First report of leaf blight caused by Phytophthora ramorum on periwinkle (Vinca minor) in Washington State, USA

Phytophthora ramorum (Werres, De Cock & Man in't Veld) was recovered from symptomatic foliage of periwinkle at a botanical garden in WA in March 2015. Symptoms were tan colored lesions with a dark brown margin visible on both surfaces of the leaf and were found on wounds or around leaf margins. Periwinkle is native to Europe and is commonly used for ground cover in ornamental landscapes. It is known to be invasive in US forests near the urban/wildland interface. Potential spread of P. ramorum into WA forests is of regulatory concern, as well as long distance spread to other states via nursery stock (7 CFR §301.92-2). Phytophthora ramorum was isolated from symptomatic foliage by excising leaf pieces 4-6 mm in diameter and surface-sterilizing in 0.6% sodium hypochlorite followed by two rinses in sterile water. Leaf pieces were plated on PARP medium (Ferguson and Jeffers 1999) and after 2-3 days at 20°C, slow-growing dense colonies with coralloid hyphae were isolated onto V8 agar. Colony morphology and chlamydospore production were consistent with descriptions of P. ramorum (Werres et al. 2001), except that the isolate was slower growing and had irregular, non-wildtype morphology (Elliott et al. 2018) compared to other isolates of P. ramorum. ITS and COX1 regions of mycelial DNA was amplified and sequenced to confirm the identity of P. ramorum using primers ITS1/ITS4 (White et al. 1990) and COX1F1/COX1R1 (Van Poucke et al. 2012). Sequences were submitted to GenBank (accession nos. ITS MT031975, COX1 MT031974). BLAST results showed at least 98% similarity with sequences of P. ramorum (ITS, MN540640 [98%]; COX1, EU124920 [100%]), and belonged to the NA1 clonal lineage. Pathogenicity of P. ramorum to periwinkle was confirmed by completing Koch's Postulates. Inoculum was grown on V8 agar plates at 20°C for two weeks until sporangia were abundant. A zoospore suspension was produced by flooding plates with 7 ml sterile water, incubating for 2 hours at 5°C, then for an additional hour at 24°C. Zoospores were observed under the microscope and quantified with a hemocytometer, then diluted to 2 x 105 zoospores/ml. A 10 µl droplet of inoculum was placed at one wounded and one unwounded site on six leaves on each of four plants. In addition, a set of four plants was inoculated by dipping foliage on one branch per plant into the zoospore suspension for 30 seconds. A set of four control plants were mock inoculated in the same manner using sterile water. The trial was repeated once. Inoculated plant materials were incubated in a moist chamber for 3-5 days and free moisture was present on foliage upon removal. Plants were held in a biocontainment chamber (USDA-APHIS permit # 65857) at 20C and symptom development assessed after 7 days (Figure S1). . Symptoms developed on foliage inoculated using both methods in both trials. Phytophthora ramorum was isolated once from droplet inoculated foliage at a wounded site on one plant. Reisolation onto PARP and then V8 agar was conducted from surface-sterilized symptomatic tissue and the presence of P. ramorum confirmed by observation of colony morphology and chlamydospore production. The presence of P. ramorum was also confirmed with DNA extraction from symptomatic foliage from plants from each of the two trials followed by PCR and sequencing of the COX1 gene (EU124920, 100%) (Figure S2). None of the water-inoculated controls were positive for P. ramorum. Low isolation success could be attributed to reduced pathogenicity due to being a non-wildtype isolate. Acknowledgements This work was supported by the USDA National Institute of Food and Agriculture, McIntire-Stennis project 1019284 and USDA APHIS Cooperative Agreement AP17PPQS&T00C070

First report of leaf blight caused by Phytophthora ramorum on cherry laurel (Prunus laurocerasus) in Washington State, USA.

