Ml Of Sterile Distilled Water Research Articles

First Report of Xanthomonas euvesicatoria pv. sesami Causing Leaf Spot on Sesame (Sesamum indicum L.) in South Carolina, USA.

Sesame (Sesamum indicum L.) is an annual plant known as one of the first domesticated oilseed crops. It is cultivated worldwide, mostly in Asia, Africa, and the Americas (Singh, 2006). In August 2022 and September 2023, dark angular necrotic spots on leaves and stems (100% incidence), blights, and severe defoliation were observed in a 4-acre rainfed sesame field located in the Colleton County of South Carolina, USA (Fig. S1). Bacterial streaming from cut leaf lesions was observed from diseased plants in both years. Two plants were collected for pathogen isolation in 2023. Symptomatic leaves were surface sterilized with 70% ethanol for 1 min and dried in a laminar flow hood. For each isolate, four sterile toothpicks were used to poke lesion margins and stirred in 300 µl of sterile distilled water in a 2-ml sterile microcentrifuge tube and soaked at room temperature (c. 21 °C) for 10 min. Each bacterial suspension (10 µl) was streaked on nutrient agar (NA) in a Petri dish. Convex and mucoid yellow colonies formed after a 48-h incubation at 28°C in the dark. Two isolates (S813 and S814), one from each plant, were obtained by transferring single colonies to new NA plates. Both isolates were preliminarily identified as Xanthomonas [S813: X. campestris (P = 0.53); S814: X. campestris (P = 0.77)] using a Biolog Microbial Identification System (GEN III Microplate; Identification Database v.2.8.0.15G). PCR amplification of the atpD and dnaK genes was performed for both isolates using the conditions described in Félix-Gastélum et al. (2019). The sequences of both amplicons are 100% identical for each gene between the two isolates. PCR and sequencing of the gyrB gene was also done for S813 with the primers from Young et al. (2008). The atpD (S813/S814), dnaK (S813/S814), and gyrB (S813) sequences (GenBank accessions: PP507118 to PP507120) showed the best match with 100% identity to the corresponding gene sequences [GenBank accessions: KJ491167 (100% coverage), KJ491257 (99% coverage), EU285201 (100% coverage)] of the X. euvesicatoria pv. sesami (=X. campestris pv. sesami) type strain LMG865 (Constantin et al. 2015, Parkinson et al. 2009). A neighbor joining tree with the concatenated sequences of these three genes (2,210 nt) showed that S813 and LMG 865 had the closet relationship with X. euvesicatoria pv. alfalfae (CFBP3836, Fig. S2). To fulfill Koch's postulates, three healthy sesame plants (cultivar Shirogoma) were spray inoculated separately with each suspension of S813 and S814 in sterile tap water until runoff (approx. 5×108 CFU/ml). Two sesame plants were sprayed with sterile tap water and served as negative control. All plants were maintained in a greenhouse at approximately 28/20°C (day/night) with natural photoperiod. Dark leaf spots and leaf yellowing were observed on inoculated plants 7 to 14 days after inoculation. No disease symptom was observed on the control plants. Bacteria were reisolated from leaf spots of the inoculated plants and confirmed to be X. euvesicatoria pv. sesami based on atpD and dnaK sequences. The disease was first reported in Sudan (Sabet and Dowson, 1960), after which it was reported in USA (Isakeit et al., 2012) and Mexico (Félix-Gastélum et al. 2019). To the best of our knowledge, this is the first report of this disease in South Carolina, USA. Since the interest of sesame to the farmers is increasing in the southeastern USA, it is necessary to perform further research to examine the disease distribution and its economic impact.

First Report of Anthracnose on Wurfbainia villosa var. villosa Caused by Colletotrichum gloeosporioides in China.

Wurfbainia villosa var. villosa is a traditional Chinese herbal medicine under the family Zingiberaceae, and its ripe fruits (called Fructus Amomi) are widely used clinically for the treatment of gastrointestinal disorders (Yang et al. 2023; Chen et al. 2023). In September 2023, plants of W. villosa var. villosa exhibited anthracnose-like symptoms on leaf with a disease incidence of 35% (n = 100 investigated plants) in an approximately 90 m2 field in Guangning, China (N23°42'51.70″, E112°26'35.75″). Light yellowish-green spots (~2 mm diameter) initially appeared on the infected leaves, gradually formed sub-circular or irregular spots, then fused and expanded, resulting in wilting of the leaves. To identify the causal agent, 10 symptomatic leaves were collected and transferred to the laboratory. The symptomatic leaf samples were surface sterilized in 0.5% NaClO for 2 min, and in 70% ethanol for 30 s, then washed three times with sterile water and air-dried on sterile filter paper. The leaf tissues were placed on potato dextrose agar (PDA) medium containing 100 μg mL-1 of ampicillin (Sigma-Aldrich, St. Louis, MO) and incubated for 7 days at 28°C in darkness. Nine isolates with similar colony morphology were isolated from the 10 plated leaves. Three representative isolates (GNAF03, GNAF06, GNAF09 with approximately 3.5 cm in diameter after 3 days of incubation) appeared gray to dark brown with dense aerial hyphae at the front and gray to black colonies on the reverse of the plates. Conidia were cylindrical and measured 21.2 to 29.3 μm long × 7.1 to 9.6 μm wide (n = 50). Appressoria were formed by the tips of germ tubes or hyphae and were brown, ellipsoid, thick-walled, and smooth-margined, measuring 10.2 to 12.3 μm long × 6.4 to 8.2 μm wide (n = 50). Morphologically, the fungal isolates resembled Colletotrichum sp. (Weir et al. 2012). For molecular analysis, genomic DNA was extracted from fresh mycelia of the three isolates, and the primers ACT-512F/ACT-783R, CL1/CL2A, GDF/GDR, and ITS1/ITS4 were used to amplify partial regions of rDNA-ITS, actin (ACT), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regions, respectively (Weir et al. 2012). The resulting sequences with more than 99% nucleotide identity to C. gloeosporioides were submitted to GenBank (accession numbers PP552725, PP552726, and OR827444 for ACT; PP552727, PP552728, and OR827443 for CAL; PP552729, PP552730, and OR827445 for GAPDH; PP549996, PP549999, and OR841394 for ITS). A phylogenetic tree was generated by the maximum likelihood method using the concatenated sequences of ACT, CAL, GADPH, and ITS by Polysuite software (Damm et al. 2020). Based on morphological and molecular analysis, the three isolates were characterized as C. gloeosporioides. The pathogenicity of the GNAF09 isolate was assessed on W. villosa var. villosa seedling leaves inoculated by spraying with 40 μL of conidial suspension at 106 conidia mL-1 or wounded with a sterile toothpick then inoculated with mycelial agar plugs (5 mm diameter). Control leaves were inoculated with 40 μL of sterile distilled water or agar plugs without mycelia. The inoculated plants were placed in a humid chamber at 28°C with 80% humidity and a 12 h light-dark photoperiod. Symptoms similar to those seen on naturally infected leaves were observed on all inoculated leaves after 7 days inoculation. Re-isolation was performed from 80% of the inoculated leaves and isolates were confirmed as C. gloeosporioides morphologically, confirming Koch's postulates, and by sequencing the ACT, CAL, GADPH, and ITS regions. The control groups remained asymptomatic. In previous studies, C. gloeosporioides has also caused anthracnose on Chinese medicinal plants, including Baishao (Radix paeoniae alba) (Zhang et al. 2017) and Rubia cordifolia L. (Tang et al. 2020). To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on W. villosa var. villosa in China. The results of our report serve as valuable references for further research on this disease.

Efficacy of Native Bacillus thuringiensis against Mosquito Vector.

