First Report of Phytophthora nicotianae Causing Dianthus chinensis root rot and foliage blight in China

Dianthus chinensis is a popular ornamental plant that is widely cultivated in China. From May 2020 to 2021, root rot and foliage blight were observed on approximately 50% groundcover plants at several landscape sites of Xuanwuhu Park and Nanjing Railway Station, China. Symptoms of wilting and chlorosis appeared in the initial stage, and severe infection caused the whole plant to die . To recover the causal pathogen, infected root and leaf samples were cut into 5×5 mm2 squares, surface-disinfected in 70% ethanol for 30 sec, placed onto 10% clarified V8 PARP agar at 25°C . After three days, Phytophthora-like hyphae were visibly emerged from both root and leaf tissues and growing into cV8A. Individual hyphal tips were transferred to new cV8A plates to obtain a total of 10 pure isolates. Colony morphology of all isolates on cV8A had slightly radiate to stellate patterns with cottony aerial mycelia. After four or five days all isolates had identical morphological traits including papillate and noncaducous sporangia on cV8, hyphal swellings, and intercalary and terminal chlamydospores. A representative isolate Pni-dc7 was examined for morphological measurements. Sporangia were mostly ovoid and sometimes obpyriform, averaging 28.9±5.6 µm in length and 24.9±5.8 µm in width (n=30). Chlamydospores were abundant and spherical with an average diameter at 29.2 ± 0.3 µm (n=30). Oogonia were not observed. For sequence analysis, the internal transcribed spacer (ITS) regions and large subunit (LSU) of the nuclear ribosomal RNA gene complex were amplified using the primer pairs ITS1/ITS4 and NL1/NL4 , respectively, while the mitochondrial cytochrome c oxidase subunit II (coxII) gene was amplified using FM58/FM66 (Martin et al. 2003). The ITS sequence of isolate Pni-dc7 (GenBank Acc. No. MZ519893) had a 100% identity to those of P. nicotianae (MH219914, KU172524, MT065839). The LSU sequence (MZ573547) had a 100% identity to those of P. nicotianae (KX250514, MZ348950, HQ665198).The cox2 sequence (MZ519893) had a 100% identity to those of P. nicotianae (MH221078, KJ506439, JF707072). Based on morphological and molecular evidence, Pni-dc7 was identified as P. nicotianae. Pathogenicity tests were conducted using both detached leaves and whole plants. Asymptomatic leaves were collected from healthy plants.A 5×5 mm2 Pni-dc7-colonized cV8A plug was placed on each wound of five leaves. Sterile agar plugs were used for a non-inoculated control leaf. All six leaves were placed on a wet filter paper in a closed container at 25°C. All inoculated leaves had necrotic tissues around the wounds, the symptoms progressed from spots to the entire leaves after two days . The control leaves remained asymptomatic. In the whole-plant assay, a D. chinensis plant (approx. 0.3 m in height) was inoculated with 5 mL of zoospore suspension that was mixed into the potting soil(500g). Three plants were inoculated and control plants were treated with sterile distilled water. After two weeks all three inoculated plants in three repeats of the assay had root and crown rot and foliage blight, whereas all control plants remained asymptomatic. P. nicotianae was reisolated from all inoculated plants. This is the first report of P. nicotianae causing root rot and foliage blight on D. chinensis in China. Considering the importance of D. chinensis to both ornamental nursery and landscaping industries in China, diseased plants at the landscape sites were removed to prevent the spread of P. nicotianae to production sites and other landscape locations.

