Sudden Vegetation Dieback in Atlantic and Gulf Coast Salt Marshes.

Salt marshes rank as the most productive ecosystems on the planet. Biomass production can be greater than 3 kg dry matter/m2/year, which is 40% more biomass than tropical rainforests produce. Salt marshes provide multiple benefits to mankind. For example, coastal communities receive protection from storm surges and wave erosion. Salt marshes absorb excess nitrogen and phosphorus from sewage and fertilizer run-off into rivers, which, in turn, prevents algal blooms and hypoxia in coastal waters. In addition, these unique ecosystems provide habitat and shelter for many hundreds of species of shellfish, finfish, migratory and sedentary birds, and other marine animals. Despite the richness in animal species, the intertidal marshes of the salt marsh ecosystem are dominated by only a few plant species. Of these, the most prevalent plant species in a marsh are the tall and short forms of smooth cordgrass (Spartina alterniflora). The first recorded account of a dieback in a U.S. salt marsh was in the early 1990s in the Florida panhandle where patches of Sp. alterniflora as large as 1 ha died. This article explores possible causes of Sudden Vegetation Dieback.

First Report of Crown Rot of Bloodroot (Sanguinaria canadensis) Caused by Fusarium oxysporum in the United States.

Bloodroot (Sanguinaria canadensis L [Papaveraceae]) is a native herbaceous perennial in eastern North America, found from Nova Scotia to Florida. Although it is a common wildflower, rhizomes of double-flowered forms are sold commercially. Rhizomes planted into a wooded area in Guilford, CT produced healthy stems and flowers for a few years and then began to collapse and die in 2008. The same symptoms were observed with a new planting in 2011. Initially, leaves were dull green and were more leathery than healthy leaves. Eventually, the leaves collapsed at the junction of the petioles and the rhizomes. Vascular discoloration, if present, was obscured by the red pigmentation in the rhizome. A Fusarium sp. sporulated on the discolored tissue at the junction between healthy and rotted tissue. Stem pieces were surface disinfested (0.53% NaClO for 1 min), rinsed, and placed on Peptone-PCNB agar (2) at room temperature for 5 days. Colonies originating from single spores were subcultured on carnation leaf agar (2) and identified as Fusarium oxysporum based on falcate, thin-walled, three-septate macroconida borne in monophialides on doliform conidiophores (2). Four rhizomes of double-flowered bloodroot were planted in potting mix in the greenhouse in October 2010; sprouts were observed in March 2011. Two plants were inoculated in March 2011 by drenching the soil with 100 ml of a conidial suspension (106 spores/ml) and two control plants were treated with deionized water. Two months later, the inoculated plants were smaller than the controls. The treated plants subsequently collapsed and F. oxysporum was reisolated. Control plants remained healthy and F. oxysporum was not isolated. DNA extracted from the F. oxysporum isolates was used to obtain partial sequences of the translational elongation factor 1-alpha (tef1) gene, which were then blasted against the GenBank database. We observed a 100% similarity with F. oxysporum f. sp. lycopersici. The bloodroot isolates were compared with a known F. oxysporum f. sp. lycopersici isolate for their ability to cause disease on 2-week-old tomato seedlings (cv. Brandywine), using pathogenicity tests as described above. The known F. oxysporum f. sp. lycopersici isolate caused severe wilt and stunting of the tomato seedlings, but the bloodroot isolate caused no symptoms in inoculated seedling compared with those not inoculated. These results suggest that there may be more hosts for isolates in the F. oxysporum f. sp. lycopersici species complex than previously thought (1). An isolate (O-2603) has been deposited at the Fusarium Research Laboratory at Pennsylvania State University, University Park. Since bloodroot is now being sold commercially as an ornamental, disease management strategies may be needed. To our knowledge, this is the first report of a Fusarium crown rot of bloodroot. References: (1).V. Edel-Hermann et al. Online publication. doi:10.1111/j.1365-3059.2011.02551.x. Plant Pathology, 2011. (2) J. Leslie and B. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

First Report of Cylindrocladium pseudonaviculatum Causing Leaf Spot of Pachysandra terminalis.

