Phragmidium rosae-multiflorae on Rosa multiflora reported from Pennsylvania, USA.

Rosa multiflora Thunb. is a perennial shrub native to eastern Asia. It is commonly found on habitat margins, such as forest edges, streams, and roadsides (CABI n.d.). Due to its aromatic flowers, its usefulness in erosion control, and as a living livestock fence, R. multiflora was introduced to North America as an ornamental in the early 1800's (Hindal and Wong 1988). However, R. multifora, grows rapidly and frequently outcompetes native species, and is therefore considered invasive in North America (Hindal and Wong 1988). In May 2020 Phragmidium rosae-multiflorae Dietel, or rose rust, was collected from R. multiflora in Patton Woods Park, a small residential park in Patton Township, Centre Co., PA, USA (40° 47' 28.40" N; 77° 55' 33.37" W). P. rosae-multiflorae is an autoecious macrocyclic rust fungus known to occur on R. multiflora in the plant's native range. To our knowledge, there are no previously published records of P. rosae-multiflorae in North America. The host exhibited conspicuous aecia on the stems and petioles. Aecia were caeoma form, orange, irregular, erumpant, and pulverulent. Aeciospores were one-celled, verrucose, hyaline to pale yellow, variable in shape, ranging from ellipsoidal to globose (Fig.1), measuring 22.5 ± 3 × 15.5 ± 5 µm (n = 30). Disease symptomology and aecia are consistent with P. rosae-multiflorae Dietel (Wei 1988; Liu et. al 2020). Fungal DNA was extracted from infected petioles. Polymerase chain reaction and sequencing of the 28S region of the nuclear ribosomal DNA repeat was conducted with primers Rust2inv and LR6 following protocols in Aime (2006). The sequence shares 98.77% identity (900 / 487 bp) with P. Rosae-multiflorae from China (MN264739). A voucher specimen has been preserved in the Arthur Fungarium at Purdue University (PUR N23123) with corresponding 28S sequence (GenBank accession #MZ323415). The recorded occurrence of P. rosae-multiflorae on R. multiflora in North America is significant, given the ecological impact of the host plant as an invasive species and need for biocontrol. While no formally published records of this fungus in North America exist, there are putative occurrences across the northeastern United States reported on Global Biodiversity Information Facility (GBIF) and the Maryland Biodiversity Project websites.

Pythium and Phytopythium Species in Two Pennsylvania Greenhouse Irrigation Water Tanks.

Two commercial greenhouses producing potted plants in Pennsylvania using recycled irrigation water in an ebb-and-flood system have incurred significant crop losses due to Pythium aphanidermatum. In cooperation with the greenhouses, one or more of their water tanks was monitored continuously (128 tank samplings) for Pythium spp. by baiting. Nine species of Pythium and three species of Phytopythium were recovered, representing clades A, B, E, and K, but none was P. aphanidermatum. The recovered Pythium spp. were (i) P. rostratifingens, (ii) isolates identical to Pythium sp. nov. OOMYA1702-08 (clade B2), (iii) P. coloratum, (iv) P. middletonii, (v) and (vi) two new species in clade E2, (vii) a new species in clade B2, (viii) isolates very similar to Pythium sp. nov. OOMYA1646-08 (clade E2), and (ix) a new species in clade A. The Phytopythium spp. recovered were (i) Phytopythium litorale, (ii) P. helicoides, and (iii) P. chamaehyphon. This article illustrates the different communities of Pythium and Phytopythium spp. found in each greenhouse over 10 months. Some of the baited species display resistance to the oomycete fungicide active ingredient, mefenoxam. P. helicoides and the new species in clade B2 were pathogenic on seedlings in potting mix with fertilizer added.

Survival of Phytophthora alni, Phytophthora kernoviae, and Phytophthora ramorum in a simulated aquatic environment at different levels of pH.

