V8 Agar Research Articles

Identification and characterization of Botrytis cinerea causing gray mold on tomato in Malaysia

Botrytis cinerea, commonly known as gray mold, is a pervasive fungal pathogen that affects a wide range of plant species, leading to significant agricultural losses. The identification of Botrytis cinerea in Malaysia is crucial for protecting the agricultural sector, minimizing economic losses, ensuring food security, maintaining export quality, addressing environmental concerns, and advancing scientific research. In the present research, tomato fruits collected from Cameron Highlands, Pahang, Malaysia showed gray mold disease symptoms of B. cinerea. The fungal isolates were examined morphologically for colony colour, growth rate, conidiophores, conidia shape, and sclerotia on PDA and V8 agar. According to the results, conidiophores appeared in grape shape and length was range of 21.26-32.52 μm, ovoid conidial dimensions were in the range of 10.03-16.08 × 7.37-11.15 μm and sclerotia size was range 1.91-4.50 × 1.70-4.00 mm. All isolates were attributed to the morphospecies Botrytis cinerea on account of these characteristics. The resulting sequences deposited in GenBank were accessions MT012053 to MT012062, respectively. A BLAST analysis of the resulting 550-bp nucleotide sequences showed 99-100% identity closest matched to B. cinerea. The pathogenicity experiments showed P6 isolates of B. cinerea were highly pathogenic and caused gray mold development on tomato fruits that led to severe symptoms in five days. Meanwhile, the least pathogenic isolate was P9. In terms of temperature, B. cinerea grew faster on PDA at 20ºC, slower grew below 20ºC and did not grow at 25ºC. Identification and characterization of B. cinerea on tomato could potentially provide information to assist disease management strategies for B. cinerea.

Identification of Cercospora spp. on corn in North America and baseline flutriafol fungicide sensitivity.

Gray leaf spot (GLS) is an important corn disease reportedly caused by Cercospora zeae-maydis and C. zeina. Recently, flutriafol, a demethylation inhibitor (azole) fungicide received EPA registration as Xyway® LFR®, a product that is applied at planting for management of fungal diseases in corn, including suppression of GLS. In this study, 448 Cercospora spp. isolates were collected in 2020 and 2021 from symptomatic corn leaf samples submitted from the United States and Ontario, Canada. The Cercospora spp. were identified using multi-locus genotyping of the internal transcribe spacer (ITS), elongation factor 1-α (EF1), calmodulin (CAL), histone H3 (HIS), and actin (ACT) gene. Based on the multi-locus phylogenetic analyses, six species were identified; C. cf. flagellaris (n = 77), C. kikuchii (n = 4), C. zeae-maydis (n = 361), Cercospora sp. M (n = 2), Cercospora sp. Q (n = 1), and Cercospora sp. T (n = 3). In subsequent pathogenicity tests using selected isolates from each of these species, only C. zeae-maydis resulted in symptoms on corn with no disease symptoms observed after inoculation with C. cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T. While disease symptoms were observed on soybean following inoculation with C. cf. flagellaris, C. kikuchii, and Cercospora sp. Q, but not the other three species. Fungicide sensitivity of Cercospora spp. to flutriafol was assessed using a subset of 340 isolates. The minimum inhibitory concentration (MIC) to inhibit the growth of Cercospora spp. completely was determined based on growth of each species on flutriafol-amended clarified V8 agar at nine concentrations. The EC50 was also calculated from the same trial by measuring relative growth as compared to the non-amended control. Cercospora zeae-maydis was sensitive to flutriafol with mean MIC values of 2.5 µg/mL and EC50 values ranging from 0.016 to 1.020 µg/mL with a mean of 0.346 µg/mL. Cercospora cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T had mean EC50 values of 1.25 µg/mL, 7.14 µg/mL, 2.48 µg/mL, 1.81 µg/mL, and 2.24 µg/mL respectively. These findings will assist in monitoring the sensitivity to the flutriafol fungicide in Cercospora spp. populations.

First Report of Plumed Cockscomb (Celosia argentea var. plumosa) Stem Blight Caused by Phytophthora nicotianae in Taiwan.

Celosia spp. is a widely cultivated ornamental plant in gardens or parks in Taiwan. In September 2021, withering leaves and grayish-brown lesions were observed on the lower stem of plumed cockscombs (C. argentea var. plumosa) in Taichung City, with an incidence of about 22% in 136 plants after continuous precipitation, impacting the aesthetic value of the landscape. Symptomatic plants were collected, surface disinfected with 70% EtOH for ~20 sec., blotted dried, and excised diseased tissues (~ 3×3 mm2) were placed on 2% water agar. Four representative isolates were obtained after purification and the colonies were white with aerial and non-septated hyphae on V8 agar for 7 days. Sporangia were ovoid, ellipsoid or obpyriform, papillate, (26.3-55.9) 38.0 × 29.0 (20.1-40.6) µm (n = 200) (Ahonsi et al. 2007). Chlamydospores were spherical, terminal or intercalary, 26.0 (15.1-40.4) µm (n = 200). All isolates belong to A2 mating type with amphigynous antheridia and plerotic oospores, 21.0 (17.7-25.7) µm (n = 200), resembling the descriptions of Phytophthora (Erwin & Ribeiro 1996). For molecular identification, sequences of the ITS, β-tubulin (β-tub), and EF-1α regions of all isolates were amplified using ITS1/ITS4, TUBUF2/TUBUR1, and ELONGF1/ELONGR1 primers, respectively (White et al. 1990; Kroon et al. 2004). BLAST analyses of isolates cap1-2 (ITS: OQ581785; β-tub: OQ590022; EF-1α: OQ590026), cap1-3 (ITS: OQ581786; β-tub: OQ590023; EF-1α: OQ590027), cap2-1 (ITS: OQ581787; β-tub: OQ590024; EF-1α: OQ590028), and cap2-2 (ITS: OQ581788; β-tub: OQ590025; EF-1α: OQ590029) showed 100% of ITS identity, 99.5 to 99.9% of β-tub identity, and 99.4 to 99.6% of EF-1α identity with Phytophthora nicotianae (ITS: MG865551; β-tub: MH493987; EF-1α: MH359043). Phylogenetic trees were constructed using concatenated ITS, β-tub, and EF-1α sequences based on maximum likelihood with a GTR+G model in MEGA X and Bayesian inference method in Geneious Prime 2022.2. All isolates were clustered in P. nicotianae with similar topology, thereby were identified as P. nicotianae. To confirm pathogenicity, 7 to 10-day-old seedlings and 6-week-old plumed cockscomb plants were inoculated in separate trials and each experiment was conducted twice. For each seedling, the lower stem was inoculated with 50 μl of zoospore suspension (104 zoospores/ml), 3 plants per isolate, and then incubated at 30±2℃ with 12 h light. For adult plants, each was inoculated with mycelial plugs from one V8 plate of 10-day-old P. nicotianae, 5 plants per isolate, and incubated at 25±2℃ in a greenhouse. Control plants were inoculated with sterile water and V8 agar plugs, respectively. Stem and root rot were observed on seedlings 4 days after inoculation while wilting and lower stem browning were observed on adult plants 2 months after inoculation. All control plants remained healthy at the end of repeated trials and identical pathogens were re-isolated only from symptomatic plants, thus fulfilling Koch's rules. P. nicotianae has been reported causing root rot and stem necrosis not only on cockscomb (C. plumosa Hort. ex Burvenich) in Argentina (Frezzi 1950), but also infecting several ornamental plants recently in Taiwan (Ann et al. 2018). To our knowledge, this is the first report of stem blight caused by P. nicotianae on plumed cockscombs in Taiwan. This finding suggests limited options for landscaping and the host preference of the isolates obtained in this study should warrant further studies.

