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Background Bacillus velezensis is an endospore-forming, free-living soil bacterium with potential as a biopesticide against a broad spectrum of microbial pathogens of plants. Its potential for commercial development is enhanced by rapid replication and resistance to adverse environmental conditions, typical of Bacillus species. However, the use of beneficial microbes against phytopathogens has not gained dominance due to limitations that may be overcome with new biopesticidal strains and/or new biological knowledge. Results Here, we isolated B. velezensis strain 9D-6 and showed that it inhibits the in vitro growth of prokaryotic and eukaryotic pathogens, including the bacteria Bacillus cereus, Clavibacter michiganensis, Pantoea agglomerans, Ralstonia solanacearum, Xanthomonas campestris, and Xanthomonas euvesicatoria; and the fungi Alternaria solani, Cochliobolus carbonum, Fusarium oxysporum, Fusarium solani, Gibberella pulicaris, Gibberella zeae, Monilinia fructicola, Pyrenochaeta terrestris and Rhizoctonia solani. Antimicrobial compounds with activity against Clavibacter michiganensis were isolated from B. velezensis 9D-6 and characterized by high resolution LC-MS/MS, yielding formulae of C52H91N7O13 and C53H93N7O13, which correspond to [Leu7] surfactins C14 and C15 (also called surfactin B and surfactin C), respectively. We further sequenced the B. velezensis 9D-6 genome which consists of a single circular chromosome and revealed 13 gene clusters expected to participate in antimicrobial metabolite production, including surfactin and two metabolites that have not typically been found in this species - ladderane and lantipeptide. Despite being unable to inhibit the growth of Pseudomonas syringae DC3000 in an in vitro plate assay, B. velezensis 9D-6 significantly reduced root colonization by DC3000, suggesting that 9D-6 uses methods other than antimicrobials to control phytopathogens in the environment. Finally, using in silico DNA-DNA hybridization (isDDH), we confirm previous findings that many strains currently classified as B. amyloliquefaciens are actually B. velezensis. Conclusions The data presented here suggest B. velezensis 9D-6 as a candidate plant growth promoting bacterium (PGPB) and biopesticide, which uses a unique complement of antimicrobials, as well as other mechanisms, to protect plants against phytopathogens. Our results may contribute to future utilization of this strain, and will contribute to a knowledge base that will help to advance the field of microbial biocontrol. Electronic supplementary material The online version of this article (10.1186/s12866-018-1380-8) contains supplementary material, which is available to authorized users.

Hot pepper (Capsicum annum L.) anthracnose has been a destructive disease in commercial pepper production fields (Harp et al., 2008; Lewis-Ivey et al., 2004; Park and Kim, 1992), and in Korea, the disease is estimated to cause a loss of more than US$100 million annually (Kim and Park, 1998). Furthermore, since the pepper anthracnose outbreak in 2002, this disease has received high priority (Kim et al., 2008). Several Colletotrichum spp., such as C. acutatum, C. coccodes, C. dematium, and C. gloeosporioides, thought to be causal agents of anthracnose (Park and Kim, 1992). However, more recently, C. acutatum was identified as the primary Colletotrichum species for anthracnose in pepper (Kim et al., 2008). Similar outbreaks of anthracnose on peppers have occurred in Ohio (Lewis-Ivey et al., 2004) and Florida, USA (Harp et al., 2008), and C. acutatum was identified as the primary Colletotricum species for anthracnose in pepper (Lewis-Ivey et al., 2004). C. acutatum (teleomorph: Glomerella acutata) is an important anthracnose pathogen on a wide range of host plants, causing significant economic loss in various crops, including apple, almond, citrus, strawberry, tomato, and hot pepper (Sutton, 1992; Freeman et al., 1998; Peres et al., 2005). Various fungicides have been identified to control C. acutatum, including copper compounds (e.g., copper hydroxide), the quinone outside inhibitors (azoxystrobin, trifloxystrobin, or pyraclostrobin), triazoles, dithiocarbamates, and benzimidazole compounds (Harp et al., 2014; Wedge et al., 2007). Notably, C. acutatum is tolerant to benomyl and other benzimidazole fungicides (Adaskaveg and Hartin, 1997; Peres et al., 2002; Talhinhas et al., 2002; Talhinhas et al., 2005). The azole fungicides are the most effective in inhibiting in vitro growth of C. acutatum (Paredes and Munoz, 2002), but the rapid development of fungicide resistant strains has limited their use. An understanding of the fungicide-resistant mechanisms will help to enhance control of anthracnose in pepper. Phytopathogenic fungi have developed various biological mechanisms that provide resistance to fungicides or abiotic stresses. Genes responsible for this resistance include ATP-binding cassette (ABC) transporters. For example, a gene deletion mutant of an ABC transporter, ABC1in Magnaporthe oryzae, showed hypersensitivity to several drugs (Urban et al., 1999). Moreover, ABC1 (Urban et al., 1999), ABC3 (Schneider and Hunke, 1998), ABC4 (Gupta and Chattoo, 2008), and ABC5 (Kim et al., 2013) from M. oryzae, GpABC1 from Gibberella pulicaris (Fleissner et al., 2002), Mgatr4 from Mycosphaerella graminicola (Stergiopoulos et al., 2003) and BcatrB from Botrytis cinerea (Schoonbeek et al., 2001) are required for pathogenicity. ABC-transporter proteins utilize energy derived from the hydrolysis of ATP to “pump” the substrate across a membrane, thus effectively reducing intracellular concentration to less toxic levels. The proteins are defined by the presence of amino acid sequences such as the ABC-ATPase domain, ABC domain, or nucleotide-binding domain. This domain contains the two peptide motifs Walker A (p-loop) and a hydrophobic Walker B motif (Walker et al., 1982). Both motifs are involved in ATP-binding proteins and identified as ATP signatures (Hyde et al., 1990). In addition, transmembrane domains are embedded in cell membranes that consist of at least six transmembranes. Until now, no ABC transporter genes have been isolated and characterized in C. acutatum. To begin defining the functional significance of the ABC transporter gene in C. acutatum, we are the first to identify a partial cDNA that encoded an ABC transporter, CaABC1, in C. acutatum. We also present the corresponding full-length gene structure of CaABC1 with the motifs. C. acutatum CaABC1 is most closely related to the ABC transporter {"type":"entrez-protein","attrs":{"text":"XP_007590216","term_id":"615436115","term_text":"XP_007590216"}}XP_007590216 of C. fioriniae. CaABC1 also shares a high degree of homology with the other Colletotrichum spp., including C. higginsianum, C. sublineola, C. graminicola, C. orbiculare, and C. gloeosporioides. CaABC1 was up-regulated in conidiation, abiotic stresses, and multiple fungicides. To our knowledge, this is the first structural and functional analysis of an ABC transporter gene in C. acutatum. Our results will provide the basis for further study on the function of ABC transporter genes in fungicide resistance and pathogenicity in C. acutatum.

