Sterile Filter Paper Research Articles

First Report of Epicoccum nigrum Causing Leaf Spot of Eugenia involucrata in Brazil.

Eugenia involucrata (DC) is a native fruit species of forest formations in the Atlantic Complex and in the forests and savannas of the Paraná State, Brazil (Donadio, 2002). In February 2021, in the experimental area at the Universidade Tecnológica Federal do Paraná, in the Dois Vizinhos city, Paraná State, Brazil, a foliar disease was observed on twenty-two12 years old E. involucrata trees, with 20 to 80% of the leaves per tree affected. Symptoms were small, irregularly to circular shaped, reddish-brown lesions with yellow halos. As the disease progressed, the lesions increased in size and showed no distinction between mature and young tissues. Twenty symptomatic leaves (from each tree) from 11 trees grown at different locations in the orchard (50% of the total number of trees) were collected. For fungal isolation, the leaf surfaces were disinfected with 0.5% NaOCl solution for 1 min, rinsed in sterile distilled water (SDW) and dried on sterile filter paper. Five fragments (0.3 cm) of diseased leaf tissue were placed on potato dextrose agar medium. After 7 days of incubation at 25°C, orange colonies appeared, with a reddish pigment on the reverse side. Conidial were brown globular to pear solitary, verrucous and multicellular (average of 21.74 µm x 24.45 µm, n = 30). The morphological characteristics of the colony and conidia of the eight isolates matched the description of the fungal genus Epicoccum (Valenzuela-Lopez et al. 2018). Further identification of eight isolates was confirmed by amplifying and sequencing three phylogenic loci (ITS, β- tubulin and RPB2) using the ITS1/ITS4, Bt2a/Bt2b and 5F2/7cR primer pairs, respectively (White et al., 1991, Glass and Donaldson, 1995, O Donnell et al., 1998). The sequences of one representative isolate (ENcm) were submitted to GenBank (ITS, MZ442338, β-tubulin, MZ447127 and RPP2, MZ447128) respectively. A phylogenetic tree was constructed by the maximum likelihood method with 1,000 replicas of bootstrapping based on concatenated ITS, β-tubulin, and RPB2 sequences of the ENcm and strains of 14 species of the genus Epicoccum. Isolate ENcm grouped with Epicoccum nigrum stains CBS 173.73 (Chen et al., 2017). For the pathogenicity tests four young healthy branches containing 20 leaves were spray inoculated, with 1.5 mL of conidia suspension of ENcm (106 conidia/mL) covered with a punched transparent plastic bag and moistened with distilled water in orchard. The air temperature ranged from 14ºC to 25ºC. SDW was used as control. Three replicates (pathogen and control) on different trees were performed. After 7 days the fungus was re-isolated from the symptomatic lesion, showing morphological characteristics similar to those of ENcm. Control branches did not show fungal growth. The inoculation test was repeated once, confirming the symptoms described above. This is the first report of the leaf spot caused by E. nigrum on E. involucrata in Brazil as well as in the world. E. nigrum on E. involucrata leaves could pose potential threat on productivity, whose impact may affect the fruit tree's ability to perpetuate, its survival in natural conditions or in commercial orchards.

First Report of Yeast-Spot Disease of Soybean Seeds Caused by Eremothecium coryli in Serbia.

Over the last 15 years, the area planted with soybeans (Glycine max) in Serbia has increased drastically, from 131,000 hectares in 2005 to 230,000 in 2019, and the average yield reached 3.2 t/ha in 2020. The Province of Vojvodina is the most important soybean production region with 95% of the total soybean area in Serbia (www.stat.gov.rs). During the 2021 growing season, soybean seeds with various kinds of symptoms including colour changes, light and dark brown spots, blotching, necrosis, and shriveling were collected from soybean field before harvest of soybean cv. Dukat in the Tamiš locality (South Banat District, Vojvodina Province: GPS: 44°56'12.936"N 20°43'24.216"E) in Serbia. The incidence of symptomatic seeds was estimated at 6.4%. Symptomatic soybean seeds were surface disinfected with 2% NaOCl for 2 min, rinsed in sterile water, dried on sterile filter paper, placed on potato dextrose agar (PDA) and were incubated at 25°C in the dark for 10 to 14 days. The identification of fungi at the genus level based on morphological characteristics revealed the presence of species of Macrophomina, Botrytis, Cercospora and Alternaria, which were previously reported as pathogens of soybean seed in Serbia (Krsmanović et al. 2020). Also, seven white to slightly creamy colonies with yeast-like morphology were observed around seeds expressing discoloration and necrotic and sunken spots. Ten days later, microscopic observations of yeast-like colonies revealed the presence of globose budding cells (diameter of 20 to 28 μm) mostly single or rarely in short chains. Also, two to eight needle-shaped ascospores (52 to 80 μm in length) were arranged lengthwise in many cylindrical to naviculate asci (60 to 96 x 8 to 12, avg. 72.4 x 9.2 µm). Ascospores were with a unilateral, slender, flexuous, whip-like appendage. The morphology of the different fungal structures indicated that the pathogen was Eremothecium coryli (Pelgion) Kurtzman and it was further supported by molecular identification. Total DNA was extracted directly from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and PCR amplification performed with primers ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990). Sequence analysis of ITS region revealed that the Serbian isolate ND2/21 (GenBank Accession No. OL958602) shared the highest nucleotide identity of 100% with E. coryli isolate (Accession No. KY103387). For pathogenicity test, fresh soybean seeds (cv. Sava) were surface-disinfected with 2% NaOCl and rinsed in sterile water before inoculation. The seeds were pierced 3-4 times with a sterile insect pin through a drop of yeast suspension (concentration 106 ascospores/ml) of one selected single-spore isolate (ND2-21). Similarly, control seeds were pierced with sterile insect pins through a drop of sterile distilled water. Five inoculated seeds and control (five replicates per treatment) were arranged uniformly in a Petri dish (9 cm diameter) and incubated at 22 to 25°C in the dark and kept under >95% relative humidity during the first 48 h. Twenty days after inoculation, small brown necrotic lesions were visible on the soybean seeds. Re-isolation from symptomatic seeds on PDA dishes yielded yeast-like colonies with the same morphological characteristics as those used for inoculation, thus confirming Koch's postulates. The control seeds had no symptoms. This fungus is widely known as a pathogen of yeast spot disease on soybean seeds (Heinrichs et al. 1976; Kimura et al. 2008), but to our knowledge, it has never been reported in Serbia. Considering that invasive species Nezara viridula L. and Halyomorpha halys (STÅL, 1855), the vectors of this fungus, were reported in our country (Kereši et al. 2012; Šeat 2015) and that their mass appearance has been documented in recent years (Konjević et al. 2020), the presence of this pathogen has the potential to cause considerable damage and severe yield losses, resulting in significant economic impact on soybean production in Serbia.

Preliminary study of microplastics content in the digestive tract of sea cucumber from Demak Waters

As a marine delicacy, sea cucumbers were often eaten raw in many part of the world and they have been reported to ingest microplastic. This study aimed to determine the microplastics in the digestive system of sea cucumbers from Demak Waters. The ten samples of Acaudina sp were taken using a bottom dredge from Wulan Estury and put in the glass-bottles samples. In the laboratory, the digestive tract was taken out from the sea cucumber, divided into 7 parts, and pooled in the glass beaker. Microplastics were digested with KOH and incubated at 40 °C for 24 hours. Then, the digestion solution was filtered through a sterile cellulose nitrate filter paper by vacuum filtration. The microplastics obtained were observed for their physical characteristics (density, shape and colour) under the microscope. The density of microplastics were varied among the part of digestive tract. They consisted of fragments, fiber, and pellets. The colour of microplastics were varied but dominated by the blue. As a deposit feeder, the sea cucumber might ingest the microplastics in the sediment they fed on.