In April 2014, Phytophthora ramorum (Werres, De Cock & Man in't Veld) was recovered from symptomatic foliage of cherry laurel (Prunus laurocerasus) at an ornamental plant nursery in Washington State. Cherry laurel, also known as English laurel, is widely propagated in WA because it is commonly used in landscaping. It is invasive in forests near the urban/wildland interface in the western US and in Europe (Rusterholz et al. 2018). Given its popularity as an ornamental species, the potential of this host to spread P. ramorum is of regulatory concern due to possible long distance spread to other states via nursery stock. Foliar symptoms consisted of dark brown lesions near wounds or around leaf margins where water collected. Shot-hole symptoms characterized by abscission zones and dropping of infected tissues were also observed. Lesions expanded beyond the margin of the shot-hole in some cases (Figure S1A). Phytophthora was isolated from symptomatic foliage by surface-sterilizing leaf pieces in 0.6% sodium hypochlorite and 2 rinses in sterile water. They were plated on PARP medium (Ferguson and Jeffers 1999). After 2-3 days, a slow-growing dense colony with coralloid hyphae was isolated onto V8 agar. P. ramorum was identified by observing morphological features (Figure S1B). Colony and spore morphology matched that of P. ramorum (Werres et al. 2001). The isolate was confirmed as P. ramorum by PCR and sequencing of ITS and COX1 regions using primers ITS1/ITS4 (White et al. 1990) and COX1F1/COX1R1 (Van Poucke et al. 2012). Sequences were submitted to GenBank (accession nos. ITS MT031969, COX1 MT031968). BLAST results showed at least 99% similarity with sequences of P. ramorum (ITS, KJ755124 [100%]; COX1, EU124926 [99%]). Multilocus genotyping with microsatellite markers placed the isolate in the EU1 clonal lineage. Pathogenicity of P. ramorum on cherry laurel was confirmed by completing Koch's Postulates using the isolate taken from this host. Two trials were done in a biocontainment chamber (USDA-APHIS permit # 65857) since P. ramorum is a quarantine pathogen and greenhouse trials could not be conducted, using detached stems from mature, visibly healthy cherry laurel plants growing in a landscape. Phytophthora ramorum inoculum was grown on V8A plates at 20®C for 2 weeks until sporangia were abundant. A zoospore suspension was produced by flooding plates with 7 ml sterile water, incubating for 2 hours at 5®C, then 1 hour at 24®C. Zoospores were observed with light microscopy, quantified with a hemocytometer and diluted to 1 x 104 zoospores/ml. A 10 µl droplet was placed at 3 wounded and 3 unwounded sites on 4 leaves per branch. In addition, a set of samples was inoculated by dipping foliage into the zoospore suspension for 30 seconds. A set of controls was mock inoculated using sterile water. Four branches per inoculation treatment were used and the trial was repeated once. Inoculated plant materials were incubated in moist chambers for 3-5 days at 20®C. Free moisture was present on foliage upon removal. Symptom development was assessed after incubation in the biocontainment chamber at 20®C for 7 days (Figure S1C). Phytophthora ramorum was reisolated from symptomatic tissue and the recovered culture was verified morphologically and by PCR and sequencing. It was isolated more often from foliage dipped in zoospore suspension than droplet inoculated, and more from wounded than unwounded sites. None of the water-inoculated controls were positive for P. ramorum. The presence of P. ramorum was also confirmed with DNA extraction from surface-sterilized symptomatic foliage followed by PCR and sequencing of the COX1 gene (EU124926, 100%) (Figure S2). To our knowledge, this is the first report of P. ramorum naturally infecting cherry laurel in the United States. Acknowledgements This work was supported by the USDA National Institute of Food and Agriculture, McIntire-Stennis project 1019284 and USDA APHIS Cooperative Agreement AP17PPQS&T00C070 Literature cited Ferguson and Jeffers, 1999. Plant Disease 83:1129-1136 Van Poucke, K. et al. 2012. Fungal Biology 116: 1178-1191. http://dx.doi.org/10.1016/j.funbio.2012.09.003 Werres, S. et al. 2001. Mycol. Res. 105:1155-1165. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

Histopathological study reveals new insights into responses of chestnut (Castanea spp.) to root infection by Phytophthora cinnamomi