Larval source management is an effective measure to control mosquito-borne diseases. Bacillus thuringiensis produces specific insecticidal crystal proteins toxic to mosquito larvae. In many parts of the South East Asian region, Bacillus thuringiensis is used for larval source management. In Nepal, larvicidal Bacillus thuringiensis is not available. The study aims to isolate larvicidal Bacillus thuringiensis from soil samples of Nepal to control mosquitoes. Native Bacillus thuringiensis was obtained from soil samples by the acetate selection method. It was identified by observing crystal protein with Coomassie Brilliant Blue stain in a light microscope. The mosquito larvae were collected from different breeding habitats. A preliminary bioassay was performed by inoculating three loopful of 48 hours culture of spherical crystal protein producing Bacillus thuringiensis in a plastic cup containing 25 larvae and 100 ml of sterile distilled water. The cup was incubated at room temperature for 24 hours to observe the mortality of larvae. Further selective bioassay was performed with the isolate which showed 100% mortality, as described above in four replicates along with the negative and positive control. Out of 1385 Bacillus thuringiensis obtained from 454 soil samples, 766 (55.30%) were spherical crystal protein producers, among them, a single strain (14P2A) showed 100% mortality against mosquito larvae. The lethal concentration doses required to kill 50% and 90% of the larval population were 32.35 and 46.77 Parts per million respectively. The native Bacillus thuringiensis produces the crystal protein effective in killing mosquito larvae. The native Bacillus thuringiensis should be included as a tool to control mosquito-borne diseases in Nepal.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Peanut in Chongqing, China.

In July 2022, dieback and discoloration were detected on infected stems of peanut in Qijiang District of Chongqing (106.56°E,29.41°N), China, with an incidence up to 5%. These peanut stems had disease symptoms typical of anthracnose with irregular gray-brown spots with dark brown edges, sunken, and necrotic. High temperature and high humidity were favorable for the growth of the pathogen. To isolate the pathogen, we collected 10 typical infected peanuts and cut one piece from each of symptomatic stems, surface sterilized with 0.5% NaClO for 1 min, and 75% ethanol for 30 s, then rinsed three times with sterile distilled water and dried on sterilized filter paper. These pieces were incubated on potato dextrose agar (PDA) at 25°C in the dark. Pure cultures were obtained from hyphal tips of each colony. It was found that isolates with the same colony morphology were isolated from each infected stem. A representative isolate (L7) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. The colonies appeared white to gray, with white margins and aerial hyphae, and the reverse of the colonies was gray to brown. Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, 13.4 to 18.8 × 4.2 to 5.8 μm (n=50). Morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides species complex (Cannon et al., 2012). For molecular identification, genomic DNA was extracted using a CTAB method and partial sequences of β-tubulin (TUB2), actin (ACT) genes, chitin synthase (CHS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced using primers T1/T2, ACT-512F/ACT-783R, CHS-79F/CHS-345R, and GDF1/GDR1, respectively (Damm et al., 2012; Dowling et al., 2020). Using the BLAST, TUB2, ACT, CHS and GAPDH gene sequences (GenBank accession No. OR714793, OP168707, OP168708 and OR714794, respectively) were100% (429 bp out of 429 bp), 99.22% (256 bp out of 258 bp), 99.64% (276 bp out of 277 bp) and 100% (253 bp out of 253 bp) identical to C. gloeosporioides CBS:112999 (JQ005587, JQ005500, JQ005326, and JQ005239), respectively. Using Neighbor-Joining algorithm, phylogenetic analysis was conducted based on the concatenated sequences of published TUB2, ACT, CHS and GAPDH genes. The identified isolate (L7) was closely related to C. gloeosporioides. To evaluate pathogenicity, the stems of ten peanut (Zhonghua12) seedlings (2 weeks) were wounded with a sterile toothpick and mycelial plugs (5 mm in diameter) or 20 μl of conidial suspension (105/ml) were inoculated. Non-colonized agar plugs or 20 μl of sterile distilled water were treated as control. After inoculation, the peanuts were kept in a moist chamber at 28°C with 80% humidity in the dark for 24 h, and subsequently transferred to the moist chamber with 12 h light and darkness cycle for 6 days, similar symptoms were observed on all inoculated peanuts. Controls remained asymptomatic. C. gloeosporioides was reisolated from the diseased stems and confirmed using morphological features and sequence analysis of TUB2, ACT, CHS and GAPDH. Anthracnose caused by C. truncatum and C. fructicola has been reported on peanut leaves in China (Gong et al., 2023; Yu et al., 2019). To our knowledge, this is the first report of anthracnose on peanut stem caused by C. gloeosporioides in Chongqing. Our report will provide crucial information for studying on epidemiology and management of this disease.

Antibacterial Activity of the Combination of Calcium Hydroxide and Ellagic Acid towards Enterococcus faecalis and Lactobacillus acidophilus

Calcium hydroxide is a material commonly used in pulp capping because of its substantial antibacterial property. However, this antibacterial property of calcium hydroxide is less effective on Enterococcus faecalis and Lactobacillus acidophilus due to their resistance factors. Ellagic acid is a natural phenolic acid with extraordinary antibacterial properties. Combining calcium hydroxide with ellagic acid is expected to enhance its antibacterial properties towards Enterococcus faecalis and Lactobacillus acidophilus. This study aimed to determine the antibacterial activity of the combination of ellagic acid and calcium hydroxide on Enterococcus faecalis and Lactobacillus acidophilus. A suspension of Enterococcus faecalis and Lactobacillus acidophilus bacteria comparable to a McFarland standard 0.5 of 1.5 x 108CFU/ml was made. Three different calcium hydroxide and ellagic acid ratios were made by mixing 0.099g, 0.097g, and 0.095g calcium hydroxide powder with 0.001g, 0.003g, and 0.005g ellagic acid powder, respectively. These powder mixtures were then added with 0.1 mL of sterile distilled water. Four Mueller Hinton Petri dishes were divided into four sections each. The mixtures of these powders were applied to the petri dish with the diffusion method. There are significant differences (p<0.05) between every group for Lactobacillus acidophilus. Similar results are also observed between all groups of Enterococcus faecalis except for group 5 (ratio 97:3) and group 6 (ratio 95:5). The combination of ellagic acid and calcium hydroxide decreases the zone of inhibition of Enterococcus faecalis and Lactobacillus acidophilus.

First Report of Muskmelon Fruit Rot Caused by Fusarium sulawesiense in China.