First Report of Stem and Foliage Blight of Chrysanthemum Caused by Phytophthora drechsleri in the United States

Chrysanthemum (Chrysanthemum × morifolium) plants exhibiting stem and foliage blight were observed in a commercial nursery in eastern Oklahoma in June 2019. Disease symptoms were observed on ~10% of plants during a period of frequent rain and high temperatures (26-36°C). Dark brown lesions girdled the stems of symptomatic plants and leaves were wilted and necrotic. The crown and roots were asymptomatic and not discolored. A species of Phytophthora was consistently isolated from the stems of diseased plants on selective V8 agar (Lamour and Hausbeck 2000). The Phytophthora sp. produced ellipsoid to obpyriform sporangia that were non-papillate and persistent on V8 agar plugs submerged in distilled water for 8 h. Sporangia formed on long sporangiophores and measured 50.5 (45-60) × 29.8 (25-35) µm. Oospores and chlamydospores were not formed by individual isolates. Mycelium growth was present at 35°C. Isolates were tentatively identified as P. drechsleri using morphological characteristics and growth at 35°C (Erwin and Ribeiro 1996). DNA was extracted from mycelium of four isolates, and the internal transcribed spacer (ITS) region was amplified using universal primers ITS 4 and ITS 6. The PCR product was sequenced and a BLASTn search showed 100% sequence similarity to P. drechsleri (GenBank Accession Nos. KJ755118 and GU111625), a common species of Phytophthora that has been observed on ornamental and vegetable crops in the U.S. (Erwin and Ribeiro 1996). The gene sequences for each isolate were deposited in GenBank (accession Nos. MW315961, MW315962, MW315963, and MW315964). These four isolates were paired with known A1 and A2 isolates on super clarified V8 agar (Jeffers 2015), and all four were mating type A1. They also were sensitive to the fungicide mefenoxam at 100 ppm (Olson et al. 2013). To confirm pathogenicity, 4-week-old ‘Brandi Burgundy’ chrysanthemum plants were grown in 10-cm pots containing a peat potting medium. Plants (n = 7) were atomized with 1 ml of zoospore suspension containing 5 × 103 zoospores of each isolate. Control plants received sterile water. Plants were maintained at 100% RH for 24 h and then placed in a protected shade-structure where temperatures ranged from 19-32°C. All plants displayed symptoms of stem and foliage blight in 2-3 days. Symptoms that developed on infected plants were similar to those observed in the nursery. Several inoculated plants died, but stem blight, dieback, and foliar wilt were primarily observed. Disease severity averaged 50-60% on inoculated plants 15 days after inoculation. Control plants did not develop symptoms. The pathogen was consistently isolated from stems of symptomatic plants and verified as P. drechsleri based on morphology. The pathogenicity test was repeated with similar results. P. drechsleri has a broad host range (Erwin and Ribeiro 1996; Farr et al. 2021), including green beans (Phaseolus vulgaris), which are susceptible to seedling blight and pod rot in eastern Oklahoma. Previously, P. drechsleri has been reported on chrysanthemums in Argentina (Frezzi 1950), Pennsylvania (Molnar et al. 2020), and South Carolina (Camacho 2009). Chrysanthemums are widely grown in nurseries in the Midwest and other regions of the USA for local and national markets. This is the first report of P. drechsleri causing stem and foliage blight on chrysanthemum species in the United States. Identifying sources of primary inoculum may be necessary to limit economic loss from P. drechsleri.

First Report of Phytophthora pini Causing Foliage Blight and Shoot Dieback of Rhododendron pulchrum in China