First Report of Neofabraea actinidiae causing a Cranberry Fruit Rot in Oregon.

Cranberry (Vaccinium macrocarpon, L.) is a commercial small fruit that is native to North America. Oregon ranks fourth in cranberry production in the U.S.A. with 1052 Ha of cranberry beds and annual production of 23,590 metric tonnes (USDA NASS 2021). Cranberry fruit rot diseases are caused by a complex of 15 fungal pathogens belonging to 10 genera (Polashock et al. 2017). In fruit rot surveys of 'Stevens' cranberry beds in Coos and Curry Counties, Oregon, berries were collected before harvest and sorted by symptoms (e.g. softening, shriveling, or discoloration). Cranberries with field rot symptoms were surface-disinfested and lesion margin tissue placed on V8 agar. Cultures were incubated at room temperature and outgrowing fungi were transferred twice onto fresh V8 agar to obtain single isolates. Storage rots that developed on asymptomatic cranberries held at 4°C for 8 weeks were processed similarly. Since 2017, we periodically isolated Neofabraea actinidiae, which is not a member of the cranberry fruit rot complex, from rotted cranberries (Polashock et al. 2017). In 2022, N. actinidiae was isolated from 22% of 45 cranberries collected from an organically managed farm and developed storage rot and from 6% of 50 storage-rot cranberries from three conventionally managed farms. Symptoms associated with N. actinidiae on 'Stevens' cranberries often include softening and brown tissue surrounded by a yellow-colored ring. On V8 agar, N. actinidiae grew as compact white circular colonies with dense aerial hyphae near the center and accompanied by a red pigment in the agar. Pink-colored mucoidal irregular conidiomata often developed on the colony after 3 weeks. Conidia were hyaline, aseptate, and ellipsoidal to fusiform ranging from 7.5 to 12.6 µm long X 3.5 to 5.6 µm wide (n=100). Genomic DNA was extracted from N. actinidiae isolates from cranberries in 2017 and 2022. β-tubulin and the ITS 5/4 region were amplified and sequenced with primers Bt-T2m-Up/Bt-LEV-Lo1 and ITS5/ITS4 using conditions of de Jong et al. (2001) and White et al. (1990), respectively. Sequences were deposited in NCBI Genbank (Accessions: OR606303, OR606305, OR606306, & OR606309 for ITS; OR610314, OR610316, OR610317, & OR610320 for β-tubulin). BlastN analysis of β-tubulin (695 bp) and ITS (489-490 bp) had a 99.6 to 99.8% and 99.8 to 100% identity, respectively, to Neofabraea actinidiae (CBS 121403) (Accession numbers: KR859285 β-tubulin and KR859079 ITS). Phylogenetic analysis of concatenated sequences using Tamura-Nei neighbor-joining (Tamura et al. 2004) confirmed identity of cranberry isolates as N. actinidiae. Koch's Postulates were confirmed with four N. actinidiae isolates from cranberry. Agar or hyphal plugs were placed in a 3 mm wound on the side of six surface-disinfested, asymptomatic berries and incubated in a moist chamber at 20°C for 15 days or 4°C for 55 days. Similar symptoms developed on each berry inoculated with N. actinidiae, but not agar alone. The fungus was recovered from symptomatic tissues and identity confirmed by colony morphology. N. actinidiae causes a ripe rot and storage rot in kiwifruit and is one of the species causing Bull's Eye Rot of pome fruits (Tyson et al. 2019). Cryptosporiopsis actinidiae (anamorph) was isolated from cranberries roots in British Columbia, CA, but considered unlikely to be the causal agent of dieback disease of cranberry vines (Sabaratnam et al. 2009). We have demonstrated that N. actinidiae causes a cranberry fruit rot in beds and in storage. Its prevalence, associated fruit rot symptoms, and disease incidence will continue to be monitored.

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 Root and Collar Rot caused by Phytophthora nicotianae on ⨯Graptoveria 'Silver Star' in Korea.