Muskmelon(Cucumis meloL.) is a widely cultivated and economically important fruit crop worldwide. In June 2022, fruitrot symptoms were observed on ripening muskmelons (cv. Boyang) in Shouguang City (36.81°N 118.90°E) of China. To determine the causal agent, we surveyed 200 muskmelon plants in about 1000 m2 of planting area and collected diseased muskmelons. Approximately 20% of muskmelon fruits had symptoms, and yield loss averaged 20%. Water-soaked lesions were observed on the surface and the fruit rotted from inside. Lesions were covered with white mycelium. Rotted fruit were surface-disinfested with 1% NaOCl for 1 min, 75% ethanol for 30 s, and washed three times with sterile water. Pieces (1 cm3) were cut from the disinfested fruit, placed on potato dextrose agar (PDA), and incubated at 25°C for 1 week. Ten isolates with similar morphology were obtained and isolates SG66 and SG68 were selected for further characterization. Colonies maintained on PDA in the dark had an average radial growth rate of 10-12 mm/d at 25°C. Surface was white, velvety to felty mycelium. Reverse was white to pale wheat. Diffusible pigments were absent. On carnation leaf agar, sporodochia appeared as slimy dots, macroconidia were 3- to 5-septate, 20-35 × 3-5 μm, falcate, with a pronounced dorsiventral curvature, with blunt to papillate apical cell, and barely to distinctly notched basal cell. Microconidia and chlamydospores were not observed. These morphological characteristics were consistent with descriptions of Fusarium sp. DNA was extracted from isolates SG66 and SG68 using a CTAB method. Nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), calmodulin (CAM), RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α gene (TEF1) (Xia et al. 2019) were amplified using generic primers, the products sequenced, and sequences deposited in GenBank (ITS: OP251362, OP251363; CAM: OP266024, OP266025; RPB2: OP266028, OP266029; TEF1: OP266026, OP266027). Isolates SG66 and SG68 clustered with Fusarium sulawesiense (85% bootstrap) (Maryani et al. 2019). The Fusarioid-ID database pairwise alignment of ITS (526 bp), CAM (534 bp), RPB2 (861 bp), and TEF1 (636 bp) sequences from isolate SG66 showed 99.6% (98.9% coverage), 100% (100% coverage), 100% (100% coverage) and 100% (98.4% coverage) similarity with the corresponding sequences (GQ505730, LS479422, LS479855 and GQ505641), respectively, of the reference strains of F. sulawesiense (InaCC F940 and NRRL 34059). To perform a pathogenicity test, 10 μl of conidial suspensions (1 × 106 conidia/ml) were injected into ten muskmelon fruit using a syringe, and ten control fruit were inoculated with 10 μl of sterile distilled water. The test was repeated three times. After 7 days at 25°C, the pulp of all inoculated muskmelons began to rot, and the lesion expanded from the inside to the fruit surface at the injection site and became covered with white mycelia. No symptoms developed on the control fruit. The fungus was successfully re-isolated from infected tissues and confirmed as F. sulawesiense by morphological and phylogenetic analyses. F. sulawesiense has previously been reported onyellow melon (Canary) in Brazil (Lima et al. 2021) and on a range of hosts, including Luffa aegyptiaca, in China (Wang et al. 2019). To our knowledge, this is the first report of muskmelon fruit rot caused by F. sulawesiense in China.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Stevia rebaudiana in Arizona, USA.

Stevia (Stevia rebaudiana Bertoni) is an important medicinal crop grown worldwide. Leaves of stevia contain a non-caloric sweetener, stevioside, which is used as a substitute to artificial sweeteners. In August 2022, symptoms of chlorosis, wilting, and root rot were observed in about 30 % of stevia plants growing at the Agricultural Station at Yuma Agricultural Center, Yuma, AZ, USA (32.7125° N, 114.7067° W). Infected plants initially showed chlorosis and wilting, and the plants eventually died with foliage remaining intact to the plant. Cross sections of the crown tissue of affected stevia plants showed necrotic tissue and a dark brown discoloration in areas of the vascular and cortical tissues. Dark brown microsclerotia were observed on stem bases and necrotic roots of the infected plants. Five symptomatic plants were sampled to isolate the pathogen. Root and crown tissues (0.5 to 1 cm) were surface disinfested with 1% sodium hypochlorite for 2 min, rinsed three times with sterile water, and plated onto potato dextrose agar (PDA). All the five isolates displayed rapid mycelial growth on PDA at 28°C with a 12-h photoperiod. The mycelia were initially hyaline and turned from gray to black after 7 days. Masses of dark spherical to oblong microsclerotia were observed after 3 days on PDA, measuring an average of 75 µm width × 114 µm length (n=30). For molecular identification, genomic DNA was extracted from mycelia and microsclerotia of a representative isolate (Yuma) using the DNeasy Plant Pro kit (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS), translation elongation factor-1α (TEF-1α), calmodulin (CAL), and β-tubulin (β-TUB) regions were amplified using the primer sets, ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), MpCalF/MpCalR (Santos et al. 2020), and T1/T22 (O'Donnell and Cigelink 1997), respectively. A BLAST search of sequences revealed 98.7 to 100% identity to Macrophomina phaseolina sequences (MK757624, KT261797, MK447823, MK447918). Both morphological and molecular characteristics confirmed the fungus as M. phaseolina (Holliday and Punithaligam 1970). Sequences were submitted in the GenBank under accession numbers OP599770 (ITS), OP690156 (TEF-1α), OP612814 (CAL), and OP690157 (β-TUB). Pathogenicity assay was performed on 9-week-old stevia plants (var. SW2267), grown in 4-inch planters in the greenhouse. The inoculum was made from a 14-day-old culture of M. phaseolina grown in conical flasks (250 ml) in potato dextrose broth at 28°C. Mycelial mats of the fungus were blended in 250 ml of sterile distilled water, filtered through four layers of cheesecloth, and then calibrated to 105 microsclerotia/ml using a hemocytometer. Twenty healthy plants were inoculated by soil drenching 50 ml of the inoculum per pot. Soil drenching using sterile distilled water was done on 5 non-inoculated control plants. Plants were maintained in the greenhouse at 28 ± 3°C with 12 h photoperiod. After 6 weeks, necrosis at the base of petioles and chlorosis of the leaves, followed by wilting were noticed on all 20 inoculated plants, whereas all the 5 control plants remained healthy. The fungus was reisolated and identified as M. phaseolina based on the morphology and sequences of ITS, TEF-1α, CAL and β-TUB regions. Although M. phaseolina has been reported earlier on stevia in NC, USA (Koehler and Shew 2018), this is a first report from AZ, USA. M. phaseolina is known to be favored by high soil temperatures (Zveibil et al. 2011), thus represents a potential threat to stevia production in AZ, USA in coming years.

Formulation of Fungal Media from Local Plant Materials

A culture medium is a liquid or gelled substance that promotes microorganism growth in the laboratory. This study is aimed at exploring various local plant sources for fungal media formulation. The local plant materials used in this study were grains of Zea mays, seeds of Glycine max, and boiled and peeled Dioscorea dumetorum tubers. These plant materials were dried separately in the oven at 60°C, pulverized individually, measured in different proportions, and poured separately into conical flasks containing distilled water at various proportions to evaluate the potential of the fungal media formulation. The best formulation was derived from Z. mays (5 g), D. dumetorum (2.5 g), and G. max (2 g) in 50 ml of sterile distilled water (M5Y2.5S2). It was then autoclaved at 121°C for 15 mins at 15 psi and dispensed in various Petri dishes. Media characteristics such as gelling ability and consistency were compared with Sabouraud dextrose agar (SDA) as the standard media. Isolates of Aspergillus niger and Penicillium expansum were used; these were derived from deteriorating yam and bread and cultured on the media. The colony diameter, aerial growth, and sporulation of the test fungal isolates were ascertained. When compared to SDA ( 8.92 ± 0.10 ), the colony diameter of A. niger on M5Y2.5S2 ( 8.90 ± 0.10 ) did not differ significantly ( P = 0.05 ). This is also applicable in the case of P. expansum on M5Y2.5S2 ( 8.93 ± 0.06 ) when compared with SDA ( 8.92 ± 0.07 ). There was a significant difference ( P = 0.05 ) in the sporulation of A. niger (M5Y2.5S2 1.76 ± 0.06 ) when compared with SDA ( 1.70 ± 0.06 ). This is also applicable in the case of P. expansum (M5Y2.5S2 2.73 ± 0.06 ) when compared with SDA ( 2.70 ± 0.0 ). The fungal isolates showed luxuriant growth in both media. M5Y2.5S2 performed favorably in comparison to SDA in relation to all the parameters studied. M5Y2.5S2 is recommended for use in fungal culture and will save funds used for the importation of media.

First Report of Xanthomonas hydrangeae Causing Leaf Spot on Oakleaf Hydrangea (Hydrangea quercifolia) in Tuscany (Italy).