Cylindrocladium pseudonaviculatum Crous, J.Z., Groenew. & C.F. Hill 2002 was recently reported infecting common boxwood, Buxus sempervirens L., in Connecticut (2). We isolated the pathogen from leaf and stem lesions of B. sempervirens and obtained single-spored cultures on half-strength potato dextrose agar (½PDA). The pathogen was identified as C. pseudonaviculatum by morphological characteristics (1). Colony size reached 71 mm in diameter after 14 days at room temperature on ½PDA, and was fluffy with white aerial hyphae, mars brown, and reverse color chestnut brown at the center fading to pale brown forming concentric bands. Macroconidiophores were solitary or in a group of up to three, comprised a stipe, a sterile elongation, and one to three penicillate fertile branches. The stipe was up to nine septate, 90 to 250 µm long, colorless, smooth, terminating in a naviculate or broadly ellipsoidal vesicle with a pointed or papillate apex, and 27 to 50 × 6.5 to 9 µm. Primary branches were zero- to one-septate, 20 to 36 × 4 to 5 µm; secondary branches were aseptate and 11 to 20 × 3 to 4.5 µm; tertiary branches were rare, each terminal branch producing two to five phialides; phialides were doliiform or reniform, colorless, 12 to 18 µm. Conidia were cylindrical, rounded at both ends, straight, smooth, colorless, two-celled, 48 to 55 × 4.5 to 5.5 µm, and in colorless slimy cylindrical clusters. Microconidiophores were not observed. Chlamydospores were golden to dark brown, thick-walled, and smooth or rough. Microsclerotia were present on ½PDA. Primers T1 and T22 (3) were used to amplify a portion of the ß-tubulin gene from isolates Cps-CT-L1 and Cps-CT-S1. Amplified sequences were used in a BLAST search against the GenBank database to demonstrate 100% sequence identity only with other C. pseudonaviculatum strains. Both sequences were deposited in GenBank (Accession Nos. JQ866628 and JQ866629), using corresponding gene data from C. pseudonaviculatum strain STE-U 3399 (GenBank Accession No. AF449455) to distinguish coding from noncoding regions. Healthy plants of Japanese spurge, Pachysandra terminalis, with three plants per 10 cm diameter pot, were inoculated with water alone or a conidial suspension of C. pseudonaviculatum isolate Cps-CT-L1 (ATCC MYA-4891) (1.0 × 106 conidia/plant) with a handheld sprayer until runoff. Plants were kept moist in a plastic bag for 48 h at laboratory temperature and then transferred to the greenhouse. Circular lesions (1- to 4-mm diameter) were evident on leaves after 10 days. All 12 inoculated plants developed lesions, and no lesions were observed on noninoculated plants. Leaves with lesions were surface sterilized in 0.5% NaOCl for 30 s, rinsed twice in sterile water, and lesion margins plated onto water agar or ½PDA. The pathogen was reisolated from at least one leaf per plant. Koch's postulates were performed again with isolate Cps-CT-S1 (ATCC MYA-4890). After 3 weeks, many of the leaves with lesions yellowed and dropped to the soil surface and heavy sporulation of C. pseudonaviculatum and microsclerotia were observed. To our knowledge, this is the first report of C. pseudonaviculatum causing a leaf spot disease on P. terminalis. Pachysandra is a widely grown ground cover suitable for shady, humid environmental conditions that may be conducive for the development of disease. References: (1) P. Crous, et al. Sydowia 54:23, 2002. (2) K. Ivors et al. Plant Disease. 96:X, 2012. (3) K. O'Donnell and E. Cigelnik Mol. Phylogenet. Evol. 7:103, 1997.

First Report of Boxwood Blight Caused by Cylindrocladium pseudonaviculatum in the United States.

