GenBank Accession Research Articles

First report of anthracnose caused by Colletotrichum karstii on Dalbergia odorifera in China.

Dalbergia odorifera (Family: Fabaceae) is a national second-grade protected tree in China with high medicinal and economic value (Zhao et al., 2020). In July, 2022, a leaves spot disease on D. odorifera with typical anthracnose symptoms was observed in plantations in Haikou (110.319153°E, 19.072900°N), Dongfang (108.630297°E, 19.103838°N) and Qiongzhong (109.704460°E, 19.088440°N), Hainan Province, China. Disease incidence was 7.5% (n = 50 plants). Early symptoms of infected leaves were small and round dark brown spots, which developed into larger irregular necrotic lesions and leaves withered. Leaf tissues (5×5 mm) at the disease-health junction of spots from 19 leaves were sterilized with 2.5% sodium hypochlorite for 1 min, and rinsed with sterile distilled water three times. These sterilized tissues were placed on potato dextrose agar (PDA) and incubated at 28 ℃ for 5 d. 7 strains of fungi with similar morphology were isolated, and 3 single-hyphal isolates (HHL01, HHL02 and HHL03) from each location were selected for further study. Colonies on PDA were fluffy orange-yellow mycelium. Conidia were aseptate, cylindrical, smooth-walled, straight, hyaline with both ends bluntly rounded, 11.82 to 15.77 × 3.87 to 6.71 μm (n = 100; average = 13.75 × 5.52 μm). Appressoria formed on slides, measured 5.54 to 10.64 × 4.19 to 7.41 μm (n = 30; average = 8.06 × 5.97 μm) were brown to black, elliptical to irregular. For molecular biological identification, the genomic DNA of three isolates was extracted by fungal genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). The partial sequences of internal transcribed spacer region (ITS; ITS1/ITS4), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GDF1/GDR1), actin (ACT; ACT512F/ACT783R), β-tubulin (TUB2; TI/Bt2b) and calmodulin (CAL; CL1C/CL2C) were amplified and sequenced by Sangon Biotech (Shanghai) Co., Ltd (Carbone and Kohn, 1999; Weir et al., 2012). The sequences were deposited as GenBank Accession Nos. OR018110-OR018112 (ITS); OR050529-OR050537 (GAPDH, ACT and CAL) and OR192168-OR192170 (TUB). BLASTn results showed these sequences were more than 99% identity with the strain of C. karstii CORCK1 (GenBank Accession Nos. HM585406, HM585387, HM581991, HM585424 and HM582010, respectively). Multi-locus phylogenetic tree of Colletotrichum spp. showed that those three isolates were sister to C. karstii based on the maximum likelihood and bayesian inference methods. To verify pathogenicity, 2 mL spore suspension (1 × 106conidia/ml) of the isolates was sprayed on each leaves of 1-year-old D. odorifera plants, and sterile distilled water was similarly sprayed on other leaves as a negative control. The plants were incubated in a greenhouse under 90% ± 5% RH at 28 °C. Light brown small round necrotic patches developed 3 days after inoculation, while the control was asymptomatic. Photographs were taken on the fifth day after inoculation. The fungi were re-isolated from the diseased leaves and identified by morphological characterization and molecular identification, fulfilling Koch's postulates. C. karstii has been reported causing leaf rot of Carissa grandiflora in Spain (Garcia-Lopez et al., 2021), and anthracnose caused by C.tropicale was reported on D. odorifera (Yi et al., 2023). To our knowledge, this is the first report of Dalbergia odorifera leaf spot disease caused by Colletotrichum karstii. This finding provides an important basis for further research on the control of the disease.

First Report of Root Rot Caused by Pythium dissotocum on Tobacco in China.

Tobacco (Nicotiana tabacum L.) is an important economic crop that is widely grown around the world. Its annual production in China is estimated at 2.2 million tons (Berbeć and Matyka 2020). Since 2022, a root rot disease was sporadically observed on tobacco seedlings on cultivar Yunyan 87 in cultivated tobacco fields in the Hunan province of China. A disease incidence of about 10% occurred across 48 ha of tobacco fields. The affected tobacco plants had slow and stunted growth with yellowing leaves. The roots turned grayish brown, decayed, and died. Diseased roots were collected from six fields and cut into small pieces (5 mm ×5 mm) from the edge of the rotted portions, and then sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 1 min, and washed in sterilized water three times. All the sterilized tissue were placed on potato dextrose agar (PDA) medium and cultured at 26 ℃ in the dark. About 3 days later, colonies with similar morphology were removed and sub-cultured on fresh PDA. A total of six strains were obtained from six tobacco samples. Strains were white and had radial growth on PDA. Hyphae were aseptate and the sporangia were filamentous. The oogonia were subglobose, smooth, 16.04 ± 0.25 µm (n=50) in diameter, and developed on unbranched stalks. The antheridia were barrel shaped and clavate. Oospores were globose, aplerotic or nearly plerotic, measuring 6.62 ± 0.33 µm (n=50). These morphological characteristics were consistent with the description of Pythium spp. (van der Plaats-Niterink 1981). For molecular identification, the internal transcribed spacer (ITS) region of rDNA and cytochrome c oxidase subunit I (Cox I) of a representative isolate, GF-3, were amplified and sequenced (GenBank accession nos. OR228424 for ITS and OR237556 for Cox I) using universal primers ITS1/ITS4 (White et al. 1990) and FM58/FM66, respectively (Villa et al. 2006). BLASTn analysis revealed that the ITS and Cox I sequences were 99.76 % (838/840 bp) and 99.85% (671/672 bp) identical to the corresponding sequences of P. dissotocum strain CBS 166.68 (AY598634.2) and UM982 (MT981147.1), respectively. A neighbor-joining phylogenetic tree based on the Cox I sequence showed that GF-3 grouped in the P. dissotocum branch. Based on morphological and molecular characteristics, GF-3 was identified to be P. dissotocum. For pathogenicity testing, four- to five-leaf-old healthy potted tobacco seedlings of the Yunyan 87 cultivar were inoculated with a zoospore suspension (1 × 105 zoospores/ml), which was induced on V8-juice medium. The zoospore suspension was introduced into the soil around plant roots and 10 mL of inoculum was used for each plant. In the control group, plants were inoculated with sterilized water. All of the treated plants were kept in humid chambers at 26°C under a 12 h/12 h photoperiod. The pathogenicity assays were performed twice, with each treatment having three replicated plants. After 5 days, tobacco seedlings inoculated with P. dissotocum showed symptoms resembling that observed in the field. However, the control plants remained healthy. Pythium dissotocum was re-isolated from the infected plants and identified by morphological and molecular methods, thus confirming Koch's postulates. Pythium dissotocum has been reported causing root rot in other plants, including hydroponic lettuce (McGehee et al. 2018) and spinach (Huo et al. 2020). Also, many Pythium species have recently been recovered from float-bed tobacco transplant production greenhouses (Zhang et al. 2022). However, to our knowledge, this is the first report of root rot on tobacco caused by P. dissotocum in China. Since this disease could greatly affect tobacco seedling establishment in the field, appropriate management strategies need to be developed to reduce further losses in tobacco planting fields.