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.

Several fusarioid microorganisms were isolated as potential pathogens of hop (Humulus lupulus 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), Gibberella pulicaris (anamorph: Fusarium sambucinum) 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 (Hydraecia micacea) 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.

A gene encoding the ATP-binding cassette (ABC) transporter FcABC1 was characterised in Fusarium culmorum. It is homologous to two transporters, Magnaporthe grisea ABC1 and Gibberella pulicaris ABC1, that play a role in plant pathogenesis. Transcripts of FcABC1 were present during infection of barley roots. A FcABC1 replacement mutant was produced and in a point inoculation experiment of wheat heads, the mutant displayed reduced aggressiveness, indicating that FcABC1 has a function during infection of plant tissue. It is hypothesised that wheat contains an antifungal compound that inhibits the spread of the fungus in the plant and to which FcABC1 confers resistance.

▪ Abstract Gibberella species are destructive plant pathogens, although many are more familiar under their Fusarium anamorph names. The recent synthesis of phylogenetic, biological, and morphological species approaches has revitalized taxonomy of a genus that was first described almost 200 years ago. Twelve sexual species of Gibberella of agricultural importance were selected for this review to represent phylogenetic, biological, and chemical diversity of the genus. Even closely related Gibberella species can differ in reproductive mode, geographic and host distribution, plant pathogenesis, and production of toxins and other biologically active metabolites. Gibberella species have proven amenable to meiotic and molecular genetic analysis; A complete genome sequence of G. zeae should soon be available. Combining gene disruption strategies with new genomics technologies for expression profiling should help plant pathologists to understand the pathological and evolutionary significance of biological and chemical diversity in Gibberella and to identify novel strategies for disease control.

Chemical sprout inhibitors are applied to over 50% of the potato harvest to extend storage time. In the U.S.A., CIPC (1-methylethyl-3-chlorophenylcarbamate) is the only synthetic chemical registered for postharvest sprout control of stored potatoes, and it is the most widely used sprout inhibitor world-wide. Due to environmental and health safety concerns, the use of CIPC has become more restricted, and alternative sprout control methods are sought. Six bacteria strains, exhibiting superior dry rot suppressiveness in previous research, were grown in two different liquid culture media and sprayed on Russet Burbank potatoes. In growth chamber and pilot experiments repeated at two storage sites in two successive years, all six isolates demonstrated significant sprout control capabilities when applied after growth on at least one of the culture media supplied. Of the six strains tested, Pseudomonas fluorescens bv.V S11:P:12 and two strains of Enterobacter sp., S11:T:07 and S11:P:08, exhibited highest relative...

