Small Spot Research Articles

First report of Curvularia geniculata causing leave spot disease of Bletilla striata in Zhejiang of China.

Bletilla striata, a perennial herbaceous plant belonging to the family Orchidaceae, is native to China and is widely distributed in the Yangtze River basin. In China, B. striata is a popular medicinal plant that is typically used to reduce wound bleeding and inflammation. In September 2021, distinct leaf spot symptoms were observed in more than 50% of B. striata plants in a traditional Chinese medicine plantation (ca. 10 ha) in Xianju City, Zhejiang Province, China. Small, round, pale brown, necrotic spots were first observed on the leaves. Subsequently, these lesions became grayish brown in the center and dark brown with slight protuberances at the margins and eventually enlarged to 5-8 mm on the leaves. Over time, the small spots enlarged and coalesced into necrotic streaks (1-2 cm). Leaves with symptoms of disease were cut, surface-sterilized, and plated on potato dextrose agar (PDA). Fungal colonies (28×28 mm) with grayish-black mycelia from all tissues were produced after 3 days of incubation at 26 °C. The mature colonies eventually turned black in the center, with obvious rings appearing after 10 days of culture. Basal conidia ranged from pale to dark brown, whereas apical ones were pale brown, with central cells being larger and darker than basal cells. Conidia were smooth and either fusiform, cylindrical, or slightly curved with rounded tips. They ranged in length from 22.34 to 36.82 (mean = 28.63) μm with 2-4 septations and slight septal constrictions. Monospore isolation was performed to obtain a pure culture. Strain BJ2Y5 was subsequently stored in the strain Preservation Center of Wuhan University (Wuhan, China) and the strain preservation number CCTCC M 2023123 was obtained. Fresh mycelia and conidia that grew at 26 ℃ for 7 days were collected from PDA plates. DNA was extracted using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Shanghai, China). The phylogenetic position of isolate BJ2-Y5 was clarified based on DNA sequence analysis of three loci, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Berbee et al. 1999), the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of the second largest subunit of RNA polymerase II (RPB2; O'Donnell et al. 2007). A BLAST search (GenBank accession nos. OP913168, OP743380, and OP913171) showed 99% homology to the reference isolate CBS 220.52. Based on the morphological and molecular information presented in this study these isolates were identified as C. geniculata (Hosokawa et al. 2003). Furthermore, we evaluated the pathogenicity of B. striata leaves by smearing a conidial suspension (106 conidia/mL) on both sides of leaves with and without wounds. Five inoculated and three non-inoculated leaves (smeared with sterile distilled water as a negative control) were kept in a greenhouse at 26 °C under natural sunlight and covered with plastic bags for 72 h to maintain humidity. After 7 days, small round spots appeared on the wounds. Fifteen days later, the symptoms of disease on the wounded inoculated leaves were similar to those observed in the original sample, whereas the control plants remained healthy. No symptoms of infection were observed in unwounded inoculated leaves. C. geniculata was successfully re-isolated from all five inoculated leaves and was confirmed based on Koch's postulates. To the best of our knowledge, C. geniculata infection has not been previously reported in B. striata.

First Report of Corynespora cassiicola Causing Leaf Spot on Jasminum nudiflorum in China.

Winter jasmine (Jasminum nudiflorum Lindl.), a trailing, deciduous shrub, is widely used as an ornamental plant. Its flowers and leaves also has great medicinal value for treatment of inflammatory swelling, purulent eruptions, bruises and traumatic bleeding (Takenaka et al. 2002). In October 2022, leaf spot symptoms were observed on J. nudiflorum distributed in Meiling Scenic Spot (28.78°N, 115.83°E) and Jiangxi Agricultural University (28.75°N, 115.83°E), Nanchang, Jiangxi Province, China. In a week-long series of investigations, the incidences of disease could range up to 25%. Initially, the symptoms of the lesions were small yellow circular spots (0.5 to 1.8 mm), and gradually developing irregular spots (2.8 to 4.0 mm) with grayish white central parts, a dark brown inner ring, and outer yellow halo. To identify the pathogen, sixty symptomatic leaves from fifteen different plants were collected, of which twelve were randomly selected, cut into 4-mm2 pieces, and surface sterilized with 75% ethanol for 30s followed by 5% NaClO for 1 min, rinsed four times with sterile water, and then placed on potato dextrose agar (PDA) medium at 25 °C in the dark for 5 to 7 days. Six isolates with similar morphological characteristics were obtained. Aerial mycelium was vigorous, downy and exhibited white to grayish-green coloration. Conidia were solitary or catenate, pale brown, obclavate to cylindrical, apex obtuse, one to 11 pseudosepta, 24.9 to 125.7 × 7.9 to 12.9 μm (n = 50). Morphological characteristics matched Corynespora cassiicola (Ellis 1971). For molecular identification, two representative isolates (HJAUP C001 and HJAUP C002) were selected for genomic DNA extraction, and the ITS, TUB2 and TEF1-α gene were amplified, using the primer ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Lousie and Donaldson 1995) and EF1-728F/EF-986R (Carbone and Kohn 1999), respectively. The sequenced loci (GenBank accession nos. ITS: OP957070, OP957065; TUB2: OP981639, OP981640; TEF1-α: OP981637, OP981638) of the isolates were 100, 99 and 98% similar to the corresponding sequences of C. cassiicola strains (GenBank accession nos. OP593304, MW961419, MW961421, respectively). Phylogenetic analyses of combined ITS and TEF1-α sequences was performed using maximum-likelihood method in MEGA 7.0 (Kuma et al. 2016). The result showed that our isolates (HJAUP C001 and HJAUP C002) clustered with four strains of C. cassiicola at 99% bootstrap values in the bootstrap test (1000 replicates). Based on the morpho-molecular approach, the isolates were identified as C. cassiicola. The pathogenicity of one representative strain (HJAUP C001) was tested by inoculating the wounded leaves of six healthy J. nudiflorum plants under natural condition. Three leaves from each of three plants were punctured with flamed needles and sprayed with a conidial suspension (1 × 106 conidia/ml), and three wounded leaves from each of other three plants were inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and PDA plugs on three leaves each, respectively. Leaves from all treatments were incubated in a greenhouse at high relative humidity, 25°C, and 12-hour photoperiod. After one week, all wounded inoculated leaves appeared similar symptoms as described above, whereas the mock inoculated leaves were still healthy. Similar isolates with grayish white and vigorous aerial mycelium were reisolated from inoculated and symptomatic leaves and identified as C. cassiicola by DNA sequencing, fulfilling Koch's postulates. It has been reported that C. cassiicola can cause leaf spots on numerous plant species (Tsai et al. 2015; Lu et al. 2019; Farr and Crossman 2023). However, to our knowledge, this is the first report of C. cassiicola causing leaf spots on J. nudiflorum in China. This finding aids in protection of J. nudiflorum, a medicinal and ornamental plant with high economic value.

First Report of Black Spot Disease Caused by Alternaria alternata on Persimmon Fruit in China.

