Lima Bean Downy Mildew Epiphytotics Caused by New Physiological Races of Phytophthora phaseoli.

Before 1995, race D of Phytophthora phaseoli, the causal agent of downy mildew on lima bean (Phaseolus lunatus), was the prevalent physiological race in the mid-Atlantic region of the United States. Since 1995, however, new physiological races of P. phaseoli have been responsible for downy mildew outbreaks in previously resistant cultivars in this region. Cultivar differential testing of 180 isolates of P. phaseoli collected between 1994 and 2005 from Delaware and the eastern shore of Maryland has confirmed the presence of two new physiological races. The detection of race E in 1995 and race F only 5 years later in 2000, plus the lack of resistant cultivars to manage the epiphytotics in lima bean, have led to millions of dollars of crop losses. Intra- and interspecific genetic variation of Phytophthora spp. and isolates were assessed using amplified fragment length polymorphism DNA fingerprinting. Primer groups EcoRI+AG and MseI+C distinguished P. phaseoli and P. capsici from P. infestans but did not distinguish among different races of P. phaseoli.

Comparison of Colletotrichum orbiculare and Several Allied Colletotrichum spp. for mtDNA RFLPs, Intron RFLP and Sequence Variation, Vegetative Compatibility, and Host Specificity.

ABSTRACT Based on spore morphology, appressorium development, sequence similarities of the rDNA, and similarities in amplified restriction fragment length polymorphism (AFLP), it has been proposed that Colletotrichum orbiculare, C. trifolii, C. lindemuthianum, and C. malvarum represent a single phylogenetic species, C. orbiculare. In the current study, the phylogenetic relationship among isolates in the C. orbiculare species complex was reassessed. In all, 72 isolates of C. orbiculare from cultivated cucurbit or weed hosts, C. trifolii from alfalfa, C. lindemuthianum from green bean, and C. malvarum from prickly sida (Sida spinosa) were examined for mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs), RFLPs and sequence variation of a 900-bp intron of the glutamine synthetase gene and a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase gene, and vegetative compatibility. In addition, host specificity was examined in foliar inoculations on cucurbit, bean, and alfalfa hosts. Inoculations also were conducted on cucumber fruit. Genetically distinct isolates, based on vegetative compatibility, within the species complex (C. orbiculare, C. trifolii, and C. malvarum) had an identical mtDNA haplotype (haplotype A) when examined with each of three different restriction enzymes. Isolates of C. lindemuthianum had a very similar mtDNA haplotype to haplotype A, with a single polymorphism detected with the enzyme HaeIII. The four species represent a phylogenetically closely related group based on a statistical analysis of the 900- and 200-bp intron sequences. However, distinct RFLPs in the 900-bp intron were consistently associated with each species and could be used to qualitatively and quantitatively distinguish each species. Furthermore, each of the species showed distinct host specificity, with isolates of C. orbiculare (from cucurbits), C. lindemuthianum, and C. trifolii being pathogenic only on cucurbits, green bean, and alfalfa, respectively. Consequently, distinct and fixed nucleotide, or genotypic (intron sequences and RFLPs) and phenotypic (host specificity) characteristics can be used to distinguish C. orbiculare, C. lindemuthianum, and C. trifolii from one another; therefore, they should be recognized as distinct species. This species delineation is consistent with the most current species concepts in fungi. More isolates and further characterization is needed to determine whether C. orbiculare from cocklebur and C. malvarum represent distinct species. RFLPs of the 900-bp intron may represent a relatively inexpensive, reliable, and useful diagnostic tool for general species differentiation in the genus Colletotrichum.

A New Biotype of Phaeosphaeria sp. of Uncertain Affinity Causing Stagonospora Leaf Blotch Disease in Cereals in Poland.