First report of Fusarium solani causing Fusarium wilt of Bletilla striata (hyacinth orchid) in Zhejiang province of China.

Bletilla striata, a member of the family Orchidaceae, is a perennial herbaceous plant used in Chinese medicine. It is a commonly cultivated economic crop in the Yangtze River Basin provinces of China, as its roots are used to treat bleeding and inflammation. In Zhejiang province, Bletilla striata has a planting area of 1400 hectares with a total production of approximately 2.6×106 kg. In October 2021, over 40% of B. striata plants showed severe wilt in a traditional Chinese medicine plantation (ca. 10 ha) in Xianju City, Zhejiang Province, China. In July, leaf curling, crinkling, and leaf-edge browning of the diseased plants were first noticed in the field. Then, necrotic streaks gradually spread to the roots. Stems displayed chlorosis and withering and when they were cut vertically, symptoms such as vascular bundle discoloration, appeared. After October, the individual plants slowly wilted and died, their aboveground parts became filamentous, and the epidermis detached from the corm's fibrous roots. Diseased plants were easily removed as the corm root had fractured. White mycelia were clearly seen in the stem. Three symptomatic leaves and three stems were cut, their surfaces disinfected, and plated on potato dextrose agar (PDA). Six strains were subsequently isolated from all samples. Fungal colonies with white to cream-colored mycelia from all tissues appeared after 3 d of incubation at 26 °C. Pure cultures obtained after monospore isolation were examined for their morphological characteristics. The colonies grew rapidly, were fluffy and appressed, and had cottony white to pale cream coloration. Microconidia were hyaline, oval to reniform, with zero or one-septate (4.0-12.0 × 1.0-5.5 µm), and usually formed on elongated monophialidic conidiogenous cells. Macroconidia were wide, fusiform, or slightly curved with one or three septa (23.0-36.0 × 4.5-7.0 μm). Chlamydospores were spherical and were abundant on carrot agar (CA) medium within 2 wk. Fresh mycelia and conidia that grew at 26 ℃ for 7 d were collected from PDA plates. Next, DNA was extracted using the Ezup Column Fungi Genomic DNA Purification kit (Sangon Biotech, Shanghai, China). We amplified a portion of RNA polymerase II second largest subunit gene (RPB2) using primers 5f2/7cr (O'Donnell et al. 2010), the internal transcribed spacer (ITS) region using primers ITS1F/ITS4 (White et al. 1990), and the partial translation elongation factor-1α gene using primers EF1/ EF2 (O'Donnell et al. 1998) from the genomic DNA and sent the PCR amplicons for sequencing at Tsingke Biotechnology Co., Ltd., Wuhan, China. A BLAST search of the obtained sequences (GenBank accessions OP743920, OP913183, and OP913180) showed 99-100% homology with the respective sequences of the Fusarium solani reference isolate NRRL46702 (O'Donnell et al. 2008). Based on the morphological and molecular characteristics and BLAST search, the fungus was identified as F. solani (Leslie and Summerell 2006). Pathogenicity of the purified F. solani isolate was assessed by inoculateing a F. solani spore suspension of 1×106 conidia/mL (20 mL per seedling) on corm wounds made with a toothpick. Four inoculated and three non-inoculated seedlings (sterilized water as a negative control) were grown in a greenhouse at 26 °C under natural sunlight and covered with plastic bags to maintain humidity for 72 h. After 15 d, leaf browning on leaf edges, new leaf bases, and corm epidermis was observed. Symptoms, similar to those detected in the original sample, developed on the inoculated leaves, whereas the controls remained asymptomatic. Fusarium solani was successfully re-isolated from all four inoculated seedlings, and their identity confirmed by generating partial Tef1 and RPB2 sequences, thereby fulfilling the Koch's postulate. To our knowledge, F. solani has not been previously reported as a pathogen of B. striata.

First Report of Penicillium cellarum Causing Rot Disease on Dioscorea polystachya in China.

Dioscorea polystachya (Chinese yam) is a kind of medicine and food homologous crop, the tubers as its main production organ, with high potassium, low fiber, high protein and rich nutrition characteristics. In 2022, at the Chinese herbal medicine planting experimental site in Anguo, Baoding City, Hebei Province, China, we found the symptoms of Chinese yam decay during the storage, with an incidence of 15%~25%. The diseased part of Chinese yam tuber rots expands from the outside to the inside and sags, with a brown or dark brown discoloration, and the surface covered with a thick grayish green mold. The diseased tissue was first rinsed with clean water to remove dirts from the surface. Thereafter, 3 to 4 mm Chinese yam pieces were picked from rotting edge with a sterilized forceps, sterilized with 75% alcohol for 30 s followed by 0.1% mercuric chloride solution for 1min, and then rinsed three times with sterile water. The sterilized pieces were cultured on potato dextrose agar (PDA). One isolated fungus was obtained, and conidia were observed after incubation for 5 days at 26°C. Pure cultures were isolated by single-spore isolation. Conidia were single spore, round or oval, colorless. Conidiophores produce several rounds of symmetric or asymmetric small stems after multiple branches, which were shaped like brooms. The length and width of 100 conidia were measured, and size ranged from 3 to 4×3 to 4 μm. On the basis of morphological characteristics, the isolate was identified as Penicillium spp. (Uy et al. 2022). To further assess the identity of isolated species, the genomic DNA of the fungal isolate (SYRF1) was extracted by CTAB protocol. The ribosomal DNA internal transcribed spacer (ITS) region and the ribosomal large subunit (LSU) were amplified and sequenced with primers ITS1/4, LR5/LROR respectively (White et al. 1990, Xu et al. 2010). The obtained ITS-rDNA region and LSU sequences (GenBank accession OQ707937 and OQ704185) of the isolate were more than 99% identity to the corresponding sequences of Penicillium cellarum in GenBank (KM249068 and MG714818). Phylogenetic results based on a maximum-likelihood analysis revealed that SYRF1 was grouped with P. cellarum. To determine the pathogenicity of the isolated fungi, tests were carried out by aseptic inoculation of fresh and healthy tubers. Before the experiment, the healthy tubers were washed, surface disinfected and dried. The tubers were then wounded with sterile inoculation needles, and the conidium-bearing hyphal discs (5 mm) were inoculated on the surface of the wounded tubers and covered with wet sterile cotton. Three tubers were inoculated repeatedly each time as the experimental group.Inoculate sterile PDA with three tubers as the control group. Each tuber was inoculated with four mycelium disks, and the pathogenicity test was repeated four times. The inoculated tubers were incubated at 26°C for 14 days with sterile PDA as control. After ten days, the inoculated points showed symptoms similar to those of the initial infection, whereas controls remained symptomless. The reisolated fungus matched SYRF1 based on morphological and sequence analyses, thereby fulfilling Koch's postulates. To the best of our knowledge, this is the first report of Penicillium cellarum as causative agent of postharvest rot of Chinese yam tubers in China. This finding will help inform the prevention and management of postharvest diseases of Chinese yam tubers.

