V8 Agar Medium Research Articles

First report of Phytophthora niederhauserii causing root rot of carob tree (Ceratonia siliqua L.) in Spain.

Carob tree (Ceratonia siliqua L.) is a crop native to the Mediterranean Basin mainly being used as livestock feed, in pharmaceutical industry, and also a valuable source of human food due to the high dietary fibre and sugar contents. In January 2023, one-year-old 'Duraio' carob trees grafted on 'Rojal' rootstock on pots showing dark brown to necrotic lesions on the secondary roots, decline symptoms and leading to the death of some plants, were detected in a nursery located at the Valencian Community region in Spain. Disease severity was 40 to 50% of root area and disease incidence was approximately 50% on approximately 1000 plants. Six representative plants were randomly collected. Roots were washed under running tap water to rinse the soil away, and small symptomatic pieces were cut, disinfected with 70% ethanol, and then dried on absorbent paper. Two-mm-long segments were plated on CMA-PARBPH, a Phytophthora-selective medium, and incubated at 25ºC. After 2-3 days, growing colonies were transferred to potato dextrose agar (PDA) medium. A Phytophthora-like organism was consistently isolated (50% of root segments, n= 120). Colonies were whitish with irregular margins, had coenocityc mycelium, irregularly branched hyphal swellings, and chlamydospores were absent. Sporangia were non-papillate, persistent, ellipsoid and measuring 25 to 40 × 50 to 90 μm (average: 35.5 × 74.7 μm, n = 50). They proliferated internally with both nested and extended proliferation. These morphological features were similar to those of Phytophthora niederhauserii (Abad et al., 2014). Internal transcribed spacer (ITS) and cytochrome c oxidase I (COX1) regions of a representative isolate CF3 were sequenced to confirm the identity. Both sequences were deposited in GenBank under accession numbers OR763816 for ITS and OR783697 for COX1. OR763816 showed 99.87% sequence identity to P. niederhauserii strain Ex-type MG865552 and OR783697 showed 99.85% sequence identity to P. niederhauserii strain Ex-type MH136944 (Abad et al., 2023). Pathogenicity test was performed on one-year-old carob tree seedlings (Duraio/Rojal) grown in 13 cm-diameter pots to fulfill Koch's postulates. The inoculum was prepared in 1 L glass flasks with a mixture of 200 ml vermiculite, 20 ml oat grains, and 175 ml of V8 broth (20% V8 juice and 0.2% of CaCO3 in demineralized water) (Jung et al., 1996). Glass flasks with vermiculite/oat/V8 mixture were autoclaved three times for 20 min at 120 ºC. These mixtures were inoculated with the isolate CF3 which was previously grown on V8 agar medium and incubated for six weeks in the dark at room temperature (Pérez-Sierra et al., 2013). For inoculation, twenty-gram inoculum were mixed with 200 g autoclaved potting mix (peat, vermiculite and sand; 1:1:1, v/v/v), and added to the pots to plant the seedlings. Seven plants were inoculated and non-infested vermiculite/oat/V8 mixture was used to prepare seven control plants. Two months after inoculation one of the inoculated plants died. Six months after inoculation, only the inoculated plants showed decline symptoms with dry leaves and root necrosis. Isolates resembling P. niederhauserii were recovered by plating the roots from all inoculated plants on CMA-PARBPH, and the identity of the isolates was confirmed as P. niederhauserii based on ITS and COX1 sequencing. To our knowledge, this is the first report of P. niederhauserii causing root rot on carob tree. The detection of this pathogen in nurseries is relevant because its dissemination to orchards could have a negative impact in carob crop production.

First Report of Colletotrichum fructicola Causing Anthracnosis on Glycine max in Brazil

Brazil is the largest producer of soybean [Glycine max (L.) Merrill], cultivated in diverse environments and systems. This scenario can contribute to emergence of new diseases or increase the severity of secondary diseases. In March 2023, elliptical to circular, brownish lesions, 5.2–6.1 cm length and 1.1–1.5 cm width, with salmon-colored masses of conidia in the center of the lesions, were observed on the stems of soybean cultivar ‘CZ 16B17 IPRO’, in the municipality of Campos Novos, Santa Catarina, Brazil (27º25'19''S and 51º14'14''05W). The presence of 210–355 μm length and 210–232 μm width acervuli was rare, with arrows larger than the mass of conidia (Figure S1). Fragments of the infected tissues were cut, disinfected and placed in Petri dishes containing Potato Dextrose Agar (PDA) or V8-agar medium and maintained at 23 ± 2ºC and a photoperiod of 12 h dark–light cycle. After 13 days, the development of grayish-white colonies was observed on both culture media, with the formation of a mass of septate hyaline, oblong, cylindrical conidia, 13.3–15.3 µm length and 2.9–3.5 µm width, with obtuse ends. One pure monosporic isolate was selected, isolate CF1. The presence of sexual structures was observed on PDA after 13 days and in V8 after 15–20 days. Perithecia were dark brown and globose, either immersed in the culture medium or on the surface between the mycelia. Inside of perithecia, unitunicate, clavate, and cymbiform asci, 39.1–61.0 μm length and 9.6–11.7 μm width were observed, containing eight spindle-shaped and slightly curved ascospores with rounded tips 13.8–18.3 μm length and 3.0–4.2 μm width (Figure S1). Pathogenicity tests were performed on young soybean plants at V1 phenological growth stage in four repetitions. PDA disks, 7mm in diameter, with growth mycelium were placed on stems while using uninfected PDA disks as a control. Plants were incubated in a chamber at 25 ± 2°C and 90% relative humidity. Anthracnose lesions were observed only on the stems of the inoculated plants. The same pattern of symptoms was observed on the stems, and the fungus were reisolated on PDA. The colony and morphological characteristics were identical to the previously isolated fungus. For molecular characterization, the growth mycelia were collected, macerated in liquid nitrogen, and DNA was extracted using the method Doyle and Doyle (1990) with CTAB. End-point PCR was performed using the GoTaq® Flexi DNA Polymerase (Promega, USA) and the primers, ITS-1F/ITS-4, T1/Bt2b, CL1C/CL2C, GDF/GDR, and SODglo2-F/SODglo2-R (Weir et al. 2012) for the amplification of internal transcribed spacer (ITS), β-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and superoxide dismutase (SOD), respectively. Amplified fragments were sequenced and compared with the available sequences in the Genbank (www.ncbi.nlm.nih.gov/genbank/). The sequences of all five-genes (Accession numbers OR883777, OR891749, OR891750, OR891751 and OR891752, respectively) of the isolate CF1 characterized in this study showed 99% nucleotide identities whith the stand isolate ICMP 18581 of Colletotrichum fructicola. A phylogenetic tree was constructed in MEGA X (Kumar et al. 2021), containing the amplified and concatenated sequences and representative species from the Colletotrichum gloeosporioides complex. The isolate grouped only with C. fructicola clade, confirming the identity of the fungus (Figure S2). To our knowledge, this is the first study reporting the infection of C. fructicola in soybeans in Brazil, which has already been reported in China (Xu et al. 2023).