Sweet persimmon is native to Japan and valued for its fruit, which are high in sugar and vitamins. In October 2021, symptoms were observed on persimmon (Diospyros kaki L. cv. Yangfeng) fruits in cold storage room in Suiping county, Henan Province (32.59 °N, 15 113.37 °E). Initially, small circular dark-brown spots were visible on the fruit rind, turning into irregular sunken dark areas, and eventually rotting 15% of 200 fruits after four weeks of cold storage (10°C, 95% relative humidity). To isolate the causal agent, 10 fruits of symptomatic tissues (4 mm2) were surface-sterilized in 2% sodium hypochlorite (NaOCl) for 1 minute, washed three times in sterile distilled water, then aseptically transferred to potato dextrose agar (PDA) and incubated for 7 days at 25°C. Fungal colonies were isolated from plant tissue, and on three colonies of similar morphology, single-spore isolation was performed. On PDA, the isolates produced circular colonies of fluffy aerial mycelia, gray-brown in the center with gray-white margins. Conidia were dark brown, obclavate or pyriform, with 0 to 3 longitudinal septa and 1 to 5 transverse septa, and a size range of 19.2 - 35.1 × 7.9 - 14.6 μm (n=100). Conidiophores were olivaceous, septate, straight, or bent, with a length of 18 - 60 × 1 - 3 μm (n=100). These morphological characteristics identify the isolates as Alternaria alternata (Simmons. 2007). Genomic DNA was extracted from a representative isolate YX and re-isolated strain Re-YX by cetyltrimethylammonium bromide (CTAB). The primers of ITS1/4, Alt-F/R, GPD-F/R, EF1/2, EPG-F/R (Chen et al. 2022), RPB2-5F/7cR (Liu et al. 1999), and H3-1a/1b (Lousie et al. 1995) were used to amplify the partial internal transcribed spacer (ITS) region, Alternaria major allergen (Alt a1), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF), endo-polygalacturonase (endoPG), RNA polymerase second largest subunit (RPB2) and Histone 3 (His3), respectively. GenBank accession No of ITS, Alt a1, GAPDH, TEF, endoPG, RPB2, His3 were ON182066, ON160008 to ON160013 for YX and OP559163, OP575313 to OP575318 for Re-YX respectively. Sequence data of Alternaria spp. were downloaded from GenBank and the BLAST analysis showed 99%-100% homology between various A. alternata strains (ITS: MT498268; Alt a1: MF381763; GAPDH: KY814638; TEF: MW981281; endoPG: KJ146866; RPB2: MN649031; His3: MH824346). A phylogenetic analysis based on ITS, Alt a1, GAPDH, TEF, and RPB2 sequences using MEGA7 (Molecular Evolutionary Genetics Analysis) revealed that the isolate YX and Re-YX were clustered in A. alternata clade (Demers M. 2022). For the pathogenicity test, seven-day-old cultures were used to create spores suspensions (5.0 × 105 spores/mL) of each of the three isolates. Ten µL aliquots from each isolate were inoculated onto ten needle-wounded persimmon fruits; ten additional fruits were inoculated with water only to serve as controls. The pathogenicity test was three replications. Fruits were deposited in a climate box at 25°C, 95% relative humidity. Seven days post-inoculation, the wounded fruit treated with spore suspensions displayed black spot symptoms similar to the symptoms on the original fruit. There were no symptoms on the control fruits. The strain Re-YX was re-isolated from the symptomatic tissue of inoculated fruits and its identity confirmed using the morphological and molecular methods previously mentioned, fulfilling Koch's postulates. The persimmon fruit rot caused by A. alternata had been reported in Turkey and Spain (Kurt et al., 2010, Palou et al., 2012). According to our knowledge, this is the first report of black spot disease on persimmon fruits caused by A. alternata in China. The disease could infect persimmon fruits during cold storage, so more control methods should be developed to prevent postharvest disease of persimmon in the future.

First Report of Colletotrichum godetiae Causing Anthracnose on Walnut (Juglans regia and Juglans sigillata) in China.

In August 2020, anthracnose lesions were observed on fruits of Juglans regia and J. sigillata in walnut orchards, in Yijun (Shaanxi Province) and Nanhua (Yunnan Province) counties, China. Symptoms on walnut fruits first appeared as small necrotic spots that rapidly enlarged into subcircular or irregular sunken black lesions (Fig. 1a, b). Sixty diseased walnut fruits (30 fruits of J. regia and J. sigillata, respectively) were randomly sampled from six orchards (10-15 ha each orchard, three orchards were selected in each county) with severe anthracnose (incidence rate of fruit anthracnose is over 60% in the orchard.) in two counties. Twenty-six single spore isolates were obtained from diseased fruits as described by Cai et al. (2009). After seven days, isolates formed grey to milky white colony with abundant aerial hyphae on the upper surface of colony, and milky white to light olive on the back of PDA (Fig. 1c). Conidiogenous cells were hyaline, smooth-walled, and cylindrical to clavate (Fig. 1d). Conidia were smooth-walled, aseptate, cylindrical to fusiform, with both ends acute or one end round and one end slightly acute (Fig. 1e), and ranged in size from 15.5-24.3×4.9-8.1 µm (n=30). Appressoria were brown to medium brown, clavate to elliptical, with the edge entire or undulate (Fig. 1f), and ranged in size from 8.0-27.6×4.7-13.7μm (n=30). The morphological characteristics of 26 isolates were similar to those of the species complex Colletotrichum acutatum (Damm et al. 2012). Six representative isolates were randomly selected (three isolates for each province) for molecular analysis. The ribosomal internal transcribed spacers (ITS) (White et al. 1990), beta-tubulin (TUB2) (Glass and Donaldson 1995), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Templeton et al.1992) and chitin synthase 1 (CHS-1) (Carbone and Kohn 1999) genes were amplified and sequenced. Sequences of 6 of 26 isolates were submitted to GenBank (Accession Nos: ITS: MT799938-MT799943, TUB: MT816321-MT816326, GAPDH:MT816327-MT816332, CHS-1: MT816333-816338). Multi-locus phylogenetic analyses revealed that six isolates clustered together with Colletotrichum godetiae ex-type culture isolates CBS133.44 and CBS130251, and the bootstrap support value was 100% (Fig.2). The pathogenicity of two representative isolates (CFCC54247 and CFCC54244) was tested using healthy fruits of the " J. regia cv. Xiangling" and " J. sigillata cv. Yangbi" varieties. Forty sterilized fruits (20 fruits were inoculated with CFCC54247, and 20 fruits with CFCC54244) were wounded by puncturing with a sterile needle through walnut pericarp and inoculated in the wound site with 10 μl of conidial suspension (106 conidia/ml) from seven day old colonies grown on PDA at 25℃. Twenty wounded fruits were inoculated with sterile water as control. Inoculated and control fruits were incubated in containers at 25℃ in a 12/12h light/dark cycle. The experiment was repeated three times. Anthracnose symptoms (Fig. 1g-h) were observed in all inoculated fruits after 12 days, whereas controls showed no symptoms. Fungal isolates from inoculated diseased fruits showed the same morphological and molecular characteristics as the isolates obtained in this study, confirming Koch's postulates. To our knowledge, this is the first report of C. godetiae causing anthracnose on the two walnut species in China. The result will be helpful for providing a basis for further research on the control of the disease.

