A single amino acid substitution in highly similar endo-PGs from Fusarium verticillioides and related Fusarium species affects PGIP inhibition.

Endo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP.

Phylogeny and evolution of mating-type genes from Pyrenophora teres, the causal agent of barley "net blotch" disease.

The main aim of this study was to test the patterns of sequence divergence and haplotype structure at the MAT locus of Pyrenophora teres, the causal agent of barley 'net blotch' disease. P. teres is a heterothallic ascomycete that co-occurs in two symptomatological forms, the net form (NF) and the spot form (SF). The mating-type genes MAT1-1-1 and MAT1-2-1 were sequenced from 22 NF isolates (12 MAT1-1-1 and 10 MAT1-2-1 sequences) and 17 SF isolates (10 MAT1-1-1 and seven MAT1-2-1 sequences) collected from Sardinian barley landrace populations and worldwide. On the basis of a parsimony network analysis, the two forms of P. teres are phylogenetically separated. More than 85% of the total nucleotide variation was found between formae speciales. The two forms do not share any polymorphisms. Six diagnostic nucleotide polymorphisms were found in the MAT1-1-1 intron (1) and in the MAT1-1-1 (3) and MAT1-2-1 (2) exons. Three diagnostic non-synonymous mutations were found, one in MAT1-1-1 and two in MAT1-2-1. For comparison with P. teres sequence data, the mating-type genes from Pyrenophora graminea were also isolated and sequenced. Divergence between P. graminea and P. teres is of a similar magnitude to that between NF and SF of P. teres. The MAT genes of P. graminea were closer to those of SF than to NF, with the MAT1-2-1 SF peptide not different from the MAT1-2-1 peptide of P. graminea. Overall, these data suggest long genetic isolation between the two forms of P. teres and that hybridization is rare or absent under field conditions, with each form having some particular niche specialization. This indicates that research on resistance to P. teres should consider the two forms separately, as different species.

Population Subdivision of Fusarium graminearum Sensu Stricto in the Upper Midwestern United States.

ABSTRACT A collection of 712 Fusarium graminearum sensu stricto (s.s.) strains, predominantly gathered between 1999 and 2000 from nine states within the United States, was examined for population structure and polymerase chain reaction-based trichothecene type. Most strains belonged to a cohesive genetic population characterized by a 15-acetyldeoxynivalenol (15ADON) trichothecene type. However, using a Bayesian model-based clustering method, we also identified genetically divergent groups of strains in some sampled locations of Minnesota and North Dakota. Strains of the major group of divergent populations were of a 3ADON trichothecene type and formed a distinct cluster with a collection of previously gathered strains from Italy, which displayed all three trichothecene types (15ADON, 3ADON, and nivalenol). The co-existence of genetically divergent populations of F. graminearum s.s. in the Upper Midwest allows for the rejection of the hypothesis that F. graminearum s.s. in the United States consists of a single population. These results also suggest that recombination has been insufficiently frequent in this homothallic (selfing) fungal species to homogenize the divergent populations observed in the Upper Midwest.

First Report of Fusarium oxysporum f. sp. canariensis Causing Fusarium Wilt on Phoenix canariensis in Sardinia, Italy.

During the summer of 2004, severe symptoms of wilt were observed on 25-year-old plants of Canary Island Date Palm (Phoenix canariensis Hort. ex Chabaud) located at the seafront of Poetto Beach in the metropolitan area of Cagliari, southern Sardinia, Italy. Symptoms consisted of one-sided leaflet dieback of fronds, necrotic and brown streaking on the lower rachis base of older leaves, and necrosis of vascular bundles. Of 300 palms, there were 90 plants that were symptomatic and at least 4 were dead. Fusarium oxysporum Schlecht. emend. Snyder & Hansen has been consistently isolated from surface-sterilized petioles of symptomatic leaves sampled from affected palms. The opportunistic pathogen Gliocladium vermoesenii (Biourge) Thom was frequently associated with F. oxysporum in diseased samples, confirming previous reports of a disease complex between these two fungi (1). Five F. oxysporum isolates collected from different symptomatic plants were analyzed with a polymerase chain reaction (PCR)-based assay with the F. oxysporum f. sp. canariensis-specific primers HK66 + HK67 (2). The thermocycling schedule was as follows: initial denaturation at 94°C for 5 min, 35 cycles each of 1 min at 94°C, 1 min at 62°C, 1 min and 30 s at 72°C, followed by a final extension at 72°C for 5 min. A 567-bp PCR product of the expected size was obtained from all tested F. oxysporum isolates, allowing their identification as F. oxysporum f. sp. canariensis. This disease was previously reported from other Italian regions (Sicily, Marche, and Liguria), but its presence in Sardinia should be considered carefully since it represents a serious threat to ornamental palms, which are abundant all over the island. The source of this outbreak may be related to the importation of seedlings from areas where F. oxysporum f. sp. canariensis is widely established. References: (1) H. D. Ohr. Pink rot (Gliocladium Blight). Pages 24-25 in: Diseases and Disorders of Ornamental Palms. A. R. Chase and T. K. Broschat, eds. The American Phytopathological Society, St. Paul, MN, 1991. (2) T. R. Plyler et al. Phytopathology 89:407, 1999.

Population genetic structure of Pyrenophora teres Drechs. the causal agent of net blotch in Sardinian landraces of barley (Hordeum vulgare L.).