In September and October 2011, a new disease was observed on Buxus spp. in North Carolina and Connecticut, respectively. In North Carolina, over 10,000 containerized Buxus sempervirens (American boxwood) were affected at one location. A few weeks later, the disease was found in Connecticut on entire plantings of B. sempervirens 'Suffruticosa' (English boxwood) at two residential properties, and shortly thereafter on over 150,000 plants at two production nurseries. Initial foliar symptoms appeared as light to dark brown spots, often with dark borders. Spots enlarged and coalesced, often with a concentric pattern, and black streaks or cankers developed on stems. Infected leaves became brown or straw colored and dropped quickly after foliar symptoms were visible. Branch dieback and plant death were also observed in Connecticut. Cultures were isolated from symptomatic leaves and stems and identified as Cylindrocladium pseudonaviculatum Crous, Groenewald & Hill 2002 (1) (syn. Cylindrocladium buxicola Henricot 2002 [2]) on the basis of morphological characteristics. Macroconidiophores were single or in groups of up to three and comprised a stipe, stipe extension, and a penicillate arrangement of fertile branches. The stipe extension was septate, hyaline (89 to 170 × 2 to 4.5 µm), and terminated in an ellipsoidal vesicle (6 to 11 µm diameter) with a papillate or pointed apex. Conidia were cylindrical, straight, hyaline, and one septate (48 to 62 × 4 to 6 µm), occurring in slimy clusters. No microconidiophores were observed. Chlamydospores were medium to dark brown, thick walled, and smooth to rough. Microsclerotia were observed on PDA (1). A portion of ß-tubulin gene sequence from two Connecticut (Genbank Accession Nos. JQ866628 and JQ866629) and two North Carolina isolates showed 100% similarity with only C. pseudonaviculatum strains. USDA-APHIS-PPQ confirmed this new United States record on October 24, 2011. Pathogenicity was confirmed by inoculating three 1-gallon container plants of B. sempervirens 'Suffruticosa' in North Carolina and four liners of B. sinica var. insularis × B. sempervirens 'Green Velvet' in Connecticut with a spore suspension of approximately 5.0 × 106 conidia (North Carolina) or 1.0 × 106 conidia (Connecticut) on the foliage of each plant; untreated control plants were sprayed with water. After incubation at ambient temperature, all inoculated plants developed foliar and stem lesions within 3 to 4 days and blighting occurred within 2 weeks; control plants remained asymptomatic. C. pseudonaviculatum was reisolated from inoculated plants. To our knowledge, this is the first report of C. pseudonaviculatum in the United States. C. pseudonaviculatum causes a serious disease of Buxus spp. in the United Kingdom and several other European countries as well as New Zealand (1). Confirmation of boxwood blight in the United States is significant because of the popularity of boxwood as a landscape plant, and because of the potential economic impact this disease may have on commercial growers; boxwood production in the United States has an annual wholesale market value of approximately $103 million (3). References: (1) P. Crous, et al. Sydowia 54:23, 2002. (2) B. Henricot and A. Culham Mycologia 94: 980, 2002. (3) USDA-NASS, Census of Horticulture, 2010.

First Report of Pestalotiopsis paeoniicola Causing Twig Blight on Paeonia suffruticosa in North America.

