Small Pieces Of Tissue Research Articles

First Report of Pectobacterium brasiliense causing Soft Rot on Conophytum luiseae in China.

Conophytum luiseae is native to the Namaqualand region of Cape, South Africa. It is a lovely plant with many short succulent spines on ingot-shaped fleshy leaf surfaces, and a high-value ornamental plant in China. In August to October 2021, a serious soft rot disease on Conophytum luiseae plants was observed in four greenhouses at a horticultural farm in Songjiang District, Shanghai, China. 70% of Conophytum luiseae plants on this farm had severe rot symptoms. Initially, wilting and soft rot symptoms appeared on fleshy leaves, then progressed into browning and withering symptoms of all fleshy leaves. To isolate and identify the causal agent, small pieces of lesion tissues were sterilized by 75% ethanol for 30 s, and rinsed three times with sterile water. Later, the tissues were crushed in sterile 2.0 mL centrifuge tube with 100 μl of sterile water. The suspension was serially diluted and spread on Luria-Bertani agar (LB) medium. After incubation at 28°C for 48 h, the bacterial colonies were tiny and streaked on LB plate for purification. After purification, five independent representative colonies were used for further confirmation. Genomic DNA from the bacterial isolate was extracted and used as the template to amplify 16s rDNA with primers 27F/1492R (Ying et al. 2012) and the housekeeping genes, dnaX with primers dnaXF/ dnaXR (Slawiak et al. 2009), and leuS with primers leuSF/ leuSR (Portier et al. 2019), respectively, by a polymerase chain reaction (PCR). The 16S rRNA sequences of one bacterial isolate was deposited in GenBank (GenBank accession OM333246) and showed a 99% similarity to that of Pectobacterium brasiliense (syn. Pectobacterium carotovorum subsp. brasiliense, Pcb) strains HG1501090309 (KU997683), BC1(CP009769), KC08 (KY021029). The dnaX (OM320998) and leuS (OM321306) sequences showed high similarity (> 99%) to P. brasiliense sequences. To further validate this identification, Pcb-specific primers BR1f/L1r was used for PCR, and it produced a predicted amplicon of 322 bp expected for P. brasiliense (Duarte et al. 2004). All five isolates could be detected by BR1f/L1r primer. To fulfill Koch's postulates, five healthy Conophytum luiseae were inoculated by spraying bacterial inoculum (108 CFU/ml), meanwhile five additional healthy Conophytum luiseae were implemented with sterilized distilled water as a negative control. The plants were then kept at 70% humidity and 25ºC. Seven days after inoculation, the inoculated plants showed serious soft rot, while the control samples remained healthy. Bacteria were re-isolated from rot of inoculated tissues, and the isolates were identified as the original pathogen by the 16S rRNA gene sequences. P. Brasiliense has been reported to cause soft rot on diverse plant hosts, like sweet potato, radish, tobacco (Liu et al. 2019; Voronina et al., 2019; Wang et al., 2017). Best to our knowledge, this is the first report that P. Brasiliense causes soft rot on Conophytum luiseae in China.

First Report of Maize White Spot Disease Caused by Pantoea ananatis in China.

Maize (Zea mays L.) is one of the most important crops in China. Since 2020, a new leaf spot disease has occurred in southwest China in areas such as Yunnan, Sichuan and Hubei provinces. Typical symptoms appeared after tasseling. The spots are scattered on the leaf surface, round to oval in shape with diameter range 3-20 mm. Spots are initially water-soaked, gradually turning yellow or white. In 2021, diseased leaf samples with typical white spot were collected for pathogen isolation and identification in Qujing, Yunnan province. Four small pieces of leaf tissue (about 0.25 cm2 in area) were excised from the edge of the necrotic lesion of each plant, surface sterilized with 75% ethanol for 1 min, rinsed three times with sterile distilled water, and soaked in sterile distilled water for 5 min. The solution was plated on Luria Broth medium (LB) plate (Shin et al. 2022) . After incubation at 28°C for 24 h, round, smooth-edged yellow colonies appeared in the LB plate. The bacterium isolated was gram-negative, negative for oxidase, positive for peroxidase, indole, citrate (Wells et al. 1987). Three strains (PA21QJ01, PA21QJ02 and PA21QJ03) showed identical colony morphology. PA21QJ01 was used for further molecular analyses. DNA was extracted from fresh colonies cultured in LB(Shin et al. 2022), and the fragments at the 16S rDNA, gyrB and rpoB loci were amplified using primers 27F/1492R (Galkiewicz and Kellogg 2008), UP-1/UP-2r (Yamamoto and Harayama 1995) and rpoBCM81-F/rpoBCM32b-R (Brady et al. 2008), respectively. The sequences of fragments of 16S rDNA, gyrB and rpoB from isolate PA21QJ01 were was deposited in GenBank (accession number: OM184301.1, OM302500, OM302499). A search for homologous sequences using BLAST resulted in 99.9, 99.6 and 99.8% identity of 16S rDNA, gyrB and rpoB, respectively, with sequences from the NN08200 of Pantoea ananatis (GenBank accession numbers: MK415050.1 for 16S rDNA, CP035034.1 for gyrB and CP035034.1.1 for rpoB). Above molecular results indicated that PA21QJ01 isolated from maize white spot is P. ananatis. Pathogenicity tests were performed on tasseled plants of the suscptible maize variety Wugu1790. After culture in LB medium plate at 30°C for 12 h, the bacterial solution was used for inoculation at a concentration of 1 × 108 CFU ml-1. After 7 days of inoculation, the leaves of the plants appeared water-soaked. After 10 days, white spot developed with brown margin. In contrast, the control plants remained healthy and symptomless. The same P. ananatis was reisolated in the inoculated maize plants, fulfilled Koch's law. In the last decade, P. ananatis has been reported to cause maize white spot in South Africa, Mexico, Poland, Argentina, Brazil (Sauer et al. 2015), and Ecuador (Toaza et al.2021). It has caused crop diseases with other crops, such as onion, rice, pineapple, melon, and sorghum, and others (Sauer et al. 2015). It caused leaf blight and leaf steak in rice in China (Yu et al. 2021). This is the first report of maize white spot caused by P. ananatis in China. However, to our knowledge, this is the first report of maize white spot disease in China. Attentions should be paid to screening for disease-resistant resources and breeding disease-resistant hybrids. Reference: Wells, J. M. et al. 1987. Int. J. Syst. Bacteriol. 37:136-143. Shin, G. Y. et al. 2022. Plant Dis. Doi: 10.1094/PDIS-08-21-1810-SC. Brady, C., et al. 2008. Syst. Appl. Microbiol. 31:447. Galkiewicz, J. P., and Kellogg, C. A. 2008. APPL ENVIRON MICROB, 74.24: 7828-7831. Toaza, A. et al. 2021. Plant Dis. Doi:10.1094/PDIS-02-21-0298-PDN Yamamoto, S., and Harayama, S. 1995. APPL ENVIRON MICROB, 61:1104.L. Sauer, A. V. et al. 2015. Agronomy Science and Biotechnology. Doi:10.33158/ASB.2015v1i1p21 Yu et al. 2021. Plant Dis. Doi:10.1094/PDIS-05-21-0988-PDN.