⨯Graptoveria 'Silver Star' (a cross between Graptopetalum filiferum and Echeveria agavoides) from the Crassulaceae family, are an evergreen succulent with lotus constellation-shaped flowers, making it consumer favorite ornamental plant in Korea. In 2019, Korea's ornamental production was estimated at KRW 517.4 billion (EUR 382 million), from 4,244 ha of farming area according to the Ministry of Agriculture, Food and Rural Affairs of Korea. In July 2023, ⨯Graptoveria 'Silver Star' plants with chlorotic leaves, root and collar rot were observed in a greenhouse in Yongin (37°14'27.9"N, 127°10'39.19"E), Korea. To isolate the causal agent, small pieces (1 mm2) of symptomatic tissues were surface-sterilized using 1% NaOCl for 1 min, then put onto a water agar (WA) plate and incubated in the dark at 25℃ for five days. Two isolates (FD00202, FD00203) were obtained from diseased leaves, stem and roots by isolating single sporangium. To investigate the morphological characteristics of the isolates, the mycelium from potato dextrose agar (PDA) were transferred to V8 agar (V8A) followed by incubation at 25°C in the dark for 7 days. The isolates produced dense cottony mycelium, with slightly petaloid and light rossette pattern, with coralloid edges measuring 70 to 83 mm diameter. Sporangium were spheroid (30.0-48.0 µm long, 25.0-35.0 µm wide) with globose chlamydospores (17.0-50.0 µm long, 18.0-38.0 µm wide). Oogonia were not observed. Morphological and cultural characteristics of these isolates were phenotypically similar to that of Phytophthora nicotianae (Faedda et al. 2013; Abad et al. 2023). For molecular identification, genomic DNA was extracted from 5 days old cultures using the Maxwell® RSC PureFood GMO and Authentication Kit (Promega). Two gene regions, the rDNA-ITS, COX I were amplified and sequenced using primers ITS1/ITS4 and FM83/FM84, respectively (White et al. 1990; Martin and Tooley 2003). The resulting sequences were deposited in GenBank with accession no. LC783858 to LC783861. A BLASTn search of the DNA sequences from ITS, COX I showed 99.81 and 98.94% identity to P. nicotianae isolate IMI 398853, respectively. Maximum likelihood phylogenetic analyses were performed for the combined data set with ITS, COX I using MEGA7 under the Tamura-Nei model (Kumar et al. 2016). The isolates formed a monophyletic group with P. nicotianae isolate IMI 398853, CPHST BL162, and CPHST BL 44. Based on morphological characteristics and molecular analysis, the isolates were identified as P. nicotianae. T confirm their pathogenicity, inoculum was prepared in accordance with Ann (2000). Artificially wounded healthy plant roots were dipped in zoospore suspension (3.0 × 106 zoospore/ml) for 24 hours, with mock-treated plants (control) dipped in sterile distilled water (Ann. 2000). Thereafter, the plants were transplanted into new medium and kept under high humidity. Symptoms were observed after 10 days of incubation. The plants inoculated with P. nicotianae showed similar symptoms of chlorotic leaves with root and collar rot, while control remained symptomless. The pathogen was re-isolated from all inoculated plants and confirmed as P. nicotianae by morphological and molecular analysis. but not from controls, fulfilling Koch's postulates. Phytophthora nicotianae was previously report on Echeveria derenbergii and Kalanchoe blossfeldiana causing brown spot on stems and roots in California and Korea, respectively (French 1989; Oh and Son 2008). To best of our knowledge, this is the first report of P. nicotianae causing root and collar rot on ⨯Graptoveria 'Silver Star' plants in the Korea.

First Report of Root Rot Caused by Phytophthora lacustris on alder (Alnus lusitanica) in Spain.

Alder decline caused by Phytophthora species is considered one of Europe´s most important diseases of natural ecosystems. In Spain, P. x alni, P. uniformis, P. x multiformis and P. plurivora have been detected in association with root and collar rot in riparian alder populations (Pintos et al. 2010, 2012, 2017; Haque et al. 2014). During the active growth periods 2020-2021, a field survey of the newly designed species Alnus lusitanica (Vít et al. 2017) was carried out at the most symptomatic areas along the Miño-Sil river basin in Galicia and León (NW-Spain). Samples of bark, root, rizosphere soil of declining trees, and river water were collected. Symptoms, similar to those caused by other Phytophthora species, included low leaf density of the canopy, generally with smaller and chlorotic leaves, and branches with dry tips. In some cases, the presence of cankers on the trunk were observed (Figure 1). Necrotic lesions were transferred onto V8-PARPH, a Phytophthora selective medium. Soil and water were baited with carnation petals. Three isolates of one Phytophthora species was recovered: two from the roots of trees, and one from river water at three different sites. The isolates were transferred to V8 agar and incubated a 22ºC in the dark. Colonies had petaloid patterns, and their optimum growth temperature ranged from 25 to 30 °C. In soil extract, non-caducous, non-papillate and ovoid sporangia were produced. Sporangia averaged 41.0×29.4 μm with a length/breadth ratio of 1.39. Internal proliferation occurred (Figure 2). No sexual structures were observed . Genomic DNA was extracted from mycelium obtained from pure cultures of three Phytophthora isolates. The ITS region of the ribosomal DNA template was performed by nested PCR using DC6 (Cooke et al. 2000) and ITS4 (White et al. 1990) in the first round, and ITS6 (Cooke et al. 2000) and ITS4 in the second. The mitochondrial gene cox1 was amplified with primers CoxF4N and CoxR4N (Kroon et al. 2004). BLASTn analyses showed 100% identity to the type of P. lacustris (AF266793, 156 matching bp) for ITS, and 100% identity to the ex-type of P. lacustris (MH136916, 596 matching bp) for the cox1 sequence. Phylogenetic analysis indicated that our isolates were localized in the same evolutionary branch as P. lacustris based on consense sequences of ITS and cox1 sequences. The ITS and cox1 sequences generated in this study were deposited in GenBank with accession number OP588369 and OP548051, and one isolate isolated from root (CECT 21212) was submitted to the Spanish Type Culture Collection (Paterna, Valencia). Pathogenicity tests with isolate CECT 21212 were conducted on ten 3-year-old alders (A. lusitanica) growing in free draining 5 L pots. One shallow cut was made into the cambium at the root collar level. A colonized 5-mm mycelial agar plug, from a 7-day-old culture was inserted into every wound (mycelial surface face-down) and sealed with Parafilm®. Five control plants were inoculated with a sterile agar plug. Plants were kept in a controlled chamber at 25 ºC and 80% humidity. After a 3-week incubation period, inoculated plants showed dieback symptoms and necrosis of the inner bark tissue (Figure 3). Lesion lengths ranged from 2 to 8 cm. Control plants remained symptomless. P. lacustris was recovered from all inoculated plants, but not from the control ones. To our knowledge, this is the first report of P. lacustris causing root rot on alders in Spain. This new detection represents an increased threat to riparian alders by the presence of an additional Phytophthora species associated with alder decline, since P. lacustris can readily adapt to a wide variety of climatic conditions with the ability to infect different hosts (Nechwatal et al. 2013).

