Diversity of the trifunctional histidine biosynthesis gene (his) in cereal Phaeosphaeria species.

Phaeosphaeria species are important causal agents of Stagonospora leaf blotch diseases in cereals. In this study, the nucleotide sequence and deduced polypeptide of the trifunctional histidine biosynthesis gene (his) are used to investigate the phylogenetic relationships and provide molecular identification among cereal Phaeosphaeria species. The full-length sequences of the his gene were obtained by PCR amplification and compared among cereal Phaeosphaeria species. The coding sequence of the his gene in wheat-biotype P. nodorum (PN-w) was 2697 bp. The his genes in barley-biotype P. nodorum (PN-b), two P. avenaria f. sp. triticea isolates (homothallic Pat1 and Pat3), and Phaeosphaeria species from Polish rye and dallis grass were 2694 bp. The his gene in heterothallic isolate Pat2, however, was 2693 bp because the intron had one fewer base. In P. avenaria f. sp. avenaria (Paa), the his gene was only 2670 bp long. The differences in the size of the his gene contributed to the variation in amino acid sequences in the gap region located between the phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase sub-domains. Based on nucleotide and deduced amino acid sequences of the his gene, Pat1 was not closely related to either PN-w or the Paa clade. It appears that rates of evolution of the his gene were fast in cereal Phaeosphaeria species. The possible involvement of meiotic recombination in genetic diversity of the his gene in P. nodorum is discussed.

Relative Contribution of Seed-Transmitted Inoculum to Foliar Populations of Phaeosphaeria nodorum.

ABSTRACT A marked-isolate, release-recapture experiment was conducted to assess the relative contributions of seed-transmitted (released isolates) versus all other inocula to foliar and grain populations of Phaeosphaeria nodorum in winter wheat rotated with nonsusceptible crops in New York and Georgia, United States. Seed infected with two distinct groups of marked isolates of P. nodorum containing rare alleles (identified by amplified fragment length polymorphisms [AFLPs]) and balanced for mating type were planted in experimental field plots in two locations in each state. Recapture was done by isolating P. nodorum from leaves showing necrotic lesions at spring tillering and flowering stages, and mature grains from spikes showing glume blotch. Isolates from these samples were genotyped by AFLPs and categorized as released or nonreleased to infer sources of inoculum. Both infected seed and other sources of the pathogen contributed significant primary inocula to populations recovered from leaves and harvested grain. Seed-transmitted genotypes accounted for a total of 57% of all isolates recovered from inoculated plots, with a range of 15 to 90% of the populations of P. nodorum collected over the season in individual, inoculated plots at the four locations. Plants in the noninoculated control plots also became diseased and 95% or more of the isolates recovered from these plots were nonreleased genotypes. Although other potential sources of P. nodorum within and adjacent to experimental plots were not ruled out, nonreleased genotypes likely were derived from immigrant ascospores potentially from sources at a considerable distance from the plots. Our results suggest that, although reduction of seedborne inoculum of P. nodorum may delay foliar epidemics, this strategy by itself is unlikely to result in high levels of control in eastern North America because of the additional contribution from alternative sources of inoculum.

RNA polymerase II gene (RPB2) encoding the second largest protein subunit in Phaeosphaeria nodorum and P. avenaria.

A 5586 bp sequence (accession no. DQ278491), which includes the RNA polymerase II gene (RPB2) encoding the second largest protein subunit (RPB2), was obtained from the wheat biotype Phaeosphaeria nodorum (PN-w) by PCR amplification. The 3841 bp full length RPB2 gene contains two exons and a 52 bp intron, and encodes a complete 1262 amino acid protein. Similar to the C-terminals of the beta subunits of prokaryotes and yeast RNA polymerases, the deduced RPB2 protein contained many structural features needed for gene transcription. Based on the phylogenetic analysis with the deduced RPB2 polypeptide sequences, the PN-w was closely related to the maize pathogen Cochliobolus heterostrophus. Size differences were found in the full length RPB2 gene of cereal Phaeosphaeria species, mainly due to differences in intron size. No nucleotide substitutions were found in homothallic P. avenaria f.sp. triticea (Pat1) and barley biotype P. nodorum (PN-b) isolates used in this study. The nucleotide and deduced amino acid sequences of the RPB2 gene in Pat1 were closely related to that in PN-w.

Effect of Crop Rotation on Take-all of Wheat in Double-Cropping Systems.