Two heptapeptides with broad antifungal activity were identified and assessed for their ability to act synergistically with thiabendazole. The hexapeptide 66–10 was the progenitor of the heptapeptides and exhibited minimal inhibitory concentrations (MICs) of 9.3 to 9.8 μg/ml for thiabendazole (TBZ) resistant Fusarium sambucinum strains (MIC of 186 to 312 μg/ml). Heptapeptide derivatives 77–3 and 77–12 exhibited MICs between 3.8 and 7.5 μg/ml against the same strains. Incubation of conidia or mycelia with the peptide 77–3 showed that treated fungal structures were stained by the membrane impermeant dye SYTOX Green indicating disruption of membranes. Conidia incubated with peptide 77–3 at 10 μg/ml showed a 91 ± 3.6% reduction in viability in 15 min. A checkerboard method was used to test the peptides and TBZ individually and in combination to determine potential synergistic activity. The results indicate that small peptides can act synergistically with TBZ against TBZ-resistant F. sambucinum.

Gibberella pulicaris (anamorph: Fusarium sambucinum), is a common potato pathogen that produces trichothecene mycotoxins and reduces plant yield and tuber quality. Field isolates have been recovered from western Canada that are resistant to thiabendazole, a benzimidazole fungicide registered in many countries to control dry rot in storage. To characterize this resistance, homokaryons were derived from single ascospores that were produced in vitro from crosses of thiabendazole-resistant G. pulicaris field isolates with sensitive isolates of an opposite mating type. The β-tubulin gene was amplified from these homokaryons using degenerate oligonucleotides synthesized to conserved sequences of the β-tubulin gene derived from related fungi. An open reading frame containing three putative introns and a deduced amino acid sequence of 446 amino acids exhibited a high level of homology to the β-tubulin gene of other fungi. Although nucleotide and amino acid differences were observed in the β-tubulin gene, none wer...

Sulfate assimilation plays a vital role in prototrophic organisms. Orthologues of the alternative sulfate transporter (AstA) gene from Aspergillus nidulans were identified in the fungal plant pathogens Fusarium sambucinum and Fusarium graminearum. By physiological and biochemical analyses, the AstA orthologues were determined to be able to uptake sulfate from the environment. Similarly to astA in A. nidulans, the FsastA gene was found to be regulated by sulfur metabolite repression (SMR) in a sulfur-dependent manner. In contrast, the FgastA transcript was undetectable, however, when the FgastA gene was expressed heterologously in A. nidulans, the translated FgAstA protein acted as a sulfate transporter. Interestingly, F. sambucinum astA expression was remarkably augmented in infected potato tubers, despite the presence abundant sulfate and was found not to be correlated with plant resistance.

The process of selecting biological control agents for further development frequently does not involve conducting bioassays of strain effectiveness on a range of pathogen isolates or host cultivars. Additionally, though previous studies have demonstrated that the medium used to produce biomass of an antagonist can alter its efficacy, this factor is also rarely considered when selecting for the most effective antagonist. Host cultivar, pathogen isolate, and the cultivation medium used to produce the antagonists' biomass were examined as factors of potential importance for assessing the relative effectiveness of bacterial biocontrol strains accurately. Five bacterial antagonists that control Fusarium dry rot on stored potato tubers were assayed for effectiveness against 10 isolates of Gibberella pulicaris . All antagonists reduced disease severity (35-81%) regardless of the specific assays conducted. However, when the antagonists' biomass were produced on two media that differed both in nutrient composition...

ATP citrate lyase (ACL) catalyzes the formation of cytosolic acetyl-CoA, which is mainly used for the biosynthesis of fatty acids and sterols. In this paper, we show for the first time that in filamentous fungi two different subunits of ACL are encoded by two separate genes. This is in contrast to animals where ACL is encoded by a single gene. Data are presented on acl genes from the filamentous fungi Sordaria macrospora and Gibberella pulicaris. In S. macrospora, both genes, acl1 and acl2, are clustered within a region of 10 kb and are divergently transcribed.

Four newly isolated strains of endophytic fungi namely Gibberella pulicaris, Acremonium sp., `Synnematous' sp. and Nodilusporium sp. were compared for their degradative activity on raw and gelatinized starches, substrate specificity and optimum pH. Results showed that the raw starch-degrading enzyme from Acremonium sp. had a broad activity towards both small and large granule size of raw starches while the enzyme from other strains showed high activity toward starches of smaller granule size. Analysis of the end product by TLC showed that enzyme from Gibberella pulicaris, Acremonium and Nodilusporium sp. hydrolysed raw sago starch to produce solely glucose but the enzyme of `Synnematous' sp. produced glucose and maltose.