The European chestnut (Castanea sativa) is threatened by the hemibiotrophic oomycete Phytophthora cinnamomi, the causal agent of ink disease. Chestnut species have different susceptibility levels to P. cinnamomi, with the Asian species (C. crenata; C. mollissima) exhibiting the highest level of resistance. A histological approach was used to study the responses exhibited by susceptible and resistant chestnut genotypes by characterizing the early stages of P. cinnamomi infection and the cellular responses it induces in roots. C. sativa (susceptible) and C. crenata (resistant) plantlets, were inoculated with a P. cinnamomi virulent isolate with a zoospore suspension or by direct contact with mycelia agar plugs. Root samples were collected at 0.5, 3.5, 24, 48, and 72 hours after inoculation (hai) for microscopic observations. Penetration was observed, in both species, at 0.5hai and 3.5hai with mycelium and zoospore inoculations, respectively. In both inoculation methods, following the penetration into the rhizodermis, P. cinnamomi hyphae grew inter- and intracellularly through the cortex and into the vascular cylinder. C. crenata cells displayed a delay in the pattern of infection, by having fewer cell layers colonized when compared with C. sativa. At 72hai, the collapse of the first layers of C. sativa cortical cells was observed, indicating the beginning of necrotrophy. C. crenata was able to respond more efficiently to P. cinnamomi than C. sativa, by restricting the pathogen’s growth area through the early activation of resistance responses, such as callose deposition around some intracellular hyphae, HR-like cell death, cell wall thickening and accumulation of phenolic-like compounds.

KETAHANAN BEBERAPA LADA HASIL PERSILANGAN TERHADAP Phytophthora capsici ASAL LADA

<p>ABSTRAK</p><p>Busuk pangkal batang (BPB) lada yang disebabkan oleh cendawanPhytophthora capsici merupakan masalah utama pada budidaya lada diIndonesia. Penyakit ini telah ditemukan di semua areal produksi lada diIndonesia. Sampai saat ini, saran pengendalian yang dianjurkan adalahpengendalian secara terpadu untuk mengurangi kerugian ekonomi akibatpenyakit ini. Akhir-akhir ini usaha untuk mendapatkan jenis lada yangtahan dilakukan melalui persilangan. Tujuan penelitian ini adalahmengevaluasi ketahanan F1 yang diperoleh dari persilangan beberapatetua. Penelitian dilakukan di laboratorium dan rumah kaca, BalaiPenelitian Tanaman Rempah dan Obat, Bogor, dari Januari sampaiDesember 2005. Dari 400 aksesi hasil persilangan yang ada, dipilih 15aksesi yang menunjukkan hasil yang menjanjikan pada uji pendahuluan.Tiga isolat Phytophthora yang menunjukkan virulensi yang berbedadigunakan sebagai isolat uji. Di laboratorium, helaian daun ke-3 dan 4diambil dari tiap aksesi dan diletakkan dalam kotak yang telah diberi tissuebasah untuk menjaga kelembapannya. Inokulasi secara buatan dilakukandengan meletakkan potongan koloni masing-masing isolat Phytophthorapada permukaan bawah daun. Luas nekrosa yang terbentuk pada masing-masing aksesi diukur dengan leaf area meter setelah diinkubasi selama 72jam. Percobaan di rumah kaca dilakukan dengan cara menyiramkansuspensi zoospora sebanyak 50 ml pada bibit lada dari masing-masingaksesi yang telah berumur 4 bulan. Jumlah tanaman yang mati dihitungsetelah diinkubasi selama 1 bulan. Data hasil pengukuran luas serangandianalisis dengan rancangan faktorial dengan dua faktor untuk duakegiatan di atas. Hasil penelitian menunjukkan bahwa tidak ada interaksiyang nyata antara aksesi dengan isolat Phytophthora yang digunakan, baikpengujian in vitro maupun rumah kaca. Sembilan aksesi menunjukkankerusakan kurang dari 20% saat di laboratorium maupun di rumah kaca,dan aksesi 27-1, 36-31, dan 4-5L menunjukkan kerusakan kurang dari10%. Persilangan lebih lanjut perlu dilakukan pada aksesi-aksesi tersebutuntuk mendapatkan keturunan yang mempunyai ketahanan lebih baik danstabil.</p><p>Kata kunci : Piper nigrum L., Phytophthora, ketahanan, persilangan</p><p>ABSTRACT</p><p>Resistance of Black Pepper Accessions to Phytophthora capsiciFoot rot disease of black pepper caused by Phytophthora capsici ismain constraint in black pepper cultivation in Indonesia. The diseasespread widely over all pepper producing areas in Indonesia. Integratedpest managements are suggested to reduce the economic loss due to thedisease. Recently, breeding program has been developed in Indonesiathrough hybridization to find out promising accessions resistant to foot rotdisease. The objective of the present study was to evaluate the resistanceof F1 progenies obtained from polination of various parents to foot rotdisease. Among 400 accessions of black pepper obtained from breedingprogram, 15 accessions were selected based on previous evaluation. ThreePhytophthora isolates were used as tester in the study. The research wascarried out in laboratory and glass house of Indonesian Spice andMedicinal Crops Research Institute, from January to December 2005. Invitro screening was carried out by inoculating detached third and fourthleaves of each accession. The leaves were set in boxes abaxial surfacefacing up, while wet tissue papers were used to retain air humidity in thebox. The lower leaf surface of each pepper accession was inoculated witha piece of Phytophthora colony then incubated in room temperature. Thewidth of necrotic areas was measured with leaf area meter after the leaveswere incubated for 72 hours. Each treatment was replicated 5 times. Ingreen house experiment, 4 month seedlings of each accession wereinoculated with 50 ml of zoospore suspension (10 5 zoospore/ml), replicated3 times, and each replication consisted of 5 seedlings. The number ofinoculated seedlings was counted after one month of incubation. Bothexperiments were arranged using factorial design with two factors: pepperaccession and Phytophthora isolate. There was no significant interactionbetween black pepper accession and the Phytophthora isolates, neither invitro nor green house. Nine accessions showed disease severity less than20%, and accession number 27-1, 36-31, and 4-5L showed disease severitybelow 10% in both experiments. To obtain better progeny resistant to stemrot disease and more stable, it is suggested to continue this pollinationprogram by using those promising accessions.</p><p>Key words: Piper nigrum L., Phytophthora, resistance, pollination</p>