First Report of Cucurbita Blossom Blight and Fruit Rot Caused by Choanephora cucurbitarum in Mexico.

In December 2022, blossom blight, abortion, and soft rot of fruits were observed on Cucurbita pepo L. var. Zucchini in Mexico under greenhouse conditions (temperatures of 10 to 32°C and relative humidity up to 90%). The disease incidence in about 50 plants analyzed was around 70% with a severity of nearly 90%. Mycelial growth on flower petals and fruit rot with brown sporangiophores was observed. Ten disinfested fruit tissues in 1% NaClO for 5 min and then rinsed twice in distilled water from the lesion edges were placed on potato dextrose agar culture medium (PDA) supplemented with acid lactic and then, the morphological characterization was carried out in V8 agar medium. After 48 h of growth at 27°C, the colonies were pale yellow with diffuse cottony mycelia that were non-septate and hyaline and produced both sporangiophores bearing sporangiola and sporangia. The sporangiola were brown, ranged from ellipsoid to ovoid, and had longitudinal striations that measured 22.7 to 40.5 (29.8) μm x 16.08 to 21.9 (14.5) μm long and wide, respectively (n=100). The sporangia were subglobose, had a diameter of 127.2 to 281.09 (201.7) μm (n=50), and contained ovoid sporangiospores that measured 26.5 to 63.1 (46.7) μm x 20.07 to 34.7 (26.3) μm long and wide, respectively (n=100) which had hyaline appendages at the ends. Based on these characteristics, the fungus was identified as Choanephora cucurbitarum (Ji-Hyun et al. 2016). For molecular identification, DNA fragments for the internal transcribed spacer (ITS) and the large subunit rRNA 28S (LSU) regions for two representative strains (CCCFMx01 and CCCFMx02) were amplified and sequenced with the primer pairs ITS1-ITS4 and NL1-LR3 (White et al. 1990; Vilgalys and Hester 1990). The ITS and LSU sequences were deposited in GenBank database (Accession numbers OQ269823-24 and OQ269827-28, respectively) for both strains. The Blast alignment showed from 99.84 to 100% identity with Choanephora cucurbitarum strains JPC1 (MH041502, MH041504), CCUB1293 (MN897836), PLR2 (OL790293), and CBS 178.76 (JN206235, MT523842). To confirm the specie identification, the evolutionary analyses were conducted from the concatenated sequences of the ITS and LSU of C. cucurbitarum and other mucoralean species with the Maximum Likelihood method and Tamura-Nei model included in the software MEGA11. The pathogenicity test was demonstrated using five surface-sterilized zucchini fruits inoculated with a sporangiospores suspension containing a concentration of 1 x 105 esp/mL on two sites per fruit (20 µL each) that previously were wounded with a sterile needle. For fruit control, 20 µL of sterile water was used. Three days after inoculation under humidity conditions at 27°C, white mycelia and sporangiola growth with a soaked lesion were observed. That fruit damage was not observed on the control fruits. C.cucurbitarum was reisolated from lesions on PDA and V8 medium which was confirmed by morphological characterization fulfilling Koch's postulates. Blossom blight, abortion, and soft rot of fruits caused by C. cucurbitarum were observed on Cucurbita pepo and C. moschata in Slovenia and Sri Lanka (Žerjav and Schroers 2019; Emmanuel et al. 2021). This pathogen has the capability to infect a wide variety of plants worldwide (Kumar et al. 2022; Ryu et al. 2022). There are no reports of C. cucurbitarum causing agricultural losses in Mexico, and this is the first report causing the disease symptoms in Cucurbita pepo in this country; however, this fungus was found in the soil of papaya-producing areas and it is considered an important plant pathogenic fungus. Therefore, strategies for their control are highly recommended to avoid spreading the disease (Cruz-Lachica et al. 2018).

First report of Phytophthora gonapodyides causing root rot on raspberry in Canada.

Raspberry (Rubus idaeus L.) is an economically important fruit crop in Canada and about 80% of red raspberries are cultivated in British Columbia. In 2018, foliar symptoms associated with root rot and wilting complex disease were observed in raspberry field of Fraser Valley areas of British Columbia. Plants were stunted with reduced numbers of primocanes. Chlorosis and necrosis on leaves and partial wilting of branches were observed. When plants were uprooted, necrosis and browning on roots were observed. Two isolates of oomycetes pathogen were isolated using baiting with rhododendron leaves and pear fruit as described in Sapkota et al. 2022. Using FastDNA Spin kit (MP Biomedical, Burlingame, CA), genomic DNA of pathogen isolates was extracted from mycelia cultured on 20% clarified V8 agar medium amended with 10 mg pimaricin, 250 mg ampicillin, 10 mg rifampicin (V8PAR) per liter following the manufacturer's standard protocol. Pathogens were identified using colony morphology on 20% clarified V8 PAR as well as internal transcribed spacer (ITS) sequencing with ITS1 primers (White et al. 1990) and multiplex targeted-sequencing with degenerate primers of three nuclear genes: heat shock protein90 (HSP90), elongation factor 1 alpha (EF1α) and beta tubulin (βtub). BLAST searches of ITS sequences of isolates of this study (accession nos. OP180065, OP180066) in NCBI GenBank showed 98.5 to 99.6% identity with the ITS sequence of P. gonapodyides (accession nos. MN513238.1, MG753496.1). Multiplex targeted sequencing also identified both isolates as a P. gonapodyides (accession nos. SRR20227809, SRR20227807) when mapped with the reference sequences (accession nos. HSP90: KX251233.1, EF1α: KX251231.1, β-tub: KX639710.1). Pathogenicity was confirmed by inoculating mycelial suspension of one isolate of P. gonapodyides on root of intact plants and mycelial plugs of two isolates on detached stems of the raspberry plants, 'Chemainus' in the greenhouse using methods described in Sapkota et al. 2022. Two experiments were conducted with three replicates in each test. Experiments were arranged using completely randomized design. In detached stem assays, distinct dark-lesion symptom appeared at 7 to 9 days after inoculation while uninoculated control stems remained asymptomatic. Intact plants showed wilting and foliar symptoms 15 days after inoculation and progressed higher at 4 to 5 weeks after inoculation. Root infection with dark brown to black color was observed when roots were assessed at 5 weeks after inoculation. The diseased root and crown tissues tested positive for Phytophthora in Agdia ImmunoStrip and P. gonapodyides was re-isolated and confirmed with multiplex-targeted sequencing. Phytophthora gonapodyides was previously reported from raspberry in Chile (Wilcox and Latorre 2002). To our best knowledge, this is the first report of P. gonapodyides infecting red raspberry in British Columbia, Canada. The detection of new Phytophthora species on raspberry may become a new potential problem to growers in addition to P. rubi, which is already a major cause of raspberry decline in the region.