Phytophthora ramorum, Phytophthora alni, and Phytophthora kernoviae present significant threats to biosecurity. As zoosporic oomycetes, these plant pathogens may spread through natural waterways and irrigation systems. However, survival of these pathogens in aquatic systems in response to water quality is not well understood. In this study, we investigated their zoospore survival at pH 3-11 in a 10% Hoagland's solution over a 14-day period. The results showed that all three pathogens were most stable at pH 7, although the populations declined overnight irrespective of pH. Extended survival of these species depended on the tolerance of pH of their germinants. Germinants of P. alni ssp. alni and P. ramorum were more basic tolerant (pH 5-11), while those of P. kernoviae were more acidic tolerant (pH 3-9). These tolerant germinants formed compact hyphae or secondary sporangia to allow longer survival of these pathogens. Long-term survival at a broad pH range suggests that these pathogens, especially P. ramorum, are adapted to an aquatic environment and pose a threat to new production areas through water dispersal.

Phytophthora aquimorbida sp. nov. and Phytophthora taxon 'aquatilis' recovered from irrigation reservoirs and a stream in Virginia, USA.

Two distinct subgroups (L2 and A(-2)) were recovered from irrigation reservoirs and a stream in Virginia, USA. After molecular, morphological and physiological examinations, the L2 subgroup was named Phytophthora aquimorbida and the A(-2) designated as Phytophthora taxon 'aquatilis'. Both taxa are homothallic. P. aquimorbida is characterized by its noncaducous and nonpapillate sporangia, catenulate and radiating hyphal swellings and thick-walled plerotic oospores formed in globose oogonia mostly in the absence of an antheridium. P. taxon 'aquatilis' produces plerotic oospores in globose oogonia mostly with a paragynous antheridium. It has semi-papillate, caducous sporangia with variable pedicels, but it does not have hyphal swelling. Analyses of ITS, CO1, ß-tubulin and NADH1 sequences revealed that P. aquimorbida is closely related to P. hydropathica, P. irrigata and P. parsiana, and P. taxon 'aquatilis' is related to P. multivesiculata. The optimum temperature for culture growth is 30 and 20 C for P. aquimorbida and P. taxon 'aquatilis' respectively. Both taxa were pathogenic to rhododendron plants and caused root discoloration, pale leaves, wilting, tip necrosis and dieback. Their plant biosecurity risk also is discussed.

Sublethal Doses of Mefenoxam Enhance Pythium Damping-off of Geranium.

The effect of sublethal doses of fungicides on fungicide-resistant Pythium isolates is unknown but potentially relevant to disease management. Occasional grower reports of Pythium disease increases after fungicide applications and our observations of greater radial growth in vitro on fungicide-amended media than on nonamended media suggests that Pythium isolates may be stimulated by sublethal doses of fungicides. The objectives of this study were to determine whether Pythium isolates were stimulated by sublethal doses of mefenoxam in vitro and whether this stimulation had any influence on Pythium damping-off of geranium seedlings. A mefenoxam-resistant isolate of Pythium aphanidermatum displayed 10% mean radial growth increase in vitro with mefenoxam at 1 × 10-10 µg/ml compared with growth on nonamended agar (nonsignificant). Geranium seedlings treated with one of eight mefenoxam concentrations were inoculated with 5-mm-diameter colonized agar plugs and evaluated for disease severity every 24 h. The area under the disease progress curve and the survival curve were estimated for each treatment and compared. Significant increases in damping-off were observed with mefenoxam at 1 × 10-6 and 1 × 10-10 µg/ml. Our data indicate that a Pythium isolate with resistance to mefenoxam can be stimulated by sublethal doses of the fungicide, and that this stimulation can result in significantly higher rates of Pythium damping-off of geranium seedlings.

Genetic structure and distribution of pythium aphanidermatum populations in Pennsylvania greenhouses based on analysis of AFLP and SSR markers.