Hydrangeas (Hydrangea L.) are popular ornamental plants used in urban landscapes and gardens worldwide for the beauty of their large flowers. In June 2022, dark brown/purple and irregular water-soaked spots coalescing into large areas of necrosis were observed on the leaves of potted Hydrangea quercifolia Bartr. plants growing in two ornamentals nurseries in Pistoia, Tuscany, Italy. Isolations, using two symptomatic plants/nursery, were performed by excising small portions of leaf tissue from the margin of the lesions, and macerating them in 100 μl of sterile distilled water (SDW). After 15 min of incubation, a loopful of the resulting suspension was streaked on yeast extract-dextrose-CaCO3 agar (YDCA) amended with 60 mg L-1 cycloheximide. Mucoid, convex and yellow colonies appeared on YDCA after incubation at 28°C for 48h. After colony purification on yeast extract-nutrient-agar (YNA), two isolates from each nursery were subject to amplification and sequence analysis of the 16S rRNA using universal primers FD1/RD1, for genus identification (Vauterin et al. 2000; Weisburg et al. 1991). All 16S rRNA sequences (OP441051) were identical and BLASTn searches indicated that the isolates belong to the genus Xanthomonas [99.9% nucleotide identity with X. hydrangeae strain LMG 31885 (LR990741.1) and 99.8% with strain LMG 31884T (NR_181958.1)]. For classification at species level, fragments of the housekeeping genes gyrB, rpoD, dnaK, and fyuA, were amplified according to Young et al. (2008). Both strands were sequenced and the consensus sequences aligned using MUSCLE as implemented in MEGA X (Kumar et al. 2018). Homologous sequences were once again identical between the isolates. A neighbor joining phylogenetic analysis of the concatenated fragments, was carried out, using the Tuscan isolate HyQ-Tu1, the type/pathotype strains of the seven pathovars of X.hortorum, proposed by Morinière et al. (2020), the four X.hydrangeae strains characterized by Dia et al. (2021) and the type strain of X.populi as the outgroup. The analysis indicated that HyQ-Tu1 isolate clusters within the X. hydrangeae branch of the recently described X. hortorum - X. hydrangeae species complex (Morinière et al. 2020; Dia et al. 2021; 2022). In agreement with this result, isolates tested positive to the LAMP assay specific for members of the complex's clade C (X. hydrangeae) (Dia et al. 2022). Based on molecular evidence, the isolates were identified as X. hydrangeae (Dia et al. 2021; Oren and Garrity, 2022). Three healthy H. quercifolia potted plants were inoculated by rubbing a 10 µl droplet of a bacterial suspension of X. hydrangeae HyQ-Tu1 adjusted to an OD600 of 0.3 (approx. 108 CFU/ml) in SDW on the adaxial surface of two leaves per plant. Two control leaves/plant were inoculated with SDW. Each inoculated leaf was enclosed for 24h in a polyethylene bag and all plants were maintained in a greenhouse at 28°C. Nine days post inoculation (DPI), leaf spots similar to those observed on naturally infected plants started to become evident on the bacteria-inoculated leaves while control leaves remained asymptomatic throughout the trial (21 DPI). Koch's postulates were fulfilled by re-isolating the bacterium from the symptomatic tissues, obtaining a positive amplification with the clade C-specific LAMP assay (Dia et al. 2022), and confirming that the gyrB sequence was 100% identical to that of X. hydrangeae HyQ-Tu1. Housekeeping gene sequences were submitted to GenBank (OP456006-9). Members of the X. hortorum - X. hydrangeae species complex have been reported to affect H. quercifolia in the USA (Uddin et al. 1996) and H. quercifolia and H. arborescens L. in Belgium (Cottyn et al. 2021). To the best of our knowledge, this is the first documentation of X. hydrangeae causing disease on H. quercifolia in Italy. Further work is required to verify the presence of the bacterium in other European countries and to assess the economic impact that it causes within and outside nurseries.

The Phytochemical and Antifungal Efficiency of Bean Leaf and Root against Some Pathogenic Fungi Isolated from Spoilt Vegetables Sold within Anambra Metropolies

Investigated in vitro were the phytochemical and antifungal properties of bean leaf and root against certain pathogenic fungi isolated from spoiled vegetables marketed in Anambra metropolises. By combining 100g, 100ml of sterile distilled water, and ethanol with each plant part, two (2) distinct extract concentrations were produced from each plant component. The plants were subjected to phytochemical screening using several conventional techniques, which identified the presence of alkaloids, saponins, tannins, flavonoids, phytate, oxalate, and phenol in all of the plants but at various amounts. The effects of common antibiotics (Fluconazole 30 g/ml) were compared to those of plant extracts. According to a pathogenicity test, good vegetables rot was caused by Aspergillus niger, Fusarium solani, Penicillium sp, Rhizopus sp, and Mucor spp. The ethanol extract from beans root turned out to be more effective. The extraction solvent, extract concentration, and test pathogens all had an impact on the extract's effectiveness. When extract concentration rose, the amount of fungal growth inhibition also rose. All of the test fungi's mycelia development was effectively/highly inhibited by beans root, although water extracts exhibited a lower degree of inhibition. Farmers should utilise bean root ethanolic extracts instead of synthetic or commercial fungicides due to their fungitoxic potential against vegetable storage fungi.

First Report of Bacterial Brown Rot Disease Caused by Ewingella americana on Cultivated Naematelia aurantialba in China.

Naematelia aurantialba (synonym Tremella aurantialba) is one of the jelly fungi and highly valued edible and medicinal mushrooms. It has been cultivated industrially in recent years and consumed popularly in China. In September 2022, brown rot disease of fruiting bodies was observed at the N. aurantialba factory in Tongzhou district, Beijing with a disease incidence of ~10%. Symptoms initially appeared as color changing from orange to light brown. The infected area expanded gradually until covered fully the fruiting body. Meanwhile, the interior of the fruiting body became rotten and dark brown. Finally, the whole fruiting body became wrinkled and brown, resulting in significantly reduced yield and economic loss. Isolations were made from 12 infected mushroom samples. Infected tissue within the fruiting body was mashed in sterilized 1.5 mL tubes containing 1 mL of sterile distilled water. After standing for 5-10 min, the suspensions were streaked on the Luria-Bertani (LB) medium and cultured at 37°C for 24h. The physiological and biochemical reactions of isolated strains were determined using the API 20E system (Reyes et al. 2004) according to the manufacturer's instructions. All the strains showed the same reaction results. The bacterial colonies were streaked on fresh LB medium at 37°C for 24 h, and a single pure culture was obtained with round, smooth and semitransparent. The bacterial cells were gram-negative, short-rod, (0.3) 0.8-2.0 (2.5) × (0.1) 0.6-1.0 (1.5) μm, and peri-flagellate. The isolates were further confirmed by sequence analysis of the 16S rRNA and gyrB genes with primer 27F/1492R and gyrB-UP1s/gyrB-UP2sr (Liu et al. 2018). Using EzBioCloud data searches, the 16S rRNA sequence of four strains (GenBank accession OP727593, OP727595, OP727596, OP727601) matched the sequence of E. americana type strain ATCC 33852 (accession JMPJ01000013) with identity of 99.65%~99.93 and 100% completeness. The GyrB sequence matched the E. americana in GenBank (MK460250) and showed 98.71% identity and 100% completeness. Finally, the pathogen was identified as E. americana based on morphological, physiological, biochemical, and molecular characteristics. The pathogenicity test was conducted by spreading bacterial suspensions cultured 48h onto 12 healthy cultivated fruiting bodies of N. aurantialba, with sterile distilled water as a control, and then cultured in a chamber at 23°C with 85% relative humidity. Brown symptoms, similar to natural symptoms, were observed on all inoculated fruiting bodies after 48h, whereas the controls remained symptomless. Pathogenic bacteria were isolated from the inoculated fruiting body and confirmed to be E. americana based on morphological and 16S rRNA molecular characteristics, thus fulfilling Koch's postulates. E. americana caused stipe necrosis on Agaricus bisporus in Egypt (Madbouly et al. 2014), the oak tree in Thailand, and pneumonia in Humans (Doonan et al. 2016), and brown blotch on Flammulina velutipes (Liu et al. 2018). To our knowledge, this is the first worldwide report of E. americana infecting jelly fungus N. aurantialba causing brown rot disease. E. americana is an opportunistic cross-kingdom pathogen (Liu et al. 2018). That will provide a critical alert on the prevention, effective monitoring, and control of the disease.

