Gary W. Moorman

Identification and characterization of Pythium species associated with greenhouse floral crops in pennsylvania

During 1996 to 2001, samples submitted to clinics from commercial greenhouses involved 11 species and two unidentified isolates of Pythium from 110 plant samples, five potting soil tests, and five tests of irrigation water. Pythium irregulare was found in 45% of the plant samples, four of the five water samples, and three of the five potting soils. Pythium aphanidermatum accounted for 29% of all plant but 77% of the poinsettia samples. The Pelargonium samples received were infected with P. aphanidermatum, P. dissotocum, P. heterothallicum, group F, P. irregulare, P. myriotylum, and P. ultimum. The base pair sequence of the ITS1, 5.8S, and ITS2 regions of ribosomal DNA effectively differentiated the species encountered. The ras-related protein gene sequence did not differentiate P. aphanidermatum, P. arrhenomanes, and P. deliense from one another. One isolate each of P. cylindrosporum, P. dissotocum, P. heterothallicum, P. splendens, and P. ultimum exhibited resistance to the phenylamide fungicide mefenoxam, an isomer of metalaxyl, while 38% of the P. aphanidermatum and 37% of the P. irregulare isolates were resistant.

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.

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).

Zoosporic Tolerance to pH Stress and Its Implications for Phytophthora Species in Aquatic Ecosystems

ABSTRACT 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 ≤ 0.0472) after 1-day exposure for five of the seven species. In contrast, no change occurred (P ≥ 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.

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.

Containment basin water quality fluctuation and implications for crop health management

Containment basins (CB) are an integral part of recycling irrigation systems that foster agricultural sustainability through water resource conservation. However, little is known regarding this aquatic ecosystem and the lack of water quality data has become an increasingly serious liability in crop health management. Here we report on four distinct seasonal and two diurnal patterns of change in water quality in the CBs. The four seasonal patterns are (a) periodic fluctuation in chlorophyll a, pH, and dissolved oxygen (DO), (b) oxidation–reduction potential (ORP) rises with decreasing DO, (c) tendency for increase in electrical conductivity, salinity, and total dissolved solids, and (d) weather-dependent changes in turbidity and temperature. The two diurnal patterns are (1) chlorophyll a, pH, DO, and temperature consistently peak between 16:00 and 17:00 hours and bottom out around 08:00 hours, and (2) ORP peaks in the morning and bottoms in the evening. Eight of the nine parameters excluding temperature were correlated; and algal blooms appear to be a major driving force for changes in the other seven parameters. These results underscore the importance of water quality monitoring in irrigation management and provide a framework for better understanding of pathogen aquatic ecology and how changes in water quality might be employed in a manner that suppresses plant pathogens and improves crop quality and productivity.

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 Pythiumaphanidermatum, 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.

Bacterial Blight of Geranium: A History of Diagnostic Challenges

Geraniums (Pelargonium spp.) have been an important part of greenhouse potted plant and bedding plant production for almost a century. Cultivars produced by vegetative propagation and by true seeds are grown and sold worldwide. In 1996, the wholesale value of geraniums exceeded

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

205 million in the United States alone (2). Pelargonium species are affected by a number of fungal diseases, including rust (Puccinia pelargonii-zonalis), gray mold (Botrytis cinerea), and root rot (Pythium spp.). Several bacteria and numerous viruses also cause diseases on geranium. Indeed, the most destructive disease of geraniums is bacterial blight (Xanthomonas campestris pv. pelargonii). Although hybrid geraniums grown from seed are susceptible to bacterial blight, it is the vegetatively propagated cultivars of florists’ geranium (P. × hortorum) and ivy geranium (P. peltatum) that are most commonly affected, because the causal organism inhabits the vascular tissue of infected plants and is carried in the cuttings. Munnecke (22) and Nichols (24) estimated 10 to 15% annual losses due to bacterial blight in the 1950s and 1960s, and such losses continue today (22,24). These loss figures are somewhat misleading because they are based on the industry-wide production of geraniums. In individual greenhouse operations, entire geranium crops have been destroyed by this disease. The reputations of several specialty propagators of Pelargonium have been ruined or severely damaged when these growers unknowingly sold infected cuttings and distributed them throughout the bedding plant industry. Despite efforts to eliminate this disease from geranium production systems, it continues to occur annually.

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

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, β-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.