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 Leaf Spot of Sugar Beet Caused by Stemphylium vesicarium in Minnesota, USA.

In July 2021, sugar beet (Beta vulgaris L.) leaves with numerous tan to brown spots with white-bleached center and oval to irregularly shaped were collected from a field in Minnesota (MN) (46.2774° N, 96.3100° W), with 15% disease incidence and 30% disease severity. Leaves were washed with tap water then surface disinfected in 1% NaOCl aqueous solution for 1 min. Samples were rinsed thrice with sterile distilled water and dried in a laminar flow hood. A 2-cm leaf disc was plated on potato dextrose agar amended with streptomycin sulfate (200 mg/L) and incubated for four days at 25°C under 12-h light/dark cycle. Single spore cultures were obtained by suspending in sterile water spores harvested from a single colony. The suspension was streaked on a dish with V8 agar media and incubated as described. Five pure cultures were transferred to clarified V8 agar media for morphological feature observations. Colonies were uniform in appearance and developed light to olivaceous green mycelium. Conidia were dark brown to olivaceous green in color and measured 30 × 18 μm (n=20). They were oblong to broadly oval shaped muriform, and multiseptated (1 to 5 septa). Hyphae were septate and pale brown. Conidiophores were short, septate, and light to dark brown in color. Based on the morphological characteristics, isolates were identified as Stemphylium vesicarium (Simmons 1969). Genomic DNA of all five isolates were extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). PCR amplification and sequencing of the internal transcribed spacer (ITS) region (ITS1/ITS4 primers), the largest subunit of RNA polymerase II (5F2/7cR primers) (O'Donnell et al. 2009), the plasma membrane ATPase (ATPD-F1/ATPD-R1) gene (Lawrence et al. 2013), glyceraldehyde-3-phosphate-dehydrogenase gene (GAPDH) (gpd1/gpd2) (Berbee et al. 1999), and β-tubulin gene (Bt2a/Bt2b primers) (Glass and Donaldson 1995) were done using standard procedures. Sequences were submitted to GenBank under accession numbers OP584331 (ITS), OP589289 (RPB2), OP589290 (ATPase), OP994239 (GAPDH) and OP382477 (β-tubulin). The BLASTN search of the sequences showed 100% similarity with MT629829 (ITS) (525/525 bp), KC584471 (RPB2) (859/859 bp), JQ671770 (ATPase) (794/794 bp), MK105974 (GAPDH) (519/519 bp) and MN410922 (β-tubulin) (320/320 bp) reference sequences of S. vesicarium. Pathogenicity tests were done using four cv. Maribo MA 504 plants. S. vesicarium spore suspensions (1 × 106/ml) were sprayed on three leaves from each plant. This trial was repeated with three replicates. A similar group of plants were sprayed with autoclaved distilled water to serve as non-inoculated control. All plants were incubated in the mist chamber for 5 days at 25°C, under daily 14/10 light-dark cycles, and >80% relative humidity, then transferred to the greenhouse kept at 23 ± 2°C and a 12-h photoperiod. Fifteen days post-inoculation, all inoculated plants had multiple lesions with dark brown margins with a grayish center, and non-inoculated control plants were asymptomatic. The re-isolated fungus was morphologically similar to isolates retrieved from the field. S. vesicarium was reported on sugar beet in Michigan (Metheny et al. 2022). This is the first report of S. vesicarium causing disease on sugar beet in MN. Stemphylium sp. is a major problem of sugar beet in the Netherlands (Hanse et al. 2015). Efforts should be made to prevent introduction of susceptible beet cultivars so that the disease does not become widespread in the USA.

First Report of Root Rot Caused by Pythium myriotylum on Sesame in China.

