, 107, and 108 CFU/ml) decreased disease (38 to 76% versus control, P < 0.05). When four strains were assayed at 11 concentrations (range 105 to 108 CFU/ml) against G. pulicaris, linear regression of the log-dose, log-response data was significant for all four strains (P < 0.001 to 0.01, R2 = 0.50 to 0.74). Challenging G. pulicaris with all possible antagonist pairings within 2 sets of 10 antagonist strains (5 × 105 CFU of each strain per ml) resulted in 16 of 90 pairs controlling disease better than predicted based on averaging the performance of the individual strains making up the pair (P < 0.10). Successful pairs reduced disease by ~70% versus controls, a level of control comparable to that obtained with 100 times the inoculum dose of a single antagonist strain. Neither strain genus nor soil of origin were useful in predicting successful antagonist pairs. Factors potentially influencing dose-response relationships and the effectiveness of antagonist pairs in controlling disease are discussed.