Cytodifferentiation of fundic part of glandular stomach in non descript breed of Indian prenatal goat

Though the anatomy and physiology of the adult caprine (Capra hircus L.) stomach have been investigated extensively, the early development of the abomasum has not yet been fully elucidated. The glandular part of abomasum plays an important role in digestion of ingested food by action of gastric juices. Very few studies have been conducted so far regarding histogenesis of goat foetal abomasum in India. In the present study, we have investigated the embryonic and early foetal development of the goat, Capra hircus L. fundic abomasum. We collected 36 developing abomasum of healthy and normal embryos/foeti of goat and assigned them into three group viz. Gr. I (0-50 days of gestation), Gr. II (51-100 days of gestation) and Gr. III (101-150 days of gestation). Small pieces of tissues were processed by routine paraffin. The wall of glandular stomach, the fundic part, was composed of epithelium, pleuripotent blastemic tissue and serosa up to 44 days of gestation. Tunica muscularis became separable at 46 days of gestation. The epithelium was stratified type up to 50 days and gradually changed to pseudo-stratified columnar to simple columnar type from 76 days of gestation. Primary and secondary abomasal folds were observed at 51 and 76 days of gestation, respectively. Gastric pit, the fore runner of gastric gland was reported first at 70 days. The gland became branched tubular type at 145 days. The cells found in the mucosa of the abomasum were surface epithelial cells, chief cells, parietal cells, mucous neck cells and undifferentiated cells. Chief and parietal cell were observed at 76 days and mucous neck cells at 82 days of gestation. Reticular, collagen and elastic fibers came into sight at 38, 76 and 100 days of gestation, respectively. The present study is expected to supplement known data and knowledge regarding histogenesis of goat fetal abomasum and help in diagnosis and treatment of related congenital anomalies.

First Report of Alternaria alternata Inciting Leaf Spot of Dangshen (Codonopsis pilosula) in China.

As a commonly traditional Chinese medicine, the perennial herb Dangshen (Codonopsis pilosula) has superior curative effects including regulating immunity, strengthening the spleen, and tonifying lungs (Bai et al. 2020). To imitate natural ecological conditions, plants were grown on hillside fields with stems prostrate on the ground, tangle-up with each other. In August 2020, leaf spots were observed on C. pilosula in Wutai county, Shanxi province, China, and indicated a high disease incidence (70%-80%) in investigated fields (6.67 ha). Small brown necrotic spots, occasionally enclosed by chlorotic halos, were observed on leaves, stems, and sepals. For identification of the pathogen, 15 small pieces (5×5 mm) of symptomatic tissues from 5 randomly-collected diseased plants were surface sterilized, placed on potato dextrose agar plates, and incubated for 4 d in darkness at 25 °C to obtain the colonies. Cultures were purified by single spore isolation from these colonies. A total of 15 isolates named as Dcp-3, and Dcp-5~Dcp-18 were recovered. They produced ovoid or obclavate spores with 15.9-57.5×9.1-20.1 μm in size, 1-6 transverse septa, and 0-4 longitudinal septa. The conidial chains with 4 to 6 spores had numerous secondary and occasionally tertiary chains on potato carrot agar plates. Because all isolates had identical morphological traits, five genes from the representative isolate Dcp-3, actin (ACT), Alternaria major allergen (Alt a1), plasma membrane ATPase (ATP), histone 3 (H3), and rDNA ITS, were amplified with primer pairs ACTDF1/ACTDR1, Alt-for/Alt-rev, ATPDF1/ATPDR1, H3-1a/H3-1b, and ITS1/ITS4, respectively (Hong et al. 2005; Lawrence et al. 2013; Ma et al. 2020). BLASTn searches indicated species of Dcp-3 could not be accurately confirmed by rDNA ITS, ATP, ACT, and Alt a1 (GenBank accession nos. OM334894, OM362504, OM326344, OM362500). Phylogenetic analysis showed it was most closely related to Alternaria alternata, A. arborescens, and A. tenuissima based on concatenated sequences of above four genes. The H3 sequence (OM362508) shared 100% homology with that of A. alternata (MN481948). The phylogenetic tree using H3 also confirmed Dcp-3 as A. alternata. Heathly, two-year-old C. pilosula were transplanted to a greenhouse. A surface-sterilized leaf was sprayed with 50 μL spore suspension (106 conidia/mL) of Dcp-3. A leaf sprayed with isometric sterile water was used as controls. Each treatment used six plants (five leaves per plant). Plants were covered with sterilized plastic bags and incubated at 22 ℃. The test was repeated twice. A week later, control leaves were healthy, but brown necrotic spots similar to field symptoms emerged on treated leaves. The A. alternata isolates were re-isolated from the border of lesions, and confirmed by morphological and molecular characteristics mentioned above, fulfilling Koch's postulates. Leaf spot of C. pilosula caused by Septoria codonopsidis has been reported in China (Wang et al. 2011). However, to our knowledge, this is the first report of A. alternata inciting leaf spot of C. pilosula in China. Our report would promote growers to enhance the field management and consider associated strategies on controlling Alternaria leaf spot of C. pilosula.

Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development.

Whole animal perfusion is a well-established method that has been used for the past decades in multiple research fields. Particularly, it has been very important for the study of the brain. The rapid and uniform fixation of tissue is essential for the preservation of its integrity and the study of complex structures. For small tissue pieces submerging in formaldehyde solution oftentimes is sufficient to get a good fixation, larger tissues or organs with a more complicated structure present a greater difficulty. Here, we report the precise parameters to successfully perform trans-cardiac perfusion of neonatal mouse pups that allows a uniform fixation of the whole body for subsequent structural analysis and immunohistochemistry. In comparison to standard perfusion procedures of adult mice, changes in the pump velocity, the buffer volume and in the needle size lead to high quality fixation of neonatal mice pups. Further, we present a whole-body section staining, which results in a highly specific immunofluorescence signal suited for detailed analysis of multiple tissues or systems at the same time. Thus, our protocol provides a reproducible and reliable method for neonatal perfusion and staining that can rapidly be applied in any laboratory. It allows a high quality analysis of cellular structures and expression profiles at early developmental stages.

First Report of Chinese Cabbage Wilt Caused by Plectosphaerella cucumerina in Inner Mongolia of China.