Lack of effective chemicals impedes control of Fusarium dry rot of stored potato tubers destined for processed and table stock use. Biological control of dry rot incited byGibberella pulicaris (anamorph=Fusarium sambucinum) has been demonstrated in laboratory studies but not in commercial storage environments. Several Gram-negative bacterial strains that were efficacious and amenable to production in liquid culture in laboratory studies were selected for pilot studies in Idaho and for bin trials at commercial storage houses in Idaho and North Dakota. In the first year of pilot studies,Pseudomonas fluorescens S22:T:04 (≈1 x 108 cfu/ml) decreased dry rot caused byG. pulicaris by 19% when coinoculated with the pathogen compared to controls and to the fungicide thiabendazole (TBZ) applied at label rates. In second year pilot studies,P. fluorescens P22:Y:05 andEnterobacter cloacae S11:T:07 (≈4 x 108 cfu/ml) reduced the severity of disease incited byG. pulicaris (25% and 17% average disease decrease, respectively) when antagonists were applied after pathogen inoculum. Treatment effects on dry rot that developed from inoculation withNectria haematococca (anamorph=Fusariumsolani var.coeruleum) were variable and influenced by interactions between antagonists, a wetting agent, and TBZ. In commercial storage bin trials,E. cloacae S11:T:07 reduced naturally occurring levels of dry rot by an average of 21% compared to 14% for TBZ, demonstrating that this antagonist was effective when produced using a liquid culture medium and methodology that approximates commercial practices for producing biomass.

The potato pathogen Gibberella pulicaris (Fusarium sambucinum) is able to metabolize the potato saponin α-chaconine by first removing the 1,2-bound l-rhamnose. The catalyzing enzyme, α-chaconinase, is inducible by the substrate and α-solanine and α-tomatine. The protein with a molecular mass of about 95 kDa was purified by fractionated ammonium sulfate precipitation followed by concanavalin A-Sepharose chromatography and chromatofocusing. The enzyme is active over a wide pH and temperature range and is highly substrate specific for α-chaconine with a Km value of 0.97 mM and Vmax of 37.13 nkat. α-Solanine and α-tomatine are not converted by the enzyme.

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.

Two strains of the potato pathogen Gibberella pulicaris were analysed for the ability to metabolize the phytoalexin rishitin and for virulence on potato tubers. While strain R-6380 was always virulent and able to metabolize the phytoalexin, strain R-7843 showed significant variation in both traits. Analysis of eight sectors of a radially grown culture of this strain revealed that six had a virulent, rishitin-metabolizing phenotype, one had an avirulent, rishitin-nonmetabolizing phenotype, and one had an intermediate phenotype for both traits. Similar results were found for single spores isolated from one of two spore suspensions from different agar cultures. In all cases high virulence was associated with fast metabolism of rishitin; weakly virulent strains only slowly degraded the phytoalexin. Restriction-fragment-length-polymorphism (RFLP) analysis of sectors with contrasting phenotypes and measurement of the nuclear DNA content from these sectors indicate that the sectors were identical to the original strain (R-7843) with respect to these traits. It is therefore concluded that the variations observed in virulence and in the ability to metabolize rishitin are the result of spontaneous genetic changes in strain R-7843, The concurrent changes in tolerance, metabolism and virulence are further indicators of a relationship among these traits and lend support to the hypothesis that phytoalexin metabolism is important in pathogenicity. Zusammenfassung Zwei Stamme des Kartoffelpathogens Gibberella pulicaris wurden auf ihre Fahigkeit zum Abbau des Phytoalexins Rishitin und auf Vinilenz gegenuber Kartoffelknollen untersucht, Wahrend der Stamm R-6380 in alien Experimenten viruletit und in der Lage war, das Phytoalexin abzubauen, zeigte der Stamm R-7843 eine deutliche Variabilitat dieser beiden Merkmale, Die Analyse von acht Sektoren einer radial gewachsenen Kultur dieses Stammes ergab, das sechs einen virulenten, Rishitin metabolisierenden, einer einen avirulenten und Rishitin nicht-abbauenden und der achte Sektor einen intermediaren Phanotyp beider Merkmale aufwiesen, Ahnliche Ergebnisse resultierten aus der Analyse einer von zwei Sporensuspensionen verschiedener Agarkulturen. In alien Fallen war hohe Virulenz mit schnellem Abbau von Rishitin korreliert und schw ache Virulenz mit langsamem Abbau des Phytoalexins. RFLP Analysen und die Messung des nuklearen DNA-Gehaltes von Sektoren mit entgegengesetztem Phanotyp zeigten, das sich diese Sektoren in den untersuchten Merkmalen nicht von dem Ausgangsstamm (R-7843) unterschieden, Daraus ergibt sich, das die beobachtete Variabilitat der Virulenz und die Fahigkeit zum Abbau von Rishitin das Resultat spontaner genetischer Veranderungen des Stammes R-7843 sind. Die gemeinsame Veranderung von Toleranz, Rishitin- Metabolismus und Virulenz ist ein weiteres Indiz fur eine kausale Beziehung zwischen diesen Merkmalen und unterstutzt die Hypothese, das der Abbau von Phytoalexinen fur Pathogenitat von Bedeutung ist.