Monoconidial cultures of Pyrenophora teres, the causal agent of barley net blotch, were isolated from leaves collected from six populations of the barley landrace "S'orgiu sardu" growing in five agro-ecological areas of Sardinia, Italy, and genotyped using AFLPs. The 150 isolates were from lesions of either the "net form" (P. teres f. sp. teres) or the "spot form" (P. teres f. sp. maculata) of the disease. Of 121 AFLP markers, 42%, were polymorphic. Cluster analysis resolved the isolates into two strongly divergent groups (F(ST) = 0.79), corresponding to the net (45% of the isolates) and the spot (55% of the isolates) forms (designated the NFR and SFR groups, respectively). The absence of intermediate genotypes and the low number of shared markers between the two groups indicated that hybridization between the two formae is rare or absent under the field condition of Sardinia. Five of the barley populations hosted both forms but in different proportions. The SFR populations were similar in overall polymorphism to the NFR populations. However, compared to the SFR form, the NFR occurred in all fields sampled and showed a higher population divergence (F(ST) = 0.43 versus F(ST) = 0.09 with all isolates; F(ST) = 0.37 versus F(ST) = 0.06 with clone corrected samples) probably due to a lower migration rate. AFLP fingerprints resolved 117 distinct genotypes among the 150 isolates sampled (78%), 87% in SFR and 68% in NFR isolates. Although the absolute numbers may be a function of the number of AFLP markers assayed, the relative difference suggests that clonality is more prevalent among the NFR isolates (with 11 of 46 haplotypes observed more than once), compared with SFR isolates (7 of 71 haplotypes). Both digenic and multilocus linkage disequilibrium analyses suggested that sexual reproduction occurs at significant levels within the NFR and SFR populations, and that the relative contribution of sexual and asexual reproduction varies among different environments.

Occurrence and Etiology of Brown Apical Necrosis on Persian (English) Walnut Fruit.

In 1998, a severe fruit drop was observed in Italy, principally on cv. Lara Persian (English) walnut (Juglans regia). Dropped fruit showed a brown patch at the blossom end and blackening and rot of inner tissues. The disease, called brown apical necrosis (BAN), was investigated on fruit collected in Italy and France in 1999. In 2000, studies were carried out in three walnut orchards located in Italy and in France to substantiate the etiology of BAN. Isolations performed from inner diseased fruit tissues yielded several fungi, in decreasing frequency of isolation: species of Fusarium and Alternaria, and one species each of Cladosporium, Colletotrichum, and Phomopsis. However, only Fusarium spp. were recovered from stigmas of BAN-affected fruit. The fungi associated with BAN-diseased fruit and species composition differed among locations and over time, confirming results obtained in previous investigations. The species of Fusarium used in pathogenicity tests reproduced BAN-disease symptoms when inoculated on fruit, whereas an Alternaria alternata isolate caused only limited necrosis of the style. However, the role of the other fungi commonly isolated from BAN-diseased fruit remains to be defined. The walnut blight pathogen, Xanthomonas arboricola pv. juglandis, occasionally was isolated from BAN-diseased fruit. No correlation was found between the extent of external brown patches and the size of inner lesions. Repeated isolations from and inoculations of fruit demonstrated that BAN can be considered a complex disease, and the inner infections originate from the style of the fruit.

Fusarium nygamai Associated with Fusarium Foot Rot of Rice in Sardinia.

Fusarium nygamai Burgess & Trimboli was first described in 1986 in Australia (1) and subsequently reported in Africa, China, Malaysia, Thailand, Puerto Rico, and the United States. F. nygamai has been reported on sorghum, millet, bean, cotton, and in soil where it exists as a colonizer of living plants or plant debris. F. nygamai was also reported as a pathogen of the witch-weed Striga hermonthica (Del.) Benth. To our knowledge, no reports are available on its pathogenicity on crops of economic importance. In a survey of species of Fusarium causing seedling blight and foot rot of rice (Oryza sativa L.) carried out in Sardinia (Oristano, S. Lucia), F. nygamai was isolated in association with other Fusarium species-F. moniliforme, F. proliferatum, F. oxysporum, F. solani, F. compactum, and F. equiseti. Infected seedlings exhibited a reddish brown cortical discoloration, which was more intense in older plants. The identification of F. nygamai was based on monoconidial cultures grown on carnation leaf-piece agar (CLA) (2). The shape of macroconidia, the formation of microconidia in short chains and false heads, and the presence of chlamydospores were used as the criteria for identification. Two pathogenicity tests comparing one isolate of F. nygamai with one isolate of F. moniliforme were conducted on rice cv. Arborio sown in artificially infested soil in a greenhouse at 22 to 25°C. The inoculum was prepared by growing both Fusarium species in cornmeal sand (1:30 wt/wt) at 25°C for 3 weeks. This inoculum was added to soil at 20 g per 500 ml of soil. Pre- and post-emergence damping-off was assessed. Both F. nygamai and F. moniliforme reduced the emergence of seedlings (33 to 59% and 25 to 50%, respectively, compared to uninoculated control). After 25 days, the seedlings in infested soil exhibited a browning of the basal leaf sheaths, which progressed to a leaf and stem necrosis. Foot rot symptoms caused by F. nygamai and F. moniliforme were similar, but seedlings infected by F. nygamai exhibited a more intense browning on the stem base and a significant reduction of plant height at the end of the experiment. Either F. nygamai or F. moniliforme were consistently isolated from symptomatic tissue from the respective treatments. References: (1) L. W. Burgess and D. Trimboli. Mycologia 78:223,1986. (2) N. L. Fisher et al. Phytopathology 72:151,1982.