First Report of Gray Leaf Spot Caused by Pyricularia oryzae on Common Millet in Italy.

Common millet (Panicum miliaceum L.) is a niche crop cultivated in Italy for its high nutritional and gluten-free characteristics. These aspects combined with its sustainability and adaptability to paddy soils make it relevant in crop rotation with rice in some organic farms in Northwestern Italy. In September 2021, in a farm producing organic rice located in Rovasenda (VC), Italy (GPS: 45°54'82"N, 8°.31'96"E), plants of millet (var. San Marino), sown on July 1st and almost at physiological maturity, were observed with gray leaf spot symptoms with an incidence of 50% in a 12.5 ha field cultivated in a yearly rotation with rice. Oval shape lesions (between 1 and 5 cm long and 0.5 to1 cm wide) with a light gray colored centers and dark reddish-brown edges were observed on the length of the leaf blade. Gray colonies producing conidia morphologically like Pyricularia developed from 140 symptomatic leaf fragments with a 2% frequency. Monosporic cultures were obtained from five colonies and were grown on Potato Dextrose Agar for morphological and molecular identification. The colonies showed gray, fluffy mycelium, with a smooth margin, with a radius of 28 to 30 mm after 10 days at 28°C. The isolates produced hyaline, pyriform, and 2-septate conidia 17.9 to 33.5 µm (average 25.7 µm) long and 8.3 to 11.7 µm (average 10. µm) wide (n=50) on PDA. Conidiophores were solitary, erect, straight, or curved, septate, and measured 80.2 to 221.3 µm (average 150.7 µm) long and 3.6 to 5.8 µm (average 4.7 µm) wide (n=50). The molecular identification of one single-conidia isolate was confirmed by PCR and sequence comparison of the region of the transposon Pot2 (Harmon et al., 2003). BLASTn searches of GenBank using transposon Pot2 (ON843711) representative isolate 21-03-C, revealed 99.79% identity to P. oryzae (Syn. Magnaporthe oryzae) isolate MD1 (MK608664). Pathogenicity tests were carried out by suspending conidia from a 14-days old culture on PDA in sterile water at a concentration of 1×105 CFU/ml. Twenty seeds of P. miliaceum were sown in pots filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 28°C under a 12 hours day/night light cycle. Diseased leaves of 10 plants were inoculated by aspersion of 10 ml of the conidial suspension of isolate 21-03-C 2 weeks after the sowing (Harmon et al.; 2003). Plants were incubated in a near moisture-saturated environment in plastic bags at 28°C for 72h, after which symptoms were visually assessed. Ten control plants, inoculated with 10 ml of sterile distilled water remained healthy. P. oryzae was reisolated from leaves showing oblong, whitish lesions, surrounded by light brown margins and identified by resequencing of transposon Pot2. P. oryzae has been already reported on millet in South Korea as causal agent of blast (Klaubauf et al.; 2014). To our knowledge, this is the first report of P. oryzae as a pathogen of millet in Italy and in Europe. Further studies should be conducted to characterize these millet isolates by assessing their virulence on Oryza sativa and the implications in the adoption of P. miliaceum in rotation with rice, to prevent possible carry over of the pathogen on the main crop.

First report of Atractylodes lancea leaf spot caused by Fusarium acuminatum in China.

Atractylodes lancea Thunb. DC (cangzhu) is a traditional Chinese medicinal plant (Cai et al., 2020). In June 2020, leaf spots were observed in A. lancea plants at the Chongqing Institute of Medicinal Plant Cultivation located in Nanchuan District, Chongqing, China (29°8'26.46″ N, 107°13'23'21″ E). Approximately 75% of the plants displayed leaf spot, partial leaf wilting, and stunted growth, and some plants died. To determine the cause of this disease, five typical leaf spots were cut into small pieces. The pieces were successively surface-disinfected with 0.5% NaClO for 1 min and 75% ethanol for 30 s, washed thrice with sterile water, and placed on potato dextrose agar (PDA) to incubate at 25 ℃. These isolates initially formed abundant white aerial mycelium, then gradually developed a rose pigmentation with a brownish color in the center and grayish rose at the periphery of the colony (Li etal. 2019).Mycelial tipswere picked and placed on carnation leaf agar (CLA) and inoculated for 7 days. The macroconidia of the isolates were slender, distinctively curved in the bottom half of the apical cell, and sickle-shaped, with 3-4 septa. They ranged in size from 16.68-26.49 × 1.48-2.34 μm (n=50). The microconidia were fusiform with or without one septum. Their size ranged from 6.19-11.02 × 1.25-1.43 μm (n=50) (Li etal. 2019). The morphological characteristics of the isolates were consistent with those of Fusarium spp. PCR amplification and DNA sequencing of the internal transcribed spacer (ITS) region and β-tubulin (TUB2) gene were performed using the primers ITS1/ITS4 (White etal. 1990) and Bt-2a/Bt-2b (Robideau etal. 2011), respectively. BLASTn analysis revealed that the ITS sequences of the isolates were 100% identical to those of the F. acuminatum isolates from the Fusarium MLST database (http://isolate.fusariumdb.org/guide.php). Furtheranalysisrevealed that the TUB2 sequences were 99.14% identical to those of the F. acuminatum strain S16 isolates (MF662644) from the GeneBank database of the NCBI server. Based on the morphology and sequence analyses, the isolates were identified as F. acuminatum. Pathogenicity tests were conducted on 1.5-year-oldA.lanceaplants by inoculating spore suspensionsunder greenhouse conditions (25°C). For this, wound were made on leaves by piercing with sterilized toothpicks. 30 μl of spore suspension containing 2 × 106 conidia/ml was placed on each wound. Wounds on the leaves of control plants were inoculated with 10 μl of sterile distilled water. There were three plants for each treatment. After incubation at 25 °C for 5 days in a greenhouse, the leaves of the treated plants all showed partial wilting, consistent with the field observations. No symptoms were observed in controlled plants. The fungi were again isolated from the symptomatic tissues and were identical to the original isolate. The experiment was repeated twice with similar results. Pathogenicity symptoms were similar to what was first observed in the field and the isolated fungi were verified based on morphological characteristics, thus fulfillingKoch'spostulate. To the best of our knowledge, this is the first time that A. lancea leaf spot caused by F. acuminatum has been discovered in China. The leaf spot caused by F. acuminatum on A. lancea has serious yield loss, and proper control measures should be applied.

