Patricia A. Richardson

Single-strand-conformation polymorphism of ribosomal DNA for rapid species differentiation in genus Phytophthora

Single-strand-conformation polymorphism (SSCP) of ribosomal DNA of 29 species (282 isolates) of Phytophthora was characterized in this study. Phytophthora boehmeriae, Phytophthora botryosa, Phytophthora cactorum, Phytophthora cambivora, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora colocasiae, Phytophthora fragariae, Phytophthora heveae, Phytophthora hibernalis, Phytophthora ilicis, Phytophthora infestans, Phytophthora katsurae, Phytophthora lateralis, Phytophthora meadii, Phytophthora medicaginis, Phytophthora megakarya, Phytophthora nicotianae, Phytophthora palmivora, Phytophthora phaseoli, Phytophthora pseudotsugae, Phytophthora sojae, Phytophthora syringae, and Phytophthora tropicalis each showed a unique SSCP pattern. Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, and Phytophthora megasperma each had more than one distinct pattern. A single-stranded DNA ladder also was developed, which facilitates comparison of SSCP patterns within and between gels. With a single DNA fingerprint, 277 isolates of Phytophthora recovered from irrigation water and plant tissues in Virginia were all correctly identified into eight species at substantially reduced time, labor, and cost. The SSCP analysis presented in this work will aid in studies on taxonomy, genetics, and ecology of the genus Phytophthora.

A Species-Specific Polymerase Chain Reaction Assay for Rapid Detection of Phytophthora nicotianae in Irrigation Water

ABSTRACT Phytophthora nicotianae is a common and destructive pathogen of numerous ornamental, agronomic, and horticultural crops such as tobacco, tomato, and citrus. We have developed a species-specific polymerase chain reaction (PCR) assay for rapid and accurate detection of this pathogen in irrigation water, a primary source of inoculum and an efficient means of propagule dissemination. This PCR assay consists of a pair of species-specific primers (PN), customization of a commercial soil DNA extraction kit for purification of DNA from propagules in irrigation water, and efficient PCR protocols for primer tests and sample detection. The PN primers proved adequately specific for P. nicotianae in evaluations with 131 isolates of P. nicotianae, 102 isolates from 15 other species of Phytophthora, and 64 isolates from a variety of other oomycetes, true fungi, and bacteria. These isolates originated from a wide range of host plants, three substrates (plant tissue, soil, and irrigation water), and numerous geographic locations. The detection sensitivity is between 80 and 800 fg DNA/mul. The assay detected the pathogen in naturally infested water samples from Virginia and South Carolina nurseries more rapidly and accurately than standard isolation methods. Use of this PCR assay can assist growers in making timely disease management decisions with confidence.

Efficacy of Chlorine on Multiple Species of Phytophthora in Recycled Nursery Irrigation Water

Recycled irrigation water is a primary source of inoculum for Phytophthora spp. and is capable of spreading propagules throughout nursery production. Chlorination commonly is used by the industry to disinfest recycled irrigation water; however, chlorine has not been fully researched as a disinfestant for this purpose. In this study, zoospores of seven species and eight isolates of Phytophthora were exposed for 2 min to free available chlorine at 0.25, 0.5, 1.0, 2.0, and 4.0 mg/liter. Zoospores, mycelial fragments, and culture plugs of P. nicotianae also were exposed to chlorine concentrations ranging from 0.25 to 8.0 mg/liter for periods ranging from 15 s to 8 min. In addition, chlorinated water was assayed monthly in 2000 and 2001 at two commercial nurseries, and quarterly in the first year at four other nurseries in Virginia, for chlorine and survival of pythiaceous species using a selective medium. No zoospores of any species tested survived endpoint free chlorine at 2 mg/liter, while limited mycelial fragments of P. nicotianae survived at 8 mg/liter, and mycelial plugs treated at the same level of chlorine were able to produce few sporangia. Phytophthora spp. were recovered only from nursery irrigation water with levels of free chlorine at 0.77 mg/liter or lower. The results of this study are essential for improving current chlorination protocols.

Comparison of membrane filters as a tool for isolating pythiaceous species from irrigation water.

ABSTRACT Filter-based isolation is the primary approach for quantifying plant pathogens in irrigation water; however, the performance of various membranes is largely unknown. This study evaluated nine hydrophilic membranes for recovery of pythiaceous species, a group of very common and destructive pathogens on numerous ornamental plants and many agronomic crops. Three sources of water samples and three plating methods were used for the membrane comparison. Durapore5, Millipore5, and Osmonics5 filtered 100 ml of irrigation water or 50 ml of irrigation runoff in 10 s or less, whereas the other membranes required 34 s to 13.5 min or even a second membrane to filter the same water sample volume. Millipore5 and Durapore5 showed greater recovery rates than other membranes for all water sources when the membranes were directly inverted onto agar plates. Durapore5 was also one of the top two membranes for spore suspension and irrigation water samples when membrane washings were spread over the agar surface. Durapore5 was the only membrane that consistently performed well for both spore suspension and irrigation water samples. These data suggest that use of Durapore5 not only increases the sensitivity of filter-based isolation for quantifying pythiaceous species in irrigation water but also saves filtering time.

Phytophthora pini Leonian resurrected to distinct species status

Phytophthora pini was named by Leonian in 1925, but this species was largely ignored until 1956 and then merged with P. citricola by Waterhouse in 1963. This study compared the ex-type and ex-authentic cultures of these two species with isolates of P. plurivora and the P. citricola subgroups Cil I and III reported previously. Examination of these isolates revealed that the ex-type culture of P. pini is identical to P. citricola I. Phytophthora pini Leonian therefore is resurrected to distinct species status and redescribed here with a Latin description, replacing P. citricola I. Molecular, physiological and morphological descriptions of this species are presented. The molecular description includes DNA sequences of five nuclear and mitochondrial regions as well as PCR-SSCP fingerprints. The relationship among the above species and other species recently segregated from the P. citricola complex also is discussed.