Sesame (Sesamum indicum L.) is a very important oilseed crop and cultivated on 11.7 million hectares, producing 6.02 million tons of seeds with an average seed yield of 512 kg ha-1 in the world (Yadav et al. 2022). In June of 2021, diseased roots were observed on sesame in the villages of Mada and Hanba, Xiangcheng city (114.88°N, 33.13°E), Henan province, China. The diseased plants appeared stunted and wilted at the seedling stage. Approximately 7.1% to 17.7% of plants were affected in two fields, 0.6 ha in total, and disease severity in each affected plant ranged from 50% to 80%. Twenty-four disease plants were collected to confirm the pathogen. The diseased roots were cut into small fragments (2 to 5 mm long), surface sterilized with 75% ethanol (for 1 min), and 10% sodium hypochlorite (for 1 min) and then rinsed in sterilized water three times (1 min each rinse). The fragments were blotted dry and transferred to a potato dextrose agar (PDA) medium (potato 200 g/L, glucose 20 g/L, agar 18 g/L) amended with streptomycin (50 μg/mL). After incubation at 28°C for 24 h, white mycelium grew out from plant fragments. Then, a total of seven morphologically similar strains were transferred onto fresh V8 agar by hyphal tip transfer (Rollins 2003). By light microscope observations, the sporangia were filamentous or digitated, and undifferentiated or inflated lobulate. The oospores were mostly aplerotic, globose or subglobose in shape, and 20.4 to 42.6 μm in diameter (n = 90, n: Total number of oospores measured). Furthermore, antheridia were bulbous-like or clavate-like and were observed attached to the surface of the oospores. The zoospores were abundant and ranged from 8.5 to 14.2 μm in diameter. The morphology characteristics of all strains were consistent with those of Pythium myriotylum (Watanabe et al. 2007). Genomic DNA was extracted from the representative strain 20210628 using the CTAB method (Wangsomboondee et al. 2002). The complete internal transcribed spacer (ITS) and cytochrome oxidase subunit I gene (COI, COX1) can be valid and useful barcodes for accurate identification of many oomycetes (Robideau et al. 2011). The ITS and COI were amplified with the primers ITS1/ITS4 (Riit et al. 2016) and primers OomCox-Levup/OomCox-Levlo (Robideau et al. 2011), respectively. The nucleotide sequences obtained were deposited in the GenBank database under the accession numbers OM230138.2 (ITS) and ON500503.1 (COI). GenBank BLAST search identified the sequences as P. myriotylum ITS and COI sequences (e.g., HQ237488.1 and MK510848.1, respectively) with 100% coverage and 100% identity. To evaluate the pathogenicity, sesame seeds (cultivar: Jinzhi No.3) were planted in 12-cm-diameter plastic pots containing a mixture of sterilized soil, vermiculite and peat mossat a ratio of 3:1:1. Oospores were collected following the procedure of Raftoyannis et al. (2006) with minor modifications. Three-leaf stage sesame roots were soaked with 5 mL of oospore suspension at 1 × 106/mL of the 20210628 strain, and the control plants were inoculated with sterilized water. All plants were maintained in a greenhouse (28±2°C, > 80% R. H.). The experiment was repeated twice with three replications. The plants inoculated with P. myriotylum showed the water soak symptom on the stem base 7 days after inoculation, while control plants were symptomless. Three weeks after inoculation, the plants showed root tissue necrosis, root rot, and dwarfing symptoms that were similar to those observed on sesame plants in the field, while control plants remained healthy. P. myriotylum was re-isolated from the inoculated plants and the morphology was the same as the original strain 20210628. These results suggest that P. myriotylum is the causal agent of sesame root rot. Previous studies have revealed that P. myriotylum can cause root rot in peanuts (Yu et al. 2019), chili pepper (Hyder et al. 2018), green bean (Serrano et al. 2008) and aerial blight of tomato (Roberts et al. 1999). To the best of our knowledge, this is the first report of P. myriotylum causing root rot on sesame. This pathogen can infect plant roots and develop rapidly if no effective control measures are implemented. Once the disease breaks out in a large area, the yield of sesame will be seriously threatened. The results provide important implications for the prevention and management of this disease.

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.

First Report of Phytophthora occultans Causing Dieback of Buxus sempervirens in the Czech Republic.

In Nov 2011, and then recurrently since Sep 2020, an extensive decline has been recorded in boxwood (Buxus sempervirens), sometimes with several dozens of damaged individuals planted in private gardens and public areas and purchased in amateur markets in the Czech Republic. The leaves of the plants first showed orange-bronze discoloration, then dried and fell off, and the affected plants died. The roots, collars and stems of these plants had dark brown to black necrotic lesions. Phytophthora occultans Man in 't Veld & K. Rosend. was consistently isolated on selective medium PARPNH (Jung et al. 1996) directly from segments of symptomatic collar tissues and from rhododendron leaf pieces used to bait excised roots. On 20% V8 agar (V8A) and on carrot agar (CA), colonies had a stellate pattern. Radial growth at 25°C was 9.4 mm/day on V8A and 5.3 mm/day on CA. The cardinal growth temperatures were min. 7°C, optimum 25 to 27°C, and max. 32°C. The isolates were homothallic and produced on CA colorless globose oogonia ranging from 25.4 to 36.4 µm (n = 40) in diam. Oospores were slightly aplerotic and measured (n = 40) 22.5 to 31.9 µm in diam., with a 0.9 to 1.5 µm thick wall. The antheridia were predominantly paragynous and averaged 11.5 × 9.9 µm (height × width, n = 40). Noncaducous sporangia were obpyriform, ovoid, elongated to irregular and semipapillate, sometimes bipapillate and measured (n = 40) 31.4 to 73.4 × 17.8 to 32.1 µm, and the L:B ratio was 1.9 to 2.0. Chlamydospores and hyphal swellings were not observed. The morphological characteristics resembled those described for P. occultans (Man in't Veld et al. 2015). The isolates were deposited in the Czech Collection of Phytopathogenic Oomycetes (CCPO) under accession nos. 551.11, 1158.20, 1176.21, 1201.21, 1218.21, 1236.21 and 1261.22. For molecular identification, the internal transcribed spacer (ITS) region, cytochrome oxidase subunit 1 gene (COX1), and translation elongation factor-1α (EF) gene from all isolates were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), COXF-CIT/COXR-CIT (Man in't Veld et al. 2015), and ELONGF1/ELONGR1 (Kroon et al. 2004), respectively. The resulting sequences of representative isolates P1158.20 and P1176.21 were deposited in GenBank (accession nos. MW750576 and OP326036 for ITS, ON862131 and OP313505 for COX1 and MW762616 and ON862132 for EF). BLASTn searches of GenBank, using the partial ITS, COX1, and EF sequences, revealed 100, 100, and 99% sequence identity, respectively, to P. occultans ex-holotype culture CBS101557 accessions JX978155, JX978156 and KF650770 (Man in't Veld et al. 2015). Concatenated sequences of the three genes were used to conduct a phylogenetic analysis using the maximum likelihood method in MEGA 11 (Tamura et al. 2021). The isolates were identified as P. occultans based on morphology and a multigene phylogenetic analysis. Koch´s postulates were confirmed by a soil infestation test. Healthy 2-year-old B. sempervirens plants were inoculated (9 plants per isolate and control, isolates no. 1158.20, 1176.21, 1261.22) with three 5-mm-diam. V8A mycelial plugs by inserting into the substrate near the collar. Control plants were treated with sterile agar plugs. All plants were kept in a greenhouse at 25°C and exposed to 24 h of flooding up to collar once a week. All inoculated plants showed wilting, collar lesions and root rot occurred after 21 days, while control plants remained healthy. The pathogen was reisolated from infected plants and confirmed by molecular identification. P. occultans was found for the first time in 1998 on Buxus sempervirens in the Netherlands and later in Belgium, the United Kingdom, Germany and Romania (Man in´t Veld et al. 2015, Nechwatal et al. 2014), as well as in the USA (Reeser et al. 2015, Gitto et al. 2018). This is the first report of P. occultans in the Czech Republic. This pathogen likely poses another significant threat to boxwood cultivation in addition to the previously invaded Cydalima perspectalis and Calonectria pseudonaviculata.