Pythium aphanidermatum is one of the most aggressive species in the genus and has a wide host range, but little is known about its population genetic structure. We tested 123 P. aphanidermatum isolates with six AFLP primer combinations and four SSR markers. The genetic diversity of P. aphanidermatum was 0.34 with AFLP and 0.55 with SSR markers. SSR genotypes totaled 3-8 for each locus, and a total of 14 SSR genotypes were found among all isolates. Three major genetic groups were identified with the combination of AFLP and SSR marker data. The genetic structure observed among P. aphanidermatum isolates was related to location and mefenoxam fungicide resistance instead of host. Four genotypes (PA1, PA2, PA5 and PA7) were found in the population from a commercial greenhouse, and the genetic diversity of a greenhouse population was similar to that found in the whole sample. The molecular tools for P. aphanidermatum isolates identified the possible gene flow within and among populations in Pennsylvania greenhouses.

Detection of Elm Yellows Phytoplasma in Elms and Insects Using Real-Time PCR.

A rapid and accurate method to detect the common strain of elm yellows (EY) phytoplasma in elm and insect samples was developed using a real-time polymerase chain reaction (PCR) procedure based on the TaqMan minor-groove-binder probe. Primers and probe were designed based on the EY phytoplasma-specific translocation protein secY gene DNA sequence. Success of the DNA extraction procedure was evaluated by amplifying the chloroplast trnL gene of Ulmus americana. The real-time PCR assay reacted positively with EY and EY phytoplasma strain ULW DNA, an isolate which occurs in Europe. It did not cross-react with Illinois EY or aster yellows phytoplasma DNA, both of which are known to occur in elm trees in the United States, nor did it amplify several other phytoplasmas belonging to the 16SrV and other phylogenetic groups. The real-time PCR protocol was used to identify 30 EY-positive elm trees on The Pennsylvania State University, University Park campus. Threshold cycle (CT) values obtained from the EY phytoplasma-infected elm trees ranged from 15 to 37. EY phytoplasma was detected in several leafhopper taxa. This real-time PCR method can be used for the diagnostic screening of elm trees and for the identification of possible insect vectors of EY phytoplasma.

Mefenoxam Sensitivity in Phytophthora cinnamomi Isolates.

Phytophthora cinnamomi is a destructive root pathogen of numerous woody plant species in the ornamental plant nursery. Sixty-five isolates of P. cinnamomi were evaluated for mefenoxam sensitivity on 20% clarified V8 agar amended with mefenoxam at 0 or 100 µg/ml. In the presence of mefenoxam at 100 µg/ml, eight isolates were intermediately sensitive, with mycelium growth ranging between 11 and 18% of the nonamended control, and 57 isolates were highly sensitive, with little or no mycelium growth. Five intermediately sensitive and five sensitive isolates were chosen to characterize their responses to mefenoxam at 0, 0.1, 1, 10, and 100 µg/ml. For intermediately sensitive isolates, the mefenoxam concentration causing 50% inhibition of mycelium growth (EC50 values) ranged between 0.03 and 0.08 µg/ml; EC50 values for sensitive isolates varied from 0.01 to 0.02 µg/ml. Five intermediately sensitive and seven sensitive isolates were selected further to assess in vivo sensitivity to mefenoxam using Lupinus angustifolius 'Russell Hybrids'. Lupine seedlings were treated with distilled water or mefenoxam at label rate (Subdue MAXX, 1 fl. oz. of product per 100 gal.) and then, 2 days later, inoculated with a 5-mm-diameter mycelial plug of P. cinnamomi on each cotyledon. Mefenoxam-treated plants averaged more than 96% less disease than water-treated plants. Mefenoxam provided adequate protection of lupines from infection by all 12 isolates regardless of their in vitro levels of sensitivity to mefenoxam. The ability to develop mefenoxam resistance was assessed in P. cinnamomi isolates with different mefenoxam sensitivity by UV mutagenesis and adapting mycelium to increasing concentrations of mefenoxam. Both UV mutagenesis and mycelium adaptation generated isolates with reduced sensitivity to mefenoxam. These isolates, however, did not grow as quickly as their corresponding parent. This study suggests that P. cinnamomi populations from ornamental nurseries in Virginia are sensitive to mefenoxam.