A new Phaeosphaeria sp. biotype was isolated from winter ryes in Poland during 1995. Two isolates, Sn23-1 and Sn48-1, were obtained from diseased leaves of cvs. Motto and Dankowskie, respectively. The rye Phaeosphaeria sp. represented by isolate Sn48-1 has similar pycnidiospore morphology and induces disease symptoms in cereals similar to Phaeosphaeria nodorum, the causal agent of Stagonospora nodorum blotch disease (4). The pathogen (Sn48-1) produces hyaline, cylindrical pycnidiospores that are mostly three-septate and measure 12.8 to 23.7 × 2.1 to 3.2 µm (average size = 16 × 2.6 µm) on water agar. A molecular comparison of several genes in isolates Sn23-1 and Sn48-1 revealed that the rye Phaeosphaeria sp. was different from P. nodorum. In the conserved alpha-box sequence (1,93 bp) of the MAT1-1 gene, a four nucleotide difference occurred between the wheat-biotype P. nodorum and isolates Sn23-1 and Sn48-1 (GenBank Accession Nos. AY072933 and AF322008). In addition, the length of the internal transcribed spacer (ITS) region of the nuclear rDNA was the same for the wheat-biotype P. nodorum and the two rye Phaeosphaeria sp. isolates. However, a six nucleotide discrepancy was found in the ITS region (GenBank Accession Nos. U77362 and AF321323). The beta-glucosidase (bgl1) and beta-tubulin (tubA) genes differ in length between the wheat-biotype P. nodorum and two rye Phaeosphaeria sp. isolates (2,3). The main difference was due to the intron sizes of these two genes. One extra nucleotide was found in the intron2 of the bgl1 gene (GenBank Accession Nos. AY683619 and AY683620) and the intron1 of the tubA gene (GenBank Accession Nos. AY786337 and AY786331), respectively, in these two rye Phaeosphaeria sp. isolates. Disease severity on the fifth leaf (GS15) of Polish wheat (Alba, Begra, and Liwilla), triticale (Bogo and Pinokio), and rye (Zduno) cultivars was assessed with one (resistant) to nine (susceptible) scales 14 days after inoculation. Aggressiveness of wheat-biotype P. nodorum isolate Sn26-1 and rye Phaeosphaeria sp. isolate Sn48-1 was significant (P < 0.01) in five cultivars except in the moderately resistant wheat cv. Liwilla. The rye Phaeosphaeria sp. isolate Sn48-1 severely affected Polish rye Zduno (8.3) and two triticale cultivars (6.5), while the infection by isolate Sn26-1 was moderate (3-4). On the contrary, the wheat-biotype P. nodorum isolate Sn26-1 was more aggressive on wheat (4.1 on moderately resistant Alba and 6.2 on highly susceptible Begra) than the rye Phaeosphaeria sp. isolate Sn48-1, which had a scale of 2.2 and 4.3, respectively. Under laboratory conditions, the rye isolate Sn48-1 was able to cross with the wheat-biotype P. nodorum isolate Sn26-1 that has an opposite mating-type (MAT1-2) gene, but few viable ascospores were produced (1). References: (1) P. C. Czembor and E. Arseniuk. Mycol. Res. 104:919, 2000. (2) A. Malkus et al. FEMS (Fed. Eur. Microbiol. Soc.) Lett. 249:49, 2005. (3) E. Reszka et al. Can. J. Bot. 83:1001, 2005. (4) M. J. Richardson and M. Noble. Plant Pathol. 19:159, 1970.

Genetic Diversity of Sclerotinia homoeocarpa Isolates from Turfgrasses from Various Regions in North America.

Sixty-seven isolates of Sclerotinia homoeocarpa, causing dollar spot disease in creeping bentgrass, annual bluegrass, Bermudagrass, and perennial ryegrass turf, collected from 23 golf courses in various geographical regions of the United States and Canada between 1972 and 2001, were characterized by vegetative compatibility, genetic diversity, and pathogenicity. Eleven vegetative compatibility groups (VCGs A to K) were identified among the isolates tested in this study, and five of them (VCGs G to K) were new. VCG B was the most predominant group, typifying 33 isolates (51%) tested. S. homoeocarpa isolates collected from golf courses in Pennsylvania belonged to seven VCGs (A, B, E, F, G, I, and K), whereas three groups were observed in those collected from New York (B, E, and G) and New Jersey (E, H, and I). Two isolates, one each from Pennsylvania and Canada, were incompatible when paired with the tester isolates in all possible combinations, and did not fall into any known VCG. An isolate collected from Canada was compatible with tester isolates from two VCGs (C and D). Genetic analyses using amplified fragment length polymorphism (AFLP) showed the presence of two genetically distinct groups, designated as major group and the minor group. The major group included 36 isolates collected from various golf courses in the United States and Canada. Two isolates collected from bermudagrass in Florida formed a separate cluster, the minor group. Isolates that belonged to the major group were further divided into two subgroups (1 and 2). Subgroup 1 consisted of all the isolates that belonged to VCGs A, E, G, H, and I. Three of the four isolates that belonged to VCG K also were clustered with isolates of subgroup 1. Subgroup 2 consisted of all the isolates from VCG B, and one each from VCGs F and K. Pathogenicity assays on Penncross creeping bentgrass showed significant differences (P = 0.05) in virulence among the isolates. Overall, a relationship between virulence and VCGs was observed, in which certain virulence groups corresponded to specific VCGs; however, such a relationship was not observed between virulence and AFLPs. Close similarity among isolates of S. homoeocarpa collected from different locations in the United States and Canada suggests that isolates of the same genotype could be involved in outbreaks of dollar spot epidemics at multiple locations.

