Cowger, C and HV Silva-Rojas. 2006. Frequency of Phaeosphaeria
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Population Biology Frequency of Phaeosphaeria nodorum, the Sexual Stage of Stagonospora nodorum, on Winter Wheat in North Carolina Christina Cowger and Hilda Victoria Silva-Rojas First author: U.S. Department of Agriculture–Agricultural Research Service, Department of Plant Pathology, North Carolina State University, Raleigh 27695; and second author: Instituto de Recursos Genéticos y Productividad, Colegio de Postgraduados, Montecillo, Estado de México, México. Accepted for publication 25 March 2006. ABSTRACT Cowger, C., and Silva-Rojas, H. V. 2006. Frequency of Phaeosphaeria bodies from wheat spikes than of those from lower stems and leaves. nodorum, the sexual stage of Stagonospora nodorum, on winter wheat in Ascocarps also were found at frequencies <10% in some wheat debris North Carolina. Phytopathology 96:860-866. samples from 2004 and 2005. Analysis of the nucleotide sequences of internally transcribed spacer regions of 18 genetically distinct North Ascocarps of Phaeosphaeria nodorum, which causes Stagonospora Carolina isolates from 2003 suggested that all were P. nodorum, not the nodorum blotch (SNB) of wheat, have not been found by others in the morphologically similar P. avenaria f. sp. triticea. Neither the 18 isolates eastern United States despite extensive searches. We sampled tissues from from 2003 nor a set of 77 isolates derived from 2004 Kinston leaf living wheat plants or wheat debris in Kinston, NC, each month except samples gave reason to suspect a mating-type imbalance in the larger June from May to October 2003. Additional wheat samples were gathered P. nodorum population (P ≥ 0.4). We conclude that, in North Carolina, in Kinston, Salisbury, and Plymouth, NC, in 2004 and 2005. For the sexual reproduction plays a role in initiation of SNB epidemics and the 3 years, in all, 2,781 fruiting bodies were dissected from the wheat tissues creation of adaptively useful genetic variability, although its relative and examined microscopically. Fruiting bodies were tallied as P. nodorum importance in structuring this population is uncertain. pycnidia or ascocarps, “unknown” (not containing spores, potentially P. nodorum or other fungi), or “other fungi.” In the 2003 sample, asco- Additional keywords: Leptosphaeria nodorum, pseudothecia, Septoria carps of P. nodorum were present each month after May at a frequency of nodorum. 0.8 to 5.4%, and comprised a significantly higher percentage of fruiting Phaeosphaeria nodorum (E. Müller) Hedjaroude, syn. Leptos- hypotheses that epidemics are initiated by ascospores and patho- phaeria nodorum E. Müller (anamorph = Stagonospora nodorum gen populations are random-mating in Texas, Oregon, and (Berk.) Castellani & E. G. Germano, syn. Septoria nodorum Switzerland (21,22,29). (Berk.)) is the causal agent of Stagonospora nodorum blotch However, Shah et al. (39) have argued that such results also (SNB) of wheat, a widespread and yield-reducing disease in could be produced by genetically heterogeneous seedborne fungal wheat-growing regions with relatively high rainfall. populations. These researchers have shown that the percentage of P. nodorum is a bipolar, heterothallic fungus (19). The teleo- artificially infected seed is correlated with later disease incidence morph has been found in many parts of the world, including and severity in plots in New York state sown with that seed (39), Brazil (31), France (34), Germany (32), Ireland (33), Italy (16), and that wheat cultivars differ in relative susceptibility to seed New Zealand (36), and South Africa (23). However, in some parts infection (38). They suggest that, in at least some production sys- of the United States, the search for ascocarps of P. nodorum, tems, such as that in New York, primary inoculum for epidemics which are pseudothecia, has proven elusive. Researchers in Mon- may be mainly seedborne (38). tana (37) and Oregon (8) have observed abundant P. nodorum Mating-type ratios have been studied to obtain evidence on the ascocarps. But researchers in the northeastern (39) and south- extent of sexual reproduction by P. nodorum. Among 56 P. no- eastern (9) United States, where disease levels are often substan- dorum isolates from Europe, Australia, the United States, Mor- tial, have not found P. nodorum ascocarps or ascospores despite occo, and Israel that could be assigned a mating type, Halama intensive searches over many years. (18) found that the MAT1-1 allele was present in 92% of isolates Controversy has existed over the degree to which sexual repro- and the MAT1-2 allele in the other 8%. Such an imbalance could duction affects the P. nodorum population structure. Populations suggest a departure from random mating in the populations giving collected in 1984 and 1985 from small areas of wheat fields in the rise to those isolates. Bennett et al. (3) sampled two fields in New United Kingdom revealed a spatially fine-grained polymorphism York