Zoosporic tolerance to pH stress and its implications for Phytophthora species in aquatic ecosystems.

Phytophthora species, a group of destructive plant pathogens, are commonly referred to as water molds, but little is known about their aquatic ecology. Here we show the effect of pH on zoospore survival of seven Phytophthora species commonly isolated from irrigation reservoirs and natural waterways and dissect zoospore survival strategy. Zoospores were incubated in a basal salt liquid medium at pH 3 to 11 for up to 7 days and then plated on a selective medium to determine their survival. The optimal pHs differed among Phytophthora species, with the optimal pH for P. citricola at pH 9, the optimal pH for P. tropicalis at pH 5, and the optimal pH for the five other species, P. citrophthora, P. insolita, P. irrigata, P. megasperma, and P. nicotianae, at pH 7. The greatest number of colonies was recovered from zoospores of all species plated immediately after being exposed to different levels of pH. At pH 5 to 11, the recovery rate decreased sharply (P < or = 0.0472) after 1-day exposure for five of the seven species. In contrast, no change occurred (P > or = 0.1125) in the recovery of any species even after a 7-day exposure at pH 3. Overall, P. megasperma and P. citricola survived longer at higher rates in a wider range of pHs than other species did. These results are generally applicable to field conditions as indicated by additional examination of P. citrophthora and P. megasperma in irrigation water at different levels of pH. These results challenge the notion that all Phytophthora species inhabit aquatic environments as water molds and have significant implications in the management of plant diseases resulting from waterborne microbial contamination.

Phytophthora irrigata, a new species isolated from irrigation reservoirs and rivers in Eastern United States of America.

A new species of Phytophthora, previously referred to as the 'Dre I' taxon, is named Phytophthora irrigata. Isolates of P. irrigata morphologically and physiologically resemble Phytophthora drechsleri. They are heterothallic, produce nonpapillate sporangia, and grow well at 35 degrees C. The above two species differ significantly in uniformity of mycelium, presence of chlamydospores, DNA fingerprint, and sequences of multiple nuclear and mtDNA regions. Phytophthora irrigata produces smaller sporangia and a distinct DNA fingerprint. Sequence alignments in the ribosomal DNA internal transcribed spacer regions place Phytophthora fallax and Phytophthora captisoa as its closest relatives. The optimal temperature for culture growth is above 30 degrees C and the maximum temperature is 40 degrees C. This new species is abundant in irrigation reservoirs and natural waterways in Virginia and was also isolated in Pennsylvania.

Identification and characterization of simple sequence repeat markers for Pythium aphanidermatum, P. cryptoirregulare, and P. irregulare and the potential use in Pythium population genetics.

Six simple sequence repeat (SSR)-enriched genome libraries from Pythium aphanidermatum, P. irregulare, and P. cryptoirregulare were constructed to develop SSR markers. One hundred six SSR primer pairs for P. aphanidermatum, 73 for P. cryptoirregulare, and 82 for P. irregulare were initially identified. After examining primers, the most polymorphic and reproducible SSR markers were selected for each Pythium species; 14 in P. aphanidermatum, 21 in P. irregulare, and 22 in P. cryptoirregulare. Analysis of isolates from each Pythium species using SSR markers showed the high degree of gene diversity and polymorphic information content (PIC) value in the three species. The average number of alleles was 3.5-5.3 in the three Pythium species. Seven SSR loci from P. cryptoirregulare and P. irregualre showed the distinct genetic separations of P. irregualre complex isolates. SSR markers identified for the three Pythium target species were highly transferable to other closely related Pythium species. Cross-amplification was found in all SSR markers between P. cryptoirregulare and P. irregulare. SSR loci were successfully amplified by direct PCR from mycelia of P. aphanidermatum, P. cryptoirregulare, and P. irregulare. These newly developed SSR markers can be used for population genetic studies and monitoring the movement of isolates in crop production systems or in nature.

Pythium cryptoirregulare, a new species within the P. irregulare complex.