Pathogenicity to Ornamental Plants of Some Existing Species and New Taxa of Phytophthora from Irrigation Water

Eighteen isolates from 12 species of Phytophthora, including several new taxa, were tested for pathogenicity to six ornamental and four vegetable species. The following three inoculation methods were used depending on infection court targeted: vermiculite culture inoculation for roots, agar block inoculation for fruit, and zoospore inoculation for foliage. All six new taxa (P. irrigata, P. hydropathica, Dre III, Cil I, Cip-like, and Gon I) are pathogenic to one or more test plants. Specifically, taxon Cil I was identified as a growing threat to horticultural crops, particularly ornamental crops in container production nurseries. The potential host list of P. tropicalis was expanded to four new families (Apocynaceae, Asteraceae, Begoniaceae, and Fabaceae) and one additional genus within each of three existing families (Ericaceae, Cucurbitaceae, and Solanaceae). New potential hosts were also identified for other existing species of Phytophthora. The practical implications of these results in crop health management programs for both ornamental and vegetable crops locally, and for development and implementation of agricultural biosecurity programs globally, are discussed.

Phytophthora tropicalis Isolated from Diseased Leaves of Pieris japonica and Rhododendron catawbiense and Found in Irrigation Water and Soil in Virginia

An unidentified species of Phytophthora was isolated from irrigation water at a production nursery in Suffolk, VA in 2000 and 2001. Water samples were assayed using a filtration method (3). A similar species was recovered from soil samples collected in two mixed-hardwood forests in Fairfax County in 2002. Soil samples were air dried, remoistened, flooded, and then baited with rhododendron and camellia leaf pieces at room temperature (22 to 24°C) (2). A Phytophthora sp. was recovered from bait pieces cultured on PARPH-V8 selective medium (2). This same species also was isolated from symptomatic leaves of Pieris japonica cv. Temple Bells and Rhododendron catawbiense cv. Maximum Roseum at a garden center in Virginia Beach in 2004. On P. japonica, symptoms appeared as water-soaked, necrotic lesions and marginal necrosis on leaves and necrosis of shoot tips; on R. catawbiense, symptoms were wilting, dieback, and death of shoots. Representative isolates produced semipapillate to papillate sporangia with tapered bases that were caducous and had long pedicels (16 to 120 μm). Sporangia on four isolates were measured: mean lengths were 40.6 to 48.4 μm, mean widths were 26.9 to 31.4 μm, and length/width ratios consistently were 1.5. Sporangia occasionally were distorted and had dual apices, and they often contained a large globule after zoospore release. Chlamydospores ranged from 25 to 32 μm in diameter. All isolates were heterothallic; four isolates paired with known isolates of P. nicotianae were found to be mating type A1. Optimum temperature for mycelium growth on cornmeal agar was 25°C with slight growth at 35°C by some isolates and no growth at 4°C. These morphological characteristics were mostly consistent with those of P. tropicalis (1). P. tropicalis is reported to have sporangia that are papillate, have lengths of 40 to 55 μm, widths of 19 to 27 μm, and length/width ratios of 1.8 to 2.4 (1). The identity of these isolates as P. tropicalis was confirmed using single-strand conformational polymorphism analysis with comparison to a reference isolate (4). These isolates have been retained in permanent collections in the Hong and Jeffers labs. One isolate from each host plant and one isolate from irrigation water were tested for pathogenicity; agar blocks of mycelium (4 × 4 mm) were placed on wounded and nonwounded leaves of P. japonica cv. Mountain Fire and R. catawbiense cv. Olga plants and wrapped with Parafilm to prevent desiccation. Lesions formed on wounded and nonwounded leaves after 4 days at 20 to 30°C, and P. tropicalis was reisolated; no lesions formed on noninoculated control leaves. To our knowledge, this is the first report of P. tropicalis in the continental United States, in irrigation water systems, and as a cause of Phytophthora foliage blight on P. japonica and R. catawbiense (1). This study suggests that the host range of this pathogen is not limited to tropical plants. Although this pathogen did not cause significant economic loss in the garden center surveyed, it was isolated in irrigation water at the production nursery from late spring through fall. An investigation of its impact on nursery crops is warranted. References: (1) M. Aragaki and J. Y. Uchida. Mycologia 93:137, 2001. (2) A. J. Ferguson and S. N. Jeffers. Plant Dis. 83:1129, 1999. (3) C. X. Hong et al. Phytopathology 92:610, 2002. (4) P. Kong et al. Fungal Genet. Biol. 39:238, 2003.

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.

The avocado subgroup of Phytophthora citricola constitutes a distinct species, Phytophthora mengei sp. nov.

Isolates from avocado tree cankers have been recognized as a distinct subgroup within the P. citricola complex since 1974, both morphologically and molecularly (isozyme and amplified fragment length polymorphism [AFLP] analyses). This subgroup is formally separated from P. citricola after comparative DNA fingerprinting and sequence analyses of the ITS region, as well as by morphological examinations. This new taxon is homothallic, produces plerotic oospores with paragynous antheridia and noncaducous semipapillate sporangia. Morphologically it differs from other species of Waterhouse group III by producing many large bizarre-shaped sporangia and smaller oogonia with asymmetric capitate antheridia. It belongs to clade 2 and is phylogenetically closer to P. siskiyouensis, P. capsici and P. tropicalis than to P. citricola. P. mengei can be easily differentiated from its relatives in the same clade and other species of this morpho-group by DNA fingerprints and sequence analysis. This new taxon is named Phytophthora mengei sp. nov.

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.