Fusarium sambucinum causes dry rot in tubers of Solanum tuberosum . Since relatively little is known about the nature of dry rot disease development, infection of F. sambucinum on tissue discs prepared from tubers of potato varieties that are susceptible or moderately resistant to this disease was studied. Increases in lignin content, and polyphenol oxidase and peroxidase activities were detected after infection. Peroxidase increased most strongly in tuber tissue that was just ahead of the advancing hyphae. Constitutive polyphenol oxidase activity was highest in the more resistant as compared to susceptible varieties. Polyphenol oxidase and peroxidase activities as well as content of chorogenic acid and the sesquiterpenes rishitin, lubimin, phytuberin and phytuberol increased as a result of infection. These increases correlated with the amount of tissue infected rather than the resistance of the tuber tissue to infection.

Potato tubers accumulate varying amounts of several saponins preferentially in the peel. These compounds are toxic to living cells containing sterols in their plasma membrane and are therefore thought to be preformed chemical defence compounds. Two strains of the potato pathogen Gibberella pulicaris (Fusarium sambucinum) , R-6380 and R-7843, were analysed for their ability to metabolize the most predominant saponins found in tubers, α-chaconine and α-solanine. The first compound is degraded by both strains via removal of α-1,2- l -rhamnose leading to β 2 -chaconine. This product is converted to the aglycone, solanidine, which is further metabolized to unknown products. The release of α-1,2- l -rhamnose is also the first step in the break down of α-solanine by strain R-6380, followed by the removal of the β-1,3-bound glucose molecule leading to γ-solanine, which is not metabolized any further. Strain R-7843 is not able to metabolize α-solanine. Crude protein extracts of the culture fluid of both strains contained enzymes able to convert α-chaconine to β 2 -chaconine, but with no α-solanine metabolic activity. This result indicates that G. pulicaris excretes enzymes specific for different saponins.

A pair of primers was desingned from conserved regions of the Tri5 genes of Fusarium sporotrichioides, F. poae, Gibberella pulicaris, and G. zeae. A test panel of 64 different species and varieties of Fusarium, as well as isolates belonging to other fungal genera, bacteria, and plants was tested for a positive reaction in an optimized PCR assay. A fragment of the appropriate size (650 bp) was produced with DNA of 18 species and varieties of Fusarium and of Cladobotryum dendroides NRRL 2903. The method was used to detect trichothecene producing fusaria in contaminated wheat malt samples. 7W5-PCR product sequences were used for the development of primers specific to subgroups of trichotecene producers.

Field-grown tubers of 22 progenies ofSolanum tuberosum L. generated in a crossing programme involving seven parents differing in resistance toFusarium coeruleum Lib. ex Sacc. andF. sulphureum Schlect. (=F. sambucinum Fuckel, teleomorphGibberella pulicaris (Fr.) Sacc.) were wound-inoculated with a cornmeal + sand culture of each pathogen. Parental genotypes were also included. The mean lesion size of each progeny was compared in 2 years of tests, as well as with published data on glasshouse-grown tubers. ForF. coeruleum there was a high correlation between years as well as with the glasshouse data, but no such correlations were apparent withG. pulicaris. Furthermore, parental and GCA values, as well as progeny means and mid-parent scores, also correlated highly forF. coeruleum but not forG. pulicaris. Glasshouse-grown tubers of 11 wildSolanum spp. were also inoculated with both pathogens. Some resistance to one or other, or both, was apparent, particularly inS. chacoense.

Two genomic fragments capable of driving the expression of the hygromycin B resistance gene (hph) were isolated from the phytopathogenic ascomycete Gibberella pulicaris (anamorph Fusarium sambucinum) using a "promoter-probe library" strategy. Two libraries consisting of random, 0.5-2.0-kb fragments of genomic DNA inserted 5' of a promoterless hph gene were constructed and used for transformation of G. pulicaris. Both libraries transformed G. pulicaris at a low frequency. Transformants tolerated up to 800 micrograms/ml of hygromycin B, while untransformed colonies were inhibited completely by 50 micrograms/ml of the antibiotic. Plasmids were re-isolated from transformants by simply digesting, the genomic DNA with KpnI, which cuts once in the polylinker 5' to the insert, and transforming E. coli with the re-ligated DNA. The recovered plasmids transformed G. pulicaris with a frequency of up to 4.4 transformants/micrograms of DNA. Both promoter fragments were sequenced and found to contain TATA and CAAT boxes as well as CT-rich sequences. This method makes it possible to easily isolate many fragments with promoter activity from filamentous fungi, and should facilitate the investigation of the promoter structures necessary for the expression of fungal genes.