Mosquitoes as a potential vector for the transmission of the amphibian chytrid fungus

The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is an infectious disease responsible for the worldwide decline of amphibian species. To mitigate these declines, it is necessary to identify the various vectors by which the fungus can be transmitted between individuals and populations. The objective of this study was to determine whether adult female mosquitoes can carry and transfer Bd fungal cells. Mosquitoes were exposed to netting soaked in a live Bd zoospore suspension to determine whether they are able to externally acquire the fungus. Another group was placed into containers with a sterile and Bd-inoculated agar plate to determine whether mosquitoes could transfer Bd between these surfaces. Bd DNA was found to be present on mosquito legs exposed to inoculated netting and agar plates suggesting that Bd can be transmitted by the mosquito over short distances. This is the first study to demonstrate that an insect host may be a mechanical vector of Bd and suggests that we should begin to consider the role of mosquitoes in the dissemination and control of the fungus.

Mosquitoes as a Potential Vector for the Transmission of the Amphibian Chytrid Fungus

The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is an infectious disease responsible for the worldwide decline of amphibian species. To mitigate these declines, it is necessary to identify the various vectors by which the fungus can be transmitted between individuals and populations. The objective of this study was to determine whether adult female mosquitoes can carry and transfer Bd fungal cells. Mosquitoes were exposed to net soaked in a live Bd zoospore suspension to determine whether they are able to externally acquire the fungus. Another group was placed into containers with a sterile and Bd-inoculated agar plate to determine whether mosquitoes could transfer Bd between these surfaces. Bd DNA was found to be present on mosquito legs exposed to inoculated netting and agar plates suggesting that Bd can be transmitted by the mosquito over short distances This is the first study to demonstrate that an insect host may be a mechanical vector of Bd and suggests that we should begin to consider the role of mosquitoes in the dissemination and control of the fungus.