Chinese cabbage [Brassica rapa L. subsp. pekinensis (Lour.) Hanelt] has been grown commercially for many decades in Huade County, Inner Mongolia. In 2018 and 2019, an unusual stem and leaf wilt disease with an average disease incidence of approximately 3% was observed. Diseased plants with spindle-shaped stem lesions were collected and small pieces (0.3 × 0.3 cm) of the diseased tissues were cut from the margins of stem lesions, surface sterilized with 75% alcohol for 3 to 5 s, 0.1% NaClO for 2 to 3 min, and washed three times with sterilized water. The treated tissues were placed on 1.5% (w/v) water agar plates and incubated at 25°C for 3 days. The mycelia were cut and transferred onto potato dextrose agar (PDA) for culture purification. Three isolates with similar morphology were obtained and named as BC-2, BC3-2 and BG2. To confirm their pathogenicity, Chinese cabbage (cv. Chunqiuhuang) seed was planted into plugs. After 30 days, the fibrous roots were wounded with a fruit knife and root-dipped in the conidium suspension (1 × 106 conidia/ml) for 20 min. Inoculated seedlings were transplanted in pots (30 × 25 cm) with sterilized nursery soil, with one seeding per pot. The roots of control plants were also wounded and dipped in sterilized water. Five seedlings were inoculated with each isolate and the experiment was repeated three times. Treated seedlings were maintained in a greenhouse at 25 to 28°C under a 12-h photoperiod. Chlorosis and wilting were observed approximately 4 weeks after inoculation, and the outer layer of leaves of the inoculated seedlings developed discoloration and wilted symptoms after 50 days. The symptoms induced by all three isolates were the same as the symptoms observed in the field, whereas no symptoms developed on the control plants. To confirm the Koch's postulates, the fungus was successfully re-isolated from the infected leaves and had similar growth and morphology as the original isolates. The three isolates were cultured for both morphology and molecular identification. The 14-day-old colonies on PDA were buff or salmon pink with few aerial hyphae, and slimy surfaces. Aerial hyphae were sparse with simple or branched conidiophores. Conidia were ellipsoidal, hyaline, surface smooth, septate or aseptate, and (4.0 to 9.7 μm × 2.0 to 3.9 μm). Such characteristics are typical of Plectosphaerella spp. (Palm et al. 1995). For molecular identification, the internal transcribed spacer (ITS) region of rDNA was amplified using the primer pair ITS1/ITS4 (White et al. 1990), and the products were directly sequenced. BLAST analysis showed that the sequences of Isolates BC-2 (511 bp out of 515 bp), BC3-2 (512 bp out of 516 bp) and BG2 (503 bp out of 505 bp) showed 99% identity to an isolate of P. cucumerina (acc. no. KT826571.1) from Bottle gourd [Lagenaria siceraria (Molina) Standl.] (Yan et al. 2016). The sequences of Isolates BC-2, BC3-2 and BG2 were deposited in GenBank (acc. nos. MW320463, MW320462 and MW320464). Although P. cucumerina was reported causing root rot of cabbage (B. oleracea) in Gansu, China (Li et al. 2017), to our knowledge, this is the first report of P. cucumerina causing Chinese cabbage wilt in Inner Mongolia, China. The presence of the disease could cause significant economic losses in Chinese cabbage production. For this reason, strategies for the management and control of this disease should be implemented.

First Report of Target Spot Caused by Rhizoctonia solani AG-6 in Tobacco in China.

Tobacco is an annual and solanaceous crop, which is widely produced in China. In July 2020, tobacco target spot was observed on 50% of tobacco plants in a 5-ha commercial field of Bijie (27.32° N, 105.29° E), Guizhou province, China. Typical symptoms firstly appeared on the old leaves as round watery spots. Then the spots became a diameter of 2 to 20 cm, with concentric ring lines and dead spots. Fifteen small pieces (5 × 5 mm) of leaf tissue were cut from the edge of the lesions, surface sterilized and placed on potato dextrose agar (PDA) medium amended with kanamycin (0.1 mg/ml). Isolate J136, one of five isolates with similar morphology, was selected for pathogen identification. The culture of the isolate on PDA was brown and exhibited radial mycelial growth after incubation at 28 oC in darkness for 5 days. Hyphae of the fungus were white at the beginning, turned light brown to brown at the later stages, and finally became thick and separated. Sclerotia were brown and produced on PDA after 25 days of incubation in the dark. These characteristics were similar to the colony characteristics of R. solani. The genomic DNA of Isolate J136 was extracted using the CTAB method. PCR analyses were conducted using the following primers specifically designed for the detection of individual AGs or subgroups of R. solani: AG-1 IA, IB and IC (Kuninaga 2003), AG-2-1, AG-2-2, IIIB, IV and LP (Carling et al. 2002), AG-3 PT (Misawa 2015), AG-4 HG-I and HG-II (Kuninaga 2003), and AGs-5-6 (Arakawa and Inagaki 2014). Among the 12 specific primer pairs, only AG-6-specific primers amplified a fragment of ca. 230 bp product, indicating that the tested strain belonged to R. solani AG-6. The sequence was deposited in GenBank with accession no. MZ379468. Using BLASTN search, the sequence of the gene was aligned with the voucher specimen, R. solani AG-6. A phylogenetic tree was constructed based on these sequences. After wards, Isolate J136 was tested for hyphal anastomosis reaction using the R. solani AG-6 standard strain according to the method described by Ogoshi (1987). The hyphal diameter at the point of anastomosis was reduced, with obvious anastomosis point, and the death of adjacent cells, indicating their anastomosis reactions (Anderson 1982). Thus, based on the morphological and genetic analyses, the fungus was identified as R. solani AG-6. To verify its pathogenicity, six plants (cv. Yunyan87) at the 5-to-6 leaf stage were inoculated with mycelial PDA plugs (5 mm in diameter). Leaves inoculated with PDA-only plugs served as the controls. Treated tobacco plants were maintained at a temperature range of 15 to 25 oC in a greenhouse with 85% relative humidity. After 5 days inoculation, typical symptoms were observed on the inoculated leaves, whereas no symptoms were observed on the control leaves. Koch's postulates were fulfilled by re-isolation of the pathogen from the diseased leaves. R. solani AG-2-2 is the only previously reported group of R. solani, which causes tobacco target spot in the field (Gonzalez et al. 2011). Therefore, to our knowledge, this is the first report of R. solani AG-6 causing target spot of tobacco in the field in China. Since considerable losses caused by the disease have frequently happened in this region, addition of this new group pathogen in the disease pool can be more problematic. Proper disease control strategies are in need to be developed to prevent further losses.