Colletotrichum fructicola Causal agent of Shot-Hole Symptoms on Leavesof Prunus sibirica in China.

Prunus sibiricaL. (Siberian apricot) is a member of the Rosaceae family and an ecologically important tree species in China (Buer et al., 2022). Shot hole symptoms on the leaves were observed in five Siberian apricot groves in Chengdu (103.81 E, 30.97 N), Sichuan province in July 2020. The symptoms first appeared as small purplish-brown spots with yellow rings around them. As the disease progressed, the damaged area (diameter 1.5-3.0 cm) became necrotic and fell off. The disease incidence was about 60% and the disease index was 28.6 of leaves in the grove. in most severe cases. Fifteen symptomatic leaves were collected from 5 different trees in an orchard. Pathogen isolation was performed from symptomatic leaf tissue (5 × 5 mm) though surface disinfection (in 70% ethanol and 2% NaClO) and incubation on Potato Dextrose Agar (PDA) at 28℃ for 3 days. Overall 10 isolates with similar colony morphology wereobtained from the 15 infected tissue pieces, and three representative isolates (XCK 2-4) were selected for further study. Colonies of the isolates on PDA were initially cottony, pale white to grayish-green with abundant aerial hyphae and produced conidial masses after 7 days. Conidiogenous cells were clavate and aggregated in acervuli. Conidia were smooth-walled, single-celled, straight, and slightly obtusely rounded at both ends, 12.8 to 18.7 × 4.3 to 5.7 μm in size (Fig. 1). The morphological characteristics of the three isolates were consistent with the description of species in the Colletotrichum gloeosporioides complex. DNA was amplified using the following primers pairs for the internal transcribed spacer (ITS) region of rDNA and partial sequences of beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and translation elongation factor (TEF-1), respectively: ITS1/ITS4, T1/Bt2b, GDF/GDR, CHS-F/CHS-R, and EF-F/EF-R (Vieira et al., 2014). Accession numbers (MW228049, MW284974, MW284976, MW284975 and MW284977, respectively) were obtained afterepositing all the resulting sequences in GenBank. Nucleotide blast showed 99 to 100% identities with Colletotrichum fructicola (GenBank accessions nos. MZ961683, MW284974, MN525881, MN525860, MF627961). Phylogenetic analysis of combined ITS-TUB-GAPDH genes using the Mrbayes inference method showed that the three isolates clustered with three reference isolates of C. fructicola as a distinct clade(Fig. 2). To verify Koch's postulates, ten 3-year-old healthy potted plants ofP.sibirica were inoculated by spraying a conidial suspension (6 × 105 conidia/mL) of isolate XCK2 on both sides of leaves, and the control leaves were sprayed with sterile water. Then, all treatments were placed in a moist environment (25±2°C, 80% relative humidity, natural light). The inoculated plants showed typical symptoms of plants with natural infections, while the controls remained asymptomatic after 14 days. The pathogen C. fructicola was re-isolated from all inoculated plants, and the culture and fungus characteristics were the same as those of the original isolate. Colletotrichum fructicola was not isolated from the control plants. The results indicated that C. fructicola is the causal agent of the disease. Colletotrichum fructicola was reported as a leaf pathogen on Camellia chrysanthain China (Zhao et al., 2021). This is the first report of C. fructicola causing P. sibirica leaf shot-hole in the world. The identification of C. fructicola could provide relevant information for applying management strategies and research on the Siberian apricot disease.

First Report of Sharp Eyespot of Oat (Avena sativa) Caused by Ceratobasidium cereale in Korea.

Along with barley and wheat, oats (Avena sativa) are cultivated as winter crops in Korea, and the total area for oat cultivation is 103 ha in 2021. From late March to early April 2021, sharp eyespot symptoms on oat (cv. Choyang) leaf sheaths and straws were observed in two commercial fields located in Haenam (N34°38'35.04588/E126°38'31.00668) and Gangjin (N34°38'9.46788/E126°37'19.44984), Jeollanam-do, Korea. The incidence was 5% and 7%, respectively. Small brown spots were irregular circles that began to appear on the lower sheaths, and the spots gradually enlarged in the upper part of the sheaths. The center of each lesion turned whitish-brown with dark brown margins, resulting in a blight of the sheaths. Three plants displaying typical sharp eyespot lesions were collected from each of two individual regions, Haenam and Gangjin. To isolate the causal pathogen, two infected tissues (5  5 mm) from the collected plants were surface-sterilized by treating them with 70% ethanol for 1 min and 1% NaClO for 1 min immediately after being treated with 95% ethanol for 1 min. Subsequently, the samples were rinsed three times with distilled water, dried with sterile filter paper, transferred to 1.5% water agar supplemented with 100 ppm streptomycin, and then incubated in the dark at 25°C. Hyphae emerging from the randomly selected three independent tissues from each location were subcultured on potato dextrose agar (PDA, Sparks, MD 21152, USA), resulting in three independent isolates (HNO-1, HNO-2, HNO-3) from Haenam and three (KJO1-1, KJO1-2, KJO1-3) from Ganjin after single-hypha-tip purification. Colonies on the PDA were pigmented white at first and subsequently changed to light brown after 2 weeks. All collected isolates formed globose and irregular dark brown to black sclerotia on PDA after 2 weeks. Binuclear hyphae were white to dark brown in color, branched at right angles with a septum near the branch, and multinucleate cells, suggesting that these isolates belonged to Ceratobasidium cereale (Boerema et al., 1977; Burpee, 1980; Sharon et al.,2008). For molecular identification, the ITS (GenBank accession nos. MW691851-53 for HNO-1 to HNO-3; MW691857-59 for KJO1-1 to KJO1-3), LSU (OQ397530-35), rpb2 (OQ409878-83), tef1 (OQ409884-89), and atp6 (OQ409890-95) regions of six isolates were amplified using the primer pairs ITS4/5 (White et al., 1990), LROR/LR5 (Vilgalys and Hester, 1990), bRPB2-6F/bRPB2-7.1R (Matheny, 2005; Reeb et al., 2004), TEF1-F/TEF1-R (Litvintseva et al., 2006), and ATP61/ATP62 (Kretzer and Bruns, 1999), respectively. The sequences of ITS region showed 99.7% identity with C. cereale strain WK137-56 (KY379365) and 99.8% with Ceratobasidium sp. AG-D (KP171639). Using the MEGA X program (Kumar et al. 2018), a maximum likelihood phylogenetic analysis based on the concatenated ITS-LSU, rpb2, tef1 and atp6 sequences placed the six isolates within a clade comprising C. cereale (Gónzalez et al.,2016; Ji et al., 2017; Tomioka et al., 2021; Li et al., 2014). Two representative isolate, HNO-1 and KJO1-1, were deposited in the Korean Agriculture Culture Collection (Accession No. KACC 49887 and 410268, respectively). For pathogenicity, the six isolates were cultured on sterilized ray grains at 25°C in the dark for 3 weeks as the inoculum. Five oat (cv. Choyang) seeds were sown per pot containing 80 g of the infected ray grains mixed with 150 g of composite soil and 150 ml of water (Baroker Garden Soil, Seoul Bio Co., LTD). The control was treated with 80 g of the sterilized ray grains mixed with 150 g of composite soil and 150 ml of water. All inoculated and control pots were placed in a 20°C growth chamber with a 12-h photoperiod and 65% humidity. Typical sharp eyespot symptoms were observed on the oat sheath of seedlings three weeks post-inoculation. No symptoms were observed in the control seedlings. The infection assays were repeated thrice, with similar results. The pathogen was successfully re-isolated, and its identity was confirmed via morphological and molecular analyses. In Korea, few etiological studies have been conducted on oats because they are less economical than barley and wheat. Sharp eyespot disease caused by C. cereale has already been reported in barley and wheat (Kim et al., 1991); however, this is the first report of this disease in oats in Korea.