Antifungal Potential of Acetone and Ethyl Acetate Extracts of Thevetia peruviana on Development of Phytophthora colocasiae, Causal Agent of Late Blight of Taro (Colocasia esculenta (L.) Schott) from Three Agro-Ecological Zones of Cameroon

Aims: This study was aimed to evaluate the antifungal activities of acetone and ethyl acetate extracts of Thevetia peruviana seeds on the in vitro growth of the fungus.
 Study Design: A randomized sample block design containing four treatments (T- = Negative control; T2= Ethyl acetate extract; T3= Acetone extract; T+=Callomil Plus) with three repetitions was used. Plant extracts were used at three concentrations: C1: 12.5 µl/ml; C2: 25 µl/ml and C3: 50 µl/ml; the chemical fungicide at the dose of 12.5 μL/ml.
 Place and Duration of Study: The study was conducted in the University of Yaoundé 1, Faculty of Sciences, Department of Plant Biology, Laboratory of Phytopathology and Crop Protection, and in the Institute of Agricultural Research for Development (IARD) of Yaoundé, Laboratory of Phytopathology, during the year 2019-2020.
 Methodology: acetone and ethyl acetate extracts of T. peruviana were prepared and used at concentrations of 12.5, 25 and 50 µl/ml. P. colocasiae was isolated from infected taro leaf cultivars "Macumba or Ibo coco" located in three different regions: west, Littoral and Centre. The various explants were were put in V8 agar medium and maintained in pure culture. Mycelial fragments of P. colocasiae of about 0.8 cm in diameter were cut and placed in sterile Petri dishes containing Potato Dextrose Agar (PDA) medium supplemented with different concentrations of plant extracts and incubated at 23±1°C for seven days for the evaluation of the radial growth.
 Results: The results obtained showed that the acetone and ethyl acetate extracts have completely inhibited the growth of the strain of West at 25 μ/ml while total inhibition of the pathogen was not obtained with strain of Centre region. The lowest inhibition was obtained with the strain of Littoral region: 93.88 % for acetone extract and 90.78 % for ethyl acetate extract compare to 100 % for west and Centre region at highest concentration.
 Conclusion: The acetone and ethyl acetate extracts at the concentration of 25 μ/ml totally inhibited the in vitro radial growth of some strains of P. colocasiae. These extracts, which are effective against P. colocasiae, may substitute fungicides in the fight against taro leaf blight.

Cladosporium Leaf Spot Caused by Cladosporium variabile in Winter High Tunnel Production of Spinach (Spinacia oleracea) in Maine, United States.

In January 2021, leaf spots were observed on 1 of 22 proprietary spinach cultivars in a trial under high tunnels in Albion, ME. Approximately 1% of plants were symptomatic with <30% leaf area affected. Some spinach growers reported ≤100% incidence in high tunnels in the northeastern USA. Leaf spots on the cultivar in Albion were <5 mm in diameter and tan-colored with dematiaceous spores in short chains on long conidiophores at the center of older lesions. Lesions were surface-sterilized in 0.6% NaOCl for 90 s, rinsed in sterile water, dried, and placed on clarified V8 agar medium amended with chloramphenicol (100 µg/ml). Three Cladosporium isolates (Cds053, Cds055, and Cds057) produced olive-green colonies with dematiaceous conidia, 0- to 2-septate, catenulate, and 8.0-28.0 µm (16.6 ± 0.6 µm, mean ± standard error) x 4.0-12.0 µm (8.3 ± 0.2 µm) (n = 50/isolate). The partial internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α gene (tef1-α), and actin gene (act) were amplified and sequenced with primer sets UNUP18S42/UNLO28S576B (Bakkeren et al. 2000), EF1-728F/EF1-986R (Carbone and Kohn 1999), and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. The sequence of each locus was identical among the isolates. The tef1-α (GenBank Accession No. MZ905363) and act (MZ905362) sequences were identical to accessions of tef1-α (EF679480.1) and act (EF679556.1) of C. variabile (Cooke) G. A. de Vries, whereas the ITS rDNA sequence (MZ893461) was identical to that of many Cladosporium spp., including C. variabile. Each isolate was inoculated onto four replicate 42-day-old plants of each of the spinach cultivars Viroflay and Mandolin. The plants were enclosed in plastic bags for 24 h, atomized with a spore suspension (106 conidia/ml) prepared from potato dextrose agar colonies of each isolate, and returned to the bags for 24 h. Four plants of each cultivar were atomized with deionized water as a control treatment. Plants were arranged in a randomized complete block design. By 5 days, tan spots had developed on Cladosporium-treated plants of each cultivar. By 14 days, Mandolin had developed more severe lesions (20.0 ± 2.8%, mean ± standard error of leaf area with symptoms) than Viroflay (5.4 ± 0.4%). No control plants treated with water developed lesions. After 15 days, a leaf from a plant of each treatment was incubated in a moist chamber at 20˚C with a natural diurnal light cycle. Sporulation was observed at the center of lesions from Cladosporium-inoculated plants <24 h after incubation, and Cladosporium was re-isolated. The act locus amplified and sequenced from the re-isolates, as described above, had an identical sequence to that of the original isolates, fulfilling Koch's postulates. Empirically, Cladosporium leaf spot has established in winter high tunnel production of spinach in the northeastern USA (R. Katz, personal observation). To our knowledge, this is the first official documentation of the disease in Maine. The disease is favored by cool, humid conditions (Inglis et al. 1997). Management strategies under these conditions in Maine and greater New England include foliar applications of effective fungicides (du Toit et al. 2004), selection of partially resistant cultivars, cultural practices to reduce relative humidity and durations of leaf wetness, and planting pathogen-free or treated seed (du Toit and Hernandez-Perez 2005; Hernandez-Perez and du Toit 2006).

Identification and Management of Phytophthora Aerial Blight Caused by Phytophthora nicotianae on Catharanthus roseus.

Phytophthora nicotianae is the most common pathogen in nurseries and gardens, infecting both woody and herbaceous ornamental plants. Phytophthora aerial blight symptoms such dull water-soaked lesions on shoot tips and leaf petioles, girdling on the main stem, necrosis, and wilting of annual vinca were observed in a commercial greenhouse in Warren County, TN, U.S.A., in May 2016. The objective of this study was to identify the causal agent of Phytophthora aerial blight and develop a fungicide management recommendation for ornamental producers. Attempts to isolate the pathogen from symptomatic leaf tissue were conducted, and excised leaf pieces were embedded in V8 agar medium. Morphological characterization, PCR, sequencing, and pathogenicity test of the isolate FBG2016_444 were conducted to confirm the pathogen identification. The sequence identity was 100% identical to P. nicotianae, and a combined phylogenetic tree (internal transcribed spacer, large subunit of rDNA, and ras-related protein gene) grouped isolate FBG2016_444 within the clade of P. nicotianae. In the pathogenicity study, all inoculated annual vinca plant showed Phytophthora aerial blight symptoms, and P. nicotianae was reisolated, whereas noninoculated annual vinca plant remained symptomless. These findings confirmed P. nicotianae as the causal agent of Phytophthora aerial blight of annual vinca. In addition, two rates (0.078 and 0.156 ml·liter-1) and three application intervals (7, 14, and 21 days before inoculation [DBI]) of oxathiapiprolin (Segovis) were evaluated for their ability to reduce the Phytophthora aerial blight severity on annual vinca plants. The control groups were positive (nontreated inoculated) and negative (nontreated noninoculated) plants. Both rates and application timings of oxathiapiprolin significantly reduced Phytophthora aerial blight severity and disease progress (area under disease progress curve [AUDPC]) on annual vinca plants compared with the positive control. However, 0.078 and 0.156 ml·liter-1 of oxathiapiprolin applied at 7 or 14 DBI were the most effective treatments in reducing the disease severity and AUDPC on annual vinca plants. The plant growth parameters such as increase in height and width, total plant weight, and root weight were not influenced by the application of oxathiapiprolin. The findings reported in this study will help ornamental producers with better management of Phytophthora aerial blight of annual vinca.