First Report of Leaf Spot and Blight on Crown Daisy Caused by Cercospora apii in China.

Crown daisy (Glebionis coronaria L.), also known as chrysanthemum greens, is a popular vegetable in Asia, especially in China. The leaves have been used in folk medicine as a tonic for the liver, blood, intestines and to control anemia and high blood pressure. In November 2020, severe leaf spot and blight was observed with 80% to 95% incidence on crown daisy growing in greenhouses in Fengxian, Shanghai, China(121°22'E, 30°53'N). Irregular rounded spots appeared with a light gray center and water-soaked margins. Round lesions enlarged and merged with age, followed by the development of a necrotic area resulting in the typical "frog-eye" and causing a continuous deterioration of crown daisy. Diseased leaves were washed in running water for 30 min. Small fragments (5 × 5 mm) taken from the margin of lesions were disinfected with 1% NaClO for 3 min, rinsed three times with sterile water, cultured on potato sucrose agar (PSA) augmented with 50 mg streptomycin/liter at 26 oC,and incubated in the dark. Colonies had identical morphology, and TH11290202 was selected and deposited in the plant pathology lab of Shanghai Academy of Agricultural Sciences. Mycelium was initially cottony and white and became appressed to the medium and dark brown with time. Conidia did not form on any media, including PSA, PDA, V8 agar (V8A), maize leaf carbonate agar (MLPCA), pepper leaf carbonate agar (PLPCA), etc. To confirm the identity of the pathogen, genomic DNA was extracted from TH11290202 with the cetyltrimethylammonium ammonium bromide (CTAB) method from the mycelia. Five loci were PCR amplified, namely, the internal transcribed spacer (ITS), translation elongation factor (TEF), calmodulin (cmdA), histone (H3) and actin (ACT), using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Jaklitsch et al. 2005), CAL-F/CAL-R (O'Donnell et al. 2000), cylh3f/cylh3r (Glass and Donaldson 1995), and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. The resulting sequences were deposited in GenBank (MW819910, MW981277, MW981278, ON798723, and MW981279). Analysis of the ITS, TEF, cmdA, H3 and ACT gene sequences of isolate TH11290202 revealed that it was a member of the genus Cercospora, sharing 99.79%, 99.66%, 98.10% 99.74% and 100% sequence similarity with type strain of Cercospora apii CBS 116455. A multilocus phylogenetic analysis was performed using sequences from other closely related taxa obtained from GenBank. Based on morphological and molecular characteristics, TH11290202 was identified as C. apii (Crous and Braun 2003; Groenewald et al. 2006; Milosavljević et al. 2014). To confirm pathogenicity, Koch's postulates were fulfilled on 30 mature plants, which were maintained in a growth chamber (at 26 °C, relative humidity 90%, 12/12 h light/dark). Surface-sterilized leaves were sprayed with a mycelial suspension. Brown lesions were formed 7 days after inoculation on 15 plants, whereas the noninoculated controls remained asymptomatic on the other 15 plants. To our knowledge, this is the first report of C. apii causing leaf spot and blight on G. coronaria in China and will provide useful information for developing effective control strategies.

First Report of Pythium myriotylum Causing Stem and Root Rot of Kidney Bean in Taiwan

HomePlant DiseaseVol. 107, No. 3First Report of Pythium myriotylum Causing Stem and Root Rot of Kidney Bean in Taiwan PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Pythium myriotylum Causing Stem and Root Rot of Kidney Bean in TaiwanY. R. Liang and F. C. LiaoY. R. Liang†Corresponding author: Y. R. Liang; E-mail Address: [email protected]https://orcid.org/0000-0002-9232-3936Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung 41358, TaiwanSearch for more papers by this author and F. C. LiaoTaiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung 41358, TaiwanSearch for more papers by this authorAffiliationsAuthors and Affiliations Y. R. Liang † F. C. Liao Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung 41358, Taiwan Published Online:14 Feb 2023https://doi.org/10.1094/PDIS-06-22-1464-PDNAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleKidney beans (Phaseolus vulgaris Linn.) are widely grown in central and southern Taiwan. In June 2021, kidney beans showing stem and root rot symptoms were observed in a 3,000 m2 plot in Xinyi Township, Nantou County, Taiwan (23.62°N, 120.87°E; with around 600 plants). About 15% of the plants on site displayed similar symptoms with stem and root rot. Small pieces (3 to 4 mm) of affected tissue were surface disinfected by immersion in 0.6% sodium hypochlorite for 5 min, rinsed twice in sterile distilled water, and placed on a V8 agar plate. Plates were incubated at 28°C for 24 h in the dark. A Pythium-like organism was consistently isolated. Genomic DNA was extracted from one of the isolates using a DNA extraction kit (Kaneka Easy DNA Extraction Kit version 2; Takasago, Japan). The identities were confirmed by the internal transcribed spacer (ITS) sequence (accession no. MZ994503), which had 100% homology with KY019264.1; and the cytochrome c oxidase subunit I (COI) (Robideau et al. 2011) sequence (accession no. OK513021), which had 100% homology with MT222434.1. For morphological characterization, the pathogen was cultured on V8 agar to produce oogonia (24 to 27 μm), lobulate sporangia, and zoospores. Isolates were identified as P. myriotylum based on morphological characteristics (Watanabe 2002) and ITS and COI sequences. Koch’s postulates were completed in the greenhouse on kidney beans at the four- to five-leaf stage. Six plants were inoculated with a 3-mm mycelial plug of 3-day-old V8 plates and the plugs were placed beside the collar of these plants without wounding. Four control plants were inoculated with a 3-mm water agar plug using the same method. All plants were kept in wet plastic bags (near 100% relative humidity) at 25 ± 2°C for 48 h and maintained in a growth chamber with a 12-h photoperiod. The experiment was repeated twice. Two days after inoculation, symptoms similar to the original were observed on the inoculated plants. Koch’s postulates were confirmed by reisolating the same pathogen from diseased plants and sequencing the same ITS region. Control plants remained symptomless. P. myriotylum has previously been described as infecting a wide range of plants in temperate and tropical regions (Perneel et al. 2006). It has been described occurring on ginger (Zingiber officinale), peanut (Arachis hypogaea), rape (Brassica rapa var. rapa), lettuce (Lactuca sp.), tobacco (Nicotiana tabacum L.), and royal paulownia (Paulownia tomentosa Steud.) in Taiwan. To our knowledge, this is the first report of root rot on kidney bean caused by P. myriotylum in Taiwan.The author(s) declare no conflict of interest.