Effect of Beam Oscillation Amplitude on Microstructure and Mechanical Properties of Small Laser Spot Welded QP980 Steel

In this study, butt welds of QP980 steel were produced using small laser spot (0.1 mm) oscillating welding. The effect of beam oscillation with a circular trajectory on weld morphologies, microstructures, and mechanical properties was characterized. As the oscillating amplitude rose, the energy accumulation range enlarged, and the energy peak value was decreased, leading to the appearance of the cross-section changing from a nail-like shape to a cup-cone-like shape and then to a W-type shape. The weld zone is divided into the fusion zone, inner heat-affected zone, and outer soften zone. The fusion zone and inner heat-affected zone are full of typical lath martensite and have the highest hardness. The soften zone is composed of pre-existing martensite, temper martensite, ferrite, and retained austenite and has the lowest hardness. Compared to laser welding, beam oscillation could reduce the pre-existing block martensite to decompose, leading to a narrower width and higher hardness soften zone. Although the width of the fusion zone and soften zone increases with the oscillation amplitude, all welded samples failed at the base metal with 97% joint efficiency.

Detection of aroid leaf rust Pseudocerradoa paullula on Swiss cheese plant Monstera deliciosa in the continental USA.

Monstera deliciosa Liebm. (Araceae, Monocots), sometimes referred to as Swiss cheese plant, is one of the most common aroids used as an indoor and landscape ornamental plant (Cedeño et al. 2020). Production of M. deliciosa and other closely related Araceae species represents an important sector of the ornamental nursery business worldwide. Swiss cheese plant is believed to have originated in the tropical forests of southern Mexico, where its fruit is considered a delicacy due to its sweet, exotic flavor (Cedeño et al. 2020). Since 2019, symptomatic Monstera plants from two plant nurseries and residential properties in South Florida were submitted for disease diagnosis to the Florida Department of Agriculture and Consumer Services, Division of Plant Industry (FDACS-DPI) in Gainesville, Florida, and to the University of Florida, Tropical Research and Education Center Plant Clinic in Homestead, Florida. Symptoms included small chlorotic spots on the leaf surface, which expanded and became brown to reddish-brown often with a yellow halo and produced uredinia with abundant urediniospores. The pathogen was identified morphologically as the rust fungus Pseudocerradoa (=Puccinia) paullula (Syd. & P. Syd.) M. Ebinghaus & Dianese (Pucciniaceae, Basidiomycota) (Ebinghaus et al. 2022), characterized by the production of pseudosuprastomatal uredinia. Uredinospores light-brown and globose, echinulate (1 µm height), reddish to light brown, 24 - 31 µm diameter, with thick walls, 1.5 - 2.5 µm height (n=15). Teliospores 2-celled, light-yellow and ellipsoidal, 23 - 28 × 19 - 24 µm (n =15) were observed in sori appearing as dark-brown leaf spots on the adaxial side of the leaves (e-Xtra Fig. 1). Molecular characterization of the fungal pathogen was based on the small subunit (SSU), internal transcribed spacer (ITS), and large subunit (LSU) of the ribosomal RNA genes (Aime 2006) with the addition of a LSU internal primer specific for the rust species Ppaullula_int-forward 5'ATAGTTATTGGCTTTGATTTACA-3' designed in this study to increase the quality and the sequence read length due to a 3'- ~21-Ts-homopolymer (e-Xtra Fig. 2) (GenBank accession number ON887196, ON887197, OQ275200, OQ275201). In addition to morphological identification, the host plant was identified using the Ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL) and Maturase K (matK) genes (Fazekas et al. 2012) (GenBank accession numbers ON887189, ON887193, respectively). MegaBlast searches confirmed the morphological identification with 100% identity to M. deliciosa vouchers GQ436772 and MK206496, respectively (Chen et al. 2015). Dried specimens were deposited in the Plant Industry Herbarium Gainesville (PIHG 16226, 16227, 17154, 17155). Molecular identification of the rust pathogen P. paullula was carried out through megaBlast (Chen et al. 2015) searches together with a phylogenetic analysis performed in RAxML v8 (Stamatakis 2014) (e-Xtra Fig. 3). Koch's postulates were performed by using urediniospores, collected from an infected sample and were kept for 7 days at 4 C, as an inoculum source. Healthy rooted M. deliciosa plants were inoculated by rubbing the inoculum on both leaf surfaces at >90% RH, room temperature, 12/12 light cycle. After the incubation period (48 h), plants were placed in a climate-controlled greenhouse and watered twice a week, ~30 C, ~65 RH, 12/12 light cycle. After three weeks, all inoculated plants developed symptoms resembling those observed on the samples submitted for disease diagnosis. Controls did not show symptoms. Spores from the pustules of inoculated plants were identified as P. paullula by both morphology and molecular means. The genus Pseudocerradoa comprises P. paullula and its sister species P. rhaphidophorae (Syd.) M. Ebinghaus & Dianese. Both species can be distinguished by size and coloration of urediniospores and their host range within the Araceae. Pseudocerradoa rhaphidophorae produces smaller urediniospores and only occurs on Rhaphidophora species (Shaw 1995). Pseudocerradoa paullula is not considered fully established in Florida, since the host distribution is mainly restricted to indoors and M. deliciosa is rarely used as an outdoor ornamental (Wunderlin et al. 2023). Here we name the disease caused by P. paullula as "aroid leaf rust", due to its ability to infect several species in this plant family. Other closely related hosts reported as susceptible to this pathogen are Monstera standleyana G.S.Bunting (as M.s. cv. variegata), Monstera adansonii var. laniata (Schott) Mayo & I.M. Andrade, Monstera subpinnata (Schott) Engl., Typhonodorum lindleyanum Schott, and Stenospermation sp. (Shaw 1991, 1992, 1995). To date, the aroid leaf rust was only known from Australia, China, Japan, Malaysia, and Philippines (Lee et al. 2012; Shaw 1991). Based on our review, P. paullulla was intercepted once from Malaysia in 2014 at the port of Los Angeles, USA (BPI voucher 893085). This present study reports the establishment of P. paullula in Florida, USA infecting M. deliciosa.