The first USA continental record of coffee leaf rust (Hemileia vastatrix) on coffee (Coffea arabica) in southwest Florida, USA.

Coffee leaf rust (CLR), caused by Hemileia vastatrix Berk. & Broome (Zaghouaniaceae) is considered the most significant fungal disease of Coffea arabica L. (Rubiaceae), from which berries are harvested and processed to obtain coffee beverage (Talhinhas et al. 2017). In Florida, coffee plants are mainly used as ornamentals due to their fragrant flowers; however, there are ongoing field trials evaluating the adaptability of plants for coffee production to climate conditions in the state (Crane et al. 2005). In November 2021, young seedlings of C. arabica var. caturra from a residence in Naples (Collier County) in southwest Florida were discovered with signs of rust fungus. Minute, yellow, suprastomatal sori 53-81 µm in diam were formed on the abaxial leaf surface, forming blotches. Light-yellow urediniospores measured 29-31 × 20-29 µm, with a reniform or "hunchbacked" shape, had thick walls measuring 1.5-2.5 µm in height, and were dorsally echinulate, the individual spikes measuring 2.5-3.3 µm in height. Spikes were scattered over most of the dorsal surface and form a dense ridge separating the dorsal from the smooth ventral side. (e-Xtra Fig. 1). Symptoms and signs are consistent with published descriptions of CLR produced by H. vastatrix (Ritschel 2005). To confirm the identification, DNA sequencing of the large subunit (LSU) of the ribosomal repeat was done following the protocols of Aime (2006) (GenBank accession number OR296753-OR296754). The Florida specimen shares 100% sequence identity (887/728 bp) with other accessions of H. vastatrix in congruence with maximum likelihood phylogenetic analysis performed in RAxMLv8.0.0 (Stamatakis 2014) (e-Xtra Fig. 2). In addition to CLR, Hemileia coffeicola Maubl. & Roger, causal agent of powdery rust of coffee, produces similar leaf spots on coffee but has a restricted geographical distribution. This agent is found only above 500 m a.s.l. in central Africa (Silva et al. 2006) and produces larger urediniospores (34-40 × 20-28 µm) (Maublanc & Roger 1934) in sori are scattered in abaxial leaf surface giving a powdery appearance. Hemileia vastatrix has been reported from almost every major coffee growing country of the world as well as Hawaii and Puerto Rico (Keith et al. 2022, Ramirez-Camejo et al., 2022). This is the first report of CLR in the continental USA, however, CLR poses a limited threat to the USA agriculture in view of the fact coffee is not commercially grown within the continental USA. A voucher was made of dried symptomatic leaves and deposited at Plant Industry Gainesville Herbarium (PIHG 15712, 16332) and the Arthur Fungarium at Purdue University (PUR N23473). The remaining infested coffee seedlings were destroyed after phytopathological diagnosis, and the pathogen has been absent from all additional screenings since November 2021.

First Report of Leaf Spot Caused by Corynespora cassiicola on Jasminum sambac in Pakistan.

Jasminum sambac L. is a species of jasmine native to a small region in the eastern Himalayas and is cultivated worldwide as an ornamental plant (USDA-ARS 2016). In Pakistan, it is cultivated for ornamental purposes throughout the country. The flowers of this plant are traditionally used in the preparation of essential oils and for making jasmine tea. The flowers and leaves also have been used in folk medicine to treat breast cancer, epilepsy, ulcers and promote wound healing (Al-Snafi 2018). In December, 2017, almost 10 leaves of 3 plants of J. sambac growing plant nursery of Gehlan, Pattoki, Punjab a province of Pakistan were observed with leaf spot disease. Infected leaves exhibited circular to sub-circular spots with indistinct margins and grey papery centers delimited by dark brown rims. For further microscopic study, the infected leaves were examined under a stereomicroscope. For the isolation and cultural studies of infecting fungus, infected parts of leaves were surface sterilized in 1% NaOCl for about 10 seconds, washed twice in sterilized distilled water, plated on potato dextrose agar (PDA) medium and incubated at 25°C for 4 days. Pure cultures were obtained having colonies of light to dark brown color. Conidia (n=20) were light brown to pale olivaceous brown, smooth, obclavate to cylindrical in shape, 99.5-118.5 μm in length and 12.5-15.0 μm in width, with mostly 3 to 14 pseudosepta. Conidiophores (n=20) were straight to slightly curved, unbranched, and pale to light brown in color. Based on the morphological characteristics of the colonies and conidiophores and conidia, the pathogen was identified as Corynespora cassiicola (Berk and M.A. Curtis) C.T. Wei. (Berkeley & Curtis 1968; Lu et al. 2021; Wei 1950). Genomic DNA was extracted following using modified CTAB method (Gardes and Bruns 1993) and internal transcribed spacer (ITS) region was amplified with ITS1 and ITS4 primers (White et al. 1990). The ITS sequence generated of about 553 bp and deposited in GenBank (accession no. MN954556), was found more than 99% similar to previously deposited sequences of C. cassiicola (GenBank accession nos. MN339671, EU364535, FJ852574, MK139711, EU131374) as verified through BLASTn and phylogenetic tree construction. A pathogenicity test was performed for fulfilling Koch'spostulates. Conidial suspension (105 conidia/ml) of the recovered isolate was sprayed on the 5 healthy leaves of 2-month-old seedling of J. sambac. Mock inoculated plants sprayed sterile distilled water were used as a control. The seedlings were covered with plastic bags to maintain high humidity at 24 to 28°C for a week. Identical disease symptoms to those observed in nursery plants were observed on the leaves of the inoculated plants in 7 days but not mock inoculated plants and results were reconfirmed. The reoccurred fungus was isolated from the diseased spots of the inoculated leaves to complete Koch's postulates and identified microscopically. A representative sample of leaves with lesions was deposited in the LAH herbarium, Department of Botany University of the Punjab, Pakistan (LAH35691). Previously, C. cassicola has been found infecting Jasminum mesnyi in China and Jasminum sp. in Florida (Alfieri et al. 1984; Zhang et al. 2018). The best of our knowledge, this is the first report of leaf spot caused by C. cassiicola on J. sambac in Pakistan. It will establish a foundation for future studies of management strategies for this plant disease caused by C. cassiicola.