Reactivation of Bentgrass Dead Spot and Growth, Pseudothecia Production, and Ascospore Germination of Ophiosphaerella agrostis.

Ophiosphaerella agrostis incites bentgrass dead spot (BDS) of creeping bentgrass. Little is known about the biology of O. agrostis; hence the primary goal of this study was to determine some basic biological properties of the pathogen and epidemiological components of the disease. Winter-dormant creeping bentgrass field samples showing symptoms of BDS were incubated at temperatures ranging from 15 to 30°C. Between 12 and 28 days of incubation, reactivation of BDS symptoms occurred at temperatures ≥20°C, but the greatest expansion in BDS patch diameter occurred at 25 and 30°C. The optimum temperatures for growth of hyphae among 10 O. agrostis isolates ranged from 25 to 30°C, and growth was suppressed at 35°C. Pseudothecia of O. agrostis were produced in vitro on a mixture of tall fescue seed and wheat bran. Pseudothecia developed under constant fluorescent light at 13 to 28°C, but no pseudothecia developed in darkness at any temperature. Pseudothecia developed in as few as 4 days, but the highest numbers appeared about 30 days after incubation began. Ascospores incubated at 25°C germinated in as little as 2 h, with germ tubes generally emerging from the terminal rather than interior cells of ascospores. Germination during the first 4 h of incubation was enhanced by both light and the presence of bentgrass leaves or roots. After 18 h of incubation, however, there were few differences in the percentage of ascospores germinated regardless of light treatment or presence of plant tissue.

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.

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.

Evaluation of Infection Processes and Resulting Disease Caused by Dendryphion penicillatum and Pleospora papaveracea on Papaver somniferum.

ABSTRACT Two pathogenic fungi of opium poppy, Pleospora papaveracea and Dendryphion penicillatum, were isolated from field material in Beltsville, MD. The processes of infection by these two fungi were studied to determine the optimal environmental conditions for infection. Both fungi formed appressoria capable of penetrating directly through the plant epidermal layer. Of the two fungi, P. papaveracea was more aggressive, causing more rapid necrosis. Appressorial formation by P. papaveracea occurred as early as 4 h after application of a conidial suspension to poppy leaves. P. papaveracea formed more appressoria than did D. penicillatum, especially at cool temperatures (7 to 13 degrees C). In greenhouse studies, P. papaveracea caused more damage to opium poppy than did D. penicillatum when applied in 10% unrefined corn oil. In the field, P. papaveracea was more consistent in its effects on opium poppy from a local seed source designated Indian Grocery. P. papaveracea caused higher disease ratings, more stem lesions, and equal or greater yield losses than did D. penicillatum on Indian Grocery. The late-maturing opium poppy variety White Cloud was severely damaged by disease, regardless of formulation or fungal treatment. P. papaveracea was the predominant fungus isolated from poppy seed capsules and the only fungus reisolated from the field the following year. These studies provide a better understanding of the infection process and the differences between these two pathogenic fungi and will be beneficial for the development of the fungi as biological control agents.

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.

A New Disease of Agrostis palustris Incited by an Undescribed Species of Ophiosphaerella.

Creeping bentgrass (Agrostis palustris; syn. Agrostis stolonifera) is widely used on golf course putting greens. In September and October 1998, samples of diseased creeping bentgrass were received from golf courses in Maryland, Virginia, and Ohio. Disease symptoms developed in August or September 1998, and appeared initially as 1.0- to 2.0-cm-diameter, reddish brown spots that enlarged to about 8.0 cm in diameter. Leaves of plants in the center of diseased patches were tan and those on the periphery were reddish brown. Dark, ectotrophic hyphae were not observed on roots. Numerous pseudothecia were embedded in necrotic leaf and stolon tissues. A fungus was isolated from leaves, stems, and roots, and single-spore isolates were obtained from pseudothecia. Colonies of all isolates were identical in appearance and were initially rose-quartz to pinkish brown, developing a gray color as they aged. Inoculum was prepared by placing mycelium from a single-spore isolate on an autoclaved medium consisting of 50% tall fescue (Festuca arundinacea) seed, and 50% wheat (Triticum aestivum) bran (vol/vol) and grown at 28°C for 8 days. Putter and Crenshaw creeping bentgrass seedlings were grown for 14 days in 12 cm2 pots containing an autoclaved topdressing mix with a mechanical analysis of 95% sand, 1% silt, and 4% clay. The inoculum (200 mg) was mixed into the upper 5 mm of the sandy soil. Pots were placed in plastic bags and incubated during the daytime on a windowsill bench (20 to 24°C), and were maintained at 25°C at night in a darkened growth chamber. After 7 days, 2.0-cm-diameter patches of blighted leaves were observed on both cultivars in nearly all pots, and pseudothecia were found on the inoculum or on blighted foliage in some pots after 20 days. Blighted leaves were covered with a pale pinkish white mycelium and newly infected leaves at the periphery of the dead spot were a pale reddish brown. Most plants were dead 20 days after inoculation. The fungus was reisolated from blighted leaves of both cultivars and all isolates produced colonies identical in appearance and growth rate to those produced by the single-spore isolate. Pseudothecia produced in vivo were sectioned with a freezing microtome and examined microscopically. Bitunicate asci were observed and contained light-brown, 6- to 15-septate, filiform ascospores that were usually spirally twisted in the ascus and measured 70 to 150 × 2.0 to 2.5 µm. Characteristics of the pseudothecia and the ascospores fit those of the genus Ophiosphaerella Speg. (1). Based on morphometric studies of 12 collections from three different states, this fungus can be distinguished from O. graminicola by the lack of periphyses and fewer septa in ascospores (i.e., 12 to 20 septa in O. graminicola). It was distinguished from O. herpotricha by characteristics of the pseudothecia neck, ascospores, and colony color. Because of these differences, we suggest that this fungus represents a new species attacking creeping bentgrass, which will be described after further morphometric and molecular analyses. Reference: (1) J. Walker. Mycotaxon 11:1, 1980.