Development of an Antimicrobial Topical Skin Cream with Using Wood Apple (Limonia acidissima L.) Pulp Against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans

Skin inhabiting microbes mostly non-pathogenic and commensals on the skin. Among them Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans often responsible for symptomatic skin infections. Cellulites, impetigo, ecthyma, red lesions and candidiasis are some of the common microbial skin diseases caused by skin pathogens. Wood apple (Limonia acidissima L.) is an underutilized fruit in Sri Lanka and, all parts of this plant have medicinal value. Considering the high therapeutic value, the aqueous extract of wood apple pulp was used. Antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa and antifungal activity against Candida albicans was evaluated by using the standard well diffusion method. Above tested bacterial and fungal species were suspended in 3 mL of sterile distilled water separately and 108 CFU/mL population count was prepared. Aliquots of 100 µL of each suspension were inoculated and uniformly spread on Muller Hinton agar plates separately in triplicates. Wells were filled aseptically placing 50 µL of wood apple extracts, antimicrobial compounds specifically as the positive control for bacteria and fungi and sterile distilled water as the negative control. The plates were incubated at 37 °C for 24 - 48 hours and the diameter of the growth inhibition zones around the wells were measured. Furthermore, minimum inhibition concentrations of each extract were also evaluated. Comparisons were performed using one-way ANOVA followed by Tukey’s Pairwise Comparisons. The antimicrobial topical skin cream was prepared using 0.5 g mL-1 of wood apple pulp with sterile distilled water, mixed with the formulated standard ingredients. The results revealed that the aqueous extracts of wood apple pulp concentrations range from 0.15 g mL-1 to 1g mL-1 showed the antimicrobial potential of above skin pathogens variably ranged inhibition zone diameter 16 mm to 34 mm. The antimicrobial effect of the formulated cream against these pathogens was repeatedly tested. Further testing procedures needed to be followed before recommending wood apple topical antimicrobial skin cream as a marketable product.

First Report of Leaf Blight on Dumasia villosa Caused by Boeremia exigua in China.

Dumasia (Fabaceae, tribe Phaseoleae, subtribe Glycininae), a genus of trifoliate vines, is widely distributed in tropical and subtropical regions of Asia and Africa (Pan & Zhu 2010). In October 2021, lesions were observed on Dumasia villosa leaves on Longwen mountain of Guizhou Normal University, Guiyang City, Guizhou Province, China. The incidence of leaf blight on observed D. villosa leaves was 10%. All necrotic spots were close to insect-feeding sites. Necrotic spots were grey or black, and circular (2 to 10 mm in diameter) or irregular (2 to 20 mm long) in shape. Stems and pods showed no disease symptoms. Pathogen isolation was conducted following surface sterilization with ethanol. After growth for 5 days on potato dextrose agar in a moist climate chamber at 26°C under a 16 h/8 h light/dark cycle, colonies were 5.2 to 5.6 cm in diameter, olivaceous grey in colour, and sparse, white, aerial mycelia with irregular margins were evident. Conidia were hyaline, ellipsoidal to oblong, mostly 1-septate, and occasionally aseptate with dimensions ranging from 3.5 to 7.0 × 1.5 to 3.5 μm (n = 30). To confirm the species of the isolate, ribosomal DNA internal transcribed spacer (ITS) and β-tubulin (tub2) genes were amplified and sequenced using primers ITS1/ITS4 and Btub2Fd/Btub4Rd (White et al. 1990), respectively. The obtained 529 bp ITS sequence (GenBank accession no. OL872186) shares 99.61% identity with the sequences of Boeremia exigua (MF662797 and GU395499). The obtained 299 bp tub2 sequence (OM830712) shares 100% identity with the sequence of B. exigua (KR653201). Based on morphology and DNA sequence analysis, the isolate was identified as B. exigua. To fulfil Koch's postulates, healthy leaves of 8-week-old D. villosa plants (n = 6) were wounded with a sterilized hypodermic needle and inoculated with 2 μL of a conidial suspension (106 conidia/mL). Six plants were inoculated with 2 μL of sterile distilled water as controls. After 3 days in a moist climate chamber at 26°C under a 16 h/8 h light/dark cycle, dark spots were only present on conidia-inoculated leaves, while controls remained healthy. Boeremia exigua was reisolated from disease spots and confirmed using the same morphological and molecular methods described above. To our knowledge, this is the first report of leaf blight on D. villosa caused by B. exigua, although this fungus has been reported to infect leguminous plants, including field pea (Li et al. 2012), white clover (Wang et al. 2020), and soybean (Schaffrath et al. 2020). In China, B. exigua has also been reported to infect other plants, such as walnut (Cai et al. 2021; Wang et al. 2021) and Japanese ginseng (You et al. 2015). This identification suggests that D. villosa could be a potential reservoir for the pathogen affecting other leguminous crops that might be economically important.

The first report of Phytophthora multivesiculata causing black rot of Cymbidium and Ansellia africana from South Africa.

Globally, various species and hybrids of Cymbidium are of horticultural importance. In January 2022, we visited a private orchidarium near Pretoria (25° 54' 30" S, 28° 24' 34" E). During this visit, the owner reported mortality among various pure and hybrid Cymbidium and Ansellia africana, an indigenous South African ornamental orchid. Phytophthora was identified as a possible causative agent based on an initial examination of the affected orchids. The infected orchids exhibited vascular wilting. Brown, water-soaked lesions covered the roots. The pseudobulbs showed black rot symptoms. Necrotic lesions were also visible towards the basal part of the leaves. For isolation of the causal agent, pieces of infected tissues from roots, pseudobulbs and leaves were surface sterilised using 70% ethanol and plated into Phytophthora selective medium, NARPH. All the plates were incubated at 21°C in darkness. After three days, Phytophthora-like mycelia emerged from all three tissue types. These colonies were transferred onto the PDA medium. For molecular identification, genomic DNA was extracted from four representative isolates (CMW58027-30) using a Zymo Research Fungal/Bacterial DNA MiniPrep kit. The complete ITS, and partial beta-tubulin (BT) and cytochrome oxidase 1 (COX1) gene regions were amplified using the primers DC6/ITS4 (Cooke et al. 2000; White et al. 1990), Btub_F1A/Btub_R1 (Blair et al. 2008; Kroon et al. 2004) and FM84/FM83 (Martin and Tooley 2003), respectively. BLAST searches in NCBI showed that the four isolates were from Phytophthora ITS Clades 2. ITS, BT, and COX1 datasets from Bose et al. (2021) were used for the phylogenetic identification of our isolates. Single gene and concatenated datasets were analysed using both maximum likelihood and Bayesian approaches, which confirmed the identity of the isolates as Phytophthora multivesiculata. All the sequences were submitted to the GenBank: ITS (OM967212-15), BT (OM966588-91), and COX1 (OM966592-95). Measurements of sporangia and gametangia overlapped with those from Ilieva et al. (1998): sporangia (28.3 - 56.3) 41.6 x 31.3 (21.5 -3 9.6) µm; L:B 1.42 (1.08 - 1.69); exit pore 11.2 (7.1 - 14.6) µm; oogonia 44.2 (24.5 - 56.3) µm; oospore 34.2 (21.6 - 53.2) µm; antheridia (5.8 - 14.6) 11.8 x 15.3 (6.2-14.9) µm. The pathogenicity trial was conducted following the protocol suggested by Ilieva et al. (1998). Five A. africana roots and the cut ends of seven Cymbidium leaves were immersed in separate beakers containing 100 ml of sterile distilled water and ten 5 mm agar discs excised from a 7-day-old culture of P. multivesiculata (CMW58027) grown on PDA. Sterile distilled water was used as the control, with an equal number of plants and leaves. All of the sets were incubated at 21°C. After seven days, the plants and leaves developed lesions similar to those observed on the symptomatic plants at the orchidarium. Trials were repeated once and the pathogen was reisolated from both trials and the identity was confirmed by amplifying the complete ITS gene region. Phytophthora multivesiculata has been previously reported from the Netherlands (Ilieva et al. 1998), Taiwan (Chern et al. 2011), Australia (Cunnington et al. 2009), New Zealand (Hill 2004) and elsewhere causing black rot of Cymbidium. However, this is the first report of P. multivesiculata causing black rot of Cymbidium and A. africana from Africa. We are now conducting follow-up surveys to determine the distribution range of this pathogen in South Africa.