Pythium identification is based on several characteristics with considerable variation, particularly in Pythium irregulare Buis. as currently recognized. Thirty-one isolates of Pythium irregulare Buis. from various hosts and geographic regions were compared by genetic analysis of multiloci DNA fingerprints, sequence analysis of nuclear and mitochondrial genes and morphological and growth rate studies. Previous research indicated two distinct groupings within the species, P. irregulare sensu stricto and a clade referred to here as Pythium sp. Parsimony analyses of 338 AFLP markers divided P. irregulare s.l. into two clades. Comparison of the allele frequencies of 236 polymorphic AFLP loci revealed significant differences between them. The two clades differed in the frequencies of 182 (77%) alleles. P. irregulare s.s. had 122 (52%) polymorphic loci while Pythium sp. had 205 (87%). Pythium sp. had one fixed allele and 79 polymorphic loci absent in P. irregulare s.s. P. irregulare s.s. displayed 16 polymorphic loci absent in Pythium sp. Parsimony and distance analyses of the ribosomal intergenic transcribed spacers (ITS) and the cox II gene sequences support the separation of P. irregulare s.s. and Pythium sp. Amplicon length in P. irregulare s.s. ITS sequences were 936-938 bp and 936-949 bp in Pythium sp. The two clades were separated by two fixed insertion/deletion mutations, nine fixed nucleotide substitutions in the ITS region and three fixed single nucleotide substitutions in the cox II sequences. Average growth rates of the groups differed at 10, 30 and 36 C but not at 15, 21 or 25 C. Statistically significant differences were found in oogonium, oospore and ooplast diameters, antheridial cell length and in ooplast index. We propose that a new species, Pythium cryptoirregulare, be delineated from Pythium irregulare sensu stricto.

Effects of Propamocarb Hydrochloride on Mycelial Growth, Sporulation, and Infection by Phytophthora nicotianae Isolates from Virginia Nurseries.

Propamocarb hydrochloride is a systemic fungicide commonly used for control of Phytophthora diseases of nursery crops. Here we report on the effect of this compound on different growth stages of Phytophthora nicotianae, a major pathogen of numerous herbaceous and some woody ornamental plants. A total of 71 isolates were assayed for sensitivity to propamocarb at two concentrations of 1.8 mg/ml (label rate) and 10 mg/ml using clarified V8 agar as a base medium. All isolates grew at 10 mg/ml with the most sensitive isolate having 34.8% relative growth compared with growth on nonamended medium. Nine isolates were selected and further tested for mycelial growth at 0, 1, 10, 25, 50, and 100 mg/ml, and for sporangium production, zoospore motility, and germination at 0, 5, 50, 500, 5,000, and 50,000 µg/ml. EC50 values ranged from 2.2 to 90.1 mg/ml for mycelial growth, 133.8 to 481.3 µg/ml for sporangium production, 88.1 to 249.8 µg/ml for zoospore motility, and 1.9 to 184.6 µg/ml for zoospore germination, respectively. In addition, 17 selected isolates were evaluated for propamocarb sensitivity on Pelargonium × hortorum cv. White Orbit. Two days after seedlings were treated with propamocarb at 3.6 mg/ml, they were inoculated by either inverting one 5-mm-diameter plug of a 3-day-old culture or applying a 10-µl drop containing 20 zoospores onto each cotyledon. Propamocarb hydrochloride provided good protection of geranium seedlings from zoospore infections but not from mycelial infections. These results suggest that this fungicide must be used preventively for Phytophthora disease management and that mycelial growth may not be the most reliable measurement to determine the development of fungicide resistance to this compound in Phytophthora species at production facilities and in the landscape.

Amplified Fragment Length Polymorphism Analysis and Internal Transcribed Spacer and coxII Sequences Reveal a Species Boundary Within Pythium irregulare.