Alfalfa (Medicago sativa L.) resistance to the root-lesion nematode, Pratylenchus penetrans: defense-response gene mRNA and isoflavonoid phytoalexin levels in roots.

Alfalfa (Medicago sativa) varieties with antibiosis-based resistance to the root-lesion nematode (Pratylenchus penetrans), a migratory endoparasite of many crops, have been developed by recurrent selection. Individual plants from these varieties that support significantly lower nematode reproduction were identified for molecular and biochemical characterization of defense responses. Before nematode infection, RNA blot analysis revealed 1.3-1.8-fold higher phenylpropanoid pathway mRNA levels in roots of three resistant plants as compared to three susceptible alfalfa plants. The mRNAs encoded the first enzyme in the pathway (phenylalanine ammonia-lyase), the first in the pathway branch for flavonoid biosynthesis (chalcone synthase), a key enzyme in medicarpin biosynthesis (isoflavone reductase) and a key enzyme in the pathway branch for biosynthesis of lignin cell wall precursors (caffeic acid O-methyltransferase). After nematode infection, the mRNAs declined over 48 h in resistant roots but rose in susceptible plants during the first 12 h after-infection and then declined. Acidic beta-1,3-glucanase mRNA levels were initially similar in both root types but accumulated more rapidly in resistant than in susceptible roots after nematode infection. Levels of a class I chitinase mRNA were similar in both root types. Histone H3.2 mRNA levels, initially 1.3-fold higher in resistant roots, declined over 6-12 h to levels found in susceptible roots and remained stable in both root types thereafter. Defense-response gene transcripts in roots of nematode-resistant and susceptible alfalfa plants thus differed both constitutively and in inductive responses to nematode infection. HPLC analysis of isoflavonoid-derived metabolites of the phenylpropanoid pathway revealed similar total constitutive levels, but varying relative proportions and types, in roots of the resistant and susceptible plants. Nematode infection had no effect on isoflavonoid levels. Constitutive levels of the phytoalexin medicarpin were highest in roots of the two most resistant plants. Medicarpin inhibited motility of P. penetrans in vitro.

Application of amplified restriction fragment length polymorphism for genetic characterization of colletotrichum pathogens of alfalfa.

ABSTRACT Amplified restriction fragment length polymorphism (AFLP) was used to assess the levels of genomic variations among species and isolates of the genus Colletotrichum. Our objective was to characterize at the molecular level two alfalfa pathogens, isolates Arl-NW and 57RR, which are unusually aggressive to anthracnose-resistant alfalfa cultivars and whose taxa has been uncertain based on morphological criteria. The fingerprint patterns obtained were complex but did enable us to place these two isolates within the species C. trifolii and C. gloeosporioides, respectively. The diversity detected with AFLP among and within Colletotrichum species from alfalfa and other crops corroborated their published taxonomy based on morphology, ribosomal DNA sequence, and random amplified polymorphic DNA analyses. Similarity matrices generated with three primer pairs were highly correlated and, thus, were combined to determine the similarity among the fungal species and isolates that were analyzed. Analysis of the data generated with each of the primer pairs individually and application of either distance or parsimony methods supported the placement of these two isolates. The parsimony method of data analysis was more confirmatory in the placement of Phoma medicaginis as an out-group than the distance method, using either simple matching or Jaccard's coefficients to generate the similarity matrices. Our conclusion is that the AFLP technique will be useful for identification of individual isolates within complex genera such as Colletotrichum because of its ability to generate large numbers of polymorphisms and the consistency of polymerase chain reaction amplification.