Nichole R. O'Neill

Elevated atmospheric carbon dioxide concentrations amplify Alternaria alternata sporulation and total antigen production.

Background Although the effect of elevated carbon dioxide (CO2) concentration on pollen production has been established in some plant species, impacts on fungal sporulation and antigen production have not been elucidated. Objective Our purpose was to examine the effects of rising atmospheric CO2 concentrations on the quantity and quality of fungal spores produced on timothy (Phleum pratense) leaves. Methods Timothy plants were grown at four CO2 concentrations (300, 400, 500, and 600 μmol/mol). Leaves were used as growth substrate for Alternaria alternata and Cladosporium phlei. The spore abundance produced by both fungi, as well as the size (microscopy) and antigenic protein content (ELISA) of A. alternata, were quantified. Results Leaf carbon-to-nitrogen ratio was greater at 500 and 600 μmol/mol, and leaf biomass was greater at 600 μmol/mol than at the lower CO2 concentrations. Leaf carbon-to-nitrogen ratio was positively correlated with A. alternata spore production per gram of leaf but negatively correlated with antigenic protein content per spore. At 500 and 600 μmol/mol CO2 concentrations, A. alternata produced nearly three times the number of spores and more than twice the total antigenic protein per plant than at lower concentrations. C. phlei spore production was positively correlated with leaf carbon-to-nitrogen ratio, but overall spore production was much lower than in A. alternata, and total per-plant production did not vary among CO2 concentrations. Conclusions Elevated CO2 concentrations often increase plant leaf biomass and carbon-to-nitrogen ratio. Here we demonstrate for the first time that these leaf changes are associated with increased spore production by A. alternata, a ubiquitous allergenic fungus. This response may contribute to the increasing prevalence of allergies and asthma.

Assessment of Diversity in Claviceps africana and Other Claviceps Species by RAM and AFLP Analyses

ABSTRACT Genetic diversity among isolates of Claviceps africana, the sorghum ergot pathogen, and isolates of other Claviceps spp. causing ergot on sorghum or other hosts, was analyzed by random amplified microsatellite (RAM) and amplified fragment length polymorphism (AFLP) analyses. Of the RAM primer sets tested, one revealed polymorphism in C. africana isolates, with Australian and Indian isolates possessing a unique fragment. AFLP analysis, in addition to clearly distinguishing Claviceps spp., revealed polymorphisms in C. africana. A group of isolates from the United States, Puerto Rico, and South Africa exhibited 95 to 100% similarity with one another. Several isolates from Isabela, Puerto Rico were 100% similar to an isolate from Texas, and another isolate from Puerto Rico was identical with one from Nebraska. Australian and Indian isolates showed greater than 90% similarity with isolates from the United States., Puerto Rico, and South Africa. A number of polymorphisms existed in the United States group, indicating that the recently introduced population contains multiple genotypes. Isolates of C. sorghicola, a newly described sorghum pathogen from Japan, were very distinct from other species via RAM and AFLP analyses, as were isolates from outgroups C. purpurea and C. fusiformis. Both RAM and AFLP analysis will be useful in determining future patterns of intercontinental migration of the sorghum ergot pathogen, with the AFLP method showing greater ability to characterize levels of intraspecific variation.

Nep1 Protein from Fusarium oxysporum Enhances Biological Control of Opium Poppy by Pleospora papaveracea

ABSTRACT The fungus Pleospora papaveracea and Nep1, a phytotoxic protein from Fusarium oxysporum, were evaluated for their biocontrol potential on opium poppy (Papaver somniferum). Four treatments consisting of a control, P. papaveracea conidia, Nep1 (5 mug/ml), and P. papaveracea conidia plus Nep1 (5 mug/ml) were used in detached-leaf and whole-plant studies. Conidia of P. papaveracea remained viable for 38 days when stored at 20 or 4 degrees C. Nep1 was stable in the presence of conidia for 38 days when stored at 4 degrees C or for 28 days at 20 degrees C. The presence of Nep1 did not affect conidia germination or appressoria formation. Nep1 was recovered from drops applied to opium poppy leaves in greenhouse and field studies 24 h after treatment. Opium poppy treated with the combination of Nep1 and P. papaveracea had higher necrosis ratings than the other treatments. There were changes in the intercellular protein profiles, determined by sodium dodecyl sulfate gel electrophoresis and silver staining, due to application of treatments; the most intense occurred in response to the combination of Nep1 and P. papaveracea. The combination of Nep1 and P. papaveracea enhanced the damage caused to opium poppy more than either component alone.

Sources of resistance to anthracnose in the annual Medicago core collection.