Characterization and pathogenicity of a Pythium aphanidermatum isolate causing ‘damping off’ in pepper seedlings

&lt;p&gt;The production of the pepper seedling (&lt;em&gt;Capsicum annuum&lt;/em&gt;) is affected by the fungal complex that causes the ‘damping-off’, in which some species of the oomycete &lt;em&gt;Pythium&lt;/em&gt; spp., stand out. The objective of the present study was to identify the causal agent of the death of pepper plants and evaluate its pathogenicity in pepper seeds and seedlings. A fast and aggressive growing oomycete was isolate from pepper plants, morphologically identified as P. aphanidermatum based on its sexual and asexual reproduction structures and, by molecular techniques. This isolate had a high degree of&lt;em&gt; in vitro&lt;/em&gt; pathogenicity in pre-emergence and post-emergence in chile, showing 100% mortality. In addition, it presented a high rate of mycelial growth in different culture media (V8-Agar, Corn Agar, Corn Potato Agar, Potato Dextrose Agar, Czapek &amp;amp; Oat Agar), being in V8-Agar medium the only medium where it developed reproduction structures sexual and asexual. The isolation presented a mycelial growth rate of 58.3 ± 0.3 mm / day at 26 ± 2 °C in PDA medium. Due to its rapid growth and its high degree of pathogenicity &lt;em&gt;in vitro&lt;/em&gt;, it is an unusual and aggressive isolate for pepper seedlings.&lt;/p&gt;

Efficacy of Methanolic and Aqueous Extracts of Thevetia peruviana (pers.) K. Schum on Growth of Phytophthora colocasiae Racib, Causal Agent of Taro Late Blight in Cameroon

This study aims to evaluate the antifungal activities of methanolic and aqueous extracts of Thevetia peruviana seeds on the in vitro growth of Phytophthora colocasiae. A randomized sample block design containing four treatments (T-: absolute control, AE, ME and Callomil Plus at the dose of 12.5 μL/ml) with three repetitions was used. Plant extracts were used at three concentrations: C1: 12.5 µl/ml; C2: 25 µl/ml and C3: 50 µl/ml. The study took place in the University of Yaoundé 1, Faculty of Sciences, Department of Plant Biology, Laboratory of Phytopathology and Crop Protection, and in the Institute of Agricultural Research for Development (IARD) of Yaoundé, Laboratory of Phytopathology, during the year 2019-2020.&#x0D; Aqueous and methanolic extracts of T. peruviana were prepared and used at the concentrations of 12.5, 25 and 50 μL/ml. P. colocasiae was isolated from infected taro leaf cultivars "Macumba/Ibo coco" located in three different regions in Cameroon: West, Littoral and Centre. The different leaf explants of taro were put in V8 agar medium and maintained in pure culture. Mycelial fragments of P. colocasiae of about 0.8 cm in diameter were cut and placed in sterile Petri dishes containing Potato Dextrose Agar (PDA) medium supplemented with different concentrations of plant extracts and incubated at 23±1°C for seven days for the evaluation of the radial growth. Methanolic and aqueous extracts have completely inhibited the growth of West and Littoral strains at 25 μL/mL while total inhibition of the pathogen was not obtained with strain of Centre region. The lowest inhibition was obtained with the strain of Centre region (85.1%) for aqueous extract and (70.95%) for methanolic extract compare to 100% for West and Littoral region at highest concentration. The aqueous extract at the concentration of 25 μL/ml totally inhibited the in vitro radial growth of some strains of P. colocasiae. This extract, active against P. colocasiae could be used as alternative to fungicides for the control of taro leaf blight. In other hand, the strain of Littoral region was most sensible to extracts than the others. This strain could be used to provide a genetic resource for future trials in natural conditions in greenhouse and in the field.

Identification and Chemical and Biological Management of Phytopythium vexans, the Causal Agent of Phytopythium Root and Crown Rot of Woody Ornamentals.

Soilborne diseases caused by pathogens such as Phytophthora, Rhizoctonia, Fusarium, Verticillium, and Pythium species are the most important diseases of woody ornamentals. Ginkgo (Ginkgo biloba) and red maple (Acer rubrum 'October Glory') plants grown in containers and fields in Tennessee showed root and crown rot symptoms with dark brown to black lesions in 2017 and 2018. The objective of this research was to isolate and identify pathogens affecting ginkgo and red maple plants in Tennessee nurseries and to develop fungicide/biofungicide management recommendations for nursery producers. Isolations were made from the infected roots. Several Phytophthora-like colonies with spherical zoospores, filamentous to globose oogoni, and whitish mycelium were isolated on V8-PARPH medium. To confirm identity, total genomic DNA was extracted, followed by sequence analysis of the internal transcribed spacer regions, large subunit of nuclear rRNA, and cytochrome c oxidase subunits I and II of mitochondrial DNA. Based on morphological and molecular analysis, Phytopythium vexans was described as a causal agent of crown and root rot from the infected ginkgo and red maple plants. To complete Koch's postulates, a pathogenicity test was performed by drenching 100 ml of V8 agar medium slurry of Phytopythium vexans inoculum on 1-year-old potted ginkgo plant root systems as well as red maple October Glory. Necrotic lesion development was observed in the root system 45 days after inoculation and Phytopythium vexans was reisolated from the roots of both ginkgo and red maple. All control ginkgo and red maple plants remained disease free and no pathogen was reisolated. In addition, the efficacy of fungicides, biofungicides, fertilizer, and host plant defense inducers (traditionally recommended for management of oomycete diseases) for control of Phytopythium crown and root rot was evaluated on ginkgo and red maple October Glory seedlings in greenhouse and field trials. Fungicides such as Empress Intrinsic, Pageant Intrinsic, Segovis, and Subdue MAXX were effective in both greenhouse and field trials, and the biofungicide Stargus reduced disease severity caused by pathogen Phytopythium vexans on ginkgo and red maple plants in greenhouse trials. These results will help nursery producers make proper management decisions for newly reported Phytopythium crown and root rot disease of ginkgo and red maple plants.