Morphological and molecular characterization of the rust fungus, Phakopsora apoda causing leaf rust on Brachiaria grass in Rwanda

Brachiaria grass is one of the major forages that plays a key role in push pull technology. Different diseases including leaf rust caused by rust fungus, Phakopsora apoda have recently become an important challenge resulting in yield loss of Brachiaria grass in different countries including Rwanda. The objective of this study was to provide morphological and molecular characteristics of Phakopsora apoda causing Brachiaria leaf rust. Spores isolated from rusted Brachiaria leaves collected in five districts of Rwanda were analyzed. Samples were put in paper bags and left at room temperature for two days. Thereafter, spores were removed using brushes and put in eppendorf tubes, stored at 4oC in darkness for further characterization. For morphological characteristics, colour and measurements of spores were recorded using Optika B-350 microscope with installed camera and calibrated micrometer. Multigene analysis using rust specific primers and large subunit (LSU) of nuclear ribosomal RNA was performed for molecular characterization. The results indicated that the colour of spores was yellowish or brownish while the shape was ellipsoidal or circular with average measurements of 25.1µm and 16.8µm for length and width respectively. Primers amplified the size of DNA between 1291bp and 1381bp and the size between 874bp to 882bp for rust specific primers and LSU respectively. The identity of the sequence was 96% with e-value of 0, sequence coverage of 60% for rust primer and the sequence identity was 94.97% to 95.37%, sequence coverage of 99% to 100% and e-value of 0 for LSU primer. All sequences recovered from five isolates of leaf rust matched to the genebank accession number MG461668.1 for both rust specific and LSU sequences. The results of this study provide useful information to develop effective options for leaf rust disease management in Rwanda. Key words: Internal transcribed spacer, large subunit, nuclear ribosomal RNA, Phakopsora apoda, sequence, spore.

Isolation, and characterization of Cladosporium alboflavescens for Acetaminophen biodegradation

One of the pharmaceutical micropollutants with a detrimental effect on the environment is paracetamol. Mycoremediation of the pollutants is a widely accepted concept based on cost and eco-friendly nature. Fungi isolated from pharmaceutical industry effluent have been used to target the biodegradation of paracetamol. The fungus’s internal transcribed spacer (ITS 1) sequencing matched that of Cladosporium alboflavescens (GenBank accession number OQ977005) by 99.81%. The strain demonstrated 89% biodegradation of paracetamol (1000 ppm) after 96 hours of incubation with 4-aminophenol as the predominant biodegradative metabolite, according to the spectrophotometric and high-performance liquid chromatographic analysis. The half-life of 1.44 days and simple first order kinetics were proposed by the Computer Assisted Kinetic Evaluation (CAKE) tool used to examinethe biodegradation kinetics.
 Key words: Acetaminophen, 4-aminophenol, Cladosporium, Mycoremediation

Overcoming dormancy in Desmanthus virgatus seeds for improved viability monitoring protocols of genebank accessions

Desmanthus virgatus is a perennial forage legume used as a livestock feed source. Seeds of this species are physically dormant and thus, propagation is difficult. The dormancy also impacts the routine viability monitoring of genebank accessions. The present study investigated different methods to break dormancy in seeds collected from various locations at different times. The result showed that most pre-treatments significantly increased germination percentage. Accessions treated with concentrated sulphuric acid (25 or 30 minutes) followed by sandpaper scarification showed the highest proportion of normal seedlings (94.6 and 96%). Boiling water (2.3 or 3 minutes) was also effective in all accessions except for accession 331. However, accessions treated with hot water (3, 5 or 8 minutes) had a considerable proportion of hard seeds. The proportion of dead/abnormal seedlings was high in seeds treated with a hot wire in all accessions, ranging from 27.5 (accession 15489) to 75.5% (accession 16). The untreated seeds remained hard at the final germination count. We conclude that it is important to use concentrated sulphuric acid and mechanical scarification with sandpaper to break the dormancy for the species to enhance germination, obtain uniform field stands and make wise decisions during storage.

Analisis Molekuler Tilapia Lake Virus (TiLV) di Sentra Budidaya Benih Ikan Gurami (Osphronemus goramy) Jawa Timur

Tilapia Lake Virus (TiLV) is a New Emerging Disease that has been defined and included in the WOAH disease list (WOAH, 2022). This virus can infect both freshwater and marine fish, causing high mortality in Ttilapia up to 90%. Reverse transcyptase method - Semi Nested Polymerase Chain Reaction (RT-snPCR) for detection of TiLV in Osphronemus goramy. TiLV was discovered for the first time in Thailand and was also found in Jogjakarta in 2021. Further research is needed to see the damage and genetic variation caused by TiLV in Osphronemus goramy. This study aims to study the genetic variability and homology of TiLV isolates in Osphronemus goramy as definitive hosts in East Java. The results showed clinical symptoms of lethargic fish, decreased appetite, abnormal behavior, reduced reflexes, sluggish movements, darker body color, congestion, thinning fins and exopthalmia. The PCR results showed that the Osphronemus goramy showing clinical symptoms of TiLV infection were all positive. The sequencing results were further identified using the BLAST program, showing the highest similarity of the genetic identity of the sample at 96.8% with TiLV from Israel (Genebank Accession Number NC 029927.1).