First report of leaf blight caused by Apoharknessia eucalyptorum on Eucalyptus grandis × Eucalyptus urophylla in China.

Eucalypt GL-9 (Eucalyptus grandis × Eucalyptus urophylla) is one of the most widely grown genotypes of Eucalyptus in China. Each year, leaf blight causes serious economic losses in the eucalyptus industry in the south of China. In December 2019, a leaf blight disease was found to be widespread on eucalyptus GL-9 in Hechi in Guangxi, China (25°22'17"N, 108°15'32"E). Symptomatic lesions were usually brown at the early stage of infection and then turned off-white at the late stage. They had a large number of black round pycnidia randomly dispersed on the surface. Most of the lesions initially started from the leaf tip and then gradually expanded to the base of the leaf. Three randomly sampled leaves were washed using sterile water. Next, small pieces of tissue (5×10 mm) were removed from the margins of the lesions, surface disinfected with 75% ethanol for 1 min and 0.1% HgCl2 for 3 min, and then washed three times with sterile water. The tissues were placed on potato dextrose agar (PDA) and incubated at 28°C for 5 days to observe the fungus morphological characteristics. The hyphae on the PDA were milky yellow, and the PDA was light yellow when viewed from the bottom, with few aerial hyphae. The colonies had petal-like edges. In the later stage, hyphae in the center of the colonies turned brown. Three representative isolate (EC7, EC8, EC10) were selected for further study. Their conidia were olive-shaped, spindle-shaped, or obliquely globose, 8.80-11.93 μm in length (10.35 μm in average), 4.69-7.33 μm in width (6.06 μm in average) (n=100 in each isolate), with a conical apiculus and a hyaline basal appendage that was tubular, smooth, and thin-walled. For molecular identification, their genomic DNA was extracted using a Genomic DNA Kit (Tiangen, China). The internal transcribed spacer (ITS) region of rDNA and β-tubulin (TUB) genes were amplified using ITS5/ITS4 and βt2a/βt2b primer sets, respectively (White et al. 1990). BLASTn searches showed that the ITS and TUB sequences had the highest identity with Apoharknessia eucalyptorum strains, with 100% (586/586 in EC7 and EC8, 590/590 in EC10) identity for ITS (KY979752.1) and 99% (502/505 in EC7, 506/508 in EC8 and 504/507 in EC10) identity for TUB (KY979919.1) of ex-type CBS 142519. The ITS and TUB sequences of three isolates were submitted to GenBank (EC7: OM060439 and OM103586, EC8: OM679378 and OM715153, EC10: OM679377 and OM715152). A maximum likelihood phylogenetic tree was constructed by combining the two sequenced loci in MEGA7. Three isolates clustered in the A. eucalyptorum clade with 92% bootstrap support. Thus, based on morphological (Crous et al. 2017; Garrett et al. 2018) and molecular characteristics, the pathogen was identified as A. eucalyptorum. In a pathogenicity test, twenty healthy GL-9 seedlings were collected, and were divided into four groups. Seedlings from groups 1-3 were used to inoculate three isolates respectively, and seedlings from another group were sprayed distilled water as control. Before test, leaves were washed with sterile water, surface disinfected with 75% ethanol, and then rinsed with sterile water. After drying, an inoculation needle was used to make tiny wounds near the leaf margin on each leaf. Next, conidia solution (1×107 conidia/ml) and sterile water were sprayed to leaves in different groups and moistened with airtight bags. After 3 days, airtight bags were moved. Lesions appeared on all the pathogen-inoculated leaves, whereas only the inoculation point turned brown on the control leaves. The pathogenicity test was repeated three times and the same results were obtained. Fungi were re-isolated from symptomatic leaves and identified as A. eucalyptorum following the same methodologies used for the initial identification. To our knowledge, this is the first report of A. eucalyptorum causing leaf blight on E. grandis × E. urophylla in China. This study expands the understanding of the pathogen of leaf blight on E. grandis × E. urophylla. More research is needed to develop effective strategies to manage this disease.

First Report of Leaf Spot on Panax notoginseng Caused by Caryophylloseptoria pseudolychnidis in Yunnan, China.

Panax notoginseng is a unique traditional medicinal plant in China, which has the effects of improving myocardial ischemia, protecting liver and preventing cardiovascular diseases (Jiang, 2020). In July 2021, gray-brown round spots were found on the leaves of P. notoginseng in the plantations of Lincang City (23º43´10˝N, 100º7´32˝E). By September, the symptoms were observed on more P. notoginseng plants, with incidence reaching 31%. Initial symptoms on leaves were small, brown spots that expanded, with black granular bulges on the lesions, often surrounded with yellow halo. As the disease progressed, multiple lesions merged, leaves became yellow, and abscission occurred. To isolate the causal pathogen, twelve symptomatic leaves were randomly obtained from twelve P. notoginseng plants. Small pieces of infected leaf tissues (about 5 mm2) were disinfected with 75% ethanol for 30 s, soaked in 2% sodium hypochlorite for 3 min, and then rinsed 3 times with sterile water and blotted dry. Sample tissues were plated on potato dextrose agar (PDA) plates incubated at 25℃ for 5 days with 12 h light/dark photoperiod. Hyphal-tips from the growing edge of colonies were transferred to fresh PDA to obtain pure cultures. Eight isolates were obtained with similar colony morphology, gray (top view) or black (back view) coloration, with a villous surface, and slow-growing on PDA. Conidia were hyaline, slender and obtuse to subobtuse at both ends, 10.3 to 52.62 (av. 25.2) µm × 1.4 to 4.0 (av. 2.4) µm (n=200) in size. Characteristics of the colonies and conidia were consistent with Caryophylloseptoria pseudolychnidis as described by Quaedvlieg et al. (2013) and Verkley et al. (2013). Genomic DNA of three representative isolates (LINC-4 to LINC-6) was extracted, and the rDNA-ITS region, ACT, and LSU gene regions were amplified and sequenced using the primer pairs ITS4/ITS5, 512F/783R, and LSU1Fd/LR5, respectively. Sequences have been deposited in GenBank (OK614104-OK614106 for ITS, OK614109-OK614111 for LSU, OK628350-OK628352 for ACT). BLAST search showed that all sequences were 98% to 100% homology with the corresponding sequences of C. pseudolychnidis. ITS sequences of the three isolates (LINC-4 to LINC-6) showed 99.21% identity (500/504 bp) to C. pseudolychnidis strain CBS 128630 (GenBank accession no. NR156266). LSU sequences of the three isolates showed 99.76% identity (823/825 bp) to C. pseudolychnidis strain CBS 128630 (MH876481). For ACT sequences, LINC-4 and LINC-5 showed 98.53% identity (201/204 bp) to C. pseudolychnidis strain 128614 (KF253599); LINC-6 showed 99.02% identity (202/204 bp) to C. pseudolychnidis strain 128614 (KF253599). Further, the neighbor-joining and maximum-likelihood method were used for multilocus phylogenetic analysis of the obtained sequences using MEGA-X (Kumar et al. 2018). The three isolates were clustered in the same clade with two C. pesudolychidis from database. Three isolates (LINC-4 to LINC-6) were tested for pathogenicity to confirm Koch's postulates. Annual potted P. notoginseng was inoculated with spore suspension (105 spores.mL-1). Each isolate was inoculated onto two leaves each of five P. notoginseng plants. The controls were similarly mock-inoculated with sterile water. To maintain high humidity (>90% RH), all plants were placed in transparent plastic boxes in a greenhouse at 25℃ with a 12 h light/dark photoperiod. Fifteen days post-inoculation, inoculated leaves showed similar symptoms to those observed in the field, and control plants remained healthy. The pathogen were reisolated from symptomatic leaf spots, and the colony characteristics were the same as those of the original isolates. Morphological characteristics, molecular data, and Koch's postulates tests confirmed C. pseudolychnidis as the cause of P. notoginseng leaf spot disease. To our knowledge, this is the first report of C. pseudolychnidis causing leaf spot on P. notoginseng in Yunnan, China. The spread of this disease might pose a serious threat to the production of P. notoginseng. The occurrence and spread of this pathogen should be further studied in order to formulate reasonable control measures.