Occurrence of Leaf Spot Caused by Alternaria alternata on Eggplant (Solanum melongena) in Pakistan.

Eggplant (Solanum melongena L.) is a popular vegetable that is grown in both tropical and subtropical regions all year long. The crop is cultivated on small family farms and is a good source of income for resource-limited farmers in Pakistan. In early May 2019, leaf spots on eggplant (cv. Bemisaal) were observed in an experimental field (31°26'14.0"N 73°04'23.4"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms were small, circular, brown, necrotic spots uniformly distributed on leaves. The spots gradually enlarged and coalesced into large, nearly circular or irregularly shaped spots that could be up to 3 cm in length. The center of the spots was light tan, surrounded by a dark brown ring, a chlorotic halo, and tended to split in the later developmental stages. Disease incidence was approximately 35% in the infected field. The causal agent of this disease was isolated consistently by plating surface sterilized (1% NaOCl) sections of symptomatic leaf tissue onto potato dextrose agar (PDA). After 6 days incubation at 25°C with a 12-h photoperiod, fungal colonies had round margins and the cottony mycelia were dark olivaceous with a mean diameter of 7.5 cm. For conidial production, the fungus was grown on potato carrot agar (PCA) and V8 agar media under a 16-h/8-h light/ dark photoperiod at 25°C. Conidiophores were septate, light to olive golden brown with a conidial scar, from which conidia were produced. Conidia were borne singly or in short chains and were obpyriform to obclavate, measured 29 ± 4.8 × 13.25 ± 2.78 μm (n=30) with zero to three longitudinal and two to six transversal septa. The morphological characters matched those of Alternaria alternata (Fr.) Keisel (Simmons et al. 2007). DNA was extracted using the DNAzol reagent (Thermo Fisher Scientific MA, USA). For molecular identification, internal transcribed spacer (ITS) region between ITS1 and ITS2, actin gene (β-Actin), translation elongation factor (TEF-1α) gene, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of two representative isolates (JLUAF1 and JLUAF2) were amplified with primers ITS1/ITS4 (White et al. 1990), β-Actin 512 F/783 R, EF1-728F/-986R (Carbone et al. 1999), and gpd1/gpd2 (Berbee et al. 1999), respectively. The sequences were deposited in GenBank (accession nos. MT228734.1 and MT228735.1 for ITS; MT260151.1 and MT260152.1 for β-Actin, MT260163.1 and MT260164.1, for TEF-1a, and MT260157.1 and MT260158.1 for GAPDH). BLASTn analysis of these sequences showed 100% identity with the sequences of A. alternata for ITS rDNA, β-Actin, TEF-1α, and GAPDH, respectively. Based on the morphological characters and DNA sequences, the leaf spot isolates of eggplant were identified as A. alternata. To confirm the pathogenicity on eggplant, six-week old healthy potted eggplants of cv. Bemisaal were sprayed at the true leaf stage with conidial suspensions of A. alternata (106 conidia/ml; obtained from 1-week-old cultures) amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant) using an atomizer in the greenhouse. Three plants were inoculated with each of the two isolates (JLUAF1 and JLUAF2), whereas three control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse, and the experiment was conducted twice. After 10 days of inoculation, each isolate induced leaf spots which were similar to typical spots observed in the field, whereas the control plants remained symptomless. The fungus was re-isolated from symptomatic tissues. Re-isolated fungal cultures were morphologically and molecularly identical to A. alternata, thus fulfilling Koch's postulates. Previously, A. alternata has been reported to cause leaf spots on eggplant in India (Raina et al. 2018). To our knowledge, this is the first report of A. alternata causing leaf spot on eggplant in Pakistan. The disease could represent a threat for eggplant crops due to its increasing cultivation. It is important to develop disease management strategies for Alternaria alternata causing leaf spot of Eggplant in Pakistan.

Virulence and genetic diversity of Phytophthora isolates associated with cocoa pod rot

Phytophthora palmivora is an important pathogen of cocoa in the world and it has a widest host-range. High adaptability to host plants and changes in environmental conditions forms a new evolutionary strain among P. palmivora populations. The purpose of this study was to analyze the virulence and genetic diversity of the isolates associated with cocoa pod rot symptoms. The isolates (infected cocoa pods) collected from Bone, Bulukumba and Pinrang regencies were characterize morphologically and molecularly. For morphological identification, Phytophthora black pod disease was determined by hyphal growth on juice V8 agar medium and for genetical and virulent identification, pathogen was tested with primer pairs of ITS4 and ITS5 and the diversity was then analyzed with the BOXA1R primer. The Hyphae of isolates were found to have stellate pattern, ovoid sporangium, smooth globose oogonium and oospores. Of 21 isolates, 14 isolates were obtained from Bone, 6 isolates from Bulukumba and 3 isolates from Pinrang respectively and testing virulence of the isolates after reinoculated onto the pod layer shown that seventeen (17) isolates expressed a much higher virulent level and only four (4) isolates performed moderately level. DNA fragments of isolates were amplified at 900 bp and the highest genetic diversity among isolates were 67% level.

Pseudocercospora exilis Causing Leaf Spot Disease on Brassica rapa subsp. parachinensis in China

Chinese flowering cabbage (Brassica rapa L. subsp. parachinensis) is an important vegetable crop of China and widely cultivated in the southern provinces. During February of 2017 to 2019, a severe leaf spot disease was seen on Chinese flowering cabbage plants in different agricultural fields around Lianzhou and Guangzhou cities of Guangdong province, China. Disease incidence reached up to 70% in severely infested fields. Early disease symptoms observed were small circular or irregular spots of brown color, 3 to 11 mm in diameter and often coalesced. Ten symptomatic leaves were cut into small pieces (5 × 5 mm), surface sterilized with 1% NaOCl, plated onto potato dextrose agar (PDA) medium, and incubated at room temperature for 7 days under continuous fluorescent light. A total of 11 pathogen isolates were purified by successively subculturing on new PDA medium plates using the single-spore technique. A cercosporoid fungus was consistently isolated from diseased leaves. The colonies were raised and greenish black in color. Conidiophores were brown, subcylindrical, multiseptate, straight or slightly curved, unbranched, and smooth. Conidiogenous cells were pale brown, smooth, and arranged terminally. Conidia were pale brown, smooth, obclavate or fusoid, straight to slightly curved, with obtuse apex, one- to seven-septate, and 44.3 to 69.12 × 4.8 to 6.2 μm (n = 20). The morphological and cultural characteristics of all isolates were consistent with Pseudocercospora exilis Hern.-Gut. & Dianese (Hernandez-Gutierrez and Dianese 2009; Silva et al. 2016). One monosporic isolate (PE-1) was obtained by plating individual spores on V8 agar medium. To further confirm the species, genomic DNA was extracted from the hyphal mass of the isolate PE-1 using the DZup fungal DNA extraction kit (Sangon Biotech, Shanghai, China). The internal transcribed spacer (ITS) and translational elongation factor (EF-1α) regions of the isolate PE-1 were amplified with primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999) and sequenced by Sangon Biotech. The obtained DNA sequences were deposited in GenBank (accessions MN971630 for the ITS sequence and MN991198 for the EF-1α). BLAST analysis showed that the ITS and EF-1α sequences shared 99% nucleotide identity with the previously submitted P. exilis sequences (GenBank accessions NR147302 and KT290193 for ITS and EF-1α, respectively). Pathogenicity testing was performed by spraying conidial suspension (1 × 10⁶ conidia/ml) of isolate PE-1 on the leaves of 40-day-old Chinese flowering cabbage plants. Five plants were used for inoculation. Another five plants were sprayed with distilled sterilized water to serve as a control. Plants were kept in transparent boxes for 2 days to retain moisture and subsequently incubated in a growth room at 28 ± 2°C with a 12-h photoperiod. Similar leaf spots were observed on the leaves of all inoculated plants after 1 week of inoculation. No symptoms were seen on control plants. The same pathogen was reisolated consistently from inoculated leaves. The species identity was confirmed by both morphological and molecular characteristics, thus fulfilling Koch’s postulates. Previously, P. exilis has been reported to cause leaf spot disease on Chamaecrista orbiculata in Brazil (Hernandez-Gutierrez and Dianese 2009). To our knowledge, this is the first report of leaf spot on Chinese flowering cabbage caused by P. exilis. This disease can cause severe economic losses, because Chinese flowering cabbage is mainly sold in the market and used in salads or cooked dishes. Timely measures must be taken to manage this disease.