ABSTRACT Pythium irregulare is a plant-pathogenic oomycete that causes significant damage to a variety of crops, including ornamentals and vegetables. Morphological as well as molecular studies have reported high levels of genetic diversity within P. irregulare sensu lato which has raised the question as to whether it is a single species or is actually a complex of morphologically similar (cryptic) species. In this study, we used amplified fragment length polymorphism (AFLP) fingerprinting and DNA sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal genes (ITS region) and a portion of the mitochondrial cytochrome oxidase II gene and the spacer region between coxI and coxII to characterize 68 isolates of P. irregulare from the United States. The ITS sequence of a P. irregulare neotype at the CBS collection as well as ITS and coxII sequences for P. irregulare, P. spinosum, and P. sylvaticum from previous studies were included in our analysis. Cluster analysis identified a 19-isolate group (IR-II) that separated itself from the rest of the sample (IR-I). Population structure and sequence analyses supported the distinction of IR-I and IR-II and identified IR-II as P. irregulare sensu stricto. IR-I was designated Pythium sp. clade IR-I. Two insertion/deletion mutations and nine nucleotide substitutions in the ITS region and three in the sequence of coxII and the adjacent spacer region separated the two species. Additionally, they differed significantly (P > 0.01) in the frequency of 182 (77%) AFLP alleles. Gene flow results suggested that P. irregulare sensu stricto and Pythium sp. clade IR-I are cryptic species capable of exchanging favorable alleles (Nm = 0.72).

Diagnosis and Population Analysis of Pythium Species Using AFLP Fingerprinting.

Accurate identification of Pythium species, the causal agents of Pythium root rot and dampingoff of seedlings, and characterization of populations within the species would greatly assist in selecting and implementing control strategies for these pathogens. Several molecular techniques offer methods for accurate and rapid identification of species, but provide little information about their populations. In this study, amplified fragment length polymorphism (AFLP) fingerprinting was used to characterize plant-pathogenic Pythium species and intraspecific populations. Species-diagnostic AFLP fingerprints for Pythium aphanidermatum, P. irregulare, and P. ultimum, and tentative fingerprints for six other species, were identified. Intraspecific distance analyses of P. aphanidermatum, P. ultimum, and P. irregulare revealed distinct patterns of intraspecific variation among the three species. P. aphanidermatum showed the smallest mean distance among isolates (15%), followed by P. ultimum (37%). P. irregulare had the largest mean distance among isolates (64%), which were divided into two populations with great genetic differentiation (FST = 0.2), suggesting the presence of a cryptic species boundary within this species.

Single-strand conformational polymorphism analysis of the ribosomal internal transcribed spacer 1 for rapid species identification within the genus Pythium.

Single-strand conformational polymorphism (SSCP) of the ribosomal internal transcribed spacer 1 (ITS-1) was characterized for 58 isolates of Pythium, representing 41 species from the five groups of Plaats-Niterink. Thirty-one species each produced a distinct SSCP pattern. Three species produced more than one unique pattern, corresponding to morphological subgrouping. The remaining seven species produced three distinct patterns with two or three morphologically similar species sharing a pattern. A successful blind test with four samples and the identification of eight previously unknown isolates from irrigation water demonstrated the reliability of this technique for species identification. Each SSCP pattern was defined and described by the positions of the top and bottom bands and the number of bands in between, which allows laboratories to use this technique without need to access the type isolates of Pythium species.

The internet-based fungal pathogen database: a proposed model.

ABSTRACT A better understanding of the phenotypic and genetic diversity of significant agricultural pathogens and how their populations change in the field is critical for designing successful, long-term disease management strategies. Although efforts to determine the genetic diversity of plant pathogens have substantially increased in recent years, mainly due to the availability of various molecular tools, complementary efforts to archive and integrate the resulting data have been very limited. As a consequence, it is often difficult to compare the available data from various laboratories because the data have been generated by diverse tools, often preventing any direct comparisons, and are saved in a format that is unsuitable for comparative studies. The establishment of an internet-based database that cross-links the digitized genotypic and phenotypic information of individual pathogens at both the species and population levels may allow us to effectively address these problems by coordinating the generation of data and its subsequent archiving. We discuss the needs, benefits, and potential structure of such a database.