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

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

Morphometrical and Histochemical study of glandular stomach (Proventriculus) in local domestic male ducks (Anase Platyrhchos)

The present work explores morphometric and histochemical foundations of the wall of proventriculus in adult domestic male ducks. To achieve this study, six healthy adult ducks were purchased from local vendors in Mosul province. All birds were sedated. Their stomachs were removed after being dissected to achieve the gross description and measurements of the proventriculus, including the length and diameter of cranial, middle, and caudal regions of the proventriculus. Small tissue pieces 6-8 mm in length from three regions of proventriculus were processed for histomorphological studies using hematoxylin and Eosin stain, combined PAS-AB pH 2.5, toluidine blue, and Masson's trichrome stains. The finding showed that the proventriculus connects with the esophagus from the cranial side and the muscular stomach from the caudal side. It was a thin elongated oval tubular organ. Histologically, the mucosa of the proventriculus revealed the presence of folds and sulci-like structures covered by simple columnar epithelium. The tunica submucosa consisted of many signs of profound and straightforward sub-mucosal glands supported by connective tissue septa and extended from the basal part of the diverged gland through the lamina propria, which was organized in a conical manner (adenomere). Tunica Muscularis appeared as a double-layer coordinated in the form of inner longitudinal and outer circular ones. Finally, serosa consists of fibrous tissue, vessels, fatty tissue, and nerve plexuses studded by the mesothelial cells. The highest thickness of the four layers of mucosal, submucosal, muscular, and serosal layers in the distal part of ‎the proventriculus. Histochemical PAS staining positively reacted with epithelial tunica mucosa and wall of proventricular glands.

First Report of Sarocladium kiliense Causing Brown Rot of Hypsizygus marmoreus in China.

Hyjpsizygus marmoreus (Peck) H.E. Bigelow is one of the most popular and widely cultivated edible mushrooms worldwide. In June 2021, an epidemic of H. marmoreus fruiting bodies infected with brown rot occurred at a cultivation facility in Yan'an (Shaanxi province), China, which resulted in a 90% economic loss. The fruiting body surface was covered with white-to-gray velvet-like mycelia, which gradually spread to the pileus, eventually covering the whole fruiting body (Fig. 1A). Brown rot, which is the most important factor limiting H. marmoreus fruiting body yield and quality, is responsible for severe economic losses in northern Shaanxi province. To identify the causal agent of this disease, small pieces of diseased tissue were collected from fruiting bodies, disinfected with 70% ethanol, and rinsed three times with sterile distilled water. They were then placed on potato dextrose agar (PDA) medium in plates and incubated at 26 °C. The colonies on the PDA medium after a 14-day incubation at 26 °C were 40-45 mm in diameter, orange-white on the surface (Fig. 1B), pale orange on the underside (Fig. 1C), slightly wrinkled or cerebriform, and glabrous or fasciculate. Vegetative hyphae were septate, hyaline, smooth, and thin-walled. The unicellular conidia were cylindrical with rounded ends (3.5 to 4.0 × 1.0 to 1.5 μm; n = 30). The cultural and morphological characteristics of the representative isolate MG1 were consistent with those of Sarocladium kiliense (Grütz) Summerbell (Giraldo et al. 2015). For the molecular identification, DNA was extracted from MG1. The internal transcribed spacer (ITS) region, a gene encoding the second largest RNA polymerase II subunit (RPB2), a β-tubulin gene (TUB2), and an actin gene (ACT) were amplified by PCR using primers ITS1/ITS4, fRPB2-5F/fRPB2-7cR, Bt2a/Bt2b, and ACT-512F/ACT-783R, respectively. The resulting ITS (MZ818340), RPB2 (MZ833454), TUB2 (MZ833455), and ACT (MZ833456) sequences from MG1 were 99.82%, 99.19%, 99.69%, and 99.22% identical to the corresponding sequences in S. kiliense isolate CBS 400.52 (ITS: KM231849, RPB2: KM232425, TUB2: KC479789, and ACT1: KM231258). On the basis of the morphological and molecular features, MG1 was identified as S. kiliense (Summerbell et al. 2011; Lombard et al. 2015; Giraldo et al. 2015). Pathogenicity tests, which were repeated three times, were conducted using conidial suspensions (approximately 1 × 105 spores/mL) prepared in sterile distilled water. The surface of 30 healthy H. marmoreus fruiting bodies maintained in a plastic box was sprayed with the MG1 conidial suspensions. Control H. marmoreus fruiting bodies were sprayed with sterile distilled water. The inoculated fruiting bodies were maintained in darkness at 25 °C and 95% relative humidity. The disease symptoms that developed in 3 days included the presence of gray mycelia on the fruiting body surface. Additionally, S. kiliense was reisolated from symptomatic pilei at 6 days post-inoculation. Disease symptoms were undetectable on the negative control. To the best of our knowledge, this is the first report of H. marmoreus infected with brown rot caused by S. kiliense in China.

Occurrence of Leaf Spot Caused by Mycocentrospora acerina on Japanese Ginseng (Panax japonicus) in China.