First report of Pythium aristosporum causing root rot on rice seedling in Taiwan.

Rice (Oryza sativa L.) is the principle staple crops in the World and its production can be severely damaged by Pythium species. Several Pythium species including P. afertile, P. arrhenomanes, P. dissotocum, P. elongatum, P. spinosum, have been recorded to cause rice seedling root rot in Taiwan (List of Plant Diseases in Taiwan edited by Tzean et al., 2019). During the survey of rice seedling diseases, we identified a new species of Pythium that causes seedling root rot on rice in commercial nursery trays in two nursery fields in 2019 in Taichung, Taiwan. Stunting and root rot symptom were found on the affected plants and up to 20% seedlings in a nursery tray showed similar symptoms. To isolate the pathogen, symptomatic roots were surface sterilized with 75% ethanol for 1 min and rinsed in sterile water. The margin of lesion was cut off, placed on 1.5% water agar and incubated at 28 ℃. After 24 h, the hyphal tips of a white colony growing from the diseased region were transferred to potato dextrose agar (PDA) medium. Koch's postulates were fulfilled by inoculating the germinated rice seeds with mycelia. Rice seeds of O. sativa var. Tainan11 (TN11) were treated with 75% ethanol and then 1.2% NaOCl for 15 min. The sterilized seeds were soaked in sterile water under dark condition for 3 days and the water was replaced every day. Five of the pre-germinated seeds with 2~5 mm embryonic shoot were placed in a sterile petri-dish and inoculated with 3-ml mycelial suspension (OD600 = 0.045) prepared by blending the mycelia of a 3-days PDA culture using an Oster 10 speed blender 6640 (Oster, USA). The seeds-mycelia were then covered with sterilized soil mixture of Akadama soil and rice husk (1:1, volume to volume) and incubated in a growth chamber at 28 ℃. Seven days post-inoculation, the inoculated seedlings showed stunting with short and necrotic roots (Fig. S1). The pathogen was reisolated from the diseased seedlings and identified with morphology and molecular methods. For morphological characterization, the pathogen was cultured on V8 agar to produce oogonia and zoospore (Chamswarng and Cook 1985). Globose oogonia with multiple antheridia (1-5 per oogonium), inflated filamentous sporangia, vesicle with abundant zoospores, main hypha with up to 6.57 μm wide and mature aplerotic oospores with diameter 24.35-30.81 μm (average= 27.22 μm; n=20) were observed (Fig. S1) that are similar to the descriptions for P. aristosporum (van der Plaats-Niterink 1981). Genomic DNA was extracted with CTAB method (Wang and White 1997) and the sequences of the internal transcribed spacer (ITS) region and gene region of β-tubulin (tub) and cytochrome c oxidase subunit II (cox II) were amplified with published primers (Villa et al., 2006). The obtained sequences were submitted to GenBank (accession nos: OL701302 (ITS), OL763269 (tub), and OL763270 (cox II); Fig. S2). Phylogenetic relationships between this Pythium pathogen and other 55 Pythium isolates, including the type species of P. aristosporum (ATCC11101), were conducted with the concatenated sequences of tub and cox II and analyzed by Bayesian interference (Fig. S3). Based on the tree built with tub and cox II sequences, this pathogen was identified as P. aristosporum that has not been reported in rice and other plants in Taiwan. It was observed in laboratory assays that this pathogen caused significant root-rot symptoms on several major rice varieties grown in Taiwan, including TN11, Tainung67 and Kaoshiung139. It may potentially cause severe crop loss in rice production, especially in nurseries. This identification provides important information on rice disease management.

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.

Nitrogen repression of deoxynivalenol biosynthesis is mediated by Mep2 ammonium permease in Fusarium graminearum.

Fusarium graminearum is an important wheat pathogen and a producer of deoxynivalenol (DON). Biosynthesis of DON is suppressed by ammonium and induced by arginine and polyamines. To better understand ammonium repression of DON biosynthesis, in this study, we functionally characterized three ammonium permease (MEP) genes in F. graminearum. All the mep deletion mutants were normal in growth on V8 agar. Whereas deletion of MEP1 had no detectable phenotypes, the mep2 and mep3 mutants had defects in hyphal growth under ammonium limiting conditions and infection of wheat heads, with the latter having less severe defects. Deletion of MEP2 but not MEP3 affected nitrogen repression of DON biosynthesis and genes involved in nitrate metabolism. The mep2 mep3 double mutant had more severe defects in nitrogen repression than the mep2 mutant and was defective in ascospore releasing. Mutant alleles of MEP2 with truncated C-terminal cytoplasmic tail (CT) failed to complement the mep2 mutant. Expression of a dominant active allele of RAS2 partially rescued the defects of mep2 in nitrogen repression. Taken together, these results suggest that Mep2 acts as the major sensor of ammonium availability in F. graminearum and its CT region functions in nitrogen repression via RAS2 and downstream signalling pathways.

First Report of Root Rot Caused by Phytophthora acerina on Metasequoia glyptostroboides in China.