The annual genus Medicago core collection, consisting of 201 accessions, represents the genetic diversity inherent in 3,159 accessions from 36 annual Medicago species. This germ plasm was evaluated for resistance to anthracnose caused by Colletotrichum trifolii. Anthracnose is a major disease in perennial alfalfa (Medicago sativa L.) grown in North America and disease control is based principally on the use of resistant varieties. Evaluation of the core collection was conducted using standardized environmental conditions in growth chambers, and included the M. sativa standard reference cvs. Arc (resistant) and Saranac (susceptible). The degree of resistance found among accessions within species was highly variable; however, most annual species and accessions were susceptible. Only 14 accessions from seven species exhibited resistance greater than 40% seedling survival. These included accessions of M. murex, M. muricoleptis, M. polymorpha var. brevispina, M. polymorpha var. polymorpha, M. radiata, M. soleirolii, M. truncatula, and M. turbinata. Of the 12 accessions of M. polymorpha var. polymorpha, 4 exhibited more than 50% resistance, but 3 accessions were 100% susceptible. Most of the M. truncatula and M. turbinata accessions exhibited significantly more resistance than accessions of other species. Plant introduction (PI) accession number PI 495401 of M. muricoleptis exhibited 90.3% resistance. Accessions of M. scutellata were uniformly susceptible. Histological examinations of 14 of the most anthracnose-resistant accessions revealed that C. trifolii spores germinated and produced typical appressoria, but failed to penetrate and produce the primary and secondary hyphae characteristic of susceptible interactions. Resistant reactions were similar to those found in incompatible interactions with C. trifolii and alfalfa, which have been associated with specific genes leading to the production of isoflavonoid phytoalexins. The large genetic variability in annual Medicago spp. offers potential for locating and utilizing disease resistance genes through breeding or genetic engineering that will enhance the utilization of Medicago spp. as a forage crop.

Pathogenic variability and host resistance in the Colletotrichum trifolii/Medicago sativa pathosystem.

Two hundred and fifty alfalfa cultivars currently grown in the U.S. were evaluated for their reaction to anthracnose caused by race 2 of Colletotrichum trifolii. Resistance was present in 30 cultivars, with seedling survival ranging from 20 to 68%. Three unusually highly virulent isolates of Colletotrichum collected from three locations were identified to species and race by morphological and cultural characteristics, and by reactions on host differential cvs. Arc, Saranac, and Saranac AR. Isolates Arl-NW and SB-2 were characterized as C. trifolii isolates of races 1 and 2, respectively. The morphology of isolate 57RR was distinct from that of C. trifolii and C. destructivum, and most closely conformed to the composite group C. gloeosporioides. Resistance to these isolates was evaluated in 40 selected and race differential cultivars. Resistance to isolate Arl-NW was highly variable, with seedling survival ranging from 0 to 77.8%. The isolate was classified as race I based on reactions with standard check cultivars. Isolate 57RR was highly virulent to anthracnose-resistant alfalfa clones and cultivars in needle inoculation assays, and was highly virulent to Saranac AR, Arc, and several race 2 resistant cultivars in seedling tests. Colletotrichum gloeosporioides is reported as a new pathogen of alfalfa and should be considered a potential disease problem. The variability of resistance reactions in commercially grown alfalfa cultivars to diverse isolates of Colletotrichum suggests that these cultivars will be useful sources for different types of anthracnose resistance.

Reactions in the annual Medicago spp. core germ plasm collection to Phoma medicaginis

The annual Medicago spp. core collection, consisting of 201 accessions, represents the genetic diversity inherent in 3,159 accessions from 36 annual Medicago spp. This germ plasm was evaluated for resistance to spring black stem and leaf spot caused by Phoma medicaginis. Spring black stem and leaf spot is a major destructive disease in perennial alfalfa (Medicago sativa) grown in North America, Europe, and other temperate regions. Disease control is based principally on the use of cultivars with moderate levels of resistance. Evaluation of the core collection was conducted using standardized environmental conditions in growth chambers, and included the M. sativa standard reference cultivars Ramsey (resistant) and Ranger (susceptible). The degree of resistance found among accessions within species was variable, but most annual species and accessions were susceptible. Most accessions from 10 species exhibited high disease resistance. These included accessions of M. constricta, M. doliata, M. heyniana, M. laciniata, M. lesinsii, M. murex, M. orbicularis, M. praecox, M. soleirolii, and M. tenoreana. Most of the accessions within M. arabica, M. minima, M. lanigera, M. rotata, M. rugosa, M. sauvagei, and M. scutellata were highly susceptible. Disease reactions among some accessions within species were highly variable. On a 0-to-5 disease severity scale, ratings ranged from 0.67 (PI 566873) to 4.29 (PI 566883) within accessions of M. polymorpha. Most of the M. truncatula accessions were susceptible, with a mean of 3.74. Resistant reactions were similar to those found in incompatible interactions with P. medicaginis and alfalfa, which have been associated with specific genes leading to the production of isoflavonoid phytoalexins. The large genetic variability in annual Medicago spp. offers potential for locating and utilizing disease resistance genes through breeding or genetic engineering that will enhance the utilization of Medicago spp. as a forage crop.

Ophiosphaerella agrostis sp. nov. and its relationship to other species of Ophiosphaerella.

Creeping bentgrass commonly is grown on golf courses in the United States. In the fall of 1998, symptoms of a new disease were observed on bentgrass putting greens in three states. The causal agent was determined to be an undescribed species of Ophiosphaerella based on morphological character- istics. This was supported by sequence analysis of the complete ITS region of the rDNA. A new species, Ophiosphaerella agrostis, is described based on these morphological and molecular analyses. Intraspecific genetic variation was assessed using amplified frag- ment length polymorphism DNA fingerprinting of 17 isolates collected from nine different locations. A 90% similarity was found among the isolates.