Novel microbial synthesis of rGO nanosheets for effective photocatalytic remediation of Acid Blue 113 dye, improved seed germination, antimicrobial and antioxidant applications

We report for the first time reduction of graphene oxide (GO) nanosheets (NS) by an isolated bacterium from Lonar lake Bacillus cabrialesii RIRD-SK (MCC 4670). Facile, rapid, ecofriendly, and cost-effective biological approach makes it advantageous over physical and chemical methods. Isolated bacterial strain is deposited in Gene Bank accession number MMZ151886. The physiochemical characterization of rGO NS revealed formation of dark black precipitate within 8 h. UV–Visible spectra of rGO shows absorption peak mainly at 269 nm. Fourier-transform infrared spectroscopy (FTIR) revealed presence of functional biomolecules involved in capping and stabilization of NS. X- ray diffraction (XRD) pattern of rGO confirms pure crystal structure. Scanning electron microscopy (SEM) of rGO showed ultrathin graphene NS morphology having thickness less than 3.5 nm. The elemental composition of graphene oxide (GO) and rGO was studied by energy dispersive spectroscopy (EDS) confirms bacterial reduction GO. Specific surface area rGO about 139.53 m2/g was measured by Brunauer-Emmett-Teller (BET) technique. Raman spectroscopy indicates decrease oxygen containing functional group after bacterial reduction with unique fingerprint of material. The rGO NS showed 90.10 % degradation of Acid Blue 113 diazo dye within 45 min. Bioremediation was confirmed with the help of UV–Visible spectroscopy, thin layer chromatography (TLC), FTIR, High-performance liquid chromatography (HPLC) and Liquid chromatography mass spectroscopy (LCMS). Reduced toxicity of the degraded products was observed by the phytotoxicity experiment. A mechanistic degradation pathway has been proposed. rGO NS also exhibit good antimicrobial and antioxidant properties emphasizing its role in biomedical field.

Nitric Oxide and Hypochlorite Assessment and Screening of some β-Lactamase Encoding Genes among Gram Negative Bacteria in Patients with Acute Leukemia

Objective: The serologic levels of both nitric oxide (NO) and hypochlorite (ClO-) were assessed in this study for Iraqi acute leukemic patients infected with Gram negative bacteria (GNB). Alongside, the phylogenetic analysis for both blaSHV and blaTEM genes of GNB was performed. Methods: The clinical samples were recovered from acute leukemic patients at hematology center in Baghdad from January 2021 to December 2021. The identification of bacterial isolates and susceptibility test were performed using Vitek 2 Compact System. Serum levels of NO and ClO- were assayed by Enzyme Linked Immunosorbent assay and colorimetric method, respectively. Gene screening was done utilizing polymerase chain reaction. DNA sequencing, GenBank accession numbers submission, and phylogenetic analysis were also conducted. Results: From 260 patients with acute leukemia, 485 clinical samples were collected from different sites. Of this total, 70 (15%) isolates of GNB were obtained, distributing as Klebsiella pneumoniae 23 (33%), Escherichia coli 21 (30%), Pseudomonas aeruginosa 18 (26%), and Acinetobacter baumannii 8 (11%). These isolates were mainly collected from urine 39 (55.71%), followed by blood 23 (32.85%), and the least from swabs 8 (11.42%). The infections of GNB were higher among acute myeloblastic leukemia (AML) patients 40 (57.14%) than these with acute lymphoblastic leukemia (ALL) 30 (42.85%). The levels of NO were higher among groups of patients than control groups. Additionally, ClO-levels were observed to be slightly increased in patients above these of controls. Most isolates of K. pneumoniae and E. coli showed high resistance rates for penicillins (ampicillin and ticarcillin), cephalosporins (ceftazidime, cefotaxime, ceftriaxone, and cefepime), followed by gentamicin and ciprofloxacin. Cefoxitin, aztreonam, imipenem and meropenem were nearly more effective against GNB isolates. On the other hand, about half isolates P. aeruginosa and all A. baumannii were resistant to the tested antibiotics. Extended Spectrum β- Lactamases (ESBLs) were released in 49 (70%) of the tested isolates of GNB. The multidrug resistant (MDR) pattern was noticed among 58 (82.85%) of GNB. Interestingly, ESBLs producing MDR isolates were determined in 34 (58.62%) of the studied GNB. Genotypic screening revealed that blaSHV and blaTEM genes were characterized in 20 (28.57%) and 32 (45.71%) of GNB isolates, respectively, while all GNB were negative for blaCTX-M. DNA sequencing of blaSHV amplicons revealed the occurrence of one point mutation (111T>A) in six isolates of K. pneumoniae with a missense effect (p.31Q>L) on the encoded protein. Forty-five GenBank accession numbers were recorded in NCBI to represent the studied variants of ESBLs. Phylogenetic analysis of both blaSHV and blaTEM displayed the possible epidemiologic routes of the tested GNB by detecting the origin host, source of collection, and geographic spread as compared with the reference sequences. Conclusion: high serum levels of both NO and ClO- indicated the potential role of these microbicidal agents to predict the serious infections of GNB in acute leukemia population. Phylogenetic analysis represented an important tool, possibly aiding in early control and prevention of pathogenic GNB.

Identification and characteristic analysis of an extracellularsignal-regulated kinase from Ostrinia furnacalis Guenée.

The extracellular signal-regulated kinase (ERK) pathway, a critical genetic determinant, controls diverse physiological functions, including innate immunity, development, and stress response. In the current study, a full-length cDNA (1592bp) encoding the ERK gene (OfERK) was cloned from Ostrinia furnacalis Guenée (GenBank accession number: MF797866). The open reading frame of the OfERK gene encoded 364 amino acids and shared 96.43%-98.08% amino acid identities with other insect mitogen-activated protein kinases. For spatiotemporal analysis of the expression pattern, OfERK exhibited a significant peak expression on the 3rd day of the pupa stage and showed the highest expression in hemocytes specifically. Indirect immunofluorescenceassays and immuno-electron microscopy revealed a wide distribution of the OfERK protein in hemocytes and epidermis. Moreover, the results demonstrated that the Bt Cry1Ab-activated toxin significantly induces the expression of OfERK. Other genes related to immune response, development, and stress response exhibited dynamic changes in expression after Cry1Ab oral treatment. The expression of OfERK was downregulated through RNA interference, and the correlation of its expression with other related genes was verified using quantitative real-time polymerase chain reaction. Our study provides valuable insights into the regulatory mechanism of ERK in insects for future studies.