During a 2019–2020 survey for plant pathogenic oomycetes in Nanjing, China, severe foliage blight and dieback were observed on approximately 20 Rhododendron pulchrum plants at three public parks and gardens. Approximately 25% of leaves and shoots were affected. Symptoms included brown to black lesions on leaves and stems, dieback of shoot tips, and wilting. Diseased tissues were collected from a five-year-old shrub with typical disease symptoms at Xuanwuhu Park. They were cut into 10×10 mm2 squares, immersed in 70% ethanol for 30 sec, and placed onto fresh clarified V8 juice agar (cV8A) containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene. Phytophthora-like hypae were transferred to new cV8A plates daily. A total of five isolates were obtained after five days of incubation at 25°C. After approximately 20 days, all isolates were identical in morphological traits including semi-papillate sporangia and gametangia (homothallic). Thirty sporangia of a representative isolate Ppi were randomly selected and examined. They were mostly ovoid and sometimes obpyriform, averaging 41.0 ± 3.9 × 24.8 ± 3.2 µm. Antheridia of 30 randomly selected gametangia were paragynous, averaging 16.7 ± 0.7 × 12.4 ± 1.5 µm. Average diameters of oogonia and plerotic oospores were 29.2 ± 0.3 µm and 26.4 ± 1.6 µm, respectively. Chlamydospores were not observed. The above morphological traits suggested the causal agent belonging to the “P. citricola-complex”. Isolate Ppi was subjected to sequencing of the rDNA internal transcribed spacer (ITS) region and the ras‐related GTP‐binding protein 1 (Ypt1) gene. ITS sequence of Ppi (GenBank ACN. MT672594) has 100% identity to that of P. pini (MG865565). It has a 3-nt difference from the ITS sequences of P. acerina (MG518642) and P. citricola (MG865475) and a 4-nt difference from that of P. plurivora (FJ665225). Ypt1 sequence of Ppi (MT680000) has 100% identity to that of P. pini (MK058416). Pathogenicity of Ppi on R. pulchrum was tested using both detached-leaf and whole-plant assays. In the former assay, each of six asymptomatic leaves was symmetrically wounded at both sides using a sterile inoculation needle. A 5×5 mm2 Ppi-colonized cV8A plug was placed on each wound of five leaves. Sterile agar plugs were used for a control leaf. All six leaves were placed on a wet filter paper in a closed container at 25°C. This assay was repeated twice. On the fifth day, all inoculated leaves had necrotic tissues around the wounds, while the control leaves remained asymptomatic. In the whole-plant assay, 20-inch-tall plants were used. Five attached leaves and the twig base of each plant were wounded. A control plant was inoculated in the same manner above, while sterile agar plugs were used. Each plant was covered with a plastic bag and maintained at 25°C. Wet cotton balls were placed in the bags to maintain humidity. After two days, the bag containing cotton balls was removed. This assay was repeated three times. After two weeks, all three inoculated plants in the three replicated trials had severe foliage blight and dieback, whereas control plants remained healthy. Phytophthora isolates recovered from artificially inoculated tissues were identical to isolate Ppi in morphological characters. Rhododendron diseases caused by P. pini were reported in the USA and Finland . This is the first report of P. pini causing foliage blight and dieback on R. pulchrum, an important nursery and landscape plant in China. Additional surveys are ongoing to determine the distribution of this pathogen in Nanjing. Management programs are under development to contain the spread of P. pini and treat diseased plants.

First Report of Phytopythium helicoides Causing Crown and Root Rot on Rhododendron pulchrum in China