state, and found a predominance of MAT1-1 among 22 for electrophoretic karyotype consistent with a prominent role for isolates from one field, but approximately equal numbers of both ascospores in initiating infections (6). Molecular tests of gene and mating types among 31 isolates from the other field. Sommerhalder genotype diversity and gametic equilibrium have supported the et al. (41) determined mating-type ratios of samples from five U.S. states, including two in the eastern soft red winter wheat region (Arkansas and New York), as well as five other countries. Corresponding author: C. Cowger; E-mail address: [email protected] They found a departure from a 1:1 ratio in only one location (Oregon). DOI: 10.1094/PHYTO-96-0860 P. nodorum is an important pathogen of soft red winter wheat This article is in the public domain and not copyrightable. It may be freely re- printed with customary crediting of the source. The American Phytopathological in North Carolina, although SNB epidemics are significant in Society, 2006. fewer than 1 of 2 years (14). The purpose of this study was to 860 PHYTOPATHOLOGY determine whether the teleomorph was present in North Carolina, A two-way χ2 test was performed to determine whether, taking and at what frequency. Due to morphological and symptom simi- only P. nodorum fruiting bodies into account, the percentages of as- larities, P. nodorum can be confused with P. avenaria (G. F. Weber) cocarps and pycnidia found in a given month of 2003 were signifi- O. Eriksson f. sp. triticea T. Johnson (25,26,44), the cause of cantly different on basal versus upper tissues. Due to small sample Stagonospora avenae blotch of cereals. Therefore, we also con- sizes, Fisher’s exact test was used for the month of September. ducted molecular tests on a sample of Phaeosphaeria isolates Ninety-five percent binomial confidence limits (BCLs) were from North Carolina to determine their species identity. calculated for frequencies of P. nodorum ascocarps and pycnidia, in each case using the proportion of that fruiting body type in MATERIALS AND METHODS contrast to all other types in the sample. PROC FREQ of SAS (SAS Institute, Cary, NC) was used to calculate asymptotic and Sample collection. In 2003, a moderately severe SNB epi- exact confidence limits; asymptotic limits were reported except demic occurred in Kinston, NC. Starting in May, wheat debris where counts were 0, when exact limits were reported. were sampled monthly (except June) for a 6-month period from Species identification. For molecular evidence on the species an experimental field of multiple soft red winter wheat genotypes of Phaeosphaeria present, a sample population of 26 isolates was at the Cunningham Research and Extension Center in Kinston. In created (STAN1 to STAN26). These isolates were derived from May, tissue samples were collected from the susceptible cultivar leaves sampled in North Carolina in May 2003, but were not the USG 3209 (PVP 200100127) and from a moderately resistant NC same genetic individuals as those that produced fruiting bodies experimental line (NC98-24182). By July, however, the post- classified in this study. In all, 21 isolates were obtained from flag harvest stubble was scattered among half-grown soybean plants, or F-1 leaves of cvs. USG 3209 and Saluda (PI 480474) sampled and it was no longer possible to identify wheat genotypes. Debris randomly in the Kinston field in which the fruiting bodies were was collected from a wide enough area to include multiple host collected, and 5 isolates were derived from F-1 leaves of cv. NK genotypes. Coker 9663 (PI 596345) sampled in an experimental field in The collected tissues were separated into “basal” and “upper.” Plymouth, NC. To make these isolations, leaf segments containing Basal tissues were lower leaves (F-4 and F-5) and the lower 10 cm pycnidia were affixed to microscope slides and placed in moist of the stems. Upper tissues were spikes (glume, rachis, and ped- chambers overnight. The cirrhi were transferred individually to uncles), except in May, when flag leaves were added to spikes in potato dextrose agar amended with kanamycin at 50 mg/liter. order to have sufficient lesioned tissue. A limited amount of tissue Monopycnidial cultures were stored on yeast-malt agar slants at was available in September 2003, leading to smaller sample sizes 4°C. in that month, and no spikes were collected in October 2003. Isolates were grown on a rotary shaker for 7 days in yeast- More temporally limited collections of wheat debris were made sucrose broth (4 g of yeast extract and 4 g of sucrose per liter of in 2004 and 2005 in an effort to confirm the results from 2003. water) amended with gentamicin at 50 mg/liter. Mycelium was Samples were gathered from Kinston (12 August and 23 October harvested by filtration through cheesecloth, frozen to –80°C, and 2004 and 16 June and 20 September 2005), Salisbury (17 June lyophilized. DNA was extracted using Qiagen DNeasy Plant Mini and 11 August 2004), and Plymouth (15 June 2004), NC.