DNA barcoding of an endangered plant species, salvadora oleoides

Salvadora oleoides Decne is an endangered plant species. This species is important as the plant is used for multiple purposes. Natives use the plants for their medicinal needs in treating rheumatic pains and coughs, and many others. This species is threatened by over-exploitation and deforestation or illegal logging, as well as its very slow growth rate, so the accurate identification of this species is important. Knowledge about threats or even distribution is limited, making identifying species more difficult. Therefore, this research aimed at DNA barcoding of endangered species Salvadora oleoides to assess their potential at the meaningful taxonomic level. This study aims to amplify the rbcL and matK genes in Salvadora oleoides collected from the different regions of Pakistan. The plant DNA isolation was carried out by the CTAB method. PCR analysis was performed, which showed amplification of both rbcL and matK regions. The amplified products were purified and sent for sequencing. The results revealed a 100% sequencing rate for rbcL; however, the matK amplified product was not sequenced due to the high heterozygosity in this region, which might result in polymerase slippage during the Sanger sequencing method. The rbcL sequence was checked for homology by BLASTn and then submitted to NCBI for GenBank accession. The accession number assigned to Salvadora oleoides was OP046316 for rbcL. Keywords: DNA Barcoding, Medicinal Plants, matk, rbcl, BLAST, Species Identification.

Genome sequence of a polerovirus isolated from wild oat in Australia.

We present the genome sequence of a polerovirus (family Solemoviridae) isolated from wild oat (Avena fatua) in Australia. The genome sequence consists of 5,631 nucleotides and shares 87% nucleotide identity with its closest relative, cereal yellow dwarf virus RPV isolate 010 (GenBank accession number EF521830).

First report of a 'Candidatus Phytoplasma australasiaticum'-related phytoplasma strain associated with shoot proliferation disease of variegated croton in Taiwan.

Codiaeum variegatum (family Euphorbiaceae) is a leaf ornamental commonly known as variegated croton, which is often found in gardens or grown as indoor plants. In December of 2022, two cutting-propagated variegated croton plants exhibiting abnormal shoot proliferation and little leaf symptoms (Fig. S1) were found in a nursery owned by a private breeder in Wanluan Township, Pingtung County, Taiwan. The plants were propagated from a single stock plant, which died during transplanting from a commercial nursery in Changzhi Township, Pingtung County. To determine the potential cause of such symptoms, leaf tissues were collected from the center of the two symptomatic plants. Their DNA were extracted with a Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics, Lebanon, NJ) and used for further testing. As controls, two symptomless stock plants were collected from the commercial nursery in Changzhi Township and used to produce cutting-propagated plants; leaf DNA was extracted from each plant as described above. The DNA samples were subjected to PCR testing using the phytoplasma-specific primer pair P1/P7 (Schneider et al. 1995), and only DNA from the symptomatic plants produced the expected 1.8-kb amplicon. The two phytoplasma isolates detected in the plants were designated as CvaA and CvaB. After sequencing and analyzing the data using the iPhyClassifier program (Zhao et al. 2009), both CvaA and CvaB were classified to subgroup 16SrII-A (GenBank accession no. L33765) with a similarity coefficient of 1.0. The near-full-length 16S rDNA fragments of the detected isolates (GenBank accession no. OR794242) were also identical (1,463/1,463 bp) to that of NCHU2014 (GenBank accession no. CP040925, bp 537768-539230), a reference 'Ca. Phytoplasma australasiaticum'-related strain (16SrII-A) found in Taiwan (Chang et al. 2015; Rodrigues Jardim et al. 2023). To validate the results, the DNA samples were also tested with 16SrII group-specific semi-nested PCR targeting the elongation factor Tu gene. The outer and inner primer pairs used were TUF-II-F1/TUF-II-R1 and TUF-II-F2/TUF-II-R1, respectively (Al-Subhi et al. 2017). An expected amplicon was detected in the symptomatic samples but not in the symptomless counterparts. The amplified fragments' sequences (GenBank accession no. OR634931) were identical to that of the elongation factor Tu gene of the 'Ca. Phytoplasma australasiaticum'-related strain NCHU2014 (989/989 bp; GenBank accession no. CP040925, bp 139344-140332). The protein translocase gene secY of the detected phytoplasma was also amplified and sequenced using semi-nested primers SecYF1(II), SecYF2(II) and SecYR1(II) (Lee et al. 2010). Again, the sequences of the detected isolates (GenBank accession no. OR862773) were identical to that of NCHU2014 (1,263/1,263; GenBank accession no. CP040925, bp 192846-194108). The quality of the DNA samples was confirmed by PCR targeting the plant host's 28S rDNA using primer pair 28KJ/28C (Cullings 1992) and all symptomatic and symptomless samples produced the target amplicon (0.7 kb). 16SrII phytoplasmas have been detected in different host plants in Taiwan (Chang et al. 2015). To our knowledge, this is the first record of this group of pathogens infecting variegated croton in Taiwan. Branch-inducing phytoplasma has been used to improve the ornamental values of poinsettias, another Euphorbiaceae species (Lee et al. 1997). Further testing is needed to determine whether the phytoplasma detected in this work could be used for similar purposes.

First Report of Vacuiphoma oculihominis Causing Fruit Rot on Navel Orange (Citrus sinensis) in China.