Panax japonicus C. A. Mey., known as Japanese ginseng or "Zhujieshen" in China, is a perennial medicinal herb (family Araliaceae) native to China and is widely grown in many provinces including Hubei, Hunan, and Sichuan. In recent years, cultivation of Japanese ginseng has increased tremendously in China because of its high value. Its root is widely used in traditional Chinese medicine for the treatment of inflammation. In early May 2020, severe necrotic lesions on leaves with 40 to 50 % disease incidences were observed on 3-year-old Japanese ginseng plants in a cultivated field in Xuanen County (30°05'N, 109°83'E), Hubei Province, China. The total area affected by the disease was approximately 30 ha. Initial symptoms showed small, circular, brown, necrotic spots uniformly distributed on leaves. The center of the spots was light tan, surrounded by a dark brown ring and a chlorotic halo. As the disease progressed, multiple lesions merged into large disease spots with visible white fungal hyphae, causing leaf wilting. Ten small pieces (0.1 × 0.1 cm in size) of leaf tissue were removed from the lesion margins, surface-sterilized with 0.5 % sodium hypochlorite for 1 min and 75 % alcohol for 20 s, washed with sterile distilled water three times, dried, and placed on Petri plates with potato dextrose agar (PDA) medium containing 10 µg/ml of ampicillin and incubated at 20 °C for 5 days. Colonies with dense mycelia were initially white and gradually becoming black. The hyphae were septate, branched, and 3 to 7 µm in width. Conidiophores were flexuous, not branched, and produced a single spore. Spores (ranged from 95.4 to 255.5 × 6.2 to 13.5 μm) were elongate, multiseptate, with a long, strongly curved beak (ranged from 25.5 to 95.4 μm), The number of septae ranged from 4 to 13. Clamydospores with smooth or slightly rough wall were spherical to ovoid and averaged 8.5 to 25.4 × 7.2 to 16.5 μm. The six isolates were preliminarily identified as Mycocentrospora acerina (R. Hartig) Deightonbased on the morphological characteristics (Gilchrist et al. 2015). To confirm the identification, isolates ZJS1, ZJS3, and ZJS5 were chosen for DNA sequencing. The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1 and ITS4 primers (White et al. 1990) and sequenced. The identical sequences of the 491 bp amplicons were deposited in GenBank (accession no. ZJS1, MZ277314; ZJS3, OL333859; and ZJS5, OL333860). BLAST analysis of the sequences showed 100 % identity to M. acerina (MH856114). Moreover, the three isolates were further confirmed as M. acerina by amplifying the large subunit (LSU) of the ribosome gene (accession no. ZJS1, MZ277321; ZJS3, OL333861; and ZJS5, OL333862), as their identical sequences exhibited 99.83 % similarities with M. acerina (MH868490). Isolate ZJS1 was chosen to fulfill Koch's postulates with 30 healthy 2-year-old P. japonicus grown in plastic pots filled with a sterilized mixture of peat moss and vermiculite (3:1). One leaf of each plant was inoculated with one 5-mm-diameter mycelium-agar disc and placed in a greenhouse at 20 ± 1 °C, with ambient lighting and relative humidity of 85 %. By 5 days after inoculation, all inoculated plants showed symptoms identical to those observed in the field, and no symptoms were observed on control plants. The fungus was reisolated from the inoculated plants and identified as M. acerina using the above method. The experiment was conducted thrice with similar results. To our knowledge, this is the first report of leaf spot caused by M. acerina on Japanese ginseng in China and the world. There is a need to develop effective management measures to reduce the occurrence of this disease.

First Report of Colletotrichum chrysophilum Causing Apple Bitter Rot in Spain.

Bitter rot of apple (Malus × domestica Borkh.) is a cosmopolitan disease affecting fruit and causes considerable losses worldwide. In September 2020, symptoms of bitter rot were observed on 'Pink Lady' apples in two orchards (~2.5 ha each) in Gualta, Catalonia, Spain (42.03803 N, 3.09831 E, and 42.03942 N, 3.10931 E). Early symptoms consisted of light brown and sunken circular lesions (1-4 mm) that enlarged over time, later becoming dark brown and water soaked, and extending cone-shaped toward the core. Sporulation was mostly noticed in larger lesions. Estimated incidence was 2% and 20% of 150 trees surveyed in each orchard, respectively. Twenty-one fungal isolates were obtained from diseased fruit by culturing small pieces of necrotic tissue on potato dextrose agar (PDA) amended with rifampicin at 50 mg/liter. Colonies on PDA looked identical. They were cottony, initially light-gray colored on top and darkening with age; colony reverse initially cream colored and darkening with age. Conidia were produced in orange acervular masses on Spezieller Nährstoffarmer Agar, and were aseptate, hyaline, cylindrical with obtuse ends, and measuring 10.1 to 14.7 × 4.5 to 7.1 μm (average 13.1 ± 1.04 × 5.3 ± 0.67 μm [mean ± SD], n = 50), with a mean length/width ratio 2.6 ± 0.39 (n = 16 isolates). Perithecia were not observed. Based on the conidial morphology, the isolates were tentatively identified as belonging to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). Total genomic DNA was extracted from all isolates and six nuclear regions were amplified and partially sequenced: the internal transcribed spacer region of rDNA (ITS), the mating type protein 1-2-1 gene and the Mat1-2-1-Apn2 intergenic spacer region (ApMAT), actin (ACT), calmodulin (CAL), glyceraldehyde 3-P dehydrogenase (GAPDH), and tubulin (TUB2). The sequences for each region were 100% identical across all isolates. BLAST searches in GenBank showed 99-100% identity with sequences of various C. chrysophilum W.A.S. Vieira, W.G. Lima, M.P.S. Câmara & V.P. Doyle strains including the ex-type CMM4268 (Vieira et al. 2017). Sequences of the representative isolate CJL1080 were deposited in GenBank (ACT, MZ488944; ApMAT, MZ442299; CAL, MZ488945; GAPDH, MZ488946; ITS, MZ443972; TUB2, MZ442300). A multilocus phylogenetic analysis through Bayesian inference conducted with the obtained sequences and reference ones (Khodadadi et al. 2020) revealed that our isolates clustered well within C. chrysophilum, as suggested by BLAST results. To confirm Koch's postulates, isolates CJL1080 and CJL1095 were inoculated on 'Pink Lady' apples. Six surface-sterilized fruits per isolate were wound-inoculated four times each with either 20 μl of a conidial suspension (105 conidia/ml) or sterile distilled water (control). After 7 days of incubation in a moist chamber at 22°C, symptoms compatible with Colletotrichum infection were observed around the wounds, whereas control inoculations remained symptomless. The fungus was reisolated from all the lesions and identified through its morphological traits and DNA sequencing (ApMat, CAL, and GAPDH). No fungus was isolated from the controls. Taxa of the C. gloeosporioides species complex causing bitter rot have been recently reported in Europe (Grammen et al. 2019; Nodet et al. 2019). This is the first report of C. chrysophilum causing apple bitter rot in Spain, which expands the knowledge on the geographic distribution of this important pathogen of apple in Europe.