First Report of Phytophthora cambivora Causing Leaf and Stem Blight and Root Rot on Taiwan Cherry (Prunus campanulata) in Taiwan

Taiwan cherry or Formosan cherry (Prunus campanulata Maxim.) is a beautiful ornamental tree that is native to Taiwan. In spring 2005, a severe disease was observed on 1- to 3-year-old seedlings of Taiwan cherry in a garden in Tungshih, Taichung, Taiwan. Infected plants showed symptoms of greenish water-soaked spots on leaves that became dark brown, 2 to 3 cm in diameter. Infected leaves withered and fell to the ground in 3 to 5 days and young shoots showed symptoms of withering and drooping. Infected roots showed symptoms of necrosis. Severely infected plants eventually died. A Phytophthora sp. was isolated consistently from diseased samples of Taiwan cherry and associated soil. Six isolates of Phytophthora, of the A1 mating type (1), were isolated from single zoospores. Two of these isolates, Tari 25141 (deposited as BCRC34932 in Bioresource Collection and Research Center, Shinchu, Taiwan) and Tari 25144 (BCRC34933), were used for pathogenicity tests on 1-year-old seedlings of Taiwan cherry to fulfill Koch's postulates. Inoculation was done by placing a cotton swab containing zoospore suspension on leaves or stem, or by soaking seedlings in the zoospore suspension. Inoculated seedlings were kept in a greenhouse at 20 to 25°C for 30 days and examined for appearance of symptoms. Results showed that both isolates were pathogenic on seedlings of Taiwan cherry, causing symptoms similar to those observed on naturally infected seedlings. The temperature range for growth of the six isolates of Phytophthora was 8 to 32°C with optimum temperature at 24°C. The linear growth rate was 72 mm per day on V8A culture (5% V8 vegetable juice, 0.02% CaCO3, and 2% Bacto agar) at 24°C. The colonies on potato dextrose agar produced sparse aerial mycelia with conspicuous radiate patterns. Sporangia were sparse on V8A agar blocks, but abundant when the agar blocks were placed in water under continuous white fluorescent light (average 2,000 lux) for 2 days. Sporangiophores branched sympodially. Sporangia were pear shaped, nonpapillate and nondeciduous, 50 to 75 (62) × 30 to 48 (40) μm, with a length/width ratio of 1.2 to 2.2 (1.6). New internal nested proliferate sporangia were formed inside the empty sac of old matured sporangia after releasing zoospores. No chlamydospores were formed on V8A. Hyphal swellings with distinctive irregular catenulation were produced on V8A and in water. The pathogen was stimulated to form its own oospores by the A2 tester using the method described by Ko (1). Oogonia were 28 to 50 (40) μm in diameter with smooth or irregularly protuberant walls. Oospores were mostly aplerotic and 18 to 42 (31) μm in diameter. Antheridia were amphigynous, mostly two-celled, and 10 to 42 (29) × 12 to 24 (19) μm. The sequence of the internal transcribed spacers (ITS) region of nuclear ribosomal DNA of isolate Tari 25141 (GenBank Accession No. GU111589) was 831 bp and had 99% sequence identity with a number of Phytophthora cambivora isolates such as GenBank Accession Nos. HM004220 (2), AY787030, and EF486692. Based on the morphological characteristics of sporangia and sexual structures and the molecular analysis of ITS sequences, the pathogen from Taiwan cherry was identified as P. cambivora (Petri) Buis. To our knowledge, this is the first report of P. cambivora on native Taiwan cherry in Taiwan and, so far, no other natural hosts have been reported. References: (1) W. H. Ko. J. Gen. Microbiol. 116:459, 1980. (2) P. W. Reeser et al. Mycologia 103:225, 2011.

A simple method for detection of Phytophthora nicotianae from soybean soil

A high detection rate of Phytophthora nicotianae was obtained from the naturally infested soil of soybean fields. Air-dried soils were firstly moistened in a flask and then pre-incubated at 25 °C for 1-4 weeks before flooding with distilled water and baiting with soybean leaf disks for 6, 12, and 24 h. The baits were then thoroughly washed, flooded with 10-15 ml of distilled water in Petri-dishes and incubated under continuous fluorescent light for 72 h. Sporangia started to emerge from the margins of leaf disks. They were easily observed under the stereomicroscope. Pure culture of the fungus was obtained by spreading zoospore suspension on a 1.5% water-agar containing anti-bacterial antibiotics. This is the first report of recovery of P. nicotianae from naturally infested soybean soils using sensitive leaf disks of soybean (Williams cultivar) as bait in Iran. All isolates were determined to be A2 mating type.