Newhall navel orange [Citrus sinensis (L.) Osbeck] is an economically important agricultural product in China. In February 2022, a rare lesion symptom was observed on Newhall navel oranges that were harvested from an orchard Ganzhou city, Jiangxi province, China (25.53° N, 114.79° E) and stored for 90 days (18±2℃, 80 to 90% RH) at the Jiangxi Key Laboratory for Postharvest Technology and Non-destructive Testing of Fruits and Vegetables (28.68° N, 115.85° E). Approximately 2% (15/750) of the oranges exhibited symptoms, with normal appearance but ink-black flesh and juice, yellowish lesions on edges of the symptoms, and no unusual odor. To isolate the pathogen, three 5 × 5 mm pieces of symptomatic tissue from a diseased orange were disinfected in 75% ethanol for 30 s, rinsed three times with sterile water, and inoculated on potato dextrose agar (PDA) at 25±1℃ and a 12:12 h photoperiod for 7 days. A pure isolate named ND-hsp was obtained. The colony was light yellow center with pale edge on the top and brown on the bottom. Conidia and pycnidia were observed on PDA medium after 2 months. Conidia were long oval, no septa, 2.9 × 3.4 μm (n = 50), and pycnidia were solitary, 39.4 × 43.9 μm (n = 20), with one or no orifice, brown to dark brown. The morphological characteristics of ND-hsp strain on PDA, oatmeal agar and malt extract agar were similar to those of the Didymellaceae (Aveskamp et al. 2010). Ulteriorly, the genomic DNA of the ND-hsp isolate was extracted from its mycelia using a fungal genomic DNA extraction kit (Solarbio, Beijing, China) for subsequent phylogenetic analyses. Four primer sets, LR0R (Rehner and Samuels 1994) /LR7 (Vilgalys and Hester 1990), V9G (Hoog and Gerrits 1998) /ITS4 (White et al. 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and RPB2-5F2 (Sung et al. 2007)/RPB2-7cR (Liu et al. 1999) were used to amplify the corresponding DNA fragments of large subunit ribosomal RNA (LSU), internal transcribed spacer region (ITS), beta-tubulin gene (TUB2) and RNA polymerase Ⅱ second largest subunit (RPB2), respectively. The obtained sequences were assigned GenBank accession numbers and showed 99 to 100% identity with their counterparts of Vacuiphoma oculihominis UTHSC DI16-308. A phylogenetic tree was constructed in MEGA 7.0 using the concatenated sequences, placing the isolate within the V. oculihominis clade by 100% bootstrap support. Pathogenicity experiments were performed in triplicate. Ten Newhall navel oranges were surface sterilized with 75% ethanol and inoculated with 15μL of a spore suspension (2×106 spores/ml) into a 3 mm-diameter wound on the equator. The control group received sterile water instead of the spore suspension. Treated and control oranges were incubated at 25±1 ℃ and about 90% relative humidity for 20 days. All oranges were cut longitudinally or transversely through the inoculated wound and examined internally. The oranges inoculated with ND-hsp exhibited ink-black flesh and juice symptoms consistent with the initial oranges. The control oranges remained asymptomatic. Under the Koch's rule, V. oculihominis was reisolated from diseased oranges and kept in Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province. GenBank database analysis confirms that V. oculihominis has been found in human eye secretions and decayed trees. This is the first report of V. oculihominis as a pathogen on navel oranges in China. Our findings contribute to understanding of citrus fruit pathogens.

First Report of Wilt Disease of Ginger Caused by Achromobacter xylosoxidans in China

Ginger (Zingiber officinale) is an important commercial crop that has been widely cultivated in China for more than 2500 years. One variety, Tongling white ginger, has been grown in the Yi'an District of Tongling city, Anhui province (30°45 N, 117°43 E), China. In August 2022, symptoms of yellowing and wilting were observed on ginger plants, with a disease incidence rate exceeding 20% in the field. The stem base of the diseased plants became soft, brown and water-soaked. Additionally, the rhizomes displayed symptoms of browning and water-soaked rot, resembling those caused by Ralstonia solanacearum and Enterobacter cloacae (Yu et al. 2003; Nishijima et al. 2004; Liu et al. 2021). Consequently, ginger bacterial wilt disease may potentially emerge from a combination of infections by diverse pathogenic bacteria. To identify novel pathogens causing the wilt disease, stem tissues of the diseased plants from different locations were sterilized with 1% sodium hypochlorite (NaOCl) for 10 min, followed by at least three time rinses with sterile water. The sterilized samples were then ground with 0.9% saline solution and left at room temperature for 30 min. A 20 μL aliquot of the suspension was serially diluted and cultured on Luria-Bertani (LB) medium at 28°C. A total of 217 isolates was picked and purified for taxonomic identification by 16S rRNA gene analyses with the primer 27F/1492R (Weisburg et al. 1991). Among these isolates, 23 single colony isolates were identified as A. xylosoxidans through NCBI BLASTn analyses. Furthermore, three single isolates from different locations were randomly selected for further experiments. The growing colonies appeared opaque white and round. Microscopic evaluation revealed that cells were rod-shaped with an average length of 1.95 µm and average width of 0.46 µm. The three isolates shared identical 16S rRNA sequences, displayed 99.72% identity with the sequence from A. xylosoxidans strain SeqID2 (GenBank accession NO. MH266081.1). The glutamate synthase (gltB), GTP-binding membrane protein (lepA), NADH:ubiquinone oxidoreductase subunit L (nuoL), RNA polymerase beta-subunit (rpoB), and the enolase (eno) genes of the three isolates were amplified by PCR using primer pairs gltB-F/gltB-R, lepA-F/lepA-R, nuoL-F/nuoL-R, rpoB-F/rpoB-R and eno-F/eno-R, respectively (Spilker et al. 2012; Vandamme et al. 2016). The gene sequences of 16S rRNA (OQ711945, OQ740153 and OR230037), gltB (OR242732, OQ737692 and OR262112), lepA (OR233727, OQ737693 and OR262113), nuoL (OR233726, OQ737694 and OR262114), ropB (OR233725, OQ737695 and OR262115) and eno (OR242733, OQ737696 and OR262116) for the isolates ZOR02, ZOR05 and ZOR12 were deposited in GenBank database. The gltB, lepA, nuoL, rpoB and eno sequences of the isolates ZOR02, ZOR05 and ZOR12 showed 98.66-99.16%, 98.9-100%, 96.28%-97.34%, 98.47-99.44% and 99.27-99.82% similarity to A. xylosoxidans strain AX27, respectively. Phylogenetic trees were constructed based on the 16S rRNA and gltB-lepA-nuoL-rpoB-eno multilocus using the Neighbor-Joining (NJ) method with 1000 bootstrap replicates in MEGA11.0 software (Álvarez et al. 2018). For pathogenicity tests, bacterial suspensions were initially prepared in sterile water at a final concentration of 108 CFU mL-1. Subsequently, 10 μL of bacterial suspensions was injected into the stem base of two-month-old ginger plants, while sterile water was used as a control (Wang et al. 2022). These plants were then incubated at 28°C and 70% relative humidity. There were three replicates for each treatment, and each replicate contained five plants. After six days of inoculation, the ginger plants injected with bacterial suspensions alone exhibited severe wilting symptoms similar to those observed in the field. However, water-soaked symptoms were not observed on rhizome tissues from the pathogen-infected plants. Bacterial pathogens were re-isolated from the diseased plants and identified using the morphological and molecular methods to meet Koch's hypothetical tests. To our knowledge, this is the first report of A. xylosoxidans causing wilt disease of ginger in China. In 2022, the average yield loss due to wilt disease in the Yi'an District of Tongling exceeded 20%, posing a major threat to local ginger cultivation. Effective disease management strategies are needed to develop for the control and prevention of the disease.