First report of rice bacterial leaf blight and grain discolouration caused by Pantoea agglomerans in India

A disease survey was carried out in Tamil Nadu, India during 2021 to assess the prevalence of diseases in major rice-growing areas and characterise the pathogens associated with infected plants. During the survey, rice plants at the seedling to grain-filling stage showing water-soaked, elongated, light brown lesions in the leaf margins and tips (Figure 1) were collected along with grains in a polythene bag, labelled and sealed. Leaves showing yellowing and drying from tips and margins were cut into small pieces, surface-sterilised in 1% sodium hypochlorite for one minute, rinsed three times with sterile distilled water and then allowed to air dry. After drying, the tissues were soaked for 30 minutes at room temperature in 0.5 ml of sterile distilled water and then a loop full of suspension was streaked onto nutrient agar (NA) medium. For the isolation of pathogen from infected seed, discoloured grain samples were surface sterilised in 3% sodium hypochlorite for one minute, rinsed thrice with sterile distilled water and allowed to air dry. Surface-sterilised grains were placed in a Petri plate containing NA medium. These plates were incubated at 28°C for one to two days. Upon incubation, straw to yellow-coloured colonies emerged. The colonies were further purified through single colony isolation on NA. The purified colonies were yellow in colour, translucent and undulate. (Figure 2.) The colonies exhibited umbonate surfaces with slightly concave centres (Al-Kidsawey et al., 2020; Delétoile et al., 2009). The pathogenicity of the isolates was demonstrated by inoculation of isolates on rice seedlings (cv. Co 39). For this, bacterial isolates were incubated in nutrient broth for 48 hours at 27°C. The bacterial suspension was adjusted to a concentration of 109 cfu/ml by adding sterile water. The bacterium was inoculated by cutting the tip (about 3 to 4 cm) of the fully expanded uppermost leaf with scissors and dipping in the bacterial suspension. Controls used sterile distilled water instead of the bacterial suspension. The inoculated plants were covered with polythene sheet overnight and incubated under laboratory conditions. The inoculated leaves developed progressive necrosis with colour shifts from pale green to brown within 3 to 14 days post inoculation, whereas the control plants exhibited no symptoms. Koch's postulates were fulfilled by isolating the bacterium on NA which produced yellow, flat to convex translucent colonies after 48 hours incubation. The bacterium was further subjected to biochemical tests using the KB002 HiAssorted Biochemical Test Kit (Himedia, Germany). The bacterium showed a positive reaction for citrate utilisation, phenylalanine deamination, nitrate reduction, glucose and arabinose utilization. It showed a negative reaction to lysine utilisation, ornithine utilization, urease, H2S production, adonitol, lactose and sorbitol utilization. Based on the morphological characters and biochemical tests, the bacterium was identified as a Pantoea sp. To further characterise the pathogen, DNA extraction was done, and a PCR assay was performed using universal primers 27F and 1492R targeting the 16S rRNA gene. Sequencing of the amplicon revealed that the bacterial isolates had 93.7% identity with Pantoea agglomerans type strain SGAir0210 (GenBank Accession No. CP028033.1). The partial 16S rRNA gene sequences of two strains of P. agglomerans were deposited in GenBank (OP784578 and OP784743). Pantoea agglomerans causing bacterial leaf blight and glume discolouration has been reported from Brazil, South Korea, Turkey and Venezuela (Aksoy & Boluk, 2019; Carrer Filho et al., 2018; González et al., 2015; Lee et al., 2010). Pantoea agglomerans also causes infection in humans (Al-Kidsawey et al., 2020; Delétoile et al., 2009)). Bacterial leaf blight in rice caused by P. stewartii subsp. indologenes has been reported from Tamil Nadu, India (Vinodhini et al., 2017). However, to our knowledge, this is the first report of P. agglomerans causing rice leaf blight and grain discolouration in India. Further studies are needed to ascertain the prevalence of P. agglomerans in commercial rice cultivars in other parts of India. We gratefully acknowledge the facilities provided by DST-FIST Lab, Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India.

First Report of Muskmelon Fruit Rot Caused by Fusarium nanum in China.

Muskmelon(Cucumis meloL.) is one of the most widely cultivated and economically important fruit crops in the world.However, many pathogens can cause decay ofmuskmelon fruit, including Fusarium asiaticum, F. equiseti, F. incarnatum and F. lateritium (Hao et al. 2021; Wang et al. 2019). Fusarium spp. are the most important pathogens affecting muskmelon fruit yield and quality (Wang et al. 2011). In August 2020, fruitrot symptoms were observed on ripening muskmelons (cv. Tianbao) in several fields in Jiyang District, Jinan City of Shandong Province, China. The incidences of infected muskmelon ranged from 15% to 30% and caused an average 20% yield loss. Symptoms appeared as pale brown, water-soaked lesions that were irregular in shape, with the lesion sizes ranging from a small spot (1 to 2 cm) to decay of the entire fruit. The core and surface of infected fruit were colonized and covered with white mycelia. Two infected muskmelons were collected from two fields, 7 km apart. Tissues removed from inside the infected fruit were surface disinfected with 75% ethanol for 30 s, and cultured on potato dextrose agar (PDA) at 25°C in the dark for 5 days. Four purified cultures were obtained using the single spore method.On carnation leaf agar (CLA), macroconidia were 1 to 5 septate, falcate, with a pronounced dorsiventral curvature with blunt to papillate apical cell, and barely to distinctly notched basal cell, measuring 12 to 35 × 3.5 to 6 μm. Microconidia and chlamydospores were not observed. These morphological characteristics were consistent with the description of Fusarium sp. Because these isolates had similar morphology, two representative isolates (XP9 and XP10) were selected for multilocus phylogenetic analyses. DNA was extracted from the representative isolates using a CTAB method. Nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), calmodulin (CAM), RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-α gene (TEF1) (Xia et al. 2019) were amplified using specific primers, sequenced, and deposited in GenBank (ITS: MW391507 and MW391508, CAM: MW392787 and MW392788, RPB2: MW392795 and MW392796, TEF1: MW392791 and MW392792). The Fusarium MLST database pairwise alignment of ITS (546 bp), CAM (628 bp), RPB2 (902 bp) and TEF1 (718 bp) sequences from isolate XP9 showed 99.63%, 99.33%, 100.00% and 99.71% similarity with the corresponding sequences (GQ505685, GQ505508, GQ505774 and GQ505596) of the reference strain of F. nanum (NRRL 22244), respectively. The overlap of ITS, CAM, RPB2 and TEF1 sequences from XP9 and NRRL 22244 were 100.00%, 95.06%, 97.45% and 94.99%, respectively. Alignments of a combined dataset of ITS, CAM, RPB2 and TEF1 were made using MAFFT v. 7, and phylogenetic analyses were conducted in MEGA v. 7.0 using the maximum likelihood method. The muskmelon isolates (XP9 and XP10) clustered together with theF. nanum reference strain CGMCC3.19498 and NRRL 22244 (100% bootstrap) (Wang et al., 2019). To perform a pathogenicity test, 10 μl of conidial suspensions (1 × 106 conidia/ml) were injected into each muskmelon fruit using a syringe, and the control fruit was inoculated with 10 μl of sterile distilled water. There were ten replicated fruits for each treatment. The test was repeated three times. After 7 days at 25°C, the interior of the inoculated muskmelons begun to rot, and the rot lesion expanded from the core towards the surface of the fruit, then white mycelia were produced on the surface. Ten isolations were re-isolated from the infected tissues and identified by morphological and phylogenetic analyses and confirmed to fulfill Koch's postulates. No symptoms were observed on the control muskmelons. To our knowledge, this is the first report of muskmelon fruit rot caused by F. nanum in China. Considering the economic value of the muskmelon crop, correct identification can help farmers select appropriate field management measures for control of this disease.