The Finite Element Analysis on the Effects of Overlapping Rate in Low-Energy Laser Shock Peening

Laser shock peening has been widely studied and pioneeringly applied in aerospace industry as a life-extension technology for structured mechanical components. However, in other promising fields such as nuclear power industry, little has been studied concerning such critical issues as long-distance transmission of the laser beam by optical fiber and optimized parameters of typically low pulse energy with micrometer-sized beam spots. In such scenario, the overlapping rate between adjacent small spots plays a critical role in obtaining homogenous residual stress and surface morphology. In this study, a three-dimensional finite element model in AISI 420 martensitic stainless steel has been developed to correlate the residual stress as well as surface morphology with varying overlapping rates. Multiple laser spots are loaded with VDLOAD user subroutine in Abaqus. The residual stress distribution is analyzed with respects of laser shocking and in-depth planes. And the surface morphology is evaluated in terms of depression depth as well as surface roughness. Combined results suggest that the overlapping rate of 61% as an optimized value, which can be used as a basis for future experimental studies and industrial applications.

First Report of Anthracnose Caused by Colletotrichum camelliae on Amorphophallus konjac in Hunan Province of China.

Amorphophallus konjac is a crop in the family Araceae, which is widely cultivated in Hunan, Yunnan, and Guizhou in China. A. konjac flour is highly valuable economically as a product for weight reduction. In June 2022, a new leaf disease was discovered in an understory A. konjac plantation in Xupu County, Hunan Province, China, where 2,000 hm2 of A. konjac had been planted. Approximately 40% of the total cultivated area exhibited symptoms. The disease outbreaks occurred during warm and wet weather (May to June). In the early stages of infection, small brown spots appeared on the leaves and then gradually spread into irregular lesions. There was a light yellow halo around the brown lesions. In severe cases, the whole plant gradually turned yellow and died. Six symptomatic leaf samples were collected from three different fields in Xupu County to isolate the causal agent. They were rinsed with sterile water, and the lesions were cut off. The lesions were rinsed in 3% hydrogen peroxide for 30 s followed by a 90 s treatment in 75% alcohol. They were then rinsed five times in sterile water, placed on water agar plates and incubated for 2-3 days at 28°C. After the mycelium had grown, they were transferred to potato dextrose agar (PDA) plates and incubated for 3-5 days at 28°C. In total, ten isolates were obtained, and seven of them were Colletotrichum (isolation frequency 70%). Three representative isolates (HY1, HY2, and HY3) were selected for further study. This fungus grew as circular white colonies, which then became grey. The older colonies looked like cotton and had dense aerial hyphae. The conidia were cylindrical, lacked a septum, and were thin-walled. They measured 14.04 to 21.58 × 5.89 to 10.40 μm (n=100). To further confirm its fungal identity, the fungus was amplified and sequenced using six genetic regions, including β-tubulin (TUB2), actin (ACT), the internal transcribed spacer (ITS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), calmodulin (CAL) and chitin synthase (CHS). The universal primers BT2a/TUB2R, ACT512F/ACT783R, ITS4/ITS5, GDF/GDR, CL1C/CL2C, CHS79F/CHS345R were used for amplification (Weir et al. 2012), sequenced by the Sanger chain termination method, and submitted to GenBank (TUB2: OQ506549, OQ506544, OP604480; ACT: OQ506551, OQ506546, OP604482; ITS: OQ457036, OQ457498, OP458555; GAPDH: OQ506553, OQ506548, OP604484; CAL: OQ506552, OQ506547, OP604483; CHS: OQ506550, OQ506545, OP604481). An analysis of a joint phylogenetic tree that was constructed using the six genes showed that the three isolates clearly clustered with Colletotrichum camelliae (syn. Glomerella cingulata f. sp. camelliae) strain ICMP 10646 (GenBank: JX010437.1, JX009563.1, JX010225.1, JX009993.1, JX009629.1, JX009892.1) and HUN1A4 (GenBank: KU252173.1, KU251646.1, KU251565.1, KU252019.1, KU251838.1, KU251913.1). HY3 was used as a representative strain for the pathogenicity test on the leaves of A. konjac from the whole plant. PDA blocks that were 6 × 6 mm and had been cultured for 5 d were placed on the leaf surface, and sterile PDA blocks were used as the control group. The climate chamber was maintained at 28°C at all times, and 90% relative humidity was maintained. The pathogenic lesions appeared after 10 days of inoculation. The pathogen that was re-isolated from the diseased tissues had the same morphological characteristics as HY3. Thus, Koch's postulates were fulfilled. C. camelliae has been shown to be the primary pathogenic fungus responsible for anthracnose in tea (Ca. sinensis (L.) O. Kuntze) (Wang et al. 2016) and camellia oleifera (Ca. oleifera Abel.) (Li et al. 2016). Anthracnose caused by Colletotrichum gloeosporioides has been reported on A. konjac (Li. 2021). However, to our knowledge, this is the first report in China and worldwide that C. camelliae causes anthracnose on A. konjac. This research lays the foundation for future research to control this disease.

Focusing of Radially Polarized Electromagnetic Waves by a Parabolic Mirror

It is well-known that a strong longitudinal electric field and a small spot size are observed when radially polarized beams are tightly focused using a high numerical aperture parabolic mirror. The longitudinal electric field component can accelerate electrons along the propagation axis at high intensities in the focal region, which opens an application in particle acceleration. In this paper, we present a rigorous derivation of the electric field obtained when a radially polarized, monochromatic, flat-top beam is focused by a parabolic mirror. The formulae were deduced from the Stratton–Chu integral known from vector diffraction theory. We examined the influence of the focusing parameters on the distribution of both the longitudinal and radial electric field components. In the small numerical aperture and short wavelength regimes, excellent agreement was found with the results obtained from the Rayleigh–Sommerfeld formula. The calculation method can be adapted for various beam types and for electromagnetic pulses as well.

First Report of Anthracnose on Tetrapanax papyriferus Caused by Colletotrichum fructicola in China.