Metasequoia glyptostroboides Hu & W. C. Cheng (Taxodiaceae), commonly called the Chinese redwood or dawn redwood, is a well-known "living fossil" and rare relict plant species endemic to China, which has been successfully cultivated throughout the world (Ma 2007). In July to September 2020, trees of Chinese redwood which were more than thirty years-old, showed symptoms of decline and death associated with branch dieback, root and collar rot (Fig. 1) in Yangtze River shelter-forests of Jiangling County in Hubei Province, China (112°15'19″E, 30°11'56″N; 40m). Diseased roots and rhizosphere soils were collected in September 2020 and April 2021. Using the baiting method, a homothallic Phytophthora sp. was recovered consistently from diseased roots and soil samples of Chinese redwood. All the isolates of this Phytophthora sp. formed similar colonies on V8 agar and corn meal agar (Fig. 2), and then three representative isolates (L4-5-4, L4-5-5 and L4-5-6) were randomly selected for morphological and molecular identification. In distilled water, semipapillate persistent sporangia were borne in simple sympodial branched sporangiophores. Sporangia were predominantly ovoid (Fig. 3a, d and f), but other shapes were observed including subglobose (Fig. 3b), limoniform (Fig. 3c) or distorted shapes (Fig. 3e), averaging 44.1 ± 7.7 µm (n=102) in length and 32.8 ± 5.2 µm (n=102) in width, with narrow exit pores of 8.0 ± 1.4 µm (n=93) and a length/breadth ratio of 1.3 ± 0.10 (n=102). Chlamydospores were not observed. Oogonia were globose or subglobose, 20.51 to 40.15 µm (av. 33.1 ± 3.9 µm) (n=119) in diameter, with smooth walls and paragynous antheridium (Fig. 3g-i). Oospores were globose or subglobose in elongated oogonia with medium wall thickness of 1.9 ± 0.5 µm (n=36), aplerotic or plerotic and 16.9 to 32.6 µm in diameter (av. 26.6 ± 3.8 µm) (n=40). According to the above morphological characteristics, this Phytophthora sp. was placed in Waterhouse's (1963) group III. The sequences of the internal transcribed spacers (ITS) region of nuclear ribosomal DNA of each isolate (GenBank Accession No. OK087320, OK087321 and OK087322) was 760 bp and had identity of 99.84% with three P. acerina isolates (JX951285, JX951291 and JX951296), while the 800 bp β-tubulin (BTUB) sequences (OK140540, OK140541 and OK140542) showed 99.97% homology to the sequence of P. acerina (KC201283) (Ginetti, Moricca and Squires 2014) (Table 1). The ML phylogenetic trees were established by comparing ITS and BTUB sequences of three Phytophthora strains (L4-5-4, L4-5-5 and L4-5-6) with reference sequences of isolates of Phytophthora in ITS and BTUB in GenBank (Fig. 4-5). Based on the morphological and molecular characteristics, the strains were identified as namely P. acerina. In addition, pathogenicity assays were performed with one of the three strains (L4-5-4) on M. glyptostroboides using both one year old and three years old seedlings. Inoculum was prepared by subculturing agar plugs from edges of CMA cultures into V8 medium plates, incubating at 20 ℃ in darkness for 10 days. Six seedlings planted in pots filled with sterilized soil were inoculated by mycelium plug at root collar and stem wounded by a 8 mm diameter puncher. Six control seedlings were inoculated in the same manner as above, and sterile agar plugs were used. After 35 days, inoculated seedlings all had necrotic lesions at the inoculation sites, and some seedlings had the symptoms of foliage blight and dieback, whereas control seedlings remained healthy (Fig. 6). The number of fibrous roots after inoculation was significantly less than the control, and the roots of inoculated seedlings blackened or even rotted, while there were no obvious symptoms in the control (Fig. 7). Phytophthora isolates recovered from the symptomatic tissues of artificially inoculated plants were identical to isolate L4-5-4 in morphological characters and ITS sequencing. This is the first report of P. acerina causing root rot on the Chinese redwood in China. As only the seedlings were inoculated, further research is needed to address the epidemiology and pathogenicity of P. acerina to adult trees of Chinese red wood.

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).

First Report of Dieback Caused by Phytophthora ramorum on Golden Chinquapin, Chrysolepis chrysophylla, in California.

Golden or giant chinquapin, Chrysolepis chrysophylla (Fagaceae), is a slow growing, evergreen shrub or tree, native to the west coast of the US. In April 2015, several declining chinquapin trees were identified on Bolinas Ridge near Mt. Tamalpais in Marin Co. CA, a Phytophthora ramorum infested region. Branch dieback was observed but no bole or branch cankers were observed. Discolored xylem tissue (5-mm2) was cultured on corn meal agar selective medium, CMA-PARP. An organism morphologically resembling P. ramorum was isolated from one water sprout piece. The ITS sequences obtained from pure cultures, symptomatic branches, and water sprouts were identical, and one was deposited into GenBank. The ITS sequence and cox2 sequence obtained from a culture were a 100% match to the ex-type of P. ramorum. Koch's postulates were tested on 3 small plants (0.5m tall, 3.8-liter pots). Plants were inoculated with 6-mm plugs taken from the margin of a 7-day-old P. ramorum colony, growing on V8 juice agar. Plugs were placed on wounded stems, approximately 2 cm above the soil line, and wrapped in Parafilm. An equal number of plants were treated with uncolonized V8 agar plugs as controls. The potting mix of 2 additional plants of the same size was infested with 200 ml of a zoospore suspension (105 spores ml-1) which was drenched around the base of the stem. Each base was scored with a razor blade before applying the suspension. Two plants were scored and drenched with sterile water as controls. After 7 days, plug-inoculated plants began to wilt and leaves began to turn grayish green. After 15 days, plants began to collapse and isolations were made from stem cankers and roots onto CMA-PARP. After 12 days, leaves of plants that were soil-drenched with zoospores were a dull green color and after 38 days, dry and brittle. Lesions were seen on the roots and stems when isolations were made at 35 days. P. ramorum was consistently isolated from stems of wound-inoculated plants and from roots and stems of soil-inoculated plants. No P. ramorum was isolated, nor symptoms were observed on any control plants. P. ramorum causes sudden oak death on many members of the Fagaceae as well as ramorum blight on more than 130 hosts (Cobb et al., 2020). To our knowledge, this is the first official detection of P. ramorum causing disease on C. chrysophylla in wildlands. Unlike other Fagaceae hosts of P. ramorum, external bole cankers were not seen on infected chinquapin trees. Branch isolations onto PARP media was difficult and detection by PCR from symptomatic vascular tissue was more efficacious. Losses of chinquapin in US forests due to P. ramorum would reduce forest diversity and could cause the loss of habitat for many animal species.