During a 2019–2020 survey of plant pathogenic oomycetes in Nanjing, China, a cluster of five adjacent Rhododendron pulchrum plants in Xuanwuhu Park exhibited symptoms including crown and root rot and wilting. foliage blight caused due to collar and had rotting crown and root tissues resultingrot foliage blight. Diseased roots were rinsed in water, cut into 10 mm pieces, immersed in 70% ethanol for 60 sec, and plated onto clarified V8 juice agar (cV8A) containingamended with pimaricin (20 mg/liter), ampicillin (125 mg/liter), rifampicin (10 mg/liter), and pentachloronitrobenzene (20 mg/liter). After three3 days of incubation at 26°C, Ffive Pythium-like isolatescoloniesisolates were obtained using hypalhyphal-tipping after 3 days of incubation at 25°C. Ten agar plugs (2×2 mm2) of each isolate were growntransferred into 10 mLl of 10% clarified V8 juice (cV8) in a 100 -mm plate at 26°C to produce mycelial mats. After 3three days, cV8 was replaced with sterile water. To stimulate sporangial production, 3–5 drops of soil extract solution were added to each plate. Five isolates had identical morphological features. Sporangia were terminal, ovoid to globose, andmeasuring 34.2 ± 6.2 µm (24.0–42.5 µm range) in length and 30.7 ± 6.6 µm (20.9–41.1 µm range) in width. Oogonia were not observed. The following primers were used to amplify the rDNA internal transcribed spacer (ITS) region and the mitochondrial cytochrome c oxidase subunit 1 (cox1COI) and 2 (cox2COII) genes of from aA representative isolate, PH-C were amplified using the primer pairs ITS6 and ITS4 (Cooke et al. 2000), OomCoxI-Levup and OomCoxI-Levlo (Robideau et al. 2011) and Cox2-F and Cox2-RC4 (Hudspeth et al. 2000), respectivelyPhe-1. Isolate A xxx675 bp, xxx657 bp and 561xxx bp fragmentPH-C , respectively were amplified and had have identical sequences of the ITS (GenBank ACN. MT824568), and cox1 (MT834959), COI and cox2 COII genes the rDNA internal transcribed spacer (ITS) region and the mitochondrial cytochrome c oxidase subunit 1 and 2 genes (GenBank ACN. MT824568, MT834959, (MT834958, respectively) sequences identical to those of Phytopythium helicoides (MN541109, MK879709, KT595689, respectively). Based on the morphological and molecular characters, all five isolatesthe causal agent waswere identified the species represented by Phe-1 was identified as P. helicoides. One-year-old R. pulchrum plants (approx. 0.3 m in height) grown in 8×8 cm2 pots were used in to test the pathogenicity trials. Ten plants wasere carefully dug up to expose root ballsclusterballs. TenThree- days -old cultures of the isolate PH-Che-1 were used as the inoculum. Five The pplantss wereere inoculated by inserting 10 agar plugs into thee root ball of each plantcluster. For inoculatingfive control plants, sterile cV8A discsplugs were used. All inoculated plants were re-potted using original fresh potting mix and potsture .Ten 3-day-old cV8A cultural plugs (5×5 mm2) of Phe-1 were evenly insert into the root ball of each of five plants, while sterile cV8A plugs were used for five control plants. All were then planted into their original pots. Plants were maintained in a growth chamber set at 26°C with a 12/12 h light/dark cycle and irrigated as needed. After 21–25 days, the inoculated plants had symptoms identical to those in the field, while the controls remained asymptomatic. Identical outcomes were obtained from two repeated The pathogenicity trials. test was repeatedconducted twice . and the coutcome was identical. Phytopythium. helicoides (Phe-1) was reisolated from all symptomatic plants inemerging from the pathogenicity trials. Phytopythium helicoides was found causing diseases of Asian lotus (Yin et al. 2015), mandarin orange (Chen et al. 2016), and kiwifruit (Wang et al. 2015) plants in China. Phytopythium isolates with identical morphological features to those of Phe-1 were recovered from rotted crown and root tissues of all inoculated plants. In this note, P. helicoides causing crown and root rot on R. pulchrum is reported for the first time. Globally, this is the first report of P. helicoides causing crown blight and root rot of R. pulchrum. Additional surveys are being conducted forto mapping the distribution of P. helicoides in Nanjing, Province of China.

Phytophthora nicotianae Can Cause Both Crown Rot and Foliage Blight on Phlox paniculata in South Carolina

Phytophthora nicotianae is a common pathogen of many herbaceous perennial plants, and this pathogen has been found causing disease on garden phlox (Phlox paniculata) in wholesale nurseries in South Carolina for a number of years. However, the relationship between P. nicotianae and garden phlox has not been studied or reported previously. Using Koch's postulates and standard inoculation methods for Phytophthora spp., P. nicotianae was found to cause crown rot on P. paniculata when potting medium was infested with colonized vermiculite and to cause foliage blight when aerial parts of the plant were inoculated with an aqueous suspension of zoospores. Foliage blight was more similar to symptoms we observed on garden phlox plants in wholesale nurseries, but crown rot also has been observed previously on plants in these nurseries. The cause of these two diseases was confirmed, but reproduction of Phytophthora foliage blight under experimental conditions was inconsistent. Thus, other factors not yet identified may play a role in the development of Phytophthora foliage blight on garden phlox in nurseries in South Carolina. Accepted for publication 1 September 2014. Published 1 November 2014.