Native to China, tree peony (Paeonia suffruticosa Andrews) is a perennial valued for its showy, often fragrant flowers. In May 2007, samples received from two field-grown tree peonies from Torrington, CT exhibited twig blight characterized by small, black spots on the bark of living or dead branches, and associated with subsequent death and loss of the branches. Colonies on potato dextrose agar (PDA) attained 5.5 to 6.5 cm in diameter in 9 days at 25°C and produced abundant, white, aerial mycelium, later turning pink and forming scattered, wet, black, acervular conidiomata containing abundant conidia that were five-celled, fusiform, smooth, straight, or slightly curved, and 24.0 ± 1.8 × 7.1 ± 0.5 µm (n = 20); three intermediate cells were dark brown and end cells were hyaline; two to four hyaline whip-like appendages on the apical cell, 26.6 ± 4.1 µm long, and one appendage on the basal cell, 6.7 ± 1.1 µm long. We identified the fungus as Pestalotiopsis paeoniicola (Tsukam. & T. Hino) J.G. Wei & T. Xu (= Pestalotia paeoniicola Tsukam. & T. Hino) based on morphology and host and have deposited a culture with CBS (CBS 124745). Originally described from Paeonia suffruticosa in Japan (1,3), the fungus is also found in China on Paeonia lactiflora (Pall.) (2). To confirm pathogenicity, two 5-year-old potted plants of Paeonia suffruticosa cv. Shichifukujin were inoculated with the fungus as follows: a 2-week-old PDA culture was used to produce a conidial spore suspension in sterile water; incisions (5 mm) were made with a sterile scalpel; 4 µl of either the conidial suspension or water were applied; and the wounds were wrapped with Parafilm. Each plant received three replicates each of the treatment and the control. Plants were loosely covered with plastic bags, kept on laboratory benches with ambient light for 1 month, and transferred to a greenhouse for an additional 2 months. Subsequent inspection revealed irregular or elongate, grayish brown lesions at the treatment inoculations, while the controls remained symptom free. Lesions gradually girdled the branches and the infected cortex turned dark brown. At advanced stages, small, black acervuli developed. Treatment and control tissues were cut into four pieces, surface sterilized, and placed on malt extract agar in petri dishes, four per dish. These were incubated for 9 days at 25°C under ambient light. Reisolation of P. paeoniicola only from tissues that had been treated with the pathogen, not from control inoculations, confirmed that the causal agent was P. paeoniicola. DNA sequences were obtained from the ß-tubulin gene (1,512 bp) and the internal transcribed spacer (ITS1 and ITS2) and 5.8S regions of the rDNA (550 bp) and were deposited in GenBank (Nos. FJ975603 and FJ997645, respectively). Neither ITS nor ß-tubulin sequences distinguished this P. paeoniicola isolate from Pestalotiopsis spp. whose sequences are deposited in GenBank. Morphological characteristics identify the fungus as P. paeoniicola, constituting, to our knowledge, the first report of this pathogen in North America. Discovery of P. paeoniicola in field-grown plants warrants further monitoring by growers because of the uncertain level of threat this pathogen may pose to the tree peony industry. References: (1) E. F. Guba. Monograph of Monochaetia and Pestalotia: 224. Harvard University Press, Cambridge, MA, 1961. (2) F. Tai. Sylloge Fungorum Sinicorum: 1021. Sci. Acad. Sin, Peking, 1979. (3) E. Tsukamoto et al. Ann. Phytopathol. Soc. Jpn. 4:183, 1956.

The mating system of the fungus Cryphonectria parasitica: selfing and self-incompatibility.

Although the genetic components of mating systems in fungi are well understood as laboratory phenomena, surprisingly little is known about their function in nature or about their role in determining mating patterns and population genetic structure. Our study of the mating system of the haploid ascomycete fungus, Cryphonectria parasitica, resulted in the following. (1) Laboratory crosses among 20 isolates, chosen randomly from North America and China, resolved into two incompatibility groups (occurring on both continents), confirming that C. parasitica has a diallelic, bipolar sexual self-incompatibility system, typical of other self-incompatible Ascomycetes, in which mating is only successful between isolates of opposite mating type. (2) PCR-based markers for mating-type alleles correlated perfectly with mating-type phenotypes of individual isolates. (3) Three genotypes, isolated from natural populations in Virginia and West Virginia, were inoculated onto chestnut trees in two sites in West Virginia and were confirmed to have self-fertilized and outcrossed in both sites. (4) Ten isolates, of a total of over 200 assayed, were confirmed to have self-fertilized in the laboratory, albeit at very low frequency. Five of these 10 isolates were ramets of a single genet, suggesting a genetic basis underlying the proclivity to self-fertilize in the laboratory. (5) Self-fertilization could not be induced in the laboratory with exudates (ostensibly containing pheromones) from isolates of opposite mating type. These results demonstrate that, a sexual self-incompatibility system notwithstanding, self-fertilization occurs under both laboratory and field conditions in C. parasitica. The disparity between observations of frequent selfing in nature and rare selfing in the laboratory suggests that the mating system is under ecological as well as genetic control.