Gibberella pulicaris is the sexual stage ofFusarium sambucinum, the major cause of potato tuber dry rot in North America and Europe. In a previous study 42 strains from dry-rotted tubers in North America were analyzed for genetic diversity by mating type and vegetative compatibility group (VCG) tests. In the present study, an additional 96 strains from North America, 21 strains from Europe, and six strains from other geographic areas were analyzed. Both mating types, MAT1-1 and MAT1-2, occur widely outside North America. In contrast, all but two, Canadian, strains of the 138 North American strains analyzed to date are MAT1-1. VCG analysis provides additional evidence that genetic diversity ofG. pulicaris from potato tubers is high in Europe but low in the United States. These results suggest that the potential for pathogen change due to sexual recombination is low in the United States, but may be higher in Europe. Limited genetic diversity ofG. pulicaris in the United States indicates that there are likely to be few barriers to heterokaryon formation and asexual recombination in the field. Thus, the potential exists for rapid spread and persistence of advantageous mutations throughout theG. pulicaris population, which may have contributed to the recent proliferation of strains resistant to the fungicide thiabendazole which has been widely used to control tuber dry rot.

The potato phytoalexin lubimin displayed a complex pattern of metabolism by strains of the potato pathogen Gibberella pulicaris (anamorph: Fusarium sambucinum). The metabolites 15-dihydrolubimin and isolubimin were common to both lubimin-sensitive and lubimin-tolerant strains. In one lubimin-tolerant strain, several unique metabolites, including the tricyclic compounds cyclodehydroisolubimin and cyclolubimin (2-dihydrocyclodehydroisolubimin) and their epoxides, accumulated at the expense of 15-dihydrolubimin and isolubimin. These tricyclic compounds were not toxic to the lubimin-sensitive strain. These results indicate that a likely pathway for lubimin detoxification in some strains of G. pulicaris involves cyclization of isolubimin to cyclodehydroisolubimin via an unsaturated intermediate. All highly virulent strains tested were tolerant of lubimin and were able to convert lubimin to apparently nontoxic products which is indirect evidence that lubimin detoxification contributes to virulence of G. pulicaris on potato tubers.

The Tox5 gene encodes trichodiene synthase, the first unique enzyme in the trichothecene biosynthetic pathway. In Gibberella pulicaris R-6380, the level of Tox5 mRNA was found to increase 47-fold in early stationary phase. Sequence analysis of the Tox5 promoter regions from geographically distinct strains of G. pulicaris revealed the existence of two Tox5 alleles (Tox5-1 and Tox5-2). All G. pulicaris strains that produce high levels of trichothecenes in liquid culture carry a 42-nucleotide (nt) tandem repeat sequence (Tox5-1) in the Tox5 promoter region, whereas strains that produce low levels of trichothecenes carry a single copy of this sequence (Tox5-2). A genetic cross between high- and low-level trichothecene producers resulted in the cosegregation of higher-level trichothecene production with the Tox5-1 allele. To determine the importance of the 42-nt repeat sequence in the regulation of Tox5 expression, reporter gene constructs carrying either the Tox5-1 or the Tox5-2 promoter region fused to the beta-galactosidase gene of Escherichia coli were introduced into the high-level-trichothecene-producing strain, R-6380. Expression of reporter gene activity in transformants was found to be regulated in a manner similar to Tox5 expression but appeared to be independent of the 42-nt sequence copy number. These results indicate that transcriptional controls play an important role in the regulation of Tox5 expression and that genes involved in trichothecene biosynthesis in G. pulicaris may be linked to Tox5.

Variation within the internal transcribed spacers (ITS1 and ITS2) and 5.8s ribosomal DNA gene of the heterothallic phytopathogenic filamentous fungus, Fusarium sambucinum (teleomorph = Gibberella pulicaris), was examined in 86 strains from diverse geographical locations by PCR amplification and direct sequencing in order to measure intraspecific divergence within the ITS region. Sequence analysis revealed three ITS types (A, B, C), within which divergence was extremely low (0-2.3%). Surprisingly, the level of intraspecific divergence observed between ITS types, A----B = 14.3%, A----C = 15%, and B----C = 4.6%, is much greater than that reported for any other species. The degree to which transition/transversions and insertion/deletions make up the pattern of ITS sequence evolution both within and between types was analyzed. The sequences of the ITS types exhibit a C-T transition bias together with a GC insertion/deletion bias. In comparison, the genic flanking sequences, including the 5.8s rDNA gene and 5' end of the 28s large nuclear rDNA, are highly conserved. By the criteria of mating and DNA-DNA hybridization, all the strains examined represent a single species. Discordance between the ITS sequence data and other molecular and genetic data on F. sambucinum is discussed.