&lt;em&gt;In-vitro&lt;/em&gt; studies on effect of fungicides against mycelial growth and sporangial germination of &lt;em&gt;Phytophthora infestans &lt;/em&gt;(Mont) de Bary) causing late blight of potato

Late blight of potato is most destructive disease. In the present study, a total of eleven fungicides, out of which eight systemic/translaminar funcides cymoxanil 50 WP, cymoxanil 8 + mancozeb 72 WP, cymoxanil 8 + mancozeb 64 WP, metalaxyl 8 + mancozeb 64 WP, metalaxyl 32 WP, fenamidone 10 + mancozeb 50 WP, difenoconazole 25 EC, dimethomorp 50 WP at concentrations of 10, 50, 100, 200 and 500 mg ml-1and three protectants viz. propineb 70 WP, mancozeb 70 WP and chlorothalonil 70 WP at concentrations of 100, 200, 500, 1000 and 2000 mg ml-1 were tested against the mycelia growth and sporangial germination ability on V8 agar medium by using poison food technique invitro. It was found that all the tested fungicides significantly inhibited the growth of mycelium compared to the control (untreated). Among the systemic fungicides dimethomorph exhibited the minimum overall mycelial growth of 38.86 mm followed by cymoxanil 8 + mancozeb 72 WP sustaining a radial growth of 43.00 mm compared to a radial mycelial growth 85.73 mm obtained in unamended check plates. An increasing trend in inhibition of mycelial growth was observed as the concentration of each fungicide was increased, such that each fungicide at 200-500 µg/ml exhibited more than 90 per cent inhibition of mycelia growth, compared to 10 µg/ml which yielded less than 10 per cent inhibition. Among protectant fungicides mancozeb exhibited highest inhibition percent with minimum mycelial growth of 46.77 mm followed by propineb yielding the mycelial growth of 50.13 mm as compared to 85.73 mm growth obtained in un amended check. The results also revealed that all the fungicides significantly reduced the sporulation and germination ability of the sporangia at all the test concentrations. Among systemic fungicides, dimethomorph proved most effective allowing sporangia germination to the extent of only 31.80 percent followed by metalaxyl + mancozeb 64 WP and cymoxanil+ mancozeb 72 WP with sporangia germination of 36.53-37.00 per cent as compared to 80.66 per cent germination observed in un-amended check (control) plates.Among the protectants.

Leaf Spot Disease Caused by Alternaria arborescens, A. tenuissima, and A. infectoria on Brassica rapa subsp. parachinensis in China

Leaf Spot Disease Caused by <i>Alternaria arborescens</i>, <i>A. tenuissima</i>, and <i>A. infectoria</i> on <i>Brassica rapa</i> subsp. <i>parachinensis</i> in China

First Report of Lasiodiplodia theobromae and Neofusicoccum parvum/N. ribis species Complex Causing Fruit Rot on Strawberry in the United States

During the last three seasons, approximately 1% of strawberry (Fragaria × ananassa) with symptoms of fruit softening and discoloration were observed in three fields in Florida. Fruit eventually became black and mummified with pycnidia embedded in the tissue that produced hyaline, aseptate conidia. Eleven isolates recovered from infected tissue were purified by growing them on 2% water agar (WA) with sterilized pine needles (Pavlic et al. 2004) and transferring a single conidium to a new 2% WA plate, by spreading a conidial suspension over the medium and selecting a single germinating conidium 16 h later. DNA of the 11 isolates was extracted from 2-week-old colonies and PCR was performed following the conditions of White et al. (1990) and Carbone and Kohn (1999). Two sets of universal primers, ITS1F/ITS4 and EF1-728F/EF1-986R, were used to amplify and sequence the internal transcriber spacer (ITS) and partial translation elongation factor 1-alpha (EF1-α) regions, respectively. Sequences compared with the GenBank database using BLASTn showed 99 to 100% identity with ITS and EF1-α sequences within the Neofusicoccum parvum/N. ribis species complex or Lasiodiplodia theobromae: N. parvum (MH057203 and KM921768; n = 2), N. kwambonambiense (n = 3), N. ribis (n = 1), N. brasiliense (n = 2), and L. theobromae (MH049696 and KX580762; n = 3). All sequences were deposited in GenBank with following accession numbers: N. parvum/N. ribis species complex (MH809520 to MH809527 for ITS, and MH809508 to MH809515 for EF1-α), and L. theobromae (MH809517 to MH809519 for ITS, and MH809505 to MH809507 for EF1-α). Mycelia of both groups on V8 agar medium were initially white and aerial, but Lasiodiplodia mycelia became light gray after 3 to 5 days and Neofusicoccum mycelia turned dark gray to olivaceous. Conidia of the Neofusicoccum group were aseptate, hyaline, oblong, and 15.0 to 22.9 × 6.1 to 10.4 μm (n = 25). Lasiodiplodia conidia were similar but larger, 22.6 to 33.0 × 13.0 to 19.4 μm (young conidia) and when mature, became brown, one septate, with longitudinal striations, measuring 25.9 to 34.8 × 14.8 to 19.6 μm (n = 25). Similar morphology was previously reported (Alves et al. 2008; Pavlic et al. 2009). To confirm pathogenicity, four isolates (two from N. parvum/N. ribis species complex, and two L. theobromae) were selected and incubated for 2 weeks at 23°C under continuous light until formation of pycnidia. Twenty mature strawberry fruit of ‘Florida Radiance’ were inoculated in the field (five fruit per isolate), by wounding the fruit with a forceps and placing a single pycnidium inside the wound. Field conditions during the incubation period ranged from 16 to 29°C and 65 to 80% relative humidity. Disease incidence was assessed 6 to 10 days after inoculation, and the experiment was repeated once. After 10 days, fruit inoculated with the N. parvum/N. ribis were completely dark and covered with pycnidia, whereas fruit inoculated with L. theobromae had 80 to 100% of its surface covered with pycnidia. All 20 inoculated fruit showed the same symptoms as described above. Both fungi were reisolated from inoculated fruit, fulfilling Koch’s postulates. N. parvum and N. kwambonambiense have been previously reported to cause a postharvest disease of strawberry in Brazil (Lopes et al. 2014). To our knowledge, this is the first report of N. parvum/N. ribis species complex and L. theobromae causing fruit rot in strawberry fields in the U.S. This study sheds light on the possible broad distribution of these pathogens on different hosts as N. parvum was recently reported in the same area causing shoot blight and stem canker on pomegranate (KC and Vallad 2016).