First Report of 16SrII Group-Related Phytoplasma Associated with Witches’ Broom Disease on Cowpea (Vigna unguiculata) in Hainan Province, China

Cowpea (Vigna unguiculata L.), a significant vegetable crop in China, holds particular prominence in the tropical island of Hainan. This region serves as the primary production area for the winter cultivation of cowpea. Phytoplasmas are an idiopathic parasitic pathogen and cannot be cultured in vitro. It is mainly transmitted by the insect vectors with the piercing and sucking mouthparts, such as leafhoppers, plant hoppers, and psyllids. (Kumari et al. 2019). On September 11, 2023, typical characteristics of phytoplasma diseases on cowpeas were observed in the experimental base of Hainan Academy of Agricultural Sciences (20°0′38.6964″N, 110°21′35.4024″E, Haikou City, Hainan Province, China), including reduced leaf size, chlorosis, and the development of broom-like branch deformities reminiscent, as depicted in Figure 1. At the same time, we found a large number of leafhoppers near the diseased plants, and we speculated that leafhoppers are the insect carriers that spread the disease. Following an on-site investigation, it was determined that the disease incidence ranges from 10% to 15%, leading to a consequential decrease of about 10% in yield, which is a potential disease that seriously threatens the cowpea industry in Hainan. Ten disease and healthy samples were meticulously collected and subsequently preserved at -80°C within the laboratory refrigerator. Three disease samples denoted as HNNKY-1, HNNKY-2, and HNNKY-3, were randomly chosen, and total DNA extraction was carried out employing the NuClean Plant Genomic DNA Kit (CWBIO, Taizhou, China), while three healthy samples were randomly selected as control. The 16S rRNA gene was amplified by PCR using the primer pairs P1/P7 (Schneider et al. 1995) and R16F2n / R16R2 (Lee et al. 1993) and the secA gene was amplified by PCR using the primer pairs secAfor1/secArev3 (Hodgetts et al. 2008). After agarose gel electrophoresis analysis, no DNA fragments were observed in the healthy leaf samples, whereas all three disease samples yielded amplification products. The PCR products were subsequently sequenced by Hainan Nanshan Biotech Co., Ltd., Haikou, China. After sequence analysis, it was found that the 16S rRNA gene and secA gene sequences HNNKY-1, HNNKY-2, and HNNKY-3 were identical to each other. We selected two gene sequences of strain HNNKY-3 to submission to the GenBank database, The length of the 16S rRNA gene sequence is 1193 base pairs, identified by the accession number OR666421, while the secA gene sequence is 825 base pairs in length, associated with the accession number OR661282. The phytoplasma strain HNNKY-3 was named 'Vigna unguiculata' witches'-broom phytoplasma. A BLAST analysis of the 16S rRNA gene revealed that strain HNNKY-3 displayed a 100% sequence match with 'Emilia sonchifolia' witches'-broom phytoplasma (MT420682), Peanut witches'-broom phytoplasma (OR239773), and 'Raphanus sativus' witches'-broom phytoplasma (OK491387). All of these phytoplasmas were classified within the 16SrII group. Based on the BLAST analysis of partial secA gene sequences, it was discerned that sequence homogeneity ranged from 99.27% to 99.74% among the studied sequences. These sequences were collectively classified as members of the 16SrII group. In addition, a phylogenetic tree was constructed by MEGA 11 (version 11.0.13) based on the 16Sr RNA gene and secA gene by the neighbor-joining method (Tamura et al. 2004). The results demonstrated the clustering of HNNKY-3 phytoplasma strains within the 16SrII group, as illustrated in Figures 2 and 3. A virtual RFLP analysis based on the 16S rRNA gene fragment of HNNKY-3 was conducted using the interactive online phytoplasma classification tool, iPhyClassifier (Zhao et al. 2009). The results indicated that the phytoplasma strain was the same as the reference pattern of the onion yellows phytoplasma of 16SrII-A (GenBank accession: L33765), and the similarity coefficient was 1.00. To best of our knowledge, this is the inaugural documentation of 16SrII Group-related phytoplasma infecting cowpea in Hainan, China, and lays the groundwork for further research on the dissemination of cowpea phytoplasma disease within China.

Confirmatory test of active IHHNV infection in shrimp by immunohistochemistry and IHHNV-LongAmp PCR.

The presence of endogenous viral elements (EVE) in the penaeid shrimp genome has been recently reported and suggested to be involved in the host recognition of viral invaders. Our previous report of a search for EVE of infectious hypodermal and haematopoietic necrosis virus (IHHNV-EVE) in the Thai Penaeus monodon whole genome sequence project (GenBank accession no. JABERT000000000) confirmed the presence of three clusters of EVE derived from IHHNV in the shrimp genome. This study aimed to compare an immunohistochemistry method (IHC) and a PCR method to detect infectious IHHNV infection in shrimp. First, specimens collected from farms were checked for IHHNV using three PCR methods; two methods were recommended by WOAH (309 and 389 methods), and a newly established long-range PCR for IHHNV (IHHNV-LA PCR) targeting almost the whole genome (>90%) of IHHNV. Among 29 specimens tested, 24 specimens were positive for WOAH methods (at least one method). Among 24 WOAH-positive specimens (WOAH+), there were 18 specimens with positive IHHNV-LA PCR method (WOAH+/LA+), six specimens with negative IHHNV-LA PCR method (WOAH+/LA-). Six specimens were negative for all methods (WOAH-/LA-). The positive signals detected by IHC method were found only in the specimens with WOAH+/LA+. The results suggest that the WOAH+/LA- specimens were not infected with IHHNV, and the positive WOAH method might result from the EVE-IHHNV. The study recommends combining the IHHNV-LA PCR method and IHC with positive PCR results from WOAH's recommended methods to confirm IHHNV infection.