Tetrapanax papyriferus is an evergreen shrub native to China and traditionally used as a herbal medicine (Li et al., 2021). In September 2021, a serious leaf spot disease with symptoms similar to anthracnose was extensively observed on T. papyriferus in Shibing county (E 127°12'0", N 25°11'60"), Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou province, China. Field surveys were conducted in about 1000 T. papyriferus plants in Shibing in September 2021. The incidence of the leaf spot on leaves was 45% to 60%, significantly reducing the quality of medicinal materials. The symptoms began as small yellow spots, developing a brown center and dark brown to black margin, and eventually the diseased leaves were wiltered and rotted. Symptomatic leaves were collected from 20 trees. Symptomatic tissue from diseased leaves was surface desinfected (0.5 min in 75% ethanol and 1 min in 3% NaOCl, washed three times with sterilized distilled water), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on potato dextrose agar (PDA) and incubated at 25°C for about 7 days (Fang. 2007). Three single-spore isolates were obtained (GUTC37, GUTC310 and GUTC311) and deposited in the collection of the Plant Pathology Deparment, College of Agriculture, Guizhou University, China (GUCC) (with the accession numbers, GUCC220241, GUCC220242, GUCC220243 respectively). These isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS], glyceraldehy-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], actin [ACT], and calmodulin [CAL] genes (White et al. 1990; Carbone and Kohn 1999; Templeton et al. 1992). Therefore, the representative isolate GUTC37 was used for further analysis. The pathogenicity of GUTC37 was tested through a pot assay. Plants were inoculated by spraying a spore suspension (106 spores·ml-1) of isolated strains onto leaves until runoff, and the control leaves sprayed with sterile water. The inoculated plants were incubated in a growth chamber at 28 ℃ and 95% relative humidity for 10 days. Pathogenicity tests were repeated three times (Fang. 2007). The symptoms developed on the inoculated leaves, while control remained asymptomatic. The lesions were first visible 72 h after inoculation, and typical lesions like those observed on field plants appeared after 10 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses from the infected leaves but not from the non-inoculated leaves. Results of pathogenicity experiments of isolated fungi fulfilled Koch's postulates. Fungal colonies on PDA were villiform, creamy-white or greyish, aerial mycelium pale grey, dense, surface partly covered with orange conidial masses. The conidia were abundant, oval-ellipsoid, aseptate, and 13.89 (11.62 to 15.21) × 5.21 (4.39 to 5.65) µm (n=50). Appressorium were greyish green, nearly ovoid to cylindrical, 9.64 (6.62 to 14.61) × 6.33 (5.45-7.72) µm (n=50). The morphological features were consistent with the descriptions of Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Prihastuti et al. 2009). The pathogen was identified to be C. fructicola by amplification and sequencing of the five genes. The sequences of the PCR products were deposited in GenBank with accession numbers OP143657 (ITS), OP177868 (GAPDH), OP177865 (CHS-1), OP278677 (ACT) and OP177862 (CAL). BLAST searches of the obtained sequences revealed 100% (509/509 nucleotides), 99.63% (269/270 nucleotides), 99.31% (287/289 nucleotides), 99.29% (280/282 nucleotides), and 99.86% (728/729 nucleotides) homology with those of C. fructicola in GenBank (JX010165, JX010033, JX009866, FJ907426, and JX009676, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate GUTC37 in a well-supported cluster with C. fructicola. To our knowledge, this is the first report of anthracnose on T. papyriferus caused by C. fructicola in Guizhou, China. This study provides valuable information for the identification and control of the anthracnose on T. papyriferus.

Detection and location of microaneurysms in fundus images based on improved YOLOv4 with IFCM

AbstractDiabetic retinopathy (DR) is one of the leading causes of blindness for people suffering from diabetes. Microaneurysm (MA) is the initial symptom of DR. MA is a lesion based disease which starts as small red spots on the retina and increases in size as the DR progresses which finally leads to blindness. So eliminating the lesion can effectively prevent DR at an early stage. However, due to complex retinal structure, different brightness and contrast of fundus images with effects of factors such as different patients, environment changes, and difference in acquisition equipment, it is difficult for existing detection algorithms to achieve accurate results of MA detection and location. Therefore, the detection algorithm of improved YOLOv4 (YOLOv4‐Pro) was proposed. First, an improved Fuzzy C‐Means (IFCM) clustering algorithm was proposed to optimize anchor parameters of target samples to improve matching results between anchors and feature graphs. In order to control noise and improve efficiency, a median filtering method was employed to update the criterion function of the original FCM algorithm, and a K‐means algorithm was employed to initialize clustering. Second, a SENet attention module was added in the backbone of YOLOv4 to enhance key information and suppress background, improving the confidence of MA effectively. Finally, the spatial pyramid pooling (SPP) module was added to the neck to enhance the acceptance domain of the output characteristics of the backbone network, and profits separating of important context information. The improved YOLOv4 with IFCM was verified on the Kaggle DR dataset and compared with other methods. Experimental results show that optimizing the prior frame with the IFCM algorithm can make it suitable to frame the Kaggle DR dataset, which improves the detection accuracy of the network by nearly 5%, and provides a nice performance on detection and location of MA in fundus images. This would help ophthalmologists finding the exact location of MA on retina, thereby simplifying the process and eliminating any manual intervention.

Power scaling of a self-mode-locked vertical-external-cavity surface-emitting laser

A typical self-mode-locked vertical-external-cavity surface-emitting laser must operate at the edge of the stable region of the resonant cavity. Its minimal pump spot on the gain chip is used as a soft aperture. By comparing with a continuous-wave (CW) laser, the pulsed laser is focused more tightly on the gain chip due to the Kerr-lens effect. This mitigates cavity loss of the pulsed laser in comparison with the CW laser, such that successive mode-locking can be initiated. The disadvantage of the above method is that the relatively small pump spot, producing relatively large thermal effect, limits the output power of the laser. To address this issue, we propose another method with the work point of the laser moved slightly from the edge of the stable region and the pump spot moderately extended, with a spot of the pulsed laser on the gain chip that could be smaller or larger than that of the CW laser. We achieve stable self-mode-locking with a record average output power of 8.18 W in a V-type resonator, limited by the applied pump power. The pulse repetition rate and width are 0.71 GHz and 1.92 ps, respectively, and the corresponding peak power is 5.6 kW.

First Report of Leaf Spot Caused by Lasiodiplodia theobromae on Kadsura coccinea in China.