PCR amplification of the mating-type idiomorphs in Cryphonectria parasitica.

In the laboratory, the ascomycete fungus Cryphonectria parasitica is rarely self-fertile, and has a self-incompatibility system that resolves into two intersterility groups, controlled by a single locus. In natural populations, however, self-fertilization occurs frequently. In this report, we show that the C. parasitica self-incompatibility locus (MAT) comprises two idiomorphs (alleles that are highly divergent in sequence), conforming to the paradigm of self-incompatibility as described for other ascomycetes. Starting with a fragment putatively from the MAT-2 idiomorph, we used a PCR-based cloning approach to identify 3.5- and 2-kb sequences unique to MAT-1 and MAT-2 isolates, respectively. These sequences were then used to design idiomorph-specific PCR primer pairs, allowing us to efficiently identify the mating types of isolates, a crucial component of our research on the environmental and genetic factors underlying this mixed mating system.

Mixed mating in natural populations of the chestnut blight fungus, Cryphonectria parasitica.

As in plants, fungi exhibit wide variation in reproductive strategies and mating systems. Although most sexually reproducing fungi are either predominantly outcrossing or predominantly selfing, there are some notable exceptions. The haploid, ascomycete chestnut blight pathogen, Cryphonectria parasitica, has previously been shown to have a mixed mating system in one population in USA. In this report, we show that both selfing and outcrossing occur in 10 additional populations of C. parasitica sampled from Japan, Italy, Switzerland and USA. Progeny arrays from each population were assayed for segregation at vegetative incompatibility (vic) and DNA fingerprinting loci. Outcrossing rates (t(m)) were estimated as the proportion of progeny arrays showing segregation at one or more loci, corrected by the probability of nondetection of outcrossing (alpha). Estimates of t(m) varied from 0.74 to 0.97, with the lowest rates consistently detected in USA populations (0.74-0.78). Five populations (four in USA and one in Italy) had t(m) significantly less than 1, supporting the conclusion that these populations exhibit mixed mating. The underlying causes of variation in outcrossing rates among populations of C. parasitica are not known, but we speculate that--as in plants--outcrossing is a function of ecological, demographic and genetic factors.

Analysis of mating-type genes in the chestnut blight fungus, Cryphonectria parasitica.

In nature, the chestnut blight fungus, Cryphonectria parasitica, has a mixed mating system; i.e., individuals in the same population have the ability to self and outcross. In the laboratory, C. parasitica appears to have a bipolar self-incompatibility system, typical of heterothallic ascomycetes; selfing is rare, although demonstrable. In this report we describe the cloning and sequencing of both mating-type idiomorphs and their flanking regions at the MAT locus in C. parasitica. The two idiomorphs, MAT1-1 and MAT1-2, are structurally similar to those of other pyrenomycetes described to date. MAT1-1 encodes three genes (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and MAT1-2 encodes a single gene (MAT1-2-1). Unlike MAT idiomorphs in some ascomycetes, the sequences at both ends of the idiomorphs in C. parasitica show a relatively gradual, rather than abrupt, transition from identity in the flanking regions to almost complete dissimilarity in the coding regions. The flanking regions have repetitive polypyrimidine (T/C) and polypurine (A/G) tracts; the significance of these repetitive tracts is unknown. Although we found repetitive tracts in the flanks and gradual transition zones at the ends of the idiomorphs, we found no special features that would explain how selfing occurs in an otherwise self-incompatible fungus.