Prospects for using fucoxanthin as a functional component of food products and beverages

Brown algae can be used as raw materials for pharmacological and functional extracts, in particular carotenoids of brown alga Sargassum pallidum are a source for biologically active substances, as a carotenoid fucoxanthin with a wide spectrum of properties useful both for daily preventive consumption and therapeutic diets. Prospects for using S. pallidum as a raw material for extracting the carotenoid fucoxanthin and its further using as a biologically active component in the food industry are considered. The algae were collected in the coastal zone of Peter the Great Bay (Novik Bay of Russky Island) in October 2020, then frozen. Before freezing, the algae were washed and excess moisture was removed. Small pieces of the alga tissue were cut, put into closed packages, and processed for 24 hours by ethyl alcohol with 80 % concentration in a dark place in refrigerator under temperature +7 oC. The obtained alcohol extract was used as a functional addictive for beverages, as iced black tea and juice concentrates. These beverages were examined for organoleptic properties (by the group of 10 experts) and economic value. The apple and red grape juice concentrates with the fucoxanthin extract have the best organoleptic properties. As the final result, technology of functional drinks with alcohol extract of fucoxanthin from brown alga S. pallidum is developed.

Impact of tissue processing on microbiological colonization in the context of placentophagy

A mother’s postpartum ingestion of raw or processed placental tissue—referred to as human maternal placentophagy—is an emerging health trend observed in industrialized nations. Placenta is commonly consumed as small pieces of raw tissue, or as raw or steamed dehydrated pulverized and encapsulated tissue. To investigate the potential neonatal health risks of this behavior, the present study focused on microbial colonization of processed placenta preparations with potentially pathogenic bacteria Streptococcus agalactiae (Group-B-Streptococci; GBS) and Escherichia coli (E. coli). In the clinical approach placentas from 24 mothers were analyzed. Two placentas, from 13 mothers with confirmed positive maternal GBS status, showed GBS-growth on their surface (2/13; 15.4%) independent from delivery mode or antibiotic treatment. All processed samples (n = 24) were free from GBS. In the experimental approach, a standardized inoculation protocol was introduced to resemble ascending vaginal and hematogenous colonization. Six placentas from elective term C-sections of GBS negative mothers were collected and artificially inoculated with highly concentrated suspensions of GBS and E. coli. Heat processing significantly reduced the number of colony forming units (CFU) for GBS and E. coli. Our results suggest placentophagy of processed tissue is an unlikely source of clinical infection.

First Report of Diaporthe cercidis Causing Leaf Blotch of Acer pictum subsp. mono in China.

Acer pictum subsp. mono (Maxim.) H. Ohashi is a common deciduous tree species that is widely distributed in Northeast and Northern China, including all provinces of the Yangtze River Basin (Liu et al. 2014). A foliar disease, with an incidence of ~90% (19/21 trees), occurred on A. pictum subsp. mono in a community park, Nanjing, Jiangsu, China in July 2019. On average, ~80% of the leaves per individual tree were infected by this disease. The symptoms initially appeared as brown, necrotic lesions at leaf tips, and half the leaf would become dark brown with time, and finally almost all of leaves were infected. Small pieces of leaf tissue (3 to 5 mm2) cut from the lesion margins were surface-sterilized in 75% ethanol for 30 s and 1% NaClO for 90 s, rinsed with sterile H2O three times, and placed on potato dextrose agar (PDA) at 25°C in the dark. The same fungus was isolated from 92% of the samples. The pure cultures were obtained by single-spore isolation. Three representative isolates (WJF1, WJF3 and WJF4) were obtained, and WJF1 was deposited in China's Forestry Culture Collection Center (CFCC 54806), and WJF3 and WJF4 were deposited at the Nanjing Forestry University (NFU 083 and NFU 084). The culture on PDA was white, with white vigorous aerial mycelia at the edge. Black pycnidia developed on the alfalfa stems at 25°C under a 14/10 h light/dark cycle for 20 days. Conidiophores were hyaline, branched, septate, straight, 16.4-34.7 × 1.5-3.0 μm (n = 30). Conidiogenous cells were 9.0-24.6 × 1.3-2.3 μm (n = 30). Alpha conidia were 7.0 ± 0.6 × 2.2 ± 0.2 μm (n = 30), fusiform, hyaline, smooth and multi-guttulate. Beta conidia were 25.5 ± 4.3 × 1.3 ± 0.1 μm (n = 30), hyaline, smooth and hamate. Morphological characters of all three isolates matched those of Diaporthe spp. (Gomes et al. 2013). DNA of three isolates were extracted and the internal transcribed spacer region (ITS), partial sequences of elongation factor 1-alpha (EF1-α), calmodulin (CAL), beta-tubulin (β-tub) and histone H3 (HIS) genes were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R and CAL228F/CAL737R (Carbone et al. 1999), βt2a/βt2b and CYLH3F/H3-1b (Glass and Donaldson, 1995, Crous et al. 2004), respectively. The sequences of WJF1, WJF3 and WJF4 were deposited in GenBank (WJF1: Accession Nos. MW301339 for ITS, MW363932 to MW363935 for EF1-α, β-tub, HIS, and CAL; WJF3: MW453062 and MW561566 to MW561569; WJF4: MW453063 and MW561570 to MW561573). BLAST results showed that the ITS, EF1-α, β-tub, HIS, and CAL sequences of WJF1 were similar with sequences of Phomopsis liquidambari C.Q. Chang, Z.D. Jiang & P.K. Chi JQ676191 (identity = 540/540; 100%), D. huangshanensis H. Zhou & C.L. Hou MN224671 (identity = 291/292; 99%), D. pescicola Dissan., J.Y. Yan, Xing H. Li & K.D. Hyde MK691230 (identity = 438/438; 100%), D. spinosa Y.S. Guo & G.P. Wang MK726170 (identity = 437/438; 99%), D. cercidis C.M. Tian & Qin Yang MK691114 (identity = 452/452; 100%), respectively. BLAST results of WJF3 and WJF4 are list in Table 1. A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed WJF1, WJF3 and WJF4 in the clade of D. cercidis. Based on the five-locus phylogeny and morphology, WJF1, WJF3 and WJF4 were identified as D. cercidis. The pathogenicity of three isolates were tested on potted 3-yr-old seedlings of A. pictum subsp. mono, grown in a greenhouse. Healthy leaves were wounded with a sterile needle and then inoculated with 10 μL of conidial suspensions (106 conidia/mL). Control leaves were treated with sterilized H2O. A total of twelve seedlings were used for the tests, 3 seedlings per treatment, and five leaves were inoculated per seedling. Each plant was covered with a plastic bag after inoculation and sterilized H2O was sprayed into the bag twice/day to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2°C/16 ± 2°C. In 5 days, all the inoculated leaves had lesions similar to those observed in the field. D. cercidis was reisolated from the lesions of the inoculated leaves and was confirmed based on morphological characteristics and ITS sequence analysis. No symptoms were observed on the control leaves, and no fungus was isolated from them. D. cercidis was previously reported on twigs of Cercis chinensis (Yang et al. 2018) and causing pear shoot canker (Guo et al. 2020). This is the first report of D. cercidis causing leaf blotch on A. pictum subsp. mono. Identification of the pathogen is imperative for diagnosing and controlling this potentially high risk disease on A. pictum subsp. mono and also for the future studies.