Kadsura coccinea (Lem.) A. C. Smith is an evergreen liana widely cultivated in China for its economic importance in traditional medicine. Many phytochemical studies on the stems and roots of K. coccinea have shown a variety of biological activities, such as anti-hepatitis, anti-HIV, and anti-tumor (Yang et al. 2020). In July 2021, symptoms of leaf spot were observed in a plantation of K. coccinea in Longan (23°03´N, 107°54´E), Guangxi province, China. The incidence of this disease was 36%, and severity varies from approximately 20 to 40% of leaf surface coverage. Symptoms began as small brown spots that expanded into irregular to nearly flower-shaped lesions. To isolate the pathogen, leaves with spots were collected, sterilized with 75% ethanol for 15 s followed by 2% sodium hypochlorite for 120 s, rinsed three times in sterilized distilled water, cut into 5 × 5 mm pieces, and placed onto potato dextrose agar (PDA) plates. The plates were kept in an incubator at 26°C in the dark for at least 2 days. A total of 27 fungal colonies of similar morphology out of 30 pieces of infected tissues were isolated. Four representative isolates (HBB1 to HBB4) were selected to study for further characterization. Fungal colonies were initially grayish-white and then turned greenish-gray on PDA. The black pycnidium and immature conidia appeared over PDA plates after 18 days. The immature conidia were colorless and transparent, elliptical, and had a single-cell structure. After 5 days, the immature conidia gradually become black and develop into mature conidia. The mature conidia were dark brown and two-celled with longitudinal striations, 20.41-29.93 × 12.42-17.19 μm (average 26.07×14.51 μm; n = 100). For DNA-based identification, the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha (EF1-α), and β-tubulin (TUB) genes of the isolates were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences were submitted to GenBank (Accession nos. MW045412 to MW045415 for ITS, MW065559 to MW065562 for EF1-α, and MW065555 to MW065558 for TUB). A phylogenetic analysis was conducted using the Maximum Likelihood method on concatenated sequences of the three genes, which showed that the four Chinese isolates from K. coccinea were clustered with reference isolates of Lasiodiplodia theobromae including the ex-neotype CBS 164.96. Pathogenicity tests were performed on young, fully expanded leaves of 2-year seedlings. A 10 μL conidial suspension (1×106 conidia/mL) was inoculated on each wound on the left-half leaf and a 10 μL sterile water was inoculated on each wound on the right-half leaf (control). Each treatment was repeated three times. Inoculated leaves were wrapped in plastic bags for 5 days and plants were maintained in a growth chamber at 27°C, 85% relative humidity. Brown leaf spots appeared 5 to 6 days after inoculation, whereas the control leaves treated with sterile water showed no symptoms. All re-isolations from spots produced colonies with the same morphological characters as L. theobromae, completing Koch's postulates. To our knowledge, this is the first report of L. theobromae causing leaf spot on K. coccinea in China and worldwide. Severe leaf disease caused by L. theobromae threatens K. coccinea production. The disease threatens K. coccinea growth, and effective control measures should be identified to reduce losses.

First report of leaf spot on pecan caused by Choanephora cucurbitarum in China.

Pecan (Carya illinoinensis K. Koch) is an important and widely planted nut tree species in Jiangsu Province, China (Mo et al. 2018). In July 2020, leaf spot symptoms were frequently observed on pecan in Jurong, Jiangsu Province (119°15'36"E, 32°1'6"N). Disease incidences ranged from 40 to 65% among 150 mature pecan trees from three nurseries. The disease severity index (DSI, Jiang et al. 2019) reached 58.4. Symptoms began as small brown spots scattered on leaves that gradually expanded to large, circular to irregular black and brown necrotic lesions. In severe cases, lesions developed on large portions of a single leaf, and eventually the dead leaves fell from the trees. Three monoconidial isolates (Chen2346, Chen2347, Chen2348) were isolated from lesion margins and cultured on potato dextrose agar (PDA) medium. Colonies on PDA were white and cottony, later becoming light gray with abundant reproductive structures. Sporangiophores were aseptate, hyaline, unbranched, and apically dilated to form a clavate vesicle, which produced sporangia. Sporangia were globular to ellipsoid, brown to dark brown, 103 to 128 µm in length, and 88 to 114 µm in width (n = 30). Sporangiola were brown, ellipsoid to ovoid, with longitudinal striae, and measured 13.9 to 18.8 × 7.9 to 10.8 μm (n = 60). The morphological characteristics of these isolates agreed with descriptions of Choanephora cucurbitarum (Kirk 1984). Genomic DNA of these three monoconidial isolates was extracted, and the partial sequences of the internal transcribed spacer (ITS) and large subunit (LSU) of rDNA were amplified using primer pairs ITS1/ITS4 (White et al. 1990) and LR0R/LR7 (Vilgalys and Hester 1990), respectively. The consensus sequences (GenBank accession nos.: OP315248 to OP315250 for ITS and OP315251 to OP315253 for LSU) were aligned using BLASTn and showed100% identity with the sequences from C. cucurbitarum found in GenBank (accession nos.: MF942131 for ITS and ON025788 for LSU). To further confirm the identity, a phylogenetic analysis was performed with MEGA (v.7.0) and MrBayes (v.3.1.2) software, using the maximum likelihood and Bayesian inference methods, respectively. The multigene phylogeny revealed that the three isolates in this study, as well as, C. cucurbitarum specimen, clustered as a strongly supported monophyletic group (99 bootstrap value; 0.95 posterior probabilities). Based on the morphological and molecular data, the isolates were identified as C. cucurbitarum. To confirm pathogenicity, healthy pecan seedlings (2 years old) were each inoculated with a mycelial block (3 × 3 mm) excised from the margin of a colony on the vein of each leaf. Seedlings treated with non-colonized PDA blocks were used as controls. The inoculated seedlings were maintained in sterile plastic boxes with moistened sheets of filter paper at 25 ± 1°C and a 12-h photoperiod. Fifteen leaves per isolate were tested for each treatment. The experiment was repeated twice. Three days after inoculation, symptoms similar to those in the field appeared, whereas the control leaves remained symptomless. Subsequently, C. cucurbitarum was reisolated from the lesions and morphologically identified, confirming Koch's postulates. To the best of our knowledge, this is the first report of C. cucurbitarum causing leaf spot on C. illinoinensis in China. This study provides the foundation to further investigate the biology, epidemiology, and management of this disease.

First Reportof Colletotrichum fructicola Causing Anthracnose on Glycine max in China.