First report of black pepper (Piper nigrum L.) anthracnose caused by Colletotrichum siamense in north-east India.

Black pepper (Piper nigrum L.) has been commonly cultivated as a spice crop in northeast India. In August 2021, anthracnose leaf spot was observed on black pepper vines with 50 to 60% of disease incidence in Assam Agricultural University, Jorhat (26.7509° N, 94.2037° E), Assam, India. On average, 80% of the leaves per individual vine were affected by this disease. Foliar symptoms initially appeared as chlorotic circular spots, which then coalesced into larger irregular lesions. The centers of the spots were brown, papery in texture, and surrounded by a yellow halo. Numerous acervuli at the center of the spots were observed. Ten vines from the orchard were sampled to identify the causal agent. Symptomatic leaves along with some healthy portion were cut (3 to 4.5 mm2), surface-sterilized in 70% ethanol for 30 s, rinsed in sterile distilled water twice, dried on sterilized filter paper, aseptically plated on potato dextrose agar (PDA) amended with Streptomycin sulphate (30 mg/L), and then incubated at 25°C for four days. Two Colletotrichum isolates were recovered from infected tissues and purified by the hyphal tip method. Fungal colonies on PDA were cottony, dense, white to gray in color, and with salmon pink conidial masses. Conidia (n = 50) were 13.6 to 19.8 × 4.2 to 6.4 μm, cylindrical, hyaline, single-celled, smooth-walled, and with rounded ends. Conidiophores were aseptate, hyaline, short and branched. Based on morphological features, the isolates were identified in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For accurate identification of two isolates, the DNA was extracted from pure culture. The internal transcribed spacer (ITS) region, actin (ACT), β-tubulin 2 (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by polymerase chain reaction (Weir et al. 2012) and sequenced. The sequences were deposited in the GenBank database (ITS: OP297054 and OP296876; ACT: OP327082 and OP327081; TUB2; OP327086 and OP327085; GAPDH: OP327084 and OP327083). A BLAST analysis of ITS, ACT, TUB2 and GAPDH sequences revealed 99.5-100%, 99.9-100%, 99.9-100% and 99.8-100% similarity respectively to C. siamense for both isolates in NCBI database. The pathogenicity tests were carried out on potted four months old vine cuttings of P. nigrum L., which were kept in a greenhouse. Ten healthy plants were sprayed with 50 µl of conidial suspension of each isolate (107 conidia ml-1, 10 ml/plant). Five control plants were sprayed with sterile distilled water. The plants were covered with sterilized plastic bags after inoculation to maintain humidity and kept in a greenhouse at day/night temperatures of 25 ± 2°C and 17 ± 2°C (Zhang et al., 2021). Within eight days, all the inoculated plants showed symptoms similar to those observed in the field, whereas control plants were asymptomatic. The pathogenicity test was repeated twice. C. siamense was consistently reisolated from the lesions and was confirmed by morphological characterization and molecular assays as described above in this note, whereas no fungus was isolated from control leaves. To our knowledge this is the first report of C. siamense causing black pepper anthracnose in northeast India. The pathogen has significant potential for causing high losses in black pepper production. These data will help researchers to develop effective management strategies for this disease.

First Report of Red Leaf Midrib lesions on Sugarcane Caused by Lasiodiplodia theobromae in China.

Sugarcane (Saccharum officinarum) is an economically important crop and is extensively planted across China. In August 2020, leaf midribs with red lesions were observed on cultivar 'Yunzhe 081609' in Kaiyuan (103.27°E, 23.71°N), Yunnan, Southwestern China. In July to August 2021, similar symptoms were observed on cultivar 'Liucheng 05-136' in Hechi (108.48°E, 24.47°N), Guangxi, and on cultivars 'Yingyu 91-59' and 'Yunzhe 081609' in Lingcang (99.45°E, 23.33°N), Yunnan. Initially symptoms appeared as red spots on the leaf midribs, which gradually expanded, forming elongated red lesions. At high severity, the leaves broke and hung down. Disease incidence of leaves was estimated at 30 to 50% across the locations. To identify the etiology of this disease, three symptomatic leaves were collected from cultivars 'Liucheng 05-136', 'Yingyu 91-59', and 'Yunzhe 081609', respectively. Symptomatic leaf midribs were cut to small fragments (3 × 5 mm), surface sterilized with 70% ethanol for 30 s followed by 1% NaClO for 1 min, rinsed with sterilized distilled water three times, air dried on sterile filter paper, plated on potato dextrose agar (PDA), and incubated at 28°C in the dark. Ten isolates with similar morphological characteristics were obtained. Colonies on PDA were white to grayish-white with aerial mycelium growing initially upward and then forming clusters. After 10 days, mycelia turned to grayish black. Immature conidia were initially hyaline, aseptate, and ellipsoid. Mature conidia became dark brown, septate, longitudinal striate, and measured 21.2 to 25.8 × 11.4 to 16.4 µm (n = 30). Morphologically, the isolates were identified as Lasiodiplodia theobromae (Alves et al. 2008). For molecular identification, genomic DNA of four representative isolates (LTGX1, LTGX2, LTYN1 and LTYN2) was extracted using the Ezup Column Fungi Genomic DNA Purification kit. The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-alpha (TEF-1α) gene, and β-tubulin (TUB) gene were amplified with primer pairs ITS1/ITS4 for ITS, EF1-728F/EF1-986R for TEF-1α, and Bt2a/Bt2b for TUB, respectively (Glass and Donaldson 1995; Carbone and Kohn 1999; White et al. 1990), and then sequenced. The ITS (ON533336-ON533339), TEF-1α (ON939550-ON939553) and TUB (OP747306-OP747309) sequences were deposited in GenBank. BLAST searches showed >99% nucleotide identity to the sequences of ex-type isolate CBS 164.96 of L. theobromae (ITS, 99.8% to AY640255; TEF-1α, 99.9% to AY640258; TBU, 100% to EU673110). Phylogenetic analysis using maximum likelihood based on the combined ITS, TEF-1α, and TUB sequences of the isolates and reference sequences of Lasiodiplodia spp. downloaded from the GenBank indicated the isolates obtained in this study formed a clade strongly supported based on bootstrap values (100%) to the ex-type isolate CBS 164.96 sequences of L. theobromae. For pathogenicity tests, three healthy 6-month-old potted sugarcane leaf midribs of cultivar 'Yunzhe 081609' were wounded with a sterile needle, then inoculated using 8-mm mycelial agar plugs from a 10-day-old culture of strain LTYN1, and covered with wet cotton to maintain high relative humidity. Sterile PDA plugs were used as controls. Plants were placed in a greenhouse at 28 to 32°C. The test was conducted twice. Five days after inoculation, red lesions appeared on the inoculated leaf midribs. These symptoms were similar to those observed in the field. The leaves used for negative controls remained symptomless. The same fungus (L. theobromae) was re-isolated from all inoculated-symptomatic tissues; and isolates had the same morphological traits mentioned above. The DNA sequence data of these isolates was also similar than the original isolates. The association of L. theobromae with S. officinarum was recorded earlier in Cuba (Urtiaga, 1986), Myanmar (Thaung, 2008) and the Philippines (Reinking, 1919). Leaf midribs with red lesions caused by Colletotrichum falcatum has already been described around the world (Costa et al. 2021; Hossain et al. 2021; Xie et al. 2019). All together, this information indicates that L. theobromae is one of the causal agent of the red lesions symptoms on the sugarcane leaf midribs. To our knowledge, this is the first report of L. theobromae causing red lesions on leaf midribs of sugarcane in China. Further research will focus on developing management strategies to control this disease effectively.