Take-all of wheat (Triticum aestivum), caused by Gaeumannomyces graminis var. tritici, became a serious problem with the widespread adoption of wheat:soybean double-cropping and minimum tillage farming systems in the southeastern United States during the past 30 years. A long-term crop rotation study was initiated in 1994 with 12 double-cropping sequences incorporating wheat, rye, or canola as the fall-planted crop and soybean or grain pearl millet as the summer crop. Cotton and fallow were included in some summer rotations during the last 2 years of the study. The purpose was to identify sustainable alternatives to the continuous wheat:soybean system that would provide acceptable management of take-all. G. graminis var. tritici cultured on autoclaved oats was incorporated into soil prior to planting the first season's crop. Take-all was severe in rotations with continuous wheat each year. Pearl millet was compatible with the cropping system but did not affect incidence or severity of take-all in a following wheat crop. Soybean or pearl millet had little effect on yield loss due to take-all in a subsequent wheat crop. A 1-year rotation with canola significantly reduced take-all incidence and severity. At the end of the second and third seasons, in those rotations where wheat followed 1 year of canola, wheat grain yield was the same as that in control plots that had little or no take-all. Two consecutive years of canola did not suppress take-all or improve wheat yields any more than a single year of canola between wheat crops. Seedling assays for take-all incidence and severity in growth chambers were conducted using soil collected twice each year near the end of each crop's growing season. Results were similar to those observed in the field. However, canola in the rotation had a greater effect in suppressing disease severity than disease incidence. Canola can be a valuable rotational crop for management of take-all in wheat in the southeastern United States.

Sequence diversity of mating-type genes in Phaeosphaeria avenaria.

Phaeosphaeria avenaria, one of the causal agents of stagonospora leaf blotch diseases in cereals, is composed of two subspecies, P. avenaria f. sp. triticea (Pat) and P. avenaria f. sp. avenaria (Paa). The Pat subspecies was grouped into Pat1-Pat3, based on restriction fragment length polymorphism (RFLP) and ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequences in previous studies. Mating-type genes and their potential use in phylogeny and molecular classification were studied by DNA hybridization and PCR amplification. The majority of Pat1 isolates reported to be homothallic and producing sexual reproduction structures on cultural media had only the MAT1-1 gene. Minor sequence variations were found in the conserved region of MAT1-1 gene in Pat1 isolates. However, both mating-type genes, MAT1-1 and MAT1-2, were identified in P. avenaria isolates represented by ATCC12277 from oats (Paa) and the Pat2 isolates from foxtail barley ( Hordeum jubatum L.). Cluster analyses based on mating-type gene conserved regions revealed that cereal Phaeosphaeria is not phylogenetically closely related to other ascomycetes, including Mycosphaerella graminicola (anamorph Septoria tritici). The sequence diversity of mating-type genes in Pat and Paa supports our previous phylogenetic relationship and molecular classification based on RFLP fingerprinting and rDNA ITS sequences.

TAXONOMY AND IDENTIFICATION OF SEPTORIA AND STAGONOSPORA SPECIES ON SMALL-GRAIN CEREALS.

Numerous significant changes have been made in the taxonomy of both the anamorphs and teleomorphs of the Septoria group of leaf spot pathogens on small grain cereals during the past 30 years. The pathogens fall into two related but distinct groups, with anamorphic genera now placed in Septoria and Stagonospora of the Sphaeropsidales. Each of these genera has distinct teleomorphs in the Loculoascomycetes in Mycosphaerella and Phaeosphaeria. These reclassifications were based largely on fungal morphology and host pathogenicity as originally characterized. Recent studies have investigated the phylogenetic relationships among these species using the techniques of molecular genetics, and have related molecular characteristics to taxonomy based on classical morphology. A clearer understanding of the taxonomy of this group will enhance our ability to diagnose and manage these important cereal pathogens.

Tilletia walkeri on Annual Ryegrass in Wheat Fields in the Southeastern United States.

Surveys for Tilletia walkeri on annual ryegrass (Lolium multiflorum) were conducted during 1997 and 1998 in the southeastern United States, where suspect teliospores of the Karnal bunt fungus, Tilletia indica, were found in USDA-APHIS surveys of wheat (Triticum aestivum) seed in 1996. T. walkeri is morphologically similar to T. indica. Annual ryegrass is a common weed in wheat fields in the southeastern United States. Between April and June 1997, ryegrass seed samples were collected from 190 fields of wheat in 47 counties in Georgia and from 26 fields in 17 counties in Alabama and south-central Tennessee. In 1998, 70 samples were collected from 40 counties in the same regions of the three states. The teliospores from these samples were 23 to 45 µm in diameter (average about 33 µm) and ranged from light brown to dark reddish brown. They had coarse, widely spaced cerebriform ridges on the surface and were surrounded by a gelatinous sheath. The ryegrass bunt was identified as the recently described species T. walkeri, occurring on ryegrass seed from Australia and Oregon. In 1997, teliospores of T. walkeri were found in 13 samples from eight counties in central Georgia and from one field in Tennessee. In 1998, more teliospores and bunted seeds were found, possibly due to frequent rain in the region throughout the flowering period for ryegrass. Teliospores were found in 26/70 of the samples, and among these, only a small number of bunted seed were found in 12 of 13/70 samples. In one wheat field in Morgan County, Georgia, about 50% of the ryegrass seed collected was partially bunted, and a small percentage was completely bunted. Fields with teliospores were widely distributed and generally matched the locations where teliospores were found in APHIS wheat seed surveys in 1996 to 1998. T. walkeri occurs at very low levels on ryegrass in the Southeast and is the source of teliospores, initially identified as those of T. indica, associated with wheat seed in APHIS surveys. No bunted wheat seeds or teliospores of T. indica were found in the survey.