Gibberella pulicaris (Fusarium sambucinum) is a major cause of dry-rot of stored potatoes (Solanum tuberosum) worldwide. The ability of field strains ofG. pulicaris to cause dry-rot is correlated with their ability to detoxify sesquiterpene phytoalexins produced by potato. All highly virulent field strains can detoxify the sesquiterpenes rishitin and lubimin. Meiotic recombinational analysis indicates that rishitin detoxification can be controlled at two or more loci. High virulence has been associated with one of these loci, designatedRiml. Detoxification of rishitin and lubimin comprises a complex pattern of reactions involving epoxidation, dehydrogenation, and cyclization. To date, seven lubimin metabolites and one rishitin metabolite have been characterized. Genes for rishitin and lubimin detoxification are being cloned fromG. pulicaris in order to more rigorously analyze the role and regulation of sesquiterpene metabolism in potato dry-rot. Our results indirectly support a role for sesquiterpene phytoalexins in resistance of potato tubers to dry-rot and may enhance research on alternative control strategies for this economically important potato disease.

An endophytic fungus, Gibberella pulicaris, produced an amylase which degraded raw starches from cereals and other crops including raw potato, sago, tapioca, corn, wheat and rice starch. In each case, glucose was the main product. Among the raw starches used, raw potato starch gave the highest enzyme activity (85 units mg−1 protein) and raw wheat starch the lowest (49 units mg−1 protein). The highest amylase production (260 units mg−1 protein) was achieved when the concentration of raw potato starch was increased to 60 g l−1. Optimum hydrolysis was at 40 °C and pH 5.5.

La trachéomycose des caféiers, due à Gibberella xylarioides (anamorphe F.xylarioides) a été observée pour la première fois sur Coffea excelsa en République Centre Africaine en 1927. Durant les années 1940 et 1950 la maladie a causé des destructions considérables de C. canephora dans plusieurs pays d'Afrique. Un arrachage systématique des plants infectés et une replantation de variétés résistantes ont permit une "éradication" de la maladie. Au début des années 1970 la maladie est réapparue en RDC et s'est étendue à l'Ouganda (1993), puis à la Tanzanie (2000) sur C. canephora. Chez C.arabica, la maladie est signalée uniquement en Ethiopie depuis 1957. L'origine de cette maladie et les processus de dispersion de l'agent pathogène sont inconnus. Pour aborder ces questions, une analyse de la structure des populations et de la spécificité d'hôte de F. xylarioides ont été réalisées. Des populations contemporaines de F. xylarioides provenant de C. canephora et de C. arabica et des isolats historiques (provenant de la première émergence de la maladie) ont été analysés à l'aide de onze marqueurs microsatellites. Les deux populations contemporaines sont différenciées, mais respectivement constituées d'un seul génotype. Des inoculations en condition contrôlées d'isolats de ces populations montrent une spécialisation sur l'hôte d'origine. Ces résultats suggèrent une divergence ancienne des populations contemporaines. Cependant, l'aire d'origine de F. xylarioides n'a pu être identifiée avec les échantillons analysés. La reproduction sexuée est observée au champ dans chaque population et la proportion des gènes sexuels MAT1-1 et MAT1-2 est 1/1. L'absence de diversité est ainsi vraisemblablement due à des forts effets de fondation lors de l'expansion de la maladie. Dans le cas de RDC, le génotype des isolats contemporains et d'un isolat historique collectés en 1960 sont identiques. L'origine de la réermergence de la maladie peut donc être locale. (Texte intégral)

Fungus strains designated as Fusarium sambucinum, F. torulosum, or Fusarium sp. nov. were crossed with MAT1-1 and MAT1-2 tester strains of Gibberella pulicaris. Of the 40 field strains that were crossed with the tester strains, 13 strains produced fertile crosses and 27 strains did not produce fertile crosses. One strain designated as F. torulosum was fertile with a tester strain of G. pulicaris, suggesting that this is an intraspecies cross and that the strain is G. pulicaris, and, consequently, F. sambucinum rather than F. torulosum. The lack of fertile crosses between tester strains and 27 of the 40 field strains suggests that these strains are not G. pulicaris. Although the ability to form a fully fertile cross with a tester strain can determine the species of a fertile strain, it is more problematic to exclude a strain only because it is infertile.

Enterobacter cloacae S11: T:07 (NRRL B-21050) is a promising biological control agent that has significantly reduced both fungal dry rot disease and sprouting in laboratory and pilot potato storages. The metabolites phenylacetic acid (PAA), indole-3-acetic acid (IAA), and tyrosol (TSL) were isolated from S11:T:07 liquid cultures provided with three different growth media. The bioactivities of these metabolites were investigated via thin-layer chromatography bioautography of antifungal activity, wounded potato assays of dry rot suppressiveness, and cored potato eye assays of sprout inhibition. Relative accumulations of PAA, IAA, and TSL in cultures were nutrient dependent. For the first time, IAA, TSL, and PAA were shown to have antifungal activity against the dry rot causative pathogen Gibberella pulicaris, and to suppress dry rot infection of wounded potatoes. Disease suppression was optimal when all three metabolites were applied in combination. Dosages of IAA that resulted in disease suppression also resulted in sprout inhibition. These results suggest the potential for designing culture production and formulation conditions to achieve a dual purpose biological control agent able to suppress both dry rot and sprouting of stored potatoes.