First report of Taxus media branch blight caused by Neopestalotiopsis clavispora in China.

Taxus media Rehder is a high-value tree species and can be used for landscape afforestation and medicinal purposes considering its beautiful shape and secondary metabolite paclitaxel. In May 2019, branch blight of T. media was observed in plantations in Hangzhou, Zhejiang province, China. The lesions were brown, necrotic and sunken, and the branches were withering. Plants from five plantations were surveyed, and the disease incidence was 60%. Necrotic tissues from 5 plans from each plantation were cut into small pieces (5×5 mm), surface disinfected with 75% alcohol for 30 s and 2% sodium hypochlorite solution for 2-3 min, washed twice with sterile water, then dried on sterile filter paper. Branch pieces were aseptically transferred to potato dextrose agar (PDA) plates and incubated at 25℃ in the dark for 3 days. Five purified fungal cultures were obtained and the representative isolate (LA-01) that grew slightly more vigorously under the same condition was selected for morphological characterization and DNA sequence comparison. The colony was initially white and cottony, with circular growth, undulated edge, wavy surface and pale beige on the back. The conidia were long spindle-shaped, straight or slightly curved, 21-27 × 6-8 μm (n=50) in overall shape. They contained 5 cells with those at the apex and base hyaline. The second and third cells from the apex were darker (brown) than the fourth cell from the apex (pale brown). Two or three (most frequently observed) hyaline appendages were apparent at the apex, 16-31 μm in length. Morphological characteristics of the obtained isolate were consistent with those in the genus of Neopestalotiopsis (Solart et al. 2018). DNA of LA-01 was extracted from fresh mycelia using Minibest Universal Genomic DNA Extraction Kit (Takara, Toyoto, Japan) according to the manufacturer's instructions. The partial regions from the internal transcribed spacer (ITS), β-tubulin gene (TUB2), and translation elongation factor subunit 1-a gene (TEF1) were amplified and sequenced using universal primer pairs ITS5/ITS4, BT2A/BT2B, and EF1-526F/EF1-1567R (Maharachchikumbura et al. 2012), respectively. The resulting nucleotide sequences were individually subjected to BLAST searches in GenBank. The nucleotide sequences of ITS (MZ148633, 554 bp), TUB2 (MZ286974, 473 bp ), TEF1 (MZ286975, 1030 bp) of the isolate LA-01 showed 99.64% similarity to ITS sequence (MW512858.1), 99.35% similarity to TUB2 sequence (MH423961.1), 98.18% similarity to TEF1 sequence (KU096881.1). The multi-locus phylogenetic tree was constructed based on the combined ITS-TUB2-TEF1 sequences using MEGA 7 using the neighbor-joining method, indicating that LA-01 represents Neopestalotiopsis clavispora. Inoculation was done on branches of ten T. media plants in the field to verify the pathogenicity of LA-01. Two healthy branches of each plant were surface sterilized with 75% ethanol allowed to dry, and two small wounds on each branch were obtained by puncturing with a sterilized needle. One wound was inoculated with 5-mm-diameter PDA discs excised from 7-day-old cultures, and wrapped with parafilm; the other inoculated with sterile PDA plugs as control, and wrapped with parafilm. Disease symptoms developed on all N. clavispora-inoculated wounds 3 days after inoculation. The yellow brown lesions became apparent 20 days later, whereas the control wounds remained asymptomatic. The fungus N. clavispora was reisolated from the diseased lesions using the same methods above, fulfilling Koch's postulates. Previously, the species was reported to cause leaf spot disease on another Taxus tree species T. chinensis in China (Wang et al. 2019). This is the first report of T. media branch blight caused by N. clavispora worldwide. Special attention should be paid to the damage it may cause to T. media producers, and precautionary monitoring on T. media population will be a significant part of the conservation efforts in China.

Novel In Vitro Study to Assess Microbial Barrier Properties of Polyurethane-Based Tissue Adhesives in Comparison to the Gold Standard Dermabond®.

Tissue adhesives as a physical barrier to microorganism penetration provide an alternative method with many advantages for wound closure in surgical settings compared to the clinical standard. This raises the need of developing and conducting in vitro methods that are sensitive and reproducible to assess their microbial barrier properties. In this study, three different polyurethane-based tissue adhesives with different physicochemical properties were evaluated in comparison to Dermabond® as a clinical gold standard for topical wound closure. Here, physicochemical properties varied in lactide concentration, viscosity, processing, and the full polymerization time. To evaluate the microbial barrier function, a 5 μl aliquot of E. coli Lux inoculum containing at least 1 × 109 CFU/ml was applied to the surface of each test adhesive and sterile filter paper as the control that was placed on an agar plate and incubated at 37°C. Plates were observed for bacterial growth (morphology), the adhesion of the adhesive/filter paper, and bioluminescence after 24, 48, and 72 hours. The data presented in this in vitro model indicated that polyurethane-based tissue adhesives with lactide concentration ≥ 5% provided a suitable barrier against microbial penetration with 95% confidence of 99% efficacy for 72 h along with Dermabond®. Interestingly, the here described method was able to discriminate between the different physicochemical properties showing a better microbial barrier function with increasing lactide concentration of the adhesive. Overall, the results of this study showed the noninferiority between Dermabond® and the two abovementioned polyurethane-based tissue adhesives.

First Report of Crown Rot Caused by Fusarium algeriense on Wheat in Kyrgyzstan.