First Report of Phytophthora Blight Caused by Phytophthora capsici on Snap Bean in New York

Phytophthora capsici Leonian is an important pathogen of solanaceous and cucurbit crops. Phytophthora blight was first reported on snap bean (Phaseolus vulgaris L.) in Michigan in 2003 (2) and Connecticut in 2010 (3). This report documents the discovery of P. capsici on snap bean (cv. Bronco) grown in Riverhead, NY in September 2008 and August 2010 on snap bean (cv. Valentino) in Holley, NY, more than 690 km away. Disease was favored by frequent rainfall and prolonged wet periods with air temperatures of 24 to 29°C. Both locations were commercial fields previously planted to pepper or cucurbits affected by P. capsici. In Riverhead, infected pods had characteristic yeast-like growth of P. capsici, which were predominantly sporangia. In Holley, large water-soaked lesions were observed on snap bean foliage, and as the disease progressed, leaves became necrotic and detached from the plant. Reddish brown lesions were observed on stems in advance of white areas of sporulation. Infected pods displayed white mycelial growth, were shriveled, and desiccated. P. capsici was isolated from symptomatic tissues. Stems and pods were surface disinfested for 3 min in 0.525% sodium hypochlorite solution, rinsed for 3 min in sterile distilled water, transferred to PARPH (4) media, and incubated at 22°C. After 5 days, hyphae from colony margins were excised and transferred to 15% unclarified V8 agar media. Cultures consisting of white mycelia and ovoid papillate sporangia on long pedicels were identified as P. capsici. Sporangia were 25.0 to 70.0 × 10.0 to 22.5 μm (average 42.0 × 16.25 μm). Identity was further confirmed by PCR primers specific to P. capsici (1). DNA was extracted from mycelia produced on V8 agar and amplification with the species-specific primers resulted in a PCR product of the same size as that obtained from a known isolate of P. capsici. Pathogenicity of the isolate from Holley was determined by two methods on 50-day-old snap bean plants (cv. Valentino) grown in a greenhouse. In method one, four plants were inoculated with 1-cm-diameter mycelial plugs excised from 8-day-old cultures. A single plug was placed against the stem at the soil line. Four control plants were treated similarly with noncolonized agar plugs. In method two, entire plants were atomized with 10 ml of a zoospore suspension (2.6 × 105/ml). Control plants were atomized with sterile distilled water. All plants were placed in a growth chamber with continuous mist for 24 h at 24°C. After 24 h, plants were enclosed in plastic bags and placed in a greenhouse at 27°C. Stem lesions similar to those observed in affected fields were evident on plants treated with mycelia plugs 2 days after inoculation. Plants inoculated with the zoospore suspension developed stem lesions and desiccated pods. Control plants were asymptomatic. P. capsici was successfully recovered from infected plant tissue, fulfilling Koch's postulates. The Riverhead isolate was demonstrated as pathogenic on snap bean and cucumber by placing colonized plugs on pods and fruit that were subsequently incubated in moist chambers (24°C, 90 to 100% relative humidity). P. capsici was successfully recovered from symptomatic pods and fruit. To our knowledge, this is the first report of Phytophthora blight caused by P. capsici on snap bean in New York. References: (1) A. R. Dunn et al. Plant Dis. 94:1461, 2010. (2) A. J. Gevens et al. Plant Dis. 92:201, 2008. (3) J. A. LaMondia et al. Plant Dis. 94:134, 2010. (4) G. C. Papavisas et al. Phytopathology 71:129, 1981.

First Report of Phytophthora ramorum Infecting Mistletoe in California

In 2005 and 2006, white fir and Douglas-fir growing in a Christmas tree plantation near Los Gatos, CA, under a black walnut tree infected with mistletoe tested positive for Phytophthora ramorum, the cause of Sudden Oak Death. Isolation from a symptomatic mistletoe inflorescence stalk was positive for P. ramorum. In 2007, fresh mistletoe leaves, stems, inflorescence stalks, and berries were inoculated with a zoospore suspension of the mistletoe isolate. All of the plant parts developed symptoms, and P. ramorum was isolated from each of them. This is the first report of an infection of a hemiparasite with P. ramorum. Accepted for publication 20 January 2011. Published 9 February 2011.