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 .

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.

α-Tomatine is a steroidal glycoalkaloid present in Solanaceous plants, principally in a number of Lycopersicon and Solanum species. High concentrations are found in leaves, stems, rootsand green fruit of tomato plants, suggesting a possible role in resistance to pathogens. The toxic effects of α-tomatine are attributed to its ability to complex with membrane sterols, causing pore formation and leakage of cell contents. Pathogenic fungi to tomato are usually less sensitive to tomatine than pathogens of other plant species and saprophytes. Fungi have evolved two principal mechanisms to avoidthe toxic effect of tomatine, either changing the composition of the cell membrane or producing specific tomatine-detoxifying enzymes, known as tomatinases. Tomatinases have distinct mechanisms of action, and molecular and catalytic properties in different pathogen species. Up to now, the knowledge of α- tomatine metabolism by phytopathogenic fungi is almost exclusively limited to the removal of sugar residues from the molecule, although recent studies in Gibberella pulicaris demonstrate that further hydroxylations of the aglycone moiety tomatidine occur, after the action of tomatinase. Tomatinase-encoding genes have been cloned from Septoria lycopersici and Fusarium oxysporum . Both genesdo not share sequence homology, suggesting that different tomato pathogens have independently developed the ability to cleave tomatine by convergent evolution. Expression of F. oxysporum tomatinase is fully repressed in the presence of glucose, indicating a possible role in nutrition besides detoxification of tomatine, although in Botrytis cinerea glucose repression of tomatinase is not observed. Othergenes different to tomatinase are induced by a-tomatine, but the complete significance of this phenomenon is still unknown.

Tolerant strains of Gibberella pulicaris (anamorph: Fusarium sambucinum) metabolize the phytoalexin xanthotoxin to 5-(2-carboxyethyl)-6-hydroxy-7-methoxybenzofuran. This compound does not inhibit the growth of strains that are sensitive to xanthotoxin itself and all xanthotoxin-tolerant and xanthotoxin-sensitive strains tested were able to further degrade the metabolite. This is the first report of fungal detoxification of a furanocoumarin.

Gibberella pulicaris, a causal agent of potato dry rot, infects potato tubers via wounds, where it is exposed to the phytoalexins rishitin and lubimin. Incubation of mycelium on agar supplemented with phytoalexins transiently induced the transcription of a polyubiquitin gene consisting of four ubiquitin units arranged head to tail; the fourth unit contains a 54-bp intron and an additional glutamine at the C-terminus of the encoded protein. Southern analysis of the G. pulicaris genome revealed one copy of the isolated polyubiquitin gene and one or two copies of other ubiquitin genes. Increased transcription of the gene was detectable above a threshold of 100 microg/ml of rishitin and at elevated temperatures, which indicates that exposure to phytoalexins causes a stress reaction of hyphal cells similar to that after heat shock.

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.

The potato ( Solanum tuberosum ) crop is generally harvested during February and March in most regions of India. This is a time when temperature starts increasing to between 30°C and 40°C in the month of June followed by rains in …

A genetically fertile, trichothecene-producing plant pathogen, Gibberella pulicaris (Fusarium sambucinum), was transformed with three different vectors: cosHyg1, pUCH1, and pDH25. All three vectors carry hph (encoding hygromycin B phosphotransferase) as the selectable marker. Transformation frequency was 0.03 transformants per mumg of DNA for pDH25 and 0.5 for pUCH1 or cosHyg1. The vector DNA sequences integrated at different sites into the fungal genome. Transformants were classified into three types based upon distinctive integration patterns: type A contained a single, intact copy of the vector at one site per genome; type B contained multiple tandem copies or a combination of single and multiple tandem copies at one or more sites per genome; type C contained a partial vector copy at one site per genome. While the transformants with cosHyg1 and pUCH1 were type A or B, type C was unique to pDH25 transformants. Type A and C transformants were both meiotically and mitotically stable. However, type B multiple inserts were unstable in mitosis and meiosis since: (1) multiple tandem copies were deleted; (2) rearrangements occurred during premeiosis; and (3) inserts in one of the type B transformants became methylated during premeiosis. Differential expression of transforming sequences between spore germination and mycelial growth was also observed among type B transformants. The ability to transform G. pulicaris with the resulting varied features of integration patterns and the behavior of transforming DNA during mitosis and meiosis provides a means to isolate, manipulate, and study cloned genes in this mycotoxin-producing plant pathogen.