Soybean (Glycine max L.) is one of the important oilseed and vegetable crop worldwide and provides the main source of vegetable oil and proteins for human and livestock (Hartman et al. 2011). In October 2021, approximately 35% of soybean pods suffered from anthracnose in the farmer's field in Chongzhou, Sichuan Province, China (103°40'12"E, 30°37'48"N), and the occurrence area accounted for about 3.3 hm2. Symptoms of soybean were characterized by yellow spots at the initial stage, gradually expanded into dark brown spots, and eventually amounts of small black particles were densely arranged in the wheel shape on dead spots. Diseased spots of soybean pods were cut into pieces and sequentially sterilized in 75% alcohol for 30 s, 4% sodium hypochlorite for 30 s, sterile water for 3 times. After that, these pieces were placed on potato dextrose agar (PDA), and incubated at 25±2°C in the dark for 5-7 days. Single spore was separately picked and transferred to a fresh PDA plate to obtain pure culture isolates. Total six pure isolates were collected, and among them the hyphae of representative isolate 8-B were initially white, turned grey gradually on PDA medium, and the colonial reverse were radiating, whorled or a mixture of both. Conidia of 8-B were septate, hyaline, unicellular, cylindrical, obtusely rounded at both ends with 1 or 2 oil balls inside, and 10.5-17.6 µm in length and 7.0 µm-3.6 µm in width (n=100). The conidial appressoria were brown subspherical, 6.9 µm-13.3 µm in length and 5.6 µm-10.1 µm (n=50) in width. Based on morphological and cultural characteristics, the isolate 8-B was tentatively identified as Colletotrichum gloeosporioides species complex（Weir et al. 2012). To test pathogenicity,the mycelial plugs were inoculated on 20 detached soybean pods at full seed (R6) stage, and three areas of each pod were lightly scratched using a needle prior to inoculation. As controls, the PDA plugs were attached to the pinned-treated pods. Three independent replicates were conducted for control and inoculated pods, respectively. All pods were incubated in a greenhouse at 25 ± 2°C with a relative humidity of approximately 90%. After 4-5 days post-inoculation, typical anthracnose lesions were observed on the inoculated pods while the control pods remained healthy only with small wound spots. The pathogen re-isolated from all the inoculated pods were morphologically identical to the inoculation isolate (8-B). For further molecular verification, the six gene fragments including the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), β-tubulin 2 (TUB2) and calmodulin (CAL) were amplified and sequenced (Weir et al. 2012, Damm et al. 2012), and the obtained sequences were deposited in GenBank (Accession numbers ON960278, ON685214, ON964475, ON974476, ON685215 and ON964477, respectively). All six gene sequences of 8-B had a high identity to C. fructicola (the stand isolate ICMP 18581) with the accession numbers ON960278 (100%), ON974476 (96%), ON685214 (99%), ON964475 (99%), ON685215 (100%), and ON964477 100%), respectively. Anthracnose disease caused by C. fructicola has previously been reported to affect a range of plant hosts worldwide (Guarnaccia et al. 2017). However, it is still unknown on C. fructicola causing anthracnose in soybean in China. This study firstly reports C. fructicola as the causal agent of anthracnose on soybean in the country, and provides a theoretical basis for the diagnosis and control of this disease.

Comparative study of process parameters on dross properties by laser machining of AISI 316L material

The main aim of this paper is to examine the impact of process parameters on the dross height formation for CO2 and fiber laser machining of SS 316L material. CO2 and fiber laser machining has a small spot size, narrow beam, high intensity, and depth of focus that are conveniently absorbed by the metal surfaces. The work is concentrated on the experimental study of laser machining of SS 316L material for 20 mm thickness. The cut qualities of the workpiece are analysed by measuring the dross height by changing the process parameters like assist gas pressure, laser power, and cutting speed. The experimentation is performed on Bystronic laser cutting machine. In this study, the DOE technique is used implementing the response surface methodology approach. Three factors and two levels are selected, and the total numbers of test runs are 17. Mathematical calculations are carried out using ANOVA. The optimum values of performance parameters are defined to achieve the required minimum dross height. The experimental analysis identified that the CO2 laser process produces higher cut quality as compared to the fiber laser process. The major inference that is drawn from this study is change in cutting speed affects the dross height. Gas pressure did not show much effect on change in dross height formation.

First Report of Lasiodiplodia theobromae Causing Brown Leaf Spot on Bruguiera gymnorrhiza in China.

Bruguiera gymnorrhiza (Linn.) Sav. is a dominant tree species of mangrove forests in tropical coastal areas of China. This species is commonly used for the greening of tidal flats and seawalls in tropical and subtropical regions (Allen, & Duke 2000). A survey that was conducted from August to September 2020, in the mangrove national nature reserve at Zhanjiang, Guangdong Province, South China. Brown leaf spot symptoms were observed on Bruguiera gymnorrhiza and disease incidence was over 10% (200 investigated trees). Symptomatic small spots initially appeared at the middle or edges of leaves, enlarged irregularly, and developed into brown necrotic spots with dying curly edges. The color of the lesion's center changed to dark brown or gray. To identify the causative agent, twenty diseased leaves were sampled for pathogen isolation. Affected foliar tissues cut into 5 × 5 mm pieces, disinfected in 75% ethanol for 2 mins, rinsed in sterile distilled water, and then air dried under a sterilized filter paper. Leaf pieces were plated onto potato dextrose agar (PDA) in Petri dishes and then incubated at 28°C in darkness for 3-5 days. Hyphal tips of fungal colonies growing from the tissue pieces were subcultured onto fresh PDA to obtain pure single hyphae cultures. The fungal colonies were initially composed of white aerial mycelia, but turned gray after 7 days. Immature conidia were hyaline, subovoid, and aseptate while mature conidia becoming dark brown, one-septate with longitudinal stripes, the length/width ratio is 19.98 to 29.50 μm (average 24.37 µm; n = 50) × 11.99 to 14.45 μm (average 13.09 µm; n = 50). On the basis of morphological features all isolates were identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl (Alves et al. 2008). For DNA-based identification, the internal transcribed spacer (ITS) region gene and fragment of elongation factor 1-alpha (EF1-α) gene of the three isolates were amplified and sequenced following the methods described in a previous study (White et al. 1990, Carbone & Kohn 1999). The obtained sequences of ITS and EF1-α were deposited in GenBank with accession numbers OK644200 and OL345571. The BLAST results showed at least 99.60% similarity with the sequences of Lasiodiplodia theobromae (ITS, MT644474.1 [99.79%]; EF1-a, MK961975.1 [99.60%]). To fulfill Koch's postulates, PDA plugs with actively growing mycelium of the isolates were inoculated on the leaves of Bruguiera gymnorrhiza plants that were wounded by using a sterilized needle or scalpel. Inoculated leaves were covered with sterilized wet cotton, and the plants were kept at 28°C and 80% relative humidity. The inoculated plants showed leaf spot symptoms that were similar to those previously observed in the field after 1-2 days, whereas control leaves remained healthy. Lasiodiplodia theobromae was consistently isolated from inoculated leaves again. Lasiodiplodia theobromae (Botryosphaeriaceae) is a plurivorous pathogen in a wide variety of hosts, mostly prevalent in tropical and subtropical climate regions. It has been previously reported to cause brown leaf spot on Broussonetia papyrifera (Luo et al. 2020), foliar diseases on Camellia oleifera (Zhu et al. 2014) and Kadsura longipedunculata (Fan et al. 2020). To our knowledge, this is the first report of Lasiodiplodia theobromae causing brown leaf spot on Bruguiera gymnorrhiza plants in China and worldwide. Our findings will help to make management strategies for control of this disease on Bruguiera gymnorrhiza.

Bessel beam generated LIPSS on NiTi wires

Ultrashort laser pulses are used to generate Laser-Induced Periodic Surface Structures (LIPSS). To apply LIPSS on small confined areas, the laser source must be focused to a small spot diameter. Contrary to commonly used high numerical aperture objectives, we present a Bessel beam-based approach to produce LIPSS covered lines with a width of 4 to 10 µm. Therefore, an 800 nm Ti:Sa-laser with a pulse duration of 110 fs and a repetition rate of 5000 Hz was used. The setup convinces with an extended depth of focus. Thus, high reproducibility of the process was proven, while no focus control and no specific requirements for the surface quality are needed. The applicability of this method is demonstrated by structuring the shell surface of nickel–titanium wires.