First Report of Alternaria alternata Causing Leaf Spot on Chili (Capsicum annuum) in Pakistan.

Chili (Capsicum annuum L.) is an important vegetable crop in Pakistan. During summer of 2019, chili leaf spot symptoms were observed on 3-month-old plants in the fields, with 30 to 40% of disease incidence, in District Faisalabad, Punjab, Pakistan. Diseased leaves were characterized by numerous tiny round spots (0.5 to 2.0 mm in diameter, average 1 mm) that were white to grey with a sunken center, surrounded with dark brown edge and chlorotic halo. The lesions gradually enlarged and coalesced into large, nearly circular, or irregularly shaped lesions that could be as long as 3 cm. Small pieces of symptomatic leaf tissues were surface sterilized in 1% sodium hypochlorite for 1 min, rinsed in sterile water, and plated on potato dextrose agar (PDA) amended with streptomycin (100 ppm). After 5 days at 25°C with a 12-hour photoperiod, same fungal colonies developed. The colonies initially appeared white and then turned olive-green. The conidiophores were brown septate and generally branched. Conidia borne singly or in short chains were multicellular, obclavate to obpyriform, and 16.2 to 38.5 µm (average 27.35 ± 2.1 µm) in length and 8 to 16.5 µm (average 12.25 ± 1.6 µm) in width, with zero to three longitudinal and two to five transverse septa (n=35). The fungus was identified as Alternaria sp. (Fr.) Keisel based on its morphological characteristics (Simmons et al. 2007). For molecular identification, genomic DNA of two representative isolates (SSUAF1 and SSUAF2) was extracted using DNAzol reagent and PCR amplification of the internal transcribed spacer (ITS)-rDNA region, Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) gene and RNA polymerase II second largest subunit (rpb2) were performed with primers ITS1/ITS4 (White et al. 1990), gpd1 and gpd2 (Berbee et al. 1999), RPB2-5F/RPB2-7cR (Liu, et al. 1999), respectively. The obtained sequences were deposited in GenBank with acc. nos. MT249008.1 and MT249009.1 for ITS-rDNA; MT318220.1 and MT318221.1 for the GAPDH; and MT318236.1, and MT318237.1 for RPB2 gene. A BLAST search in GenBank showed 100% identity with A. alternata for both ITS region (MT279999.1), GAPDH gene (MK637438.1) and RBP2 gene (MK605900.1). To confirm pathogenicity, 2-month-old healthy potted C. annuum plants were inoculated using an atomizer in a greenhouse. A total of 12 plants at the true leaf stage in each experiment were sprayed with a conidial suspension (106 conidia/ml) of both isolates amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant). Four plants were inoculated with each of the two isolates, whereas four control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse. After 10 days of inoculation, each isolate induced leaf lesions that were similar to typical lesions observed in the field. The experiment was conducted twice with similar results. The fungus was readily reisolated from symptomatic tissues whereas the control plants remained symptomless. Re-isolated fungal cultures were morphologically and molecularly identical to A. alternata, thus fulfilling the Koch's postulates. Previously, A. alternata has been reported in Italy and India (Devappa et al. 2016; Garibaldi et al. 2019). To our knowledge, this is the first report of A. alternata causing leaf spot of C. annuum in Pakistan. This report will help the identification of leaf spot of chili and the development of management strategies for control of this disease in Pakistan.

Leaf Spot Caused by Colletotrichum fructicola on Star Anise (Illicium verum) in China.

Star anise (Illicium verum) has been cultivated for centuries in southern China, and its fruit is an important seasoning spice, and can be used as a medicine (Wang et al. 2011). It is grown mainly in Guangxi, Guangdong, Guizhou, and Yunnan provinces, in China. Anthracnose is one of the important diseases of star anise, which seriously affects the yield and quality by infecting twigs, pedicels, fruit stalks and fruits (Liao et al. 2017). When leaf spots first appear, they are round, water-stained, small, dark brown spots, which expands into round separated spots, then the spots become yellowish brown with small black acervuli arranged in a circular pattern. On 22 August 2019, four leaf spot samples of star anise were collected, with two each from Shanglin County and Jinxiu County in Guangxi Province. The plantations in this area of around 8 ha had more than 80% leaf spot incidence. Small pieces of tissues (5 mm × 5 mm) were taken from the zone between symptomatic and healthy plant tissues, surface-disinfected in 75% ethanol for 10 s and 1% NaClO (sodium hypochlorite) for 1 min, and washed three times in sterilized distilled water. The sterilized leaf tissues were placed on potato dextrose agar (PDA) and incubated at 28°C in darkness for a week. Hyphae growing from tissue pieces were subcultured onto fresh PDA. Three of the four leaves yielded cultures resembling Colletotrichum spp. Four fungal isolates were obtained by a single-spore isolation method. The isolates JX1-2 and JX1-5 were collected from Jinxiu County while SL1-2 and SL2-1 were collected from Shanglin County. Genomic DNA was extracted from these four fungal isolates, followed by PCR amplification and sequencing of the rDNA internal transcribed spacer (ITS), actin (ACT), Apn2-Mat1-2 intergenic spacer, partial mating type (Mat1-2) (ApMat), calmodulin (CAL), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Weir et al. 2012). The sequences have been deposited in GenBank (ITS: MW301215 to MW301218; ACT: MW348965 to MW348968; ApMat: MW348973 to MW348976; CAL: MW348957 to MW348960; CHS-1: MW348969 to MW348972; GAPDH: MW348961 to MW348964). For phylogenetic analysis, MEGAX (Kumar et al. 2018) was used to produce a Maximum Likelihood (ML) tree with 1000 bootstrap replicates, based on a concatenation of the sequenced genomic regions for each of the four isolates from this study as well as sequences of other Colletotrichum species obtained from GenBank. The results revealed that isolates JX1-2, JX1-5, and SL1-2 were C. horii, and SL2-1 was C. fructicola (Weir et al.2012). The resulting colonies