Fusarium crown rot of wheat is an economically important disease that leads to significant yield and quality losses, especially in many arid and semi-arid wheat-growing areas worldwide. In June 2020, winter wheat (Triticum aestivum L.) plants exhibiting crown rot symptoms were identified in a commercial field located in the Tokbay location (43.033719°N, 74.325623°E), Chuy Province, Kyrgyzstan. The diseased plants were stunted and had brown discoloration on internodes of the stem bases and roots. Disease incidence was about 3%. A total of 10 plants were sampled at the ripening stage from the field to identify the causal agent. Symptomatic tissues were excised, surface disinfected with 1% NaOCl, rinsed three times with distilled water, and placed on one-fifth strength potato dextrose agar (PDA) followed by incubation at 23°C in the dark for 5 days. A total of 8 Fusarium isolates were recovered from tissues and purified by the hyphal tips method onto fresh PDA and Spezieller-Nährstoffarmer agar (SNA) plates (Leslie and Summerell 2006). Sequence analysis of the translation elongation factor 1α (TEF1) and the RNA polymerase II beta subunit (RPB2) genes were performed with primers EF1 and EF2 (O'Donnell et al. 1998), and 5f2 (Reeb et al. 2004) and 7cr (Liu et al. 1999), respectively. The sequences of three isolates showed 100% identities with the corresponding sequences of the strain NRRL 66652 of Fusarium algeriense Laraba & O'Donnell (TEF1: MF120515 and RPB2: MF120504), and the sequences of a representative isolate (KyrFa01) were deposited in GenBank (TEF1: OM135603 and RPB2: OM135604). On PDA, fungal colonies were initially yellowish-white but gradually turned yellowish-brown. Ellipsoidal microconidia produced in false heads on monophialides were usually aseptate (8.30 ± 1.17 μm, n = 50) and occasionally one-septate (21.89 ± 2.01 μm, n = 50). Sporodochial macroconidia were mostly 3-4 septate measuring 43.41 ± 2.83 μm (n = 50), slightly curved and formed generally on monophialides on SNA. No chlamydospores formation was detected after 15 days on SNA or PDA. Morphological characteristics described above were consistent with the morphology of F. algeriense, as reported by Laraba et al. (2017). To confirm pathogenicity, seeds of wheat cultivar Seri 82, Fusarium crown rot susceptible, were treated in 1% NaOCl for 2 min, rinsed twice, and placed in plates containing a piece of sterile filter paper saturated with water to induce germination for 3 days. Five pregerminated seeds were placed on the soil surface for each 9-cm-diameter pot, which was filled with a sterile potting mix containing peat, vermiculite, and soil (1:1:1 by v/v/v). A 1-cm-diameter mycelial plug taken from the margin of actively growing colonies (PDA) of the representative isolate KyrFa01 was contacted with each seed, and then seeds were covered with the same potting mix. The seeds in control pots were treated with sterile PDA plugs. The experiment was conducted in a growth chamber in a completely randomized design with five replicated pots at 23°C with a 12-h photoperiod. Disease assessment was made after 4 weeks of fungal inoculation. The isolate KyrFa01 induced discoloration on the crown and root tissues of inoculated plants similar to those observed in the field-grown plants, whereas no symptoms were observed on plants grown in the control pots. The pathogen was successfully reisolated from the symptomatic tissues, confirming Koch's postulates. To the best of our knowledge, this is the first report of crown rot caused by F. algeriense on wheat in Kyrgyzstan. Fusarium algeriense was firstly described within the Fusarium burgessii species complex by Laraba et al. (2017) as a crown rot pathogen of wheat in Algeria. The pathogen was secondly reported from wheat-growing areas in Azerbaijan (Özer et al. 2020a) and thirdly from Kyrgyzstan in this report. Özer et al. (2020b) confirmed the coexistence of this pathogen with other Fusarium species. The result warrants the need to further investigate the potential of this species in the Fusarium crown rot complex of wheat.

First Report of Phyllosticta capitalensis Causing Black Spot on Radermachera hainanensis Merr. in China.

Radermachera hainanensis Merr. plants are native in south-central and southeast of China. Plants produce large flowers, and are widely cultivated in China as ornamentals. In April 2020, R. hainanensis Merr. plants grown in Cixi Lvpin Garden (30°26'54″N, 121°25'48″E), Zhejiang Province, were found to have many black circular necrotic lesions. In the early infection stage, the lesions appeared in lower leaves as small black circular spots which developed later into large spots (11 to 38 mm diameter) with grey centers and chlorotic edges. Ultimately, the spots spread and merged. Moreover, infected leaves showed premature leaf fall. Disease intensity reached approximately 20% of plants in the affected field (0.5 ha). After effective chemical control, this disease did not spread to other healthy plants in the same garden. To identify the causative pathogen associated with the disease, ten symptomatic leaves were collected from ten different plants. Leaf tissues were cut from the lesion margins and sterilized as follows: surface sterilized with 75% ethanol for 30 seconds and washed three times in sterile distilled water. The leaf tissues were then dipped into 10% sodium hypochlorite for 3-4 minutes, then washed three times in distilled water and dried on a sterile filter paper. After drying, the surface-sterilized leaf discs were cut to small pieces (3×3 mm) and transferred to potato dextrose agar (PDA) plates and incubated at 28°C for 2 to 3 days under 12 h photoperiod. A total of 15 isolates were obtained from the affected leaves, and all the isolates displayed the same colony characteristics. Then, three single-spore isolates were randomly selected (F2, F5 and F8) for further study. The fungal colonies were dark green with a granular surface, and irregular white edges, later turning black. Conidia were one-celled, oval, and narrow at the end with a single apical end, measuring from 7.8 to 11.1 × 4.6 to 5.9 μm (av. 9.5 × 5.2 μm, n=50). These morphological characteristics were consistent with the description of Phyllosticta capitalensis (Wikee et al. 2013; Guarnaccia et al. 2017). The identity of three representative isolates were confirmed by a multilocus approach. The DNA of three isolates were extracted and partial sequences of ribosomal internal transcribed spacer (ITS), actin (ACT), and translation elongation factor 1-alpha (TEF1-α) were amplified and sequenced as previously described (White et al. 1990; O'Donnell et al. 1998; Carbone & Kohn et al. 1999). The three selected isolates shared 100% identical sequence of ITS, ACT and TEF1-α. Then representative isolate F8 was selected for further study. BLAST analysis in GenBank showed that the obtained sequence of ITS (MZ317550) had 99% identity to P. elongata isolate eSX25240811. Other two sequences of ACT (MZ326837) and TEF1-α(MZ326839) showed 99% and 98% identity to P. capitalensis isolate YLWB01, respectively. The phylogenetic trees were constructed by Bootstrap method with 1000 replications using Maximum Likelihood model implemented in the MEGA 7. Results showed that the isolate F8 clustered with P. capitalensis with 100% bootstrap support. Pathogenicity of strain F8 was tested by Koch's postulates. A pathogenicity test was performed in a greenhouse with 80% relative humidity at 28°C. 20 healthy plants were sprayed with a 1×106 conidia ml-1 suspension (three leaves from each individual plants) and another 20 healthy plants were sprayed with sterile distilled water (three leaves from each individual plant) as control. Conidia was obtained from PDA plates after 7 days of incubation in the biochemical incubator at 28°C and concentration was counted in hemacytometer. After 15 days, disease symptoms were observed on all inoculated leaves, whereas the control plants remained asymptomatic. After that, P. capitalensis was re-isolated only from the infected leaves and identified by morphological and sequence analyses. Early identification of P. capitalensis as a causal agent for black spot is crucial to employ effective disease management strategies to control disease in the field. P. capitalensis has been reported on many crops in China (Cheng et al. 2019; Tang et al. 2020; Liao et al. 2020). However, to our knowledge, this is the first report of black spot disease caused by P. capitalensis on Radermachera hainanensis Merr. in China.

Efficacy of Entomopathogenic Fungal Formulations against Elasmolomus pallens (Dallas) (Hemiptera: Rhyparochromidae) and Their Extracellular Enzymatic Activities.

Elasmolomus pallens are post-harvest insect pests of peanuts that are becoming resistant to chemical insecticides. In this, we study evaluated the effect of conidial formulations on entomopathogenic fungi against E. pallens to reduce the adverse effects. Fungal conidia were formulated and applied on sterile filter papers at varying concentrations (1 × 104–1 × 108 conidia mL−1) inside plastic containers. The test insects were exposed and maintained in a relative humidity of 80 ± 10% for 10 d at room temperature (25 ± 2 °C). Mortality was recorded every 24 h. Dose–response bioassay (LC50 and LC90) values for Aspergillus flavus formulated in oil were 1.95 × 106 and 3.66 × 109 conidia/mL, whereas formulations in Tween 80 had 9.36 × 107 and 6.50 × 109 conidia/mL. However, oil-formulated Metarhizium anisopliae had 3.92 × 106 and 2.57 × 108 conidia/mL, with 6.85 × 106 and 5.37 × 108, for formulations in Tween 80. A. flavus had LT50 values of 3.3 and 6.6 days, whereas M. anisopliae had LT50 values of 3.6 and 5.7 d. Maximum protease, chitinase, and lipase activities of 2.51, 0.98, and 3.22 U/mL, respectively, were recorded for A. flavus, whereas values of 2.43, 0.93, and 3.46 were recorded for M. anisopliae. The investigated pathogens demonstrate potential against E. pallens; therefore, their applicability under field conditions requires further investigation.

First Report of Leaf Spot of Rice Caused by Epicoccum sorghinum in the United States.

Brown spot (Cochliobolus miyabeanus), blast (Magnaporthe oryzae) and stackburn (Alternaria padwickii) are common diseases in rice with similar leaf spot symptoms. In August 2021, a leaf spot disease, with symptoms dissimilar to these diseases, occurred on almost 100% of the leaves and sheaths of rice plants (cv. Presidio) in a 1-hectare field in Eagle Lake, Texas. Lesions started as small dark brown spots on lower leaves and sheaths. The spots enlarged to become round or oval (1.5 to 5.0 mm) spots having round ends with gray centers, dark-brown borders or rings, and slight gold halos. The spots on the sheaths were similar to those on the leaf blades, with lesion size ranging from 2 to 5 mm. Pieces of infected tissue were cut from the margin of necrotic lesions, surface disinfected with 1% NaOCl for 2 min followed by 75% ethanol for 30 s and rinsed with sterile distilled water three times. The tissues were then dried on sterilized filter paper, placed on potato dextrose agar (PDA), and incubated at 25℃ for 7 days. Two isolates (LS36 and LS37) were obtained, and their colonies were initially villose, gray at the center and pale at the margin, and then turned dark gray, with the reverse side becoming scarlet. Chlamydospores were unicellular or multicellular and massively produced in nearly spherical shape (11 to 26 × 10 to 22 μm, n=100). Pycnidia were dark and mostly spheroid (105 to 171 × 76 to 128 µm, n=100). Conidia were unicellular, hyaline, ellipsoidal, with the size of 3.6 to 5.8× 1.9 to 2.8 µm (n=100). These morphological characteristics were similar to those described for Epicoccum sorghinum (Zhou et al. 2018). The rDNA internal transcribed spacer (ITS), rRNA large subunit (LSU), and translation elongation factor 1 alpha (EF1) gene of an representative isolate (LS37) were amplified (Fell et al. 2000; Wang et al. 2014) and sequenced. The ITS sequence (OK189534) of the isolate was 96.95% identical to E. sorghinum (KX758542); the EF1 sequence (OK236518) was 98.37% identical to E. sorghinum (MN461167); and the LSU sequence (OK189535) was 99.29% identical to E. sorghinum (MK817520, MK817521, and MK817522). Rice plants (cv. Presidio) at heading were inoculated with the two isolates individually by placing a drop of conidial suspension of 1 x 106 conidia/ml or a 2-mm PDA plug of 7-day-old cultures on the wounded or unwounded leaves and sheaths (3 sites/leaf or sheath, 3 plants/treatment). The wound was made by penetrating the epidermis using a 0.5-mm-diameter pin. The plants inoculated with sterilized water or PDA-only plugs served as the controls. The treated plants were placed in a dew chamber at 26℃ for 2 days and then transferred in a greenhouse (25 to 30℃). After 5 days, typical symptoms, similar to those observed in the field, developed on all of the inoculated leaves and sheaths, with the wound inoculation inducing more rapid development of symptoms than the unwounded inoculation. No symptoms developed on the controls. The two isolates produced similar symptoms and the fungus was consistently re-isolated from the infected plants and confirmed to be E. sorghinum based on morphological characteristics. The pathogenicity test was repeated twice with similar results. To our knowledge, this is the first report of leaf spot caused by E. sorghinum in rice in the United States. This disease was first reported on rice in China in 2020 (Liu et al. 2020). This research will help identify this new disease from other leaf spot-like diseases and develop management strategies for control of this disease.

First report of anthracnose on Ophiopogon jaburan caused by Colletotrichum liriopes in China.

Ophiopogon jaburan (Liliaceae), named white lilyturf, is widely cultivated as an ornamental plant in south China. During 2017-2019, leaf spots on O. jaburan were observed all year in Zhanjiang, Guangdong, China (N21°9'3"; E110°17'47"). Almost all plants were infected and the disease incidence on affected leaves was about 80% in the field. Initially, spots were brown, round or oval, and gradually enlarged to irregular shapes. The color of the spots changed from rusty-brown to grayish-white with rusty-brown borders. Subsequently, the spots expanded until the leaves withered and died. Infected tissues were surface-sterilized with 75% ethanol for 30s followed by 1% NaClO solution for 1 min, then rinsed thrice with sterile water, before placed on potato dextrose agar (PDA) containing 50mg/L ampicillin, and incubated in darkness at 25℃ with 90% relative humidity. Colonies growing on PDA were cushion-like, pale greenish grey to grayish black on the front side and clearly dark gray on the reverse. Colony diameter was av. 86.0 mm (n = 15) grown in the dark at 25 ℃ for 10 days. Conidia with oil droplets were colorless, hyaline, smooth-walled, aseptate, slightly curved, and tapered gradually to each end, 12.3-28.9 × 2.2-6.6 μm (av. 20.9×4.2μm, n=200). Setae were brown to dark brown, 2-4 septate, with the base slightly inflated, and measured 40.0-130.3 × 2.2-5μm (av. 84.3 × 3.3μm, n=23). On PDA, scattered or loosely clustered appressoria were elliptical or irregular, smooth-walled, aseptate, and dark brown. To confirm the identification, partial regions of the internal transcribed spacer (White et al. 1990), beta-tubulin (Aveskamp et al. 2009) and actin (Carbone et al 1999) were amplified and sequenced (MW989743, MZ014461 and MZ014462). The blast results showed these sequences had >99.59% homology with sequences of Colletotrichum liriopes holotype strain CBS 119444 (NR_111449, GU228098 and GU227902). Maximum likelihood analysis and Bayesian inference were performed from concatenated sequences using RAxML v.1.0.0 and MrBayes v.3.2.1 software respectively. Several C. liriopes strains clustered in the same clade. Based on morphological-molecular characteristics, the fungus was identified as C. liriopes (Damm et al 2009; Chen et al. 2019). To confirm pathogenicity, healthy leaves were surface disinfected with 75% ethanol and rinsed thrice with sterile water. On ten leaves, three sites were wounded by pricking with needles, and inoculated 20 μL of 106 conidia/ml suspension or mycelium in contact with blade surface using 6-mm mycelial plugs. Similarly, the inoculation was done for three unwounded sites each leaf. Sterile water and medium plugs (without fungus) served as controls. All leaves were incubated on sterile wet filter paper at 25-28℃ with 90% relative humidity. After 7 days, all the inoculated leaves showed symptoms similar to those of field diseases, whereas control leaves remained healthy. The fungus with morphological-molecular features identical to the original isolate was reisolated from the disease lesions. C. liriopes causes anthracnose on Bletilla ochracea, Eria coronaria, Hemerocallis fulva, Pleione bulbocodioides (Jayawardena et al 2016) and Liriope sp. (Yang et al 2020; Chen et al 2019) in China. This is the first report of C. liriopes causing anthracnose on O. jaburan in China. Anthracnose could greatly affect ornamental value of O. jaburan, and this work can alert gardeners to prevent and control of the disease.

First report of dieback on fig (Ficus carica L.) caused by Lasiodiplodia theobromae in North Al-Batinah governorate of Oman.

Fig (Ficus carica L.) is widely grown in Oman mainly for its nutritional value and health benefits. In May 2020, survey was conducted for fig trees which were showing symptoms of decline in three farms where more than 1500 fig trees were grown. The incidence of the disease was 3 to 4 % of fig trees showing symptoms of dieback of the twigs, death of entire branch and discoloration of vascular tissues were observed in Sohar, North Al-Batinah governorate of Oman. The disease severity was range between 25 to 40 % in most of the infected tree. The death of severely affected trees was observed in about 3 % of the orchards. Symptomatic samples were collected and isolation of the causal agent was performed. Infected tissues were cut into small pieces and surface-sterilized with 1 % NaOCl for 3 min. The tissues were rinsed with sterile distilled water 3-4 times, blotted dry on sterile filter paper and placed on potato dextrose agar (PDA) medium under aseptic conditions. After 2-3 days of incubation of plates at 27°C, pure culture of the fungus was obtained by hyphal tip isolation technique. Two identical isolates were grown in the PDA medium. The fungal colony was dark grey to black colored on PDA medium and the fungus produced dense aerial mycelium and numerous dark colored pycnidia in 25 days at ambient temperature range between 23-25 ℃ and nature photoperiod. Pycnidia were raptured and 20 emerged conidia were measured. The mature conidia were 1-septate, brown, ellipsoid, with thick cell wall and having longitudinal striation. The average measurement of the length of 20 conidia was 22.5-25 (24.9 µm) however the width measured was 12.5-15 (15 µm). To identify the fungus, DNA was extracted from the mycelium using phenol-chloroform-isoamyl alcohol method, following Al-Mahmooli et al. (2015). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and translation elongation factor 1-α (TEF) gene were amplified using ITS-4 and ITS-5 primers for ITS (White et al. 1990), EF1-983F and EF1-2218R and EF1-728F and EF1-986R primers for TEF (Alves et al. 2008), respectively and the amplified products were sequenced. BLASTn analysis of the ITS sequence of the fungal isolate revealed 100% identity with reference sequences of Lasiodiplodia theobromae (Pat.) Griffon & Maubl (MT075438.1). The sequence of TEF gene was 99% identical to the sequences of L. theobromae (XM_035519539.1 and MN461169.1). The nucleotide sequences were submitted to GenBank and accessioned with (MW590660) for ITS and (MZ159970, MZ159971, OM654917) for TEF gene. For phylogenetic analysis, we constructed a combined ITS-TEF dataset, following (Alves et. 2008). Using Maximum Likelihood phylogenetic analysis of the combined ITS-TEF dataset, our specimen falls in a clade formed by the members of L. theobromae (Supplementary materials). Pathogenicity of the fungus of three replicates (three fig seedlings) was established by artificial inoculation of 2- to 3-month-old potted fig seedlings with the fungus by making pin prick injuries on the stem at 5 cm above the soil level with a sterile inoculation needle, placing mycelial discs containing spores and pycnidia (15 × 15 mm) from 20-days old colonies grown on PDA on the injured surface and wrapping with parafilm which removed 48 hrs after establishing of infection. The three inoculated plants showed withering and drying of leaves 14 days after inoculation (DAI) begins near to the inoculation site expend to the younger leaves. Eventually, seedlings were showing dieback and death of entire seedling occurs in 25-28 DAI. The mock-inoculated plants remained symptomless. The fungus re-isolated from the diseased plants was confirmed by sequencing of ITS regions. The prevalence of dieback could be a potential threat to cultivation of fig in Oman. To the best of our knowledge, this is the first report of dieback disease caused by L. theobromae on fig in Oman.

Fusarium proliferatum Associated with Basal Rot Disease of Bambusa pervariabilis × Dendrocalamopsis grandis in China.

Fusarium proliferatum Associated with Basal Rot Disease of Bambusa pervariabilis × Dendrocalamopsis grandis in China.

First Report of Anthracnose Disease Caused by Colletotrichum fioriniae on Barbary wolfberry (Lycium barbarum L.) in Gansu Province, China.

Barbary wolfberry (Lycium barbarum L.) is a well-known edible and medicinal plant, widely grown in northwest China (Gao et al. 2021). During the summer of 2019, typical anthracnose symptoms were observed on fruits of barbary wolfberry in Baiyin, Gansu province, China. Approximately 30% of the barbary wolfberry fruits had typical anthracnose symptoms. Lesions on barbary wolfberry fruits were dark, circular or irregular, sunken, and necrotic or wilted, with the presence of orange to pink conidial masses under high humidity. Small pieces cut from the margins of lesions were surface disinfested with 75% ethanol for 10 s, and 1% NaClO solution for 1 min, rinsed three times in sterile distilled water, dried on sterile filter paper, placed onto potato dextrose agar (PDA), and incubated at 25 ± 1℃ for 5 days in the dark. The pure cultures were obtained by single-spore isolation. All isolates produced pale gray and dense aerial mycelia, in reverse orange to red, at times showing concentric rings on PDA at 25℃ after 10 days in the dark. Conidia (n=100) were colorless, smooth-walled, aseptate, fusiform elliptical with one or both ends, and 8.3 to 17.6 × 3.7 to 6.2 μm. Appressoria (n = 100) were solitary, pale to medium brown, smooth-walled, subglobose to elliptical, sometimes clavate or irregular, and 5.7 to 11.7 × 4.1 to 8.5 μm. The internal transcribed spacer (ITS) region of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, bate-tubulin 2 (TUB2) gene, actin (ACT) gene, calmodulin (CAL) gene, chitin synthase 1 (CHS-1) gene, and histone H3 (HIS3) gene of the two representative isolates BY19LB02 and BY19LB06 were amplified and sequenced with primers ITS1/ITS4, GDF1/GDR1, T1/Bt2b, ACT-512F/ACT-783R, CL1/CL2A, CHS-79F/CHS-354R, CYLH3F/CYLH3R, respectively (Damm et al. 2012), and deposited on GenBank (ITS, MZ496816 and MZ505524; ACT, MZ557422 and MZ557417; CHS-1, MZ557423 and MZ557418; GAPDH, MZ557424 and MZ557419; HIS3, MZ557425 and MZ557420; TUB2, MZ557426 and MZ557421). BLAST analysis of the resulting for all the sequences showed 98 to 100% similarity with those of C. fioriniae. Based on the above, the isolates BY19LB02 and BY19LB06 were identified as C. fioriniae. To confirm the pathogenicity, detached heathy barbary wolfberry fruits were surface-sterilized with 75% ethanol for 30s, rinsed three times in sterile distilled water, allowed to dry on sterile filter paper, and then wounded using sterilized needles. Fruits were inoculated by pipetting 10 μL of conidial suspension (1×106 conidia/mL) onto each wound, and controls were inoculated with 10 μL sterile distilled water. Each treatment had 30 fruit replicates. These fruits were kept in a moist chamber at 28°C in the dark. The experiment was repeated three times. After 5 days, anthracnose symptoms were observed on all of the inoculated fruits and identical to those observed in the field, whereas control fruits did not develop symptoms. Theathogen was re-isolated from the lesions of inoculated fruits, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of C. fioriniae causing anthracnose on barbary wolfberry in Gansu Province, China. The same disease on barbary wolfberry was reported in Jilin Province, China (Liu et al. 2016). Gansu is one of the main barbary wolfberry producing areas in northwest China and its geographical area, climate and environmental conditions are different from Jilin Province. Considering that barbary wolfberry is the main source of income for growers in Gansu, this identification can aid in the selection of appropriate management measures for this disease.

First Report of Leaf Spot Disease caused by Nigrospora oryzae on Nicotiana tabacum in China.

Tobacco (Nicotiana tabacum) is an important economic crop and widely cultivated in rural areas in south of China. A previously uncharacterized disease was observed on field-grown tobacco during 2020 and 2021 around Tongren city, Guizhou province of China (27°59'25.73" N, 108°7'2.43" E). The disease mainly occurred from fast growing period (about 13-16 leaves) to leaf maturity stage. In severely diseased areas, the incidence rate was between 20%-100%. Symptoms first began as yellow-brown necrotic spots on leaves, then merged into larger irregular necrotic spots surrounded by chlorotic halos. Similar lesions were also found on the stems. Ten symptomatic leaf and stem samples were collected from the different infected plants for pathogen isolation. The small pieces of discolored tissues were surface-disinfected with 2% sodium hypochlorite for 3 min and 75% ethanol for 30 s, rinsed three times with sterile water, and blotted on sterile filter paper, placed on potato dextrose agar thenincubated at 28°C in the dark for 3-4 days. The obtained isolates were purified through single-spore culture. Colonies were initially white and fluffy in appearance, later turning gray. Hyphae were smooth, branched, septa, transparent or light brown. Spores were solitary, oblate or nearly spherical, dark brown to black, smooth, 14.3 to 16.1μm × 11.8 to 15.2 μm in diameter. DNA of fungal isolates were extracted using Fungi Genomic DNA Extraction Kit (Solarbio, Beijing, China), the internal transcribed spacer (ITS) of the ribosomal DNA, β-tubulin (TUB2) gene and translation elongation factor 1-alpha (TEF1-α) were amplified with primers ITS1/ITS4, βt2a/βt2b and EF1-1728F/EF1-986R, respectively. The resulting ITS, TUB2 and TEF1-α sequences were deposited at GenBank, NCBI under accessions MZ882151, MZ927749, MZ927747, respectively. The sequence identity of ITS, TUB2 and TEF1-α with those of Nigrospora oryzae strains HBN (KU254608), HGUP191068 (MZ724102) and LC7307 (KY019409) were 99.64%, 99.29% and 99.65%, respectively. Based on morphological features and phylogenetic analysis, the pathogen was identified as N. oryzae (Wang et al. 2017). Pathogenicity tests were conducted by placing agar plugs-containing fungal mycelia and agar blocks (control) on leaves of tobacco plants grown at 28°C with 60% humidity in greenhouse. Symptoms appeared on the pathogen inoculated leaves seven days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as N. oryzae based on morphological, molecular and phylogenetic analysis, which were fulfilling Koch's postulates. This pathogen was recently identified from watermelon and kiwifruit in the Guizhou (Far and Rossman, 2021). To our knowledge, this is the first report of leaf spot caused by N. oryzae on Nicotiana tabacum in China.

First Report of Take-All Root Rot Caused by Gaeumannomyces graminis on Hybrid Bermudagrass in China.

Take-all root rot caused by Gaeumannomyces graminis (Sacc.) Arx & Olivier is a destructive disease of hybrid bermudagrass (Cynodon dactylon × C. transvaalensis Burtt-Davy) (Elliott 1991; Stephens et al. 2020). In April 2018, multiple circular to irregular patches (10 to 200 cm in diameter) were evident on 'Tifway419' bermudagrass fairway of Wohu golf course (Huangshan city, Anhui province). Patches were found at five of the 18 fairways and showed a decrease in stand density. Individual plants in patches exhibited off-color to white foliage, dark and necrotic lesions were frequently observed on roots and associated stolons. Ectotrophic runner hyphae and lobed hyphopodia are ubiquitous on the surface of necrotic roots. Two diseased samples were collected from each patch by a soil corer (10 cm in diameter), and a total of ten patches were sampled across the five symptomatic fairways. Symptomatic roots or stolons were cut into 2 to 3 cm length and surface disinfested for 3 min in 0.6% sodium hypochlorite solution. The sterilized tissues were rinsed, air dried on sterile filter paper for 5 min and placed on potato dextrose agar (PDA) amended with 50 mg L-1 each of ampicillin, streptomycin sulfate and tetracycline. Plates were incubated in the dark at 20°C for 5 days, and similar colonies were consistently recovered from 85% of the symptomatic tissues. After hyphae purification, a total of 20 fungal isolates were obtained with morphology similar to G. graminis (Hernández-Restrepo et al. 2016). DNA was extracted and amplified with primers ITS5/ITS4 (White et al. 1990). PCR products were sequenced (deposited as MZ889119 and MZ889120 in GenBank), and showed 99.43 to 99.81% homology when compared with a known G. graminis isolate (accession no. KX306505). To complete Koch's postulates, healthy Tifway419 hybrid bermudagrass was grown in 5 × 20 cm Cone-Tainers containing a sand and Pindstrup substrate (Pindstrup Mosebrug, Pindstrup, Denmark) mixture (1:1, V/V) nine weeks after transplanting from the experimental field of the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences. Five sterile oat seeds infested with G. graminis were inserted 5 cm below the soil surface in the root zone of Tifway419. Cone-Tainers inoculated with five noninfested sterile oat seeds served as controls, and each treatment was replicate 3 times. All Cone-Tainers were transferred into a growth chamber with a 12-h day/night cycle at 25/20°C and 95% relative humidity. They were covered with plastic bags for the first week, and watered once daily to encourage disease development. Hybrid bermudagrass inoculated with G. graminis decreased in plant density and turfgrass quality, black and necrotic roots covered with lobed hyphopodia were observed eight weeks after inoculation, no symptoms were present on the roots and above-ground of noninfested control plants. G. graminis was consistently reisolated from symptomatic roots and stolons, and confirmed by sequencing the ITS region as mentioned above. To our knowledge, this is the first report of G. graminis causing take-all root rot of hybrid bermudagrass in China, which may influence the management of this important root disease.

First Report of Nut Rot Caused by Neofusicoccum parvum on Hazelnut (Corylus avellana) in Italy.

Hazelnut (Corylus avellana L.), which is native to Europe and Western Asia, is a widely distributed and economically important crop in Italy, cultivated on 82,104 ha and its production is 110,618 t (ISTAT 2021). A total of one hundred and eleven black rotted nuts (incidence: 41%) with sunken lesions from Lu and Cuccaro (45°00'21.8"N/8°28'59.6"E), north-western Italy, were collected during the ripening stage of hazelnuts during October-December 2020. Symptomatic half cut kernels were sterilized in 1% NaClO for 1 min, washed in sterile water twice, and dried on sterile filter paper. The fragments were placed onto potato dextrose agar (PDA) containing streptomycin. After 48 to 72 h of incubation at 25°C, fast-growing white colonies with abundant aerial mycelia were observed. On the opposite side of the plates, colonies were initially white, then turned to dark-grayish olive after one week of incubation. Dark colonies produced globose, hyaline, ellipsoidal, unicellular conidia ranging from 12.23-15 μm x 5-6.71 μm. Morphologically, the causal agent was identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (Crous et al. 2006; Zhang et al. 2021). The DNA from the isolates HMa-19-2 and Hwb-4b-2 was extracted with the E.Z.N.A. Fungal DNA mini kit (Omega Bio-tek) according to manufacturer instructions. Molecular identification was confirmed by sequencing of rDNA internal transcribed spacer (ITS) using primers ITS1/ITS4 (White et al. 1990) and translation elongation factor 1-alpha (tef-1α) gene by using the primers EF1-728F/EF1-986R (Carbone & Kohn, 1999). The sequences of both isolates were deposited in GenBank for ITS (accession numbers MZ848132 and MZ848133) and for tef-1α gene (accession numbers MZ913266 and MZ913267). The BLAST analysis showed 99% identity with ex-type strain of N. parvum (CMW9081) for ITS and tef-1α. Maximum likelihood method based on combined sequences of ITS and tef-1α genes was performed and the isolates of N. parvum clustered with ex-type strain of N. parvum (CMW9081; eXtra file). Pathogenicity of both isolates were tested on ripening hazelnuts (BBCH: 85) to evaluate Koch's postulates. Three nuts per isolate, and per three replicates, were surface disinfected with 1% NaClO. A piece of shell (5 mm diameter) from nuts was removed with a sterile cork borer, then nuts were inoculated with PDA mycelium plugs of the same diameter cut from 7 days old PDA colony (Seddaiu et al. 2021). The control nuts were treated with sterilized PDA plugs. All inoculated nuts produced black lesions with softening pulp (eXtra file). Additionally, abundant white-gray mycelium developed on the inoculation sites. Control nuts showed no symptoms. Neofusicoccum parvuwas recently reported in Italy on chestnut (Seddaiu et al. 2021) and blueberry (Guarnaccia et al. 2021). The fungal pathogen was also reported as agent of grey necrosis of hazelnut in Chile (Duran et al. 2020). However, this is the first report of N. parvum on hazelnut nut in Italy. The findings suggest that N. parvum could severely affect hazelnut production in Italy. Accurate identification of the pathogen will support the growers to manage the disease.

First Report of Lasiodiplodia pseudotheobromae Causing Collar Rot of Peanut in Shandong Province, China.

In August 2019, a collar rot of peanut was observed in several fields in Qingdao, Shandong province, China. Disease survey was conducted in several peanut fields. Less than 5% plants exhibited various symptoms, including brown or black stem rot, pod rot, leaf chlorotic, wilted, and even dead. Symptomatic stems were cut into small pieces, surface disinfested with 70% ethanol for 1 min, 1% NaClO for 2 minutes, rinsed three times with sterile water, and dried on sterile filter papers. Pieces then were plated on potato dextrose agar (PDA) media and incubated at 25°C in darkness. Fungal cultures were initially white, then turned gray, and eventually turned black, and aerial hyphae were dense, fluffy. Conidia were ellipsoidal, initially hyaline, unicellular, 14.3 to 21.1 × 8.7 to 13.2 µm (n = 50), and mature conidia showed dark brown, with a central septum, and longitudinal stripes. Molecular identification was performed by sequencing ITS with ITS1/ITS4 (White et al., 1990) and beta tubulin gene with Bt2a/Bt2b (Glass and Donaldson, 1995) of a representative isolate ZHX9. ITS and beta tubulin regions (OK427342 and OK489788) of ZHX9 obtained 99.62 and 100% similar to L. pseudotheobromae (KF766193 and EU673111), respectively. Phylogenetic analysis was done using Neighbor-Joining (NJ) analysis based on those gene sequences. The microorganism we have isolated was identified as L. pseudotheobromae based on molecular analysis and morphological characteristics. For pathogenicity assay, twelve ten-days-old peanut (Zhonghua No.12) seedlings were each inoculated with one mycelial plug (8 mm in diameter) by placing the inoculum on the base of the stem. Twelve plants were each inoculated with a plug of non-colonized PDA as controls. Plants were incubated in a growth chamber (30°C in the day and 25°C at night, a 12-h photoperiod and 80% RH). Necrotic lesions were observed on stems of all inoculated seedlings 5 days after inoculation, whereas control plants remained asymptomatic, and L. peudotheobromae was consistently re-isolated from symptomatic stem. In Asia, peanut collar rot caused by L. teudotheobromae has been reported in India, Indonesia, North Vietnam (Nguyen, et al., 2006) and China (Guo, et al., 2014), but collar rot caused by L. pseudotheobromae has not been reported. To our knowledge, this is the first report of L. peudotheobromae causing collar rot on peanut in China. These results will provide crucial information for studying on epidemiology and management of this disease.

BIOINSECTICIDE ACTIVITY OF SILVER NANOPARTICLES PRODUCT OF BIOSYNTHESIS USING ENTOMOPATHOGEN AGAINST THE BEET CATERPILLAR SPODOPTERA EXIGUA

Spodoptera exigua (Hubner) is a polyphagous insect pest that is detrimental to farmers. Nanotechnology exists as a renewable technology that can increase effectiveness due to the small size of particles it produces. This study aimed to determine the effectiveness of biosynthesis of silver nanoparticles from the fungi Metarhizium, Trichoderma, and The Bacillus bacteria which are biocontrol agents. The research started in February and ended in May 2020, at the Biological Research Laboratory, Faculty of Mathematics and Natural Sciences Education, University of Pendidikan Indonesia. The experiment was based on a completely randomized design method (CRD). Onion armyworm (Spodoptera exigua), two isolates from the fungi Metarhizium and Trichoderma were obtained from the Indonesia Vegetable Research Institute (IVERRI). Each extract of Metarhizium and Trichoderma fungi and 50 mL of extract from Bacillus bacteria were added to 50 mL of Ag + 60 ppm solution in a 250 mL glass tube. The mixture of Metarhizium mushroom extract with 60 ppm Ag + solution or Trichoderma mushroom with Ag + 60 ppm solution was incubated at 400 C with a rotation speed of 200 rpm for five days under a dark condition. For the mixture of bacterial extracts with Ag + 60 ppm solution, incubation was at room temperature with a rotation speed of 150 rpm for three days. Analysis of the effect of NPP on the mortality of S. exiguawas done using the treatments, Metarhizum, Trichoderma, Bacillus, NPP-Metarhizium, NPP-Trichoderma, NPP-Bacillus, NPP, and distilled water as controls. Third instar larvae of S. exigua were immersed for 30 seconds. After immersion, the larvae are placed in sterile Petri dishes that have been lined with sterile filter paper and provided with caterpillar feed. Observation of caterpillar mortality was recorded at 2, 4, 6, 9, 24, 48, 64, 72, 96 and 108 hours after treatment. There were three replicates the results showed that all the treatments caused death of the insect, both entomopathogens and entomopathogenic nanoparticles. We found the three treatments of silver nanoparticles highly lethal to S. exigua. Two treatments, NPP-Metarhizium and NPP-Bacillus even caused 100% mortality after 120 hrs treatment. The possible reason for the pathogenicity of these products is considered to be biosynthesis of nanoparticles and enzymatic substances