First Report of Leaf Spot Caused byPseudocercospora vitisonBidens pilosain Brazil

Bidens pilosa L. (Asteraceae) is an aggressive weed in annual and perennial crops with high potential to reduce crop yields in several countries (Dias et al. 2017). It also may host pathogens such as Alternaria dauci (J.G. Kuhn) J.W. Groves & Skolko (Lin et al. 2015) and Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Cho et al. 2013). In Brazil, this weed is distributed across all grape-producing areas. Leaf blight on grape, caused by Pseudocercospora vitis (Lev.) Speg., is an important disease that causes premature defoliation. In March 2018, plants of B. pilosa showing leaf spots were found in a 1-ha organic orchard of fox grape cultivar Bordo in the metropolitan region of Curitiba in Parana state, Brazil. The orchard had high leaf blight incidence (above 80%). The disease normally started in early summer; in December, and around March, complete defoliation occurred caused by leaf blight. B. pilosa was distributed throughout the orchard, and around 50% of leaves were infected. Leaf spots initially appeared slightly chlorotic, becoming pale brown to dark brown. Lesions were circular or irregular, 2 to 8 mm in diameter, and often coalesced. Microscopic examination revealed that conidia were hyaline to light brown, cylindrical, straight or slightly curved, with a rounded apex. They had 4 to 10 transverse septa, 24 to 82 μm in length and 5 to 8 μm in width, with conidiophores grouped in synnema. Isolates were recovered from lesions on tissue surfaces that were disinfested by immersion in 70% alcohol for 30 s and then in 0.5% NaClO for 30 s. Pieces were washed three times with sterile distilled water and cultured on potato dextrose agar (PDA) at room temperature for 9 days under continuous fluorescent light. Hyphal tips of the obtained colony were transferred to V8 agar medium and cultured at room temperature for 4 weeks under continuous fluorescent light. Individual spores were collected and deposited in V8 agar medium to obtain one monosporic isolate (PCLEMID02). The colony presented slow growth, with 15-mm diameter after 1 week of PDA cultivation at 25°C under a 12-h photoperiod, and was dark gray on top, black on reverse. The morphological and cultural characteristics of the isolate were consistent with those described for Pseudocercospora sp. (Ellis 1971). To confirm the species, the nuclear gene regions ITS, ACT, and EF-1α of isolate PCLEMID02 were amplified and sequenced for multigene phylogenetic analysis. The DNA sequences generated were deposited in GenBank (MH330685, MH891492, and MH891491). Phylogenetic analysis was performed with the concatenated sequence alignment of three genes using MEGA 6.0 software. The neighbor-joining phylogenetic tree showed that isolate PCLEMID02 from B. pilosa clustered in one separate clade with 100% bootstrap support with sequences of P. vitis (GenBank GU269829.1, GU320533.1, and GU384541.1). To perform the pathogenicity test, a suspension containing 1 × 10⁴ conidia/ml was prepared from one isolate (PCLEMID02) with 3-week-old cultures grown on V8 agar. The conidial suspension was sprayed on the leaf surface of B. pilosa and grapes. It used 20 leaves ranging in age from 30 to 45 days. Another 20 leaves received water instead of inoculum and served as controls. All leaves were left in a moist chamber for 24 h and subsequently stored in a greenhouse at 28 ± 2°C with a 12-h photoperiod. Only the inoculated leaves showed symptoms. Leaf blight on grape and leaf spots on B. pilosa were observed 12 and 25 days after inoculation, respectively. Both the inoculated leaves and the controls were submitted to the reisolation process. The pathogen was effectively reisolated only on symptomatic leaves and presented the same cultural, morphological, and molecular characteristics as the colony used for inoculation. Thus, B. pilosa is a host of P. vitis and may allow the survival of the pathogen in the dormancy period of the grapevine and, consequently, contribute primary inoculum for the development of leaf blight on grape.

Isolation of Cryptococcus neoformans from a Chesnut Tree (Castanea sativa), Denizli, Turkey

Cryptococcus neoformans is a basidiomycetous encapsulated yeast that can cause life-threatening infections in immunosuppressed humans and animals. C.neoformans/Cryptococcus gattii infections are considered to be acquired via inhalation of aerosolized particles from the environment. Avian guano, decaying tree hollows and soil are known as environmental niches. In recent years, colonization of the woody structures of different trees such as Eucalyptus camaldulensis, Tamarix hispida, Platanus orientalis and Punica granatum has been reported in the environmental study of the western Anatolian region. Based on the results of previous studies, our country may have intensive Cryptococcus colonization niches in the western regions. The aim of this study was to investigate the presence of the colonization of C.neoformans niche in chestnut (Castanea spp.) trees on higher altitudes. In the study, the colonization of C.neoformans was screened on chestnut trees (Castanea spp.) in Aydın-Ödemiş-Denizli geographical area. This area consists of mountainous terrain between the fertile plain formed by two major rivers.This region is one of the widespreading areas of chestnut farming in Anatolia. Two hundred and fourteen chestnut trees that had deep fissures or trunk hollows were screened during mid-summer 2017. A swabbing technique was used, and all samples were cultured on Staib agar medium containing biphenyl and antibiotics. Cultures were checked for ten days for suspicious brown colonies. Suspicious yeast colonies were tested for the identification of pathogenic Cryptococcus by conventional methods and canavanine-glycine-bromothymol agar reactions. ITS 1-4 primers were used for strain PCR tests. We determined the mating type and serotypes by PCR analysis of the STE20 genes using STE20 (Aa), STE20 (Aα), STE20 (Da), and STE20 (Dα) primers. V8 agar medium was used for mating cultivation. Only one (0.47%) strain of C.neoformans was isolated from 214 screened trees. This strain was confirmed by ITS 1-4 sequencing. The serotype A MATα mating type was observed. Basidium, basidiospores and clamp connections in hyphal structure were noted with MATα mating on V8 agar medium. In this study, the first C.neoformans isolate from a chestnut tree (Castanea sativa) was determined from Denizli region. Further studies of distribution of human pathogenic Cryptococcus will be helpful to determine the risk areas for the living organisms in our region.