First Report of Pantoea ananatis Causing Crown Necrobiosis on Strawberry in China.

Strawberry (Fragaria × ananassa) is an economically important crop in Zhejiang, China. In the autumn of 2021, crown necrobiosis and angular leaf spot was observed in commercial strawberry fields (cultivar 'fenyu') in Cixi, Ningbo, Zhejiang, China (N30°9'55″, E121°21'13″). The disease incidence ranged from 5 to 8 % in the field, but could reach 50 to 60 % in some heavily affected plastic tunnels. In the affected field, this disease could reduce strawberry production by 50%. Early symptoms were water-soaked lesions around the vein of the abaxial leaves; subsequently, reddish-brown irregular spots and coalesced lesions developed. In humid conditions, a sticky bacterial ooze exuding from lesions was observed. Finally, the crown of the diseased plant was necrotized, and several pockets were observed inside the crown after dissection. To isolate the causal agent, the infected leaves and crown tissues from six different plants were surface-sterilized with 75% ethanol for 1 min, rinsed twice with sterile distilled water, cut into small pieces, and soaked in 5 ml of sterile distilled water for 20 min. The supernatant from the cut-up pieces was serially diluted and spread on nutrient agar medium. After 2 to 3 days at 28℃, several yellow colonies were grown on the medium. The colonies from five infected plants were gram-negative, anaerobic rods, yellow, viscous, and gloss, which are typical characteristics of Erwinia anana (Wells et al. 1986). To confirm the identity of the causal bacteria, PCR was conducted for six randomly selected colonies to amplify 16S rRNA (Monciardini et al. 2002), fusA, and gyrB (Stice et al. 2002). The amplicons were sequenced and blasted, and the results showed that the six colonies were identical. The 16S rRNA, fusA, gyrB sequences of the isolate CM3 were deposited in GenBank with accession number ON754076.1, OP587277, and OP587278; BLAST search showed 99.93% (1445 bp out of 1446 bp), 100% (746 bp out of 746 bp), 99.64% (1371 bp out of 1376 bp) similarity with strains of Pantoea ananatis (KT741001.1, MH015093.1 and CP066803.1 accessions, respectively). The resulting concatenated data set of 16S rRNA-fusA-gyrB was used to build a multilocus phylogenetic analysis (MLSA) by maximum likelihood criteria. The MLSA tree indicated that the isolate CM3 belonged to Pantoea ananatis. The isolate's identity was further confirmed by P. ananatis-specific primers pagyrB-F/R (Xiao et al. 2022). Thus, this isolate was designated as P. ananatis CM3. To fulfill Koch's postulates, two old leaves were broken off each of the ten 2-month-old strawberry (cultivar 'fenyu') plants to create wounds, each plants was sprayed with a cell suspension ofP. ananatis (107CFU/ml, 0.5 ml) on the stem base. Ten plants were sprayed with water to serve as a control. All plants were kept at 28/25°C (day/night) under a 12-h/12-h photoperiod. All plants were covered with transparent plastic bags to maintain humidity. After 48 h, the bags were removed. After 2 weeks, water-soaked lesions on some leaves were observed similar to those in the field . Three to five weeks after inoculation, the crown of the inoculated plants was necrotized, which was similar to the symptoms in the field. No symptoms were observed in the control plants. The experiment was repeated three times. The bacteria were successfully reisolated from the inoculated crown tissues and leaves and confirmed as CM3 according to the same methodologies used for the initial identification. Bacterial leaf blight in strawberry caused byPantoea ananatishas been reportedin Nova Scotia, Canada, and Egypt (Bajpai et al. 2019; Abdel-Gaied et al. 2022). To our knowledge, this is the first report of Pantoea ananatis causing crown necrobiosis on strawberry in China. This report provides a basis for further research on this disease and its management and control.

First Report of Neopestalotiopsis cubana Causing leaf blight on Ixora chinensis in Malaysia.

Ixora chinensis (family Rubiaceae), locally known as 'Bunga Jejarum', is widely grown as an ornamental shrub and as sources for phytochemicals with medicinal properties in Malaysia. In May 2021, irregular brown spots were found on the leaves of some 'Bunga Jejarum' in Universiti Malaysia Sabah (6°02'01.0"N 116°07'20.2"E) located in Sabah province. As the disease progressed, the spots enlarged and coalesced into large necrotic areas giving rise to drying of infected leaves. The disease severity was about 70% with 20% incidence. Five symptomatic leaves (5 x 5 mm) from five plants were excised and sterilized based on Khoo et al. (2022) before plated on five potato dextrose agar (PDA) and cultured at 25°C. After 5 days, white to pale honey and dense mycelia with lobate edge were observed on all PDA plates. Globose, black conidiomata semi-immersed on PDA were observed after a week. Two to four hyaline filamentous appendages 7.7 to 17.6 μm long attached to fusoid conidia (11.8 to 20.9 x 5.7 to 7.6 μm, n = 20), which consisted of a hyaline apical cell, basal cell, and three versicolored median cells. The upper two median cells were dark brown, while the lowest median cell was pale brown. The isolate of the causal pathogen was characterized molecularly. Genomic DNA of isolate UMS01 was extracted based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS), tubulin (TUB) and translation elongation factor 1-α (TEF) region was performed based on Khoo et al. (2022) using primers ITS1/ITS4 (White et al. 1990), T1/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1997) and EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999), respectively. PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. The isolate's sequences were deposited in GenBank as OM320626 (ITS), OM339539 (TUB) and OM339540 (TEF). They were 99% to 100% identical to ITS(KM199347) (545 out of 545 bp), TUB (KM199438) (768 out of 769 bp) and TEF (KM199521) (480 out of 481 bp) of the type sequences (CBS 600.96). Phylogenetic analysis using the maximum likelihood method based on the combined ITS, TEF and TUB sequences placed the isolate UMS01 in the same clade as the isolate CBS 600.96 of Neopestalotiopsis cubana. Thus, the pathogen was identified as N. cubanabased on the morphological description from Pornsuriya et al. (2020), molecular data in Genbank database and multigene sequence analysis. To further confirm its pathogenicity, the first and second leaves of three 'Bunga Jejarum' plants were inoculated by pipetting 1 ml aliquots of a 1 × 106 conidia/ml spore suspension. Three additional 'Bunga Jejarum' plants were mock inoculated by pipetting 1 ml of sterile distilled water on similar age leaves. The plants were covered with plastic bags after inoculation for 48 h before placing them in a glasshouse under room temperature. The leaves were sprayed with water to keep the leaf surfaces moist along the experiment. The incubation and disease observation were performed based on Chai et al. (2017) and Iftikhar et al. (2022). After 7 days post-inoculation, all infected leaves exhibited the symptoms observed in the field, whereas the controls showed no symptoms. The same fungus was isolated from the diseased leaves and, thus confirmed Koch's postulates. The experiment was repeated two more times. The reisolated fungi were visually and genetically identical to the original isolate obtained from the field samples. To our knowledge, this is the first report of N. cubana causing leaf blight on 'Bunga Jejarum' in Malaysia, as well as the world. Our finding has broadened the distribution and host range of N. cubana, indicating that it poses potential damage to the medicinal plant Bunga Jejarum in Malaysia.