Dendryphion penicillatum and Pleospora papaveracea, destructive seedborne pathogens and potential mycoherbicides for Papaver somniferum.

ABSTRACT Dendryphion penicillatum and Pleospora papaveracea were isolated from blighted Papaver somniferum and Papaver bracteatum plants grown in growth chambers and the field in Beltsville, MD. The etiology of the diseases was determined, and the fungi are being investigated as potential mycoherbicides to control the narcotic opium poppy plant. P. papaveracea is known to be a highly destructive seedborne pathogen of Papaver somniferum, causing seedling blight, leaf blight, crown rot, and capsule rot. Single conidia and ascospores were isolated and cultures established from naturally infested seed and diseased foliage and pods of opium poppy from Iran, Colombia, Venezuela, Sweden, India, and the United States (Maryland and Washington). Mycelia and conidia of P. papaveracea and D. penicillatum produced on necrotic leaf tissues appear morphologically similar, and the fungi were previously considered to be anamorph and teleomorph. However, no anamorph/teleomorph connection could be established, and the fungi appear to be distinct taxa. P. papaveracea produced conidia, mature pseudothecia, and chlamydospores in vitro and on infected stems. D. penicillatum produced conidia, microsclerotia, and macronematous conidiophores. Although both fungi were pathogenic to three poppy cultivars, conidial inoculum from P. papaveracea cultures was more virulent than conidial inoculum from D. penicillatum. Eight-week-old plants became necrotic and died 8 days after inoculation with a conidial suspension of P. papaveracea at 2 x 10(5) spores per ml. Disease severity was significantly enhanced by inoculum formulations that contained corn oil, by higher conidial inoculum concentrations, and by increased wetness periods. Symptoms on plants inoculated with either pathogen included leaf and stem necrosis, stem girdling, stunting, necrotic leaf spots, and foliar and pod blight. Inoculated seedlings exhibited wire stem, damping-off, and root rot. Conidia, and less frequently pseudothecia, of P. papaveracea and conidia of D. penicillatum were produced abundantly on inoculated, necrotic foliage, pods, and seedlings. Cultures from conidia or ascospores reisolated from these tissues consistently produced fungi whose morphologies were typical of the fungus from which the inoculum was derived.

Morphological and molecular studies on Dendryphion penicillatum and Pleospora papaveracea, pathogens of Papaver somniferum

20705-2350 Abstract: Pleospora papaveracea and Dendryphion penicillatum are well-known pathogens of opium pop- py (Papaver somniferum). The asexual state of P1. pa- paveracea has generally been identified as D. penicil- latum. However, morphological observations and AFLP analysis reveal that the anamorph of PI. papa- veracea is a Dendryphiella-like fungus and that D. pen- icillatum is not the asexual state of PI. papaveracea. While PI. papaveracea forms a sexual state in culture, D. penicillatum lacks a known sexual state. These two species can be readily differentiated using both mor- phology and colony characteristics.

Production of Pleospora papaveracea biomass in liquid culture and its infectivity on opium poppy (Papaver somniferum)

Abstract The fungus Pleospora papaveracea is a potential biocontrol agent for opium poppy. The objective of this study was to characterize the growth and production of propagules of P. papaveracea on various substrates and determine their infectivity on opium poppy. Pleospora papaveracea was grown on agar media containing wheat bran, corn cobs, soy fiber, cottonseed meal, rice flour, cornstarch, pectin, dextrin, or molasses, all with the addition of brewers yeast (BY). Maximum radial growth of P. papaveracea occurred on molasses, soy fiber, and wheat bran media. Pleospora papaveracea produced chlamydospores on dextrin–BY and cornstarch–BY only. Pleospora papaveracea growth in liquid media with 1% (wt/v) dextrin, cornstarch, soy fiber, or wheat bran resulted in the production of greater than 106 colony-forming units (cfu) ml−1 within 3 to 5 d of incubation. Pleospora papaveracea produced less than 105 chlamydospores ml−1 after 10 d of incubation in wheat bran–BY and soy fiber–BY liquid media compared with the production of greater than 105 chlamydospores ml−1 after 5 d of incubation in dextrin–BY or cornstarch–BY liquid media. Fewer cfu were produced by P. papaveracea in 0.25% dextrin or 0.25 and 0.50% soy fiber liquid media than with 1 or 2% substrate. Greater than 107 chlamydospores g−1 dry weight and 108 cfu g−1 dry weight of P. papaveracea were produced in dextrin–BY liquid media in a commercial bench-top fermentor. After air drying biomass for 6 d, propagules of P. papaveracea remained infective on opium poppy. Mycelia and chlamydospores of P. papaveracea grew and formed appressoria during the infection process. Air-dried biomass, when rehydrated in 0.001% Tween 20, caused necrosis within 48 h after application to detached opium poppy leaves. At least 94% of the propagules from air-dried biomass that germinated and infected detached opium poppy leaves were of mycelial origin. Nomenclature: Opium poppy, Papaver somniferum L.