First Report of Leaf Spot of Spinach Caused by Stemphylium beticola in Italy.

During the spring and autumn of 2017, 40-day-old spinach (Spinacia oleracea L.) plants, cultivar Crocodile, grown in a commercial field in the Asti province in Piedmont (Northern Italy), showed symptoms of a previously unknown foliar disease. The first symptoms developed at air temperatures ranging from 10 to 22°C. Five to 25% of plants showed small circular, yellow to pale brown leaf spots (1 to 2 mm in diameter), with a well-defined border, generally surrounded with a yellow halo. As the lesions expanded, they changed from circular to elliptical or irregular, becoming necrotic and broken in the center. Small tissue fragments were excised from symptomatic leaves, dipped in a solution containing 1% sodium hypochlorite for 5 s, washed in sterile water, dried on sterile paper, and plated on potato dextrose agar. After 7 days of incubation under 12-h fluorescent light at 22°C, 80% of fragments were colonized by the same fungus. Examination of the isolates IT27, IT28, and IT50 grown on V8 agar medium (200 ml of V8, 15 g of agar, 0.5 g of CaCO₃, and 1 liter of distilled water) revealed multicellular, light brown pigmented, obclavate or subspherical conidia, measuring 19.2 to 31.9 μm long × 10.7 to 21.4 μm wide (length/width ratio 1.1 to 1.4), with two to four transverse and three to four longitudinal septa. Conidia developed on a distinctive brown conidiophore with a terminal enlargement. These morphological characters permitted identifying the fungus as Stemphylium sp. (Simmons 1969). Genomic DNA from 7-day-old pure cultures of IT27, IT28, and IT50 were extracted with E.Z.N.A. Plant DNA Kit (Omega Bio-Tek). Polymerase chain reaction (PCR) was performed using primers ITS1/ITS4 to amplify the internal transcribed spacer, the intergenic region between 28 S and 18 S sequences of the ribosomal RNA, including the 5.8 S sequence. The PCR products were purified and sent for sequencing to BMR Genomics (Padova, Italy). BLASTn analysis (Altschul et al. 1997) of the three isolates (483 bp) showed 100% similarity with Stemphylium beticola CBS 141024 (GenBank no. NR_154925.1). Additionally, alignment with the CBS isolates used by Woudenberg et al. (2017) and the isolates from spinach was carried out using Clustal W with Mega 6.0.6 (Tamura et al. 2013). Sequence analysis of the same length sequences was performed using the maximum likelihood method with the Kimura two-parameter model plus gamma distribution. The sequences from IT27, IT28, and IT50 isolates clustered with the S. beticola strains CBS 141024, CBS 141025, and CBS 141026 (GenBank nos. KU850520, KU850521, and KU850522, respectively), allowing confirmation of the identity of isolates from spinach as S. beticola. The sequences from the isolates IT50, IT27, and IT28 were deposited in GenBank with the accession numbers MG913379, MG913380, and MG91338, respectively. The pathogenicity test was carried out by spraying a conidial suspension of 10⁵conidia/ml from a single-spore culture of strain IT50 onto leaves of 30-day-old spinach cultivar Crocodile plants, maintained under >90% relative humidity conditions for 5 days after inoculation. Pots (five per treatment, 10 plants/pot) were kept in a growth chamber at an average temperature of 20°C. The first lesions developed on leaves 8 days after inoculation, whereas control plants remained healthy. The pathogenicity test was conducted three times, providing the same results. Based on morphological and molecular analysis, the same fungus was consistently reisolated from the lesions. This is to our knowledge the first report of S. beticola on spinach in Italy and probably in Europe (Farr and Rossman 2018), although the same species has been reported on spinach in the United States (Woudenberg et al. 2017). The importance of the disease is at present limited but could rapidly increase in the cultivation areas where spinach is intensively grown.

First report of Phytophthora austrocedri causing phloem lesions and bronzing on Cupressus sempervirens in northern Iran

In 2016, the Qazvin Agricultural and Natural Resources Research Center received reports of foliage bronzing of Italian cypress (Cupressus sempervirens) in a public park in Qazvin city, Iran. Symptomatic, fully-bronzed foliage of trees had an orange-brown colored lesion in the phloem around the stem collar (Figs. 1-2). Phloem samples taken from the lesion edges (Green et al., 1) were plated on a Phytophthora-selective medium (synthetic mucor agar) and incubated at a temperature of 17–27°C in the dark. Resulting mycelial growth was transferred to V8 agar medium. The colony on V8 agar was white with coralloid hyphae (Fig. 3) and very slow growing (less than 0.5 mm per day). Formation of sporangia appeared on V8 agar with a width and length (μm) of (28.4) 31.5-44.7 (46.1) and (39.9) 46.7-73.5 (79.7), respectively. Homothallic oospores appeared on corn meal agar with a diameter (μm) of (21.7) 24.1–40.2 (46.5). The internal transcribed spacer region was amplified using ITS1/ITS4 primers (White et al., 5) and sequences of a representative strain were compared with related species retrieved from GenBank, resulting in 99% sequence identity with Phytophthora austrocedri (GenBank Accession Nos. JQ346530 and KJ49066; Green et al., 2 & 1). The ITS sequence of our isolate was deposited in GenBank (MF480422). According to colony morphology and sequencing of the ITS region, the isolate was identified as P. austrocedri (syn=P. austrocedrae; Greslebin et al., 3). Pathogenicity tests were performed using 15 replicate shoots (c. 40 cm long) of Italian cypress. Mycelia plugs (roughly 5 mm), derived from the margins of an actively growing 20-day-old culture of the P. austrocedri isolate on V8 agar, were used to inoculate the shoots by placing the plug on wounded points under the bark. In order to maintain humidity, inoculation points (centre of each shoot) were coated with paraffin film and wrapped with aluminum foil. The shoots were incubated at 17°C in the dark after placing in sealed plastic bags containing a damp cotton wall ball. Fifteen shoots were inoculated with sterile V8 agar as controls. After ten weeks, orange-brown phloem lesions ranging from 20-100 mm in length appeared on the inoculated shoots. The pathogen was reisolated from all of the infected shoots, whereas control shoots remained symptom-free. Phytophthora austrocedri has been reported to be a pathogen on Juniperus communis and Chamaecyparis spp. (Green et al., 2). To the authors' knowledge, this is the first report of P. austrocedri causing lesions in the phloem on Cupressus sempervirens in northern Iran or Cupressus species worldwide. The origin of P. austrocedri in Iran is unknown. As Italian cypress is only planted in Iran in parks, the potential of this pathogen spreading to nurseries and orchards around of Qazvin city is not high. However, the host range of P. austrocedri has not been studied extensively and there is concern that other economically important species, such as surrounding pistachio gardens could be at risk, since they are susceptible to other Phytophthora spp. (Saremi et al., 4). Studies will need to be conducted to determine if these local hosts are susceptible.