GenBank Accession Research Articles

First report of Fusarium falciforme causing root rot of pomegranate (Punica granatum L.) in China.

Pomegranate (Punica granatum L.) is a deciduous shrub or small tree that is native to Iran and Afghanistan. It is also a commercially important fruit tree in China and worldwide. In the summer of 2022, a serious root rot disease occurred in some pomegranate orchards in Xichuan County(32º42´ N, 111º48´ E), Henan Province, China, with an incidence of ~30%. Symptoms included leaf yellowing and wilting, root browning and rotting, and stem-base cracking, eventually leading to defoliation and death. To isolate the causal agent, small pieces (5×5 mm) of diseased root from six trees were surface-sterilized by dipping in 2% NaClO for 8 min followed by 70% ethanol for 15 s, rinsed five times with sterile water, and plated on potato dextrose agar (PDA), then incubated at 28°C in the dark for 5 days. Fifteen pure fungal isolates with the same morphological characteristics were obtained from 24 pieces of roots. All isolates produced white fluffy mycelia. Microconidia were hyaline, oval or reniform, with zero to one septa and dimensions of 7.1 to 19.9 (average 14.5 )× 3.8 to 8.0 (average 5.6) μm (n = 100). Macroconidia were sickle-shaped, one to four septate, and 20.1 to 40.8 (average 26.5) × 4.8 to 8.6 (average 6.5) μm (n = 100). Chlamydospores were spherical, single, in pairs or chains, and 5.6 to 9.8 (average 6.8) µm in diameter (n = 100). Based on the above characteristics, the pathogens were identified as Fusarium sp. (Leslie and Summerell 2006). Genomic DNA was extracted from mycelia of two representative isolates Fs1 and Fs3. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and RNA polymerase II second largest subunit (RPB2) sequences were PCR amplified using primer pairs of ITS1/ITS4, EF1/EF2, and RPB2-5f2/RPB2-7cr, RPB2-7cf/RPB2-11ar (O'Donnell et al., 2022), respectively. BLAST analysis showed that the ITS, TEF-1α and RPB2 sequences of isolates Fs1(GenBank accession nos. OK001765, OQ921726 and OQ928396) and Fs3 (GenBank accession nos. OK001771, OQ921727 and OQ928397) showed 99%-100% identity with multiple GenBank sequences of Fusarium falciforme (KY617066, MN064683, KF255514, OQ933361, KY556711 and ON331935). A phylogenetic tree based on concatenated sequences of ITS, TEF-1α and RPB2 using maximum-likelihood analysis revealed that both isolates Fs1 and Fs3 were in the same clade with F. falciforme strains. Based on the morphological and molecular characteristics, the isolates were identified as members of F. falciforme. For pathogenicity testing, conidial suspensions (1×108 spores /mL) of isolates Fs1 and Fs3 were poured onto the roots of healthy pomegranate that had been planted in pots two months previously. Ten plants were inoculated for each isolate. Control plants were drenched with sterile water. After 3 months, inoculated plants developed leaf yellowing and wilting accompanied by root browning and rotting, much like symptoms observed in field plants. The same fungi re-isolated from the experimental plants were confirmed to be F. falciforme by morphology and sequence analysis. This is the first report of F. falciforme causing root rot on pomegranate. F. falciforme is a ubiquitous soil-borne pathogen that causes root rot on multiple plants around the world (Xu F., et al. 2022; Qiu R., et al. 2023). The results of pathogen identification are essential precursors to development of effective control of the disease.

Uromyces savulescui on statice (Limonium sinuatum) in the Americas and reevaluation of its reported geographic range.

Limonium sinuatum (Plumbaginaceae) is the most commonly cultivated recognizable cut flower crop in the genus Limonium. It is known by several common names including statice and sea lavender, due to its lilac-colored flowers and the fact that it naturally inhabits mainly coastal areas (Mellesse et al, 2013). Limonium sinuatum is native to the Mediterranean, although as a popular garden plant, has been naturalized in other parts of the world including coastal areas of California (USDA NRCS 2020). Cultivated L. sinuatum is used in fresh and dry flower arrangements in the Americas, comprising approximately 20% of the floriculture cultivated area in Ecuador (Vega and Morales 2011; Abascal Cañas 2017). In December 2014, L. sinuatum plants in the public park "Baños del Inca" in Cajamarca, Peru (S 7 9'46"; W 78 27'53"), were found infected with a rust disease. The plants were scattered in the park but infection incidence was 100% as individual plants were all found to be infected (Fig 1). Based on the percentage of symptomatic areas, including the yellow halos around pustules, calculated with ImageJ (Collins, 2007) from field photographs, the disease severity was estimated to be 58.9% in average, ranging from 19.8% up to 90.0%. Uredinia were present on both sides of the leaves as well as on stems and were roundish, oblong, pulverulent, and cinnamon brown in color; urediniospores 25.5 to 35.0 × 22.5 to 31.0 µm, were globoid to ellipsoid; urediniospore walls were cinnamon-brown, 2.5 to 3.0 µm thick, densely verrucose, with 2 to 3 equatorial germ pores. Few telia were present on leaves; these were scattered roundish or oblong, and greyish in color; teliospores 26.5 to 41.0 × 16.0 to 25.0 µm, were ellipsoid to obovoid, mostly attenuated at the apex; teliospore walls were colorless, 2-3 µm thick at sides, and up to 10 µm thick at apex. Teliospores readily germinated in sori producing basidia and basidiospores (Fig. 2). The rust features and dimensions of rust spores are consistent with available descriptions of Uromyces savulescui Rayss (Guyot 1951; Vakalounakis and Malathrakis 1987). To confirm identity, a 576 bp region of the 28S subunit of the ribosomal DNA repeat was sequenced following previously published protocols and primers (Aime 2006, Aime et al. 2018). The resulting sequence (GenBank Accession No. OR291160) shared 99.83% (573/574 bp) identity with a sequence deposited as Uromyces limonii (DC.) Lev. (accession KY764194, BPI910295, Demers et al. unpublished) from L. sinuatum in Ethiopia. However, U. limonii produces orange uredinia, thin-walled yellow-orange urediniospores, teliospores with mostly light chestnut brown wall and infects different hosts (Savile and Conners 1951). It is likely that KY764194 represents a misannotated record of U. savulescui. While Koch's postulates can be a useful tool for establishing causality in certain infectious diseases, their use may be limited when it comes to rust diseases based on old herbarium specimens. In our case, due to the age of the specimen, which is almost nine years old, various other methods were employed to identify the pathogen. These methods included microscopic examination for morphological criteria of the urediniospores and teliospores, as well as molecular techniques like 28S rDNA sequencing. Rust disease on L. sinuatum has been previously reported in Ecuador but the causal agent was identified as a Puccinia sp. and reported that the rust was able to destroy entire plots in humid conditions (Vega and Morales 2011). Whether this report also represent U. savulescui is not certain, but given that the urediniospores of Puccinia species are generally 2-celled, it is unlikely. García-Hernández et al. (2008) reported U. limonii on Limonium spp. from Chile, and Coca (2020) also reported U. limonii on Limonium sp., from Bolivia. However, judging from the photomicrographs (Coca 2020), the rust in the latter report is definitely U. savulescui and not U. limonii. Uromyces savulescui has been previously reported from the Mediterranean region and the Canary Islands (Vakalounakis and Malathrakis 1987). To our knowledge there is no report of this rust in the Americas, excepting the probable misidentifications already listed herein. The specimen has been deposited in the Arthur Fungarium at Purdue University as PURN15037.

First report of Athelia rolfsii associated with southern blight disease of Amorphophallus muelleri in China.

Amorphophallus muelleri, known as konjac, is widely used in the biomedicine and food processing due to its richness in glucomannan. Between the years of 2019 to 2022, severe outbreaks of southern blight on Am. muelleri were observed during August and September in the main planting region of Mile city. The average disease incidence was 20%, resulted in 15.3% of economic losses in approximately 10,000 m2. Infected plants showed wilting and rotting and were covered with white dense mats of mycelia and sclerotia on both petiole base and tubers. Am. muelleri petiole base covered with mycelial mats were collected for pathogen isolation. The infected tissues (n=20) were washed with sterile water and surface disinfected with 75% alcohol for 60 seconds, rinsed three times with sterile water, cultured on rose bengal agar (RBA) and incubated at 27 ℃ for two days (Adre et al. 2022). Individual hyphae were transferred to new RBA plates and incubated at 27 ℃ for 15 days to obtain purified cultures. Five representative isolates were subsequently obtained and exhibited identical morphological characteristics. All isolates produced dense, cotton-white aerial mycelia and had a daily growth rate of 1.6 ± 0.2 mm (n=5). After 10 days, all isolates formed sclerotia in spherical (diameter range 1.1 to 3.5 mm, aver. 2.0 ± 0.5 mm; n=30) and irregular shapes. The number of sclerotia per plate ranged from 58 to 113 (aver=82; n=5). These sclerotia were initially white and gradually turned brown as they matured. A representative isolate (17B-1) was selected for molecular identification and the translation elongation factor (TEF, 480 nt.), internal transcribed spacer (ITS, 629 nt.), large subunit (LSU, 922 nt.), and small subunit (SSU, 1016 nt.) regions were amplified with the primers EF595F/EF1160R (Wendland and Kothe 1997), ITS1/ITS4 (Utama et al. 2022), NS1/NS4, and LROR/LR5 (Moncalvo et al. 2000), respectively. The ITS (GenBank accession no. OP658949), LSU (OP658955), SSU (OP658952), and TEF (OP679794) sequences were 99.19%, 99.78%, 99.31%, and 99.58% similar to those of At. rolfsii isolates (MT634388, MT225781, MT103059, and MN106270, respectively). Thus, the fungus represented by isolate 17B-1 was identified as At. rolfsii, corroborating the identification of Sclerotium rolfsii Sacc., the anamorph, based on cultural and morphological features. Pathogenicity tests were performed on six-month-old asymptomatic Am. muelleri plants (n=30) grown in pots with sterile soil in a greenhouse at 27 °C and 80% relative humidity. The petiole base was scratched with a sterile blade and 20 plants were inoculated by placing a 5 mm2 mycelial plug of five-day-old isolate 17B-1 on the wound. Sterile RBA plugs were used on 10 wounded control plants. After 12 days, all inoculated plants exhibited symptoms similar to those observed in the field, while the control plants showed no symptoms. The morphological and molecular identification of the fungus reisolated from inoculated petioles confirmed its identity as At. Rolfsii, fulfilling Koch's postulates. S. rolfsii was first reported on Am. campanulatus in India (Sarma et al. 2002). As At. rolfsii causes konjac diseases in all Amorphophallus growing areas (Pravi et al. 2014), the importance of At. rolfsii as an endemic pathogen of Am. muelleri in China needs to be recognized, and its prevalence should be determined as a first step to managing this disease.

The Synergic Effect of Gut-Derived Probiotic Bacillus cereus SL1 And Ocimum sanctum on Growth, Intestinal Histopathology, Innate Immunity, and Expression of Enzymatic Antioxidant Genes in Fish, Cirrhinus mrigala (Hamilton, 1822).

An effective alternative approach to combat aquaculture challenges is the strategic application of bioresources, which not only mitigate disease ailment but also optimize fish growth. Hence, current research was undertaken to highlight the synergic role of bioresources such as plant immunostimulant Ocimum sanctum along with potent gut-derived probiotic Bacillus cereus strain SL1 (Gen Bank Accession Number: FJ627945.1) on mrigal (Cirrhinus mrigala) growth, antioxidant status, gut histopathology, and immune response. For 90days, fingerlings (average weight 6.8 ± 0.5g) were fed on diets having varying concentrations of O. sanctum and B. cereus. After the completion of the feeding trial, various growth, immunity, and histological and antioxidant metrics were evaluated according to standard procedures. In comparison to the control and other treatment groups, T3 group showed a significant (P < 0.05) increase in growth parameters, antioxidant enzymatic activity, and hematological and immunological parameters. In addition to it, supplementation of both B. cereus and O. sanctum also upregulated the antioxidant-related gene expressions, such as hepatic catalase gene by 1.89-3.00 folds, hepatic SOD-1 by 4.46-7.52 folds, and GPx-1of the liver by 1.56-1.95 folds. For 10days, fingerlings were challenged with the pathogenic bacterium Aeromonas hydrophila (MTCC-1739), and maximum survival rate (77.77%) was reported in fingerlings of T3 treatment. Further histopathological studies of gut tissues affirm that O. sanctum and B. cereus play a synergic role in the protection of digestive organs from the pathogenic bacterium A. hydrophila. These results suggest that O. sanctum and B. cereus synergically improved the growth performance, immunity, antioxidant status, and gut histology of C. mrigala leading to its sustainable culture.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Hemp in Croatia.

The production of industrial hemp (Cannabis sativa) has had a sharp increase in the past five years in Croatia (Mihelcic 2017). Production has been in constant increase, from 1560 ha in 2015 to 2476 ha in 2019 (PAAFRD). In August 2019, numerous (~1.5%) premature wilted hemp plants (cultivar Fibranova) were observed in commercial fields in Vladislavci (45.4646950° N, 18.5674770° E), around Osijek, Croatia. Diseased plants showed symptoms of chlorosis, rapidly wilting, necrosis and prematurely dying. The stalk of diseased plants was completely desiccated, while internal tissues were spongy and fluffy. Near the soil line, discoloration of the stalk with small spherical to oblong black microsclerotia was detected. Roots were necrotic with brown-gray areas. Twenty root and crown segments of the infected plants (2- to 3-mm long pieces) were surface sterilized with 2% NaOCl for 5 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar (PDA, pH 6.2) media, containing 250 mg per liter of chloramphenicol to inhibit bacterial growth (Zveibil and Freeman 2005). The culture plates were incubated at 28 ± 2°C for 7 days in the dark and purified to obtain a pure culture that produces numerous, dark, hard, ovoidal-shaped sclerotia, averaging 140 x 52 μm (n=50). The single sclerotium isolate produced flat light to dark gray colonies with entire margins. Based on field symptoms, colony and microsclerotia morphology, the fungus was identified as Macrophomina phaseolina (Tassi) Goid (Marquez et al. 2021). Total DNA from the isolate was extracted with Extract-N-Amp Plant PCR Kit (Sigma-Aldrich Co., USA). To confirm morphological identification, part of the TEF 1-alpha gene region was amplified using EF1-728F (Carbone and Kohn 1999) and EF2 (O'Donell et al. 1998). The sequence of the isolate MP1 (212 bp - GenBank accession no. OQ389757), showed 100% nucleotide sequence identity to the reference sequence of M. phaseolina GenBank sequence MG434668 (Casano et al. 2018). Eighteen hemp plants (cv. Fibranova) were sown in six plastic pots (three hemp plants per pot) for the pathogenicity test. Ten-day-old M. phaseolina culture (isolate MP1) was used for inoculum preparation. Each pot of one-week-old plant was irrigated with 100 ml of a microsclerotia suspension (105 microsclerotia/ml)(Abied et al. 2018). Plants were held at 28°C and 70% relative humidity in a growth chamber (aralab, Fitoclima 10.000 HP) with a 16-hour photoperiod. Pots with control plants were irrigated with the same amount of sterile distilled water. Ten weeks after inoculation percentage of wilted plants was 77.78%. In the control variant all plants were healthy. M. phaseolina was reisolated from inoculated plants and morphologically identified. With the liberalization of the law, hemp production in Croatia is increasing, which could result in general disease problems and the disease caused by M. phaseolina. Charcoal rot will be expressed in years with dry and warm summers (Lodha and Mawar 2020), and relatively short, cool, rainy winters (Nevo et al. 2012), which has become common in the last decade in Croatia. To our knowledge, this is the first report of M. phaseolina on hemp in Croatia. The authors declare no conflict of interest.

First report of Capsicum chlorosis virus naturally infecting Ageratum conyzoides in China.

Capsicum chlorosis virus (CaCV; family Tospoviridae, genus Orthotospovirus) was first reported to infect capsicum (Capsicum annuum) and tomato (Solanum lycopersicum) in Australia in 2002 (McMichael et al., 2002). Subsequently, its infection was detected in different plants including waxflower (Hoya calycina Schlecter) in the United States (Melzer et al. 2014), peanut (Arachis hypogaea) in India (Vijayalakshmi et al. 2016), and spider lily (Hymenocallis americana) (Huang et al. 2017), Chilli pepper (Capsicum annuum) (Zheng et al. 2020), and Feiji cao (Chromolaena odorata) (Chen et al. 2022) in China. Ageratum conyzoides L. (commonly known as goat weed, family Asteraceae) is a natural weed in crop fields distributed in subtropical and tropical areas and a reservoir host of numerous plant pathogens (She et al. 2013). In April 2022, we observed that 90% of plants of A. conyzoides in maize fields in Sanya, Hainan province, China, exhibited typical virus-like symptoms of vein yellowing, leaf chlorosis, and distortion (Fig. S1 A-C). Total RNA was extracted from one symptomatic leaf of A. conyzoides. Small RNA libraries were constructed using the small RNA Sample Pre Kit (Illumina, San Diego, USA) for sequencing with an Illumina Novaseq 6000 platform (Biomarker Technologies Corporation, Beijing, China). A total 15,848,189 clean reads were obtained after removing low-quality reads. Quality-controlled qualified reads were assembled into contigs using Velvet 1.0.5 software with a k-mer value of 17. One hundred contigs shared nucleotide identity ranging from 85.7% to 100% with the CaCV using BLASTn searches online (https://blast.ncbi.nlm.nih.gov/Blast.cgi?). Numerous contigs (45, 34, and 21) obtained in this study were mapped to the L, M, and S RNA segments of the CaCV-Hainan isolate (GenBank accession no. KX078565- KX078567) from spider lily (Hymenocallis americana) in Hainan province, China, respectively. The full-length of L, M, and S RNA segments of CaCV-AC were determined to be 8,913, 4,841, and 3,629 bp, respectively (GenBank accession no. OQ597167- OQ597169). Furthermore, five symptomatic leaf samples were tested to be positive for CaCV using a CaCV enzyme-linked immunosorbent assay (ELISA) kit (MEIMIAN, Jiangsu, China) (Fig. S1-D). Total RNA from these leaves was amplified by RT-PCR with two sets of primer pairs. Primers CaCV-F (5'-ACTTTCCATCAACCTCTGT-3') and CaCV-R (5'-GTTATGGCCATATTTCCCT-3') were used for the amplification of 828 bp fragment from nucleocapsid protein (NP) on CaCV S RNA. While another, primers gL3637 (5'-CCTTTAACAGTDGAAACAT-3') and gL4435c (5'-CATDGCRCAAGARTGRTARACAGA-3') were used for the amplification of 816 bp fragment from RNA-dependent RNA polymerase (RdRP) on CaCV L RNA (Fig. S1-E and -F) (Basavaraj et al. 2020). These amplicons were cloned into the pCE2 TA/Blunt-Zero vector (Vazyme, Nanjing, China) and three independent positive colonies of Escherichia coli DH5α carrying each viral amplicon were sequenced. These sequences were deposited in the GenBank database under accession nos. OP616700-OP616709. Pairwise sequence comparison revealed that nucleotide sequences of NP and RdRP genes of the five CaCV isolates shared 99.5% (812 bp out of 828 bp) and 99.4% (799 bp out of 816 bp) nucleotide identities, respectively. They showed 86.2-99.2% and 86.5-99.1% nucleotide identities with corresponding nucleotide sequences of other CaCV isolates derived from GenBank database, respectively. The highest nucleotide sequence identity (99%) of the CaCV isolates obtained in the study was observed with the CaCV-Hainan isolate. Phylogenetic analysis based on NP amino acid demonstrated that six CaCV isolates (this study = 5 and NCBI database = 1) clustered into one distinct clade (Fig. S2). Our data confirmed for the first time the presence of CaCV naturally infecting A. conyzoides plant in China, which enriches information on the host range and will be helpful for disease management.

First Report of Banana mild mosaic virus Infecting Banana in China.

Banana (Musa spp.) is an important fruit in tropical and subtropical regions and an essential food crop in some developing countries. China has a long history of banana cultivation and ranks second in global banana production, with a planting area exceeding 11 million hectares (FAOSTAT, 2023). Banana mild mosaic virus (BanMMV) is a flexuous filamentous virus infecting bananas and a banmivirus in the Betaflexiviridae family. Its infection often results in symptomless plants of Musa spp., and the virus has a worldwide distribution, which can explain its high prevalence (Kumar et al., 2015). BanMMV infection often causes transitory symptoms, such as mild chlorotic streaks and mosaics, on young leaves (Thomas, 2015). The mixed infection of BanMMV with other banana-infecting viruses such as banana streak viruses (BSV) and cucumber mosaic virus (CMV), can exacerbate the mosaic symptoms of BanMMV (Fidan et al., 2019). In October 2021, we collected twenty-six leaf samples of suspected viral disease of bananas from four cities (Huizhou, Qingyuan, Zhanjiang, and Yangjiang) in Guangdong province, two cities (Hekou and Jinghong) in Yunnan province, two cities (Yulin and Wuming) in Guangxi Zhuang autonomous region. After fully mixing these infected samples, we divided them into two pools and sent them to Shanghai Biotechnology Corporation (China) for metatranscriptome sequencing. Each sample contained about 5 g of leaves in total. Zymo-Seq RiboFree Total RNA Library Prep Kit (Zymo Research, USA) was used for ribosomal RNA depletion and library preparations. Illumina sequencing (Illumina NovaSeq 6000) was carried out by Shanghai Biotechnology Corporation (China). Paired-end (150 bp) sequencing of the RNA library was performed on an Illumina HiSeq 2000/2500 platform. Clean reads were assembled by a metagenomic de novo assembly using the CLC Genomics Workbench (version: 6.0.4). Then the non-redundant protein database in the National Center for Biotechnology Information (NCBI) was used for BLASTx annotation. A total of 79,528 contigs were generated from the clean reads (68,878,162) through de novo assembly. A contig of 7265 nucleotides (nts) showed the highest nucleotide sequence identity (90.08%) to the genome of BanMMV isolate EM4-2 (GenBank accession no. OL826745.1). We designed specific primers according to the BanMMV CP gene (Table S1), tested the twenty-six leaf samples collected from the above-mentioned eight cities, and found that only one sample of Fenjiao (Musa ABB Pisang Awak) in Guangzhou city was infected with this virus. The symptoms of banana leaves containing BanMMV were slight chlorosis and yellowing of leaf edges (Fig. S1). We failed to detect other banana viruses, such as BSV, CMV, and banana bunchy top virus (BBTV) in the BanMMV-infected banana leaves. RNA from the infected leaves was extracted, and the assembled contig was confirmed by overlapping PCR amplification across the whole sequence (Table S1). All ambiguous regions were amplified by PCR and RACE, and the products were subjected to Sanger sequencing. The complete genome of the virus candidate was 7310 nts in length, excluding the poly (A) tail. The sequence was deposited in GenBank under accession number ON227268 (isolate BanMMV-GZ from Guangzhou). A schematic representation of the genome organization of BanMMV-GZ is shown in Fig. S2. Its genome has five open reading frames (ORF) encoding RNA-dependent RNA polymerase (RdRp), three triple gene block proteins necessary for cell-to-cell movement (TGBp1 to TGBp3) and a coat protein (CP), similar to other BanMMV isolates (Kondo et al., 2021). Phylogenetic analyses of the complete nt sequence of the full genome and RdRp gene using the neighbor-joining (NJ) method also clearly placed the BanMMV-GZ firmly within all isolates of BanMMV (Fig. S3). To our knowledge, this is the first report of BanMMV infecting bananas in China, extending the geographical range of this viral disease around the world. Accordingly, larger-scale BanMMV investigations must be conducted to determine the distribution and prevalence of BanMMV in China.

First report of Pseudocerradoa paullula causing aroid leaf rust on Swiss cheese plant Monstera deliciosa in South Carolina, USA.

In February 2023, two Monstera deliciosa Liebm. (Araceae) plants with typical symptoms of leaf rust disease were detected at a grocery store in Oconee Co., South Carolina. Symptoms included chlorotic leaf spots and abundant brownish uredinia, mainly on the adaxial surface of more than 50% of leaves. The same disease was detected on 11 out of 481 M. deliciosa plants in a greenhouse at a plant nursery located in York Co., South Carolina, in March 2023. The first plant sample detected in February was used for morphological characterization, molecular identification, and pathogenicity confirmation of the rust fungus. Urediniospores were densely aggregated, globose, golden to golden brown in color, and measured 22.9 to 27.9 µm (aver. 26.0 ± 1.1 µm; n=50) in diameter with wall thickness at 1.3 to 2.6 µm (aver. 1.8 ± 0.3 µm; n=50). Telia were not observed. These morphological traits aligned with those of Pseudocerradoa paullula (basionym: Puccinia paullula; Ebinghaus et al. 2022; Sakamoto et al. 2023; Sydow and Sydow 1913; Urbina et al. 2023). Genomic DNA was extracted from urediniospores collected from the naturally infected plant sample and used for PCR amplification and DNA sequencing of the large subunit (LSU) genetic marker with primers LRust1R and LR3 (Vilgalys and Hester 1990; Beenken et al. 2012). The LSU sequence of the rust fungus in South Carolina (GenBank accession: OQ746460) is 99.9% identical to that of Ps. paullula voucher BPI 893085 (763/764 nt.; KY764151), 99.4% identical to that of voucher PIGH 17154 in Florida, USA (760/765 nt.; OQ275201), and 99% identical to that of voucher TNS-F-82075 in Japan (715/722 nt.; OK509071). Based on its morphological and molecular characteristics, the causal agent was identified as Ps. paullula. This pathogen identification was also corroborated by the U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Plant Pathogen Confirmatory Diagnostics Laboratory in Laurel, Maryland. To confirm the fungus's pathogenicity on M. deliciosa and M. adansonii Schott (Sakamoto et al. 2023), three plants of each Monstera species were inoculated by spraying with a suspension of urediniospores collected from the original plant sample (1 × 106 spores per ml; approx. 40 ml per plant). Three non-inoculated control plants of each host species were treated with deionized water in the same manner. Plants were placed in a plastic tray with wet paper towels to maintain moisture. The tray was placed at 22C for an 8-h photoperiod and covered for five days to facilitate infection. On 25 days after inoculation, abundant spots bearing urediniospores were produced on all leaves of inoculated M. deliciosa plants. A few uredinia were observed on two of the three inoculated M. adansonii plants. All non-inoculated control plants remained asymptomatic. Morphological features of urediniospores collected from inoculated plants matched those of Ps. paullula used as the inoculum. Aroid leaf rust on Monstera plants was officially reported in Australia, China, Japan, Malaysia, Philippines, and Florida, USA (Shaw 1991; Sakamoto et al. 2023; Urbina et al. 2023). This is the first report of Ps. paullula causing this disease on M. deliciosa in South Carolina, USA. Monstera species are popular indoor and landscape plants. Potential impact and regulatory responses regarding Ps. paullula, a newly introduced and rapidly spreading pathogen in the USA, warrant further evaluation and discussion.

Occurrence of Powdery Mildew Caused by Erysiphe astragali on Astragalus scaberrimus in Inner Mongolia, China.

Astragalus scaberrimus Bunge, a perennial herb, is widely distributed in North and central China, Russia, and Mongolia (POWO, 2023). Due to its tolerance to drought, cold, high salt, low nutrients and alkaline soil, this plant is widely cultivated in China as a forage crop, for water and soil conservation, and for its medicinal properties (Meng, 2015). In 2022, powdery mildew-like signs and symptoms were seen on leaves of A. scaberrimuns cultivated on the campus of Inner Mongolia Agricultural University, Hohhot, Inner Mongolia China. White powder-like masses covered up to 99% of the leaf area with infected plants showing weak growth and senescence. More than 70% of plants (n = 180) exhibited these powdery mildew-infected symptoms. Conidiophores were 70-120 μm long (n = 20) and composed of a basal foot cell, followed by two cells and a conidium. Cylindrical- or ovoid-shaped conidia were 30-45µm long by 9-15 µm wide (n = 20). Brown or light-brown chasmothecia were 100-140 μm in diameter, with flexuous appendages. Based on these morphological characteristics, the fungus was tentatively identified as an Erysiphe sp. (Braun and Cook, 2012; Schmidt and Braun, 2020). Fungal structures were isolated from diseased leaves and genomic DNA of the pathogen was extracted utilizing the method described by Zhu et al. (2022). The internal transcribed spacer (ITS) region was amplified by PCR employing the primers PMITS1/PMITS2 (Cunnington et al., 2003) and the amplicon sequenced by Invitrogen (Shanghai, China). The sequence for the powdery mildew fungus (GenBank accession no.: MW142495) showed 100% identity (645/645 bp) with Erysiphe astragali, which was reported on A. glycyphyllos in Golestan province, Iran (accession no. OP806834). Pathogenicity tests were conducted by brushing the conidia from infected A. scaberrimus leaves onto leaves of four healthy plants, while, the four control plants were brushed in the same manner. All the treated plants were placed in separate growth chambers maintained at 19℃, 65% humidity, with 16 h light/8 h dark photoperiod. Nine days post inoculation, powdery mildew disease signs appeared on inoculated plants, whereas control plants remained asymptomatic. The same results were obtained for two repeated pathogenicity experiments. The powdery mildew fungus was reisolated and identified as E. astragali based on morphological and molecular analysis, thereby fulfilling Koch's postulates. E. astragali causing powdery mildew on A. glycyphyllus were previously reported in Germany with Genbank accesion number of MZ265150 and MZ265151 (Bradshaw et al., 2022). This, to our knowledge, is the first report of powdery mildew caused by E. astragali on A. scaberrimus. The severe occurrence of this destructive powdery mildew disease on A. scaberrimus may adversely affect the utility of the plant for soil conservation or cultivation for medicinal purposes. Identifying the causal agent of powdery mildew will support efforts for the future control and management of diseases on A. scaberrimus.

First Report of 16SrI-B Subgroup Related Phytoplasma Associated with Yellows Symptoms of Rubus cochinchinensis in China.

Rubus cochinchinensis, an important traditional Chinese medicine in China is used to treat rheumatic arthralgia, bruises and lumbocrural pain (He et al.2005). In January 2022, yellow leaves of R. cochinchinensis were found in Tunchang City, Hainan Province, a tropical island in China. Chlorosis spread along the direction of vascular tissue while the leaf veins remain green (Fig. 1). In addition, the leaves were slightly shrunken and the growth vigor is poor (Fig. 1). By survey, we found the incidence of this disease was about 30%. Three etiolated samples and three healthy samples (0.1g each) were used to extract total DNA (TIANGEN plant genomic DNA extraction kit). Using nested PCR method, phytoplasma universal primers P1 / P7 (Schneider et al., 1995) and R16F2n / R16R2 (Lee et al. 1993) were used to amplified phytoplasma 16S rDNA gene. Primers rp F1 / R1 (Lee et al. 1998) and rp F2 / R2 (Martini et al. 2007) were used to amplified rp gene. 16S rDNA gene and rp gene fragments were amplified from three leaf etiolated samples, but not from healthy samples. The amplified fragments were cloned and sequenced, and the sequences were assembled by DNASTAR11. By sequence alignment, we found the obtained 16S rDNA and rp gene sequences of three leaf etiolated samples were same. The length of 16S rDNA fragment was 1237 bp (accession number: ON944105) and the length of rp gene fragment was 1212 bp (accession number: ON960069). The phytoplasma strain was named as 'R. cochinchinensis' yellows leaf phytoplasma (RcT), RcT-HN1 strain. The 16S rDNA gene sequence of RcT-HN1is 99.8% consistent with 16SrI-B subgroup members such as the 'Brassica napus' dwarf phytoplasma strain WH3 (MG599470.1), Chinaberry yellows phytoplasma strain LJM-1(KX683297.1) and Arecanut yellow leaf disease phytoplasma strain B165 (FJ694685.1). The rp gene sequence of RcT-HN1 is 100% consistent with rpI-B subgroup members such as the 'Salix tetradenia' witches'-broom phytoplasma strain YM-1 (KC117314.1) and Chinaberry witches'-broom phytoplasma strain Hainan (EU348781.1). The phylogenetic tree analysis, based on concatenated 16S rDNA-rp gene sequence of same group phytoplasma by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value, were performed (Kumar et al., 2016). The results showed that RcT-HN1 phytoplasma strain formed a subclade in aster yellows group B subgroup (Fig. 2). The virtual RFLP analysis based on the 16S rRNA gene fragment of RcT-HN1 phytoplasma strain was performed by the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009). The results showed that the phytoplasma strain was same as the reference pattern of the onion yellows phytoplasma of 16SrI-B (GenBank accession: AP006628), and the similarity coefficient was 1.00. This is the first report that 16SrI-B subgroup related phytoplasma infected R. cochinchinensis and caused yellows symptoms in China. The discovery of the disease is helpful to the study of the spread of phytoplasma-related diseases and protect R. cochinchinensis resources.

First report of Fusarium keratoplasticum causing strawberry root rot in Sinaloa, Mexico.

Strawberry (Fragaria × ananassa) is a fruit of economic importance for Mexico, occupying the third place in world production, with an approximate production of 861, 337 t (SIAP, 2021). In January 2021, in Culiacan, Sinaloa, Mexico (24°46'46″N; 107°27'04″ W), wilting symptoms (stunted growth, leaf yellowing and wilting, necrosis in vascular bundles, root rot and wilting) were observed on commercial strawberry crops, with an incidence of 5 to 10 %. Tissue samples from symptomatic roots were cut and disinfected with alcohol, sodium hypochlorite and sterile water, to later be plated on potato dextrose agar (PDA). Fifteen monosporic isolates were obtained by single-spore culturing (Hansen and Smith, 1932). Typical Fusarium spp. colonies were obtained from all root samples. On PDA the colonies were abundant with white aerial mycelium, hyphae were branched and septate, and light-yellow pigmentation was observed in the center of old cultures (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidia were slightly curved, showing three marked septa, broad central cells, slightly tapered apices, foot-shaped basal cells and measured 59.6 - 73.4 (x̄ = 68.7) x 10.4 - 14.9 μm (x̄ = 13.6) (n = 40). The microconidia (n = 40) were thin-walled, hyaline, ovoid unicellular that measured 19.7 - 32.2 (x̄ = 26.6) x 8.8 - 11.8 μm (x̄ = 10.2). The translation elongation factor 1 alpha (EF1-α) gene (O'Donnell et al. 1998) was amplified by polymerase chain reaction and sequenced. Maximum likelihood analysis was carried out using the EF1-α sequence from the isolate FKTFRESCULSIN (GenBank accession no. OK491929) and other Neocosmospora and Fusarium species. Phylogenetic analysis revealed the isolate was Fusarium keratoplasticum (currently named Neocosmospora keratoplastica) belonging to the Fusarium solani species complex (FSSC). Pathogenicity tests were performed on strawberry plants (cultivar Camarosa) grown on autoclaved sandy loam soil mix. Twenty plants were inoculated by drenching with 20 ml of a conidial suspension (1 × 105 CFU/ml) in an isotonic saline solution of FKTFRESCULSIN grown on PDA. Ten uninoculated plants served as controls. Plants were maintained for 60 days under greenhouse conditions (25 to 30°C). The assay was conducted twice. Root and stem rot similar to that observed on the infected plants in the field was observed. No symptoms were observed on uninoculated control plants after 60 days. The pathogen was reisolated from necrotic tissue from all inoculated plants and identified as F. keratoplasticum by sequencing the partial EF1-α gene and based on its morphological characteristics, thus fulfilling Koch's postulates. To our knowledge, this is the first report of root rot and wilt of strawberry caused by F. keratoplasticum in Mexico; it also contributes knowledge to the scientific community, since there is little information about this pathogen causing damage to plants in the world. Strawberry is an important crop in Mexico, and the occurrence of this disease could threaten strawberry production.

Stemroton Adenia globosa caused by Lasiodiplodia theobromae in Jiangsu.

Adenia globosa, as an excellent indoor ornamental plant, is planted in Tropical Botanical Museum, Nanjing Zhongshan Botanical Garden, Jiangsu Province, China.In September 2022, a new stembasalrot disease was observed on A. globosa seedlings,being planted here. Stem basalrot were observed on approximately 80% of A. globosa seedlings. The basalstem of cutting seedlings appeared decayed, and stem tip eventually turned dry due to water loss (Figure S1A). To isolate the pathogen, three diseased stems were collected from three cuttings planted in different pots of the Tropical Botanical Museum. The stem sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% ethanol for 30 s and 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃ in the dark. Pure cultures were obtained by monosporic isolation. Eight isolates were obtained, and all identified as Lasiodiplodia sp.. The colonies morphology of cultures, growing on PDA were cotton-like, the primary mycelia were black gray after 7 days, and the reverse sides of PDA plates were similar to front sides in color (Figure S1B). A representative isolate, QXM1-2 was selected for the further study. Conidia of QXM1-2 were oval or elliptic, with a mean size of 11.6 µm×6.6 µm (n=35). The conidia are colorless and transparent in the early stage, and become dark brown with one-septum in the later stage (Figure S1C). The conidiophores produced conidia after nearly four weeks of cultivation on PDA plate (Figure S1D). The conidiophore was a transparent cylindrical structure, with a size of (6.4-18.2) µm × (2.3-4.5) µm ( n = 35). These characteristics were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). The internal transcribed spacer regions (ITS), translation elongation factor 1-alpha (TEF1α) and β-tubulin (TUB) genes (GenBank Accession No.OP905639, No.OP921005, and No.OP921006, respectively) were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. They had 99.8-100% homology to the ITS (504/505 bp) of Lasiodiplodia theobromae strain NH-1 (MK696029), TEF1α (316/316 bp) of strain PaP-3 (MN840491), and TUB (459/459 bp) of isolate J4-1 (MN172230). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate QXM1-2 clustered in the L. theobromae clade with 100% bootstrap support (Figure S2). To test pathogenicity, three A. globosa cutting seedlings that previously had been wounded with a sterile needle were inoculated with 20 μL conidia suspension (1×106 conidia/mL) on the stem base. The seedlings inoculated with 20 µL sterile water was used as the control. All plants were covered with clear polyethylene bags to keep moisture in a greenhouse (25℃, 80% relative humidity). The experiment was repeated three times. After 7 days post-inoculation, typical stem rot were found on the treated cutting seedlings and the control seedlings did not have any symptoms (Figure S1E-F). The same fungus, identified by morphological characteristics and sequencing using ITS, TEF1α and TUB genes, was isolated from the diseased tissues of the inoculated stems to complete Koch's postulates. This pathogen has been reported infecting the branch of castor bean (Tang et al. 2021) androot ofCitrus (Al-Sadi et al. 2014). For our knowledge, this is the first report of L. theobromae infecting A. globosa in China. This study provides an important reference for the biology, epidemiology of L. theobromae.

Screening and Isolation of Polypropylene Degrading Fungi from Waste Dumping Site, Kolhapur, India

Polypropylene (PP) and other plastic wastes are found to accumulate in the environment, creating significant ecological issues. They are determined to be considered non-biodegradable, It has been established that once it enters the environment, it stays there permanently. The present investigation aims to biodegrade PP without physical treatment and exposing it to UV light and sunlight exposed to potential fungi isolated from the soil of solid waste dumping site based on 18SrRNA analysis and the isolated strains were identified as 98.54% similar to Cladosporium sp. The fungal strain was submitted with Gene Bank accession number ON024632 and registered as a Cladosporium halotolerans strain SUK PRAKASH. The degradation was performed for 8 months of incubation in the aqueous medium. The biodegradation of polypropylene FTIR spectroscopy was performed to further examine the sheets, and the results indicated that perhaps the bonds between the sheets were weakening and breaking. The biodegraded samples of without treated PP sheets, UV-exposed PP sheets, and sunlight-exposed PP sheets exhibit weight loss of 4.2%, 6.1%, and 8.6% respectively.

First Report of Strawberry Black Root Rot Caused by Globisporangium sylvaticum in Tennessee, USA.

Globisporangium sylvaticum (syn. Pythium sylvaticum), is an oomycete that causes root rot and damping off of field crops, ornamentals, and vegetables. Several species in Pythiaceae are associated with black root rot of strawberry [(Fragaria × ananassa) Duchesne] (Millner 2006). Mature, stunted 'Chandler' strawberry plants, with reduced shoot vigor and black necrotic roots, were collected from Rhea County (June 2018) and Cumberland County, TN (May 2019). Aboveground symptoms occurred in low incidence (<5% of plants) in the fields. Plant roots were rinsed with tap water, cut into 1 to 3 cm pieces, and surface-disinfested (70% ethanol, 1 min) followed by a sterile water rinse. Root segments were crushed, placed on 20% V8 juice agar, and incubated in the dark at 21°C for 3 days. White fluffy mycelia grew from a majority of roots and coenocytic hyphae with globose hyphal swellings, delimited from hyphae by septa, were observed with microscopy. Hyphae were initially branched, curled, hyaline, and aseptate; however, septations were observed in older cultures. Globose structures (terminal and intercalary) were identified as sporangia [11 to 32 (avg. 22.1) µm diameter] when zoospores were observed (Parikh et al. 2022). Oospores [9 to 21 (avg. 16) μm diameter] were globose, smooth, aplerotic, and thick-walled. Oogonia, with or without one or more inflated antheridia, were observed when isolates were paired in culture, characteristics consistent with descriptions of Campbell and Hendrix (1967), Pratt and Green (1971), van der Plaats-Niterink (1981), and Uzuhashi et al. (2010). Genomic DNA was extracted (Extract-N-Amp™; Sigma-Aldrich, MO) for PCR amplification of internal transcribed spacer (ITS) regions of rDNA with primers ITS1/ITS4 (White et al. 1990); ITS and large subunit rRNA regions with primers UN-up18S42/UN-lo28S22 (Robideau et al. 2011); and cytochrome c oxidase subunit I (COI) mitochondrial DNA with primers OomCoxI-Levup/OomCoxI-Levlo (Robideau et al. 2011). Primers ITS1/ITS4 were used to amplify isolate TN (GenBank Accession MW386310, which had 100% homology with reference isolate MK326528). Primers UN-up18S42/UN-lo28S22 amplified isolates SAP18 and OO1 (Accessions MZ881935 and MZ881936, which had 99.8% homology with HQ665236), and COI primers amplified isolate SAP18 (Accession OK020192, which had 100% homology with GU071816 and KT692835). To satisfy Koch's postulates, inoculum of G. sylvaticum grown on autoclaved wheat seeds was added (5% w/v) to planting mix (1 peat:1 sand, v/v). Young, rooted strawberry plants were planted in 1.2-L pots with infested (n = 6) and control (no pathogen, n = 6) mixes, which was saturated with deionized water. Pots were covered with clear plastic for 48 h to maintain high humidity. Plants were grown in a greenhouse (24°C avg.) for 8 weeks. The disease assay was repeated. All plants in infested mix died, with black, necrotic roots. Plants in the control mix were healthy and well-established. The pathogen was reisolated from roots of all inoculated plants and confirmed to be G. sylvaticum based on morphology and molecular analyses. Root disease of strawberry caused by G. sylvaticum has been reported in the USA (Campbell and Hendrix 1967; Nemec and Sanders 1970; Pratt and Green 1971). This is the first report of G. sylvaticum causing root rot of strawberry in Tennessee. With the loss of methyl bromide, sustainable disease control strategies are needed to provide effective management options for strawberry black root rot.

First Report of Colletotrichum fructicola Causing Anthracnose on Punica granatum in China.

Punica granatum L. (Pomegranate), a deciduous shrub, is widely cultivated as a fruit tree and decorative plant in China. Its flowers, leaves, roots and fruit bark also has been widely used for the treatment of different types of human disease because of the high anti-inflammatory and antibacterial activitiy (Tehranifar et al. 2011). In October 2022, leaf spot symptoms were observed on P. granatum leaves in a landscaped area on the campus of Jiangxi Agricultural University (28.75°N, 115.83°E), Nanchang, Jiangxi Province, China. A survey of 40 P. granatum of 300 m2 found that up to 20% of the foliage was infected. Infection normally starts at the tip or edge of the leaves, with the initial symptoms of lesions usually being small dark brown spots (0.8 to 1.5 mm) that gradually expand into irregular spots with grayish white central parts, and brown margins (2.3 to 3.8 mm). Ten freshly infected leaves from three different plants were collected and cut into small slices, disinfected with 75% ethanol for 30 seconds followed by 5% NaClO for 1 minute, rinsed 3 times with sterile water, and then plated on potato dextrose agar (PDA) and incubated in the dark at 25°C. After 7 days, all incubated samples produced similar morphology of aerial mycelium pale grey, dense, and cottony. Conidia were hyaline, smooth-walled, cylindrical, aseptate and measuring 12.28 to 21.05 × 3.51 to 7.37 µm (n = 50). Morphological characteristics were consistent with those of Colletotrichum gloeosporioides species complex (Weir et al. 2012; Park et al. 2018). For molecular identification, we used two representative isolates (HJAUP CH005 and HJAUP CH006) for genomic DNA extraction and amplification, using primers for ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b, GDF1/GDR1, ACT-512F/ACT-783R and CL1C /CL2C (Weir et al. 2012), respectively. The sequenced loci (GenBank accession nos. ITS: OQ625876, OQ625882; TUB2: OQ628072, OQ628073; GAPDH: OQ628076, OQ657985; ACT: OQ628070, OQ628071; CAL: OQ628074, OQ628075) exhibited 98 to 100% homology with corresponding sequences of C. fructicola strains (GenBank accession nos. OQ254737, MK514471, MZ133607, MZ463637, ON457800, respectively). A phylogenetic tree was constructed using the maximum-likelihood method in MEGA7.0 for the sequences of five concatenated genes (ITS-TUB2-GAPDH-ACT-CAL). Our two isolates clustered together with three strains of C. fructicola with 99% bootstrap support values in the bootstrap test (1000 replicates). The isolates were identified as C. fructicola based on morpho-molecular approach. The pathogenicity of HJAUP CH005 was tested indoors by inoculating the wounded leaves of four healthy P. granatum plants. Four leaves from each of two healthy plants were punctured with flamed needles and sprayed with a spore suspension (1 × 106 spores/ml), and four wounded leaves from each of other two plants were inoculated with mycelial plugs (5 × 5 mm3), respectively. Mock inoculations with sterile water and PDA plugs on four leaves each were used as controls. Treated plants were incubated in a greenhouse at high relative humidity, 25°C, and a photoperiod of 12 h. After 4 days, typical anthracnose symptoms similar to natural infection appeared on the inoculated leaves, whereas the control leaves remained asymptomatic. Based on morphological and molecular data, the fungus isolated from the inoculated and symptomatic leaves was identical to the original pathogen, confirming Koch's hypothesis. Anthracnose caused by C. fructicola has been reported to affect numerous plants worldwide, including cotton, coffee, grapes and citrus (Huang et al. 2021; Farr and Rossman 2023). This is the first report of C. fructicola causing anthracnose on P. granatum in China. This disease seriously affects the quality and yield of the fruit and should be of wide concern to us.

First report of the root-knot nematode Meloidogyne enterolobii on sweet potato in Georgia, United States.

Five species of root-knot nematodes (RKN), Meloidogyne spp. (M. haplanaria, M. incognita, M. floridensis, M. javanica, and M. arenaria) were detected in 67% of vegetable-growing regions in Georgia, United States (Marquez et al. 2021a, b; Marquez and Hajihassani 2022a, b). In October 2021, two sweet potato (Ipomoea batatas) samples (FF1 and FF2) collected at harvest from a field located in Tattnall County, Reidsville, GA were received for assessment. Symptoms of RKN damage on storage roots included a veiny appearance, surface cracking, and bumpy yellow to brown colored specks (Fig. 1). The population density of RKN second-stage juveniles (J2) was 148 and 180 J2/100 cm3 of soil for FF1 and FF2 samples, respectively. Genomic DNA was isolated by smashing females (n=10) individually in 20 µL of PCR-grade water, followed by freezing at -20°C overnight and thawing at 95°C for 1 min. Each DNA sample was first analyzed with a duplex PCR method using RKN species-specific primer sets for the most common nematode species: Mi2F4/Mi1R1 (M. incognita) and Far/Rar (M. arenaria) and SEC-1F/SEC-1R (M. incognita) and Fjav/Rjav (M. javanica) (Zijlstra et al. 2000; Hajihassani et al. 2022). Since this method failed to identify the RKN species, DNA samples were amplified with Me-F and Me-R primers, specific for diagnosing M. enterolobii (Long et al. 2006). Species-specific PCR produced a 240 bp DNA fragment for FF1 and FF2 samples (Fig. 2), corresponding to M. enterolobii. RKN species identification was confirmed by DNA sequencing using C2F3/1108 and TRNAH/MRH106 primers (Powers et al. 2018; Stanton et al. 1997). Products of C2F3/1108 (GenBank accessions ON320401 and ON320405) were 100% identical with 100% query coverage to a North Carolina M. enterolobii isolate (MN809527), while TRNAH/MHR106 sequences (ON320402 and ON320406) was 99.4-99.8% identical with 89-91% query coverage to a China isolate (MF467278). Measurements [Mean (range)] of body length of M. enterolobii J2 (BL): 454.0 (411.3-485.1) µm; maximum body width (BW): 15.1 (13.8-17.0) µm; stylet length: 14.3 (12.7-15.2) µm; total BL/greatest BW: 30.1 (28.6-31.4) µm; and BL/head end to posterior end of metacorpus: 7.3 (6.5-8.2) µm. Morphological measurements of J2 were comparable to the original description of M. enterolobii Yang and Eisenback. The host suitability of sweet potato varieties [Covington (susceptible to M. enterolobii), Beauregard (susceptible to intermediate), and Regal (resistant)] to the M. enterolobii isolate was assessed (Rutter et al. 2021). Nematode eggs were extracted from skin/bumps of samples FF1 and FF2 by blending (15 sec) and shaking in a 0.5% NaOCl solution (5 min), followed by washing and centrifugation in a standard sucrose solution. Sweet potato slips were transplanted in 10.8-cm-diam. pots filled with sand and steamed field soil (1:1 v/v), and two days after planting, were inoculated with 10,000 eggs of M. enterolobii (six replicates per plant variety). Plants were maintained in a greenhouse at 25-28°C for 85 days in a completely randomized design. Root galling index (scale of 0 to 5) of 4.4, 4.2, and 0.8 (Fig. 3) and reproduction factor (final egg numbers/initial egg number) of 8.2, 7.5, and 0.01 were obtained for Covington, Beauregard, and Regal, respectively confirming that Covington and Beauregard are susceptible to M. enterolobii while Regal is resistant. Meloidogyne enterolobii has not been reported in GA and this is the first report of the nematode on sweet potato in the state. This RKN species is an emerging pest of economic importance in many crops in the Southern United States (Brito et al. 2004; Rutter et al. 2018; Ye et al. 2013). Development of effective short- and long-term control procedures is urgently needed for managing M. enterolobii.

First Report of Leaf Blotch Caused by Phyllosticta capitalensis on Daphne odora in China.

Daphne odora Thunb. an evergreen shrub with scented flowers, is used for ornamental purposes but it also has medicinal benefits (Otsuki, et al. 2020). In August 2021, leaf blotch symptoms were observed on roughly 20% of leaves of D. odora var. marginata plants in Fenghuangzhou Citizen Park, Nanchang city (28°41'48.12″ N, 115°52'40.47″ E), Jiangxi Province, China. Brown lesions first appeared on the edges of leaves, which eventually dried and died (Fig. 1A). For fungal isolation, 12 symptomatic leaves were randomly collected, the edges between diseased area and healthy area were cut into small pieces (4×4 mm), surface-sterilized by dipping in 70% ethanol for 10 s and 1% sodium hypochlorite for 30 s, and then rinsed three times with sterile distilled water. Leaf pieces were then plated on potato dextrose agar (PDA) and incubated at 28 ℃ for 3-4 days. A total of 10 isolates were recovered from the diseased leaves. The pure colonies of all fungal isolates had similar characteristics, and three isolates were randomly selected (JFRL 03-249, JFRL 03-250 and JFRL 03-251) for further study. Colonies of this fungus were gray and uneven, with a granular surface, and irregular white edges, finally turning black on PDA (Fig. 1B, C). Pycnidia were black, globose and 54-222 μm in diameter (Fig. 1D). Conidia were hyaline, single-celled, and nearly elliptical, which ranged from 7 to 13 × 5 to 7 μm (n=40) (Fig. 1E). These morphological characteristics were consistent with those described for the fungus Phyllosticta spp. (Wikee et al. 2013a). To confirm the fungal identity, the internal transcribed spacer (ITS) region, actin (ACT), translation elongation factor 1-alpha (TEF1-a), glyceradehyde-3-phosphate dehydrogenase (GPD) and RNA polymerase II second largest subunit (RPB2) genes were amplified using primers ITS5/ITS4, ACT-512F/ACT-783R, EF-728F/EF2, Gpd1-LM/Gpd2-LM and RPB2-5F2 /fRPB2-7cR, respectively (Wikee et al. 2013b). The sequences of the selected isolates were 100% identical. Hence, sequences of one representative isolate JFRL 03-250 were deposited in GenBank (OP854673, ITS; OP867004, ACT; OP867007, TEF1-a; OP867010, GPD; and OQ559562, RPB2). BLAST search analysis in GenBank showed 100% similarity with those of P. capitalensis (GenBank accession nos. ITS, MH183391; ACT, KY855662; TEF1-a, KM816635; GPD, OM640050 and RPB2, KY855820). From a phylogenetic perspective, a maximum likelihood phylogenetic tree was constructed by using IQtree V1.5.6 based on multiple sequences (ITS, ACT, TEF1-a, GPD and RPB2) (Nguyen et al. 2015), and the cluster analysis resulted the representative isolate JFRL 03-250 within a clade comprising Phyllosticta capitalensis (Fig. 2). Based on morphological and molecular characters, the isolate was identified as P. capitalensis. To confirm pathogenicity and fulfill Koch's postulates, 6 healthy potted plants were inoculated with 1× 106 conidia/ml suspension of isolate JFRL 03-250 by spraying on the leaves, whereas 6 plants were sprayed with sterile distilled water to serve as control. All potted plants were incubated at 28°C, 80% relative humidity and 12-h light/12-h dark alternating conditions in a climate cabinet. After 15 days, similar symptoms were observed in the inoculated leaves as in the field (Fig. 1F), whereas control leaves remained asymptomatic (Fig. 1G) and P. capitalensis was successfully re-isolated from the symptomatic leaves. Previously, P. capitalensis has been reported to cause brown leaf spot disease of various host plants around the world (Wikee et al. 2013b). However, to our knowledge, this is the first report of brown leaf spot caused by P. capitalensis on D. odora in China.

First Report of Powdery Mildew Caused by Erysiphe cruciferarum on Centella asiatica in China.

Centella asiatica belongs to the Umbelliferae family of perennial herbaceous plants, which are grown worldwide for use as health supplements, edible vegetables and traditional herbs, and are of vital medicinal and edible value in China. (Biswas et al. 2021). In October 2022, the investigation in the 800 m2 garden of Lanzhou (36°06' N,103°43' E) found that more than 80% of C. asiatica plants were infected by powdery mildew, and the leaf infection rate was 90%. The disease severely affects the actual value of C. asiatica. At the beginning of the disease, thin, radial, irregular white colonies appear on the leaves and gradually spread to the stems. The white colony then expands and thickens, covering the upper surface of the whole leaf, and gradually spreading to the lower parts of the stem and leaf. In severe cases, the leaves wither and die. A small portion of fungal spores was glued from the leaf surface with adhesive tape and placed in sterile water for microscopic examination (Zhang et al. 2022). The conidiophore is upright, cylindrical, composed of 3-4 cells, and its size is 72 to 110 × 8 to 10 μm. Conidial pedicels have 16 to 26 μm long cylindrical podocytes. Monoconidia are cylindrical or oval in shape, 16 to 37 μm long, width 11 to 18 μm (n=80). Conidia lack an obvious cellulose body. The bud tube is formed from the end of conidia, and papillary appressorium develops on the epiphytic mycelia. Based on these morphological characteristics, the pathogen was initially identified as Erysiphe cruciferarum (Braun et al. 2012). To validate the identity, the internal transcribed spacer (ITS) of the pathogen (JXC) rDNA was amplified by PCR and sequenced with PM6/ITS5 and PM5/ITS4 primers (Takamatsu et al. 2001). The resulting sequences were registered to GenBank (GenBank Accession OP935627 and OQ253404). At the same time, the ITS sequence size was 535 bp and 521 bp respectively. The ITS sequence of the JXC was 99% (527/534) identical to E.cruciferarum (KT588635) on Eschscholzia californica in Slovakia and 99% (527/534) identical to E.cruciferarum (KC878683) on Chinese Cabbage in China. The ITS sequences from GenBank were subjected to conduct maximum likelihood phylogenetic analysis by MEGA 7.0. The data indicate that strain JXC and E. cruciferarum are clustered on the same branch. The pathogenicity test was performed according to Koch's postulate. By gently pressing the infected leaves on five healthy potted C. asiatica. Meanwhile, five uninoculated plants were used as controls (Zhang et al. 2022). The plants were put into a greenhouse culture (25℃, 14 h light, 10 h dark, humidity ≥ 70%). After 12 days, the inoculated plants showed symptoms of powdery mildew, while the control group had no symptoms. The fungus on the inoculated plant was re-isolated, and identified as E. cruciferarum based on morphological observations and molecular identification. The powdery mildew caused by E.cruciferarum has been reported on Indian mustard in Korea and Chinese cabbage in China, respectively (Kim et al. 2009; Zhao et al. 2014). To our knowledge, this is the first report of C. asiatica powdery mildew caused by E.cruciferarum in China. This finding poses a potential threat to the quality and yield of C. asiatica plants, while providing a preventive basis for the cultivation of C. asiatica.

Molecular characterization and phylogenetic analysis of Newcastle disease viruses isolated in southern Angola, 2016-2018.

Newcastle disease (ND) is a highly contagious viral disease that affects many bird species worldwide. This study presents the results of the molecular characterization and phylogenetic analysis of 15 virulent ND viruses (NDV) isolated from chickens during outbreaks reported in 2016 and 2018, in the provinces of Namibe and Huíla, in southern Angola. A 561-nucleotide fragment of the F gene was amplified by RT-PCR and sequenced for molecular characterization. Results showed that in all isolates the amino acid sequence comprising the cleavage site of fusion protein is characteristic of virulent viruses (RRQKR/F). Blast analysis revealed high similarity (99.2%) between two isolates from Huíla province, HLA4 and HLA6, and strain 5620 (GenBank accession number KY747479) isolated from chickens in the neighboring country Namibia, in 2016. The other isolates investigated are more related (97.0%) with strain 6195 (GenBank accession number KY747480), also isolated in Namibia in 2016. Phylogenetic analysis performed by Maximum Likelihood, Neighbor-joining and Bayesian methods revealed that like the strains isolated in Namibia, the isolates from southern Angola also belong to subgenotype 2 of genotype VII (VII.2). The network analysis revealed that NBA1 isolate from Angola is closer to a common ancestor than the isolates from Namibia, suggesting that transmission of ND viruses occurred from Angola to Namibia.

First report of tobacco rattle virus infecting Weigela florida in the USA.

Weigela (Weigela florida (Bunge) A. DC., Family: Caprifoliaceae) are woody shrubs native to North China, Korea, and Japan. In the U.S., weigela are commonly used as landscape ornamental plants (McNamara et al. 2010). Two viruses have been reported in weigela: tomato spotted wilt orthotospovirus and impatiens necrotic spot orthotospovirus (Sastry et al. 2019). Ten weigela plants, originating from commercial nurseries in Minnesota, exhibiting chlorosis, chlorotic line patterns, and necrosis (e-Xtra) were submitted for virus diagnostics as potted plants at the University of Minnesota Plant Disease Clinic and the Virology Lab in 2019 and 2020 (five plants each year). Under greenhouse conditions, symptoms progressed from chlorosis to necrosis and even plant death in two of the five plants in 2019. Electron microscopy revealed rod-shaped particles of ≈20 nm in diameter and lengths between 40-200 nm with similar morphology to members of the genus Tobravirus (e-Xtra). Virus-like particles were enriched by ultracentrifugation and total nucleic acids were extracted from partial purifications using a phenol:chloroform extraction method (Lockhart et al. 1997). Tobacco rattle virus (TRV) was identified by cloning and sequencing of the 463bp amplicon obtained with the TRV detection primers described in Robinson, 1992. High-throughput sequencing (HTS) was done to confirm the TRV detection. A cDNA library was prepared from purified viral RNA using the TruSeq Stranded mRNA kit and sequenced on Illumina NovaSeq 6000 platform as 150 bp-paired end reads. A total of 44,316,446 raw data reads were obtained, preprocessed using the BBDuk plugin, and de novo assembled using SPAdes assembler. Viral contigs were identified using the NCBI BLASTX tool. The assembly of TRV RNA1 was 6,842 nt with 20,627,348 reads (47% of total reads) mapped to it and an average coverage per nucleotide at 323,639X. The assembly of TRV RNA2 was 3,033 nt with 22,769,253 reads (52% of total reads) mapped to it and an average coverage per nucleotide at 798,660X. NCBI GenBank accession numbers for the assemblies representing RNA1 and RNA2 are OQ408335 and OQ408336, respectively. NCBI BLASTn analysis showed the highest level of nucleotide identity to TRV genomic RNA segments 1 and 2, with 97% and 99% identity to the TRV isolate RNA1 (GQ903771) and RNA2 (GQ903772), respectively, that originated from Michigan potato. No other viral contigs were detected from the virion nucleic acid extraction by HTS, however this enrichment method doesn't exclude other viruses. In addition to using the detection primers by Robinson 1992, we designed primers based on our HTS data: TRV-WG-DetF3 5'- GACGAAGGAGGCTGTCATTGC-3' and TRV-WG-DetR3 5'-CGGACTATCGTGATGCCCATGC- 3'. RT-PCR amplicons from each of the 10 symptomatic plants were cloned and sequenced. Among these clones, Sanger sequence identities ranged between 96-100% compared to the HTS data and 98-99% to the TRV potato isolate from MI. To our knowledge, this is the first report of TRV infecting the ornamental host W. florida worldwide. TRV is a nematode-transmitted viral pathogen of economic importance, most notably in potatoes (Sastry et al. 2019). In the US, TRV has been reported on several landscape ornamentals, horticultural crops, and native habitats. Further research is needed to investigate the impact of TRV on the ornamental industry and the role of ornamentals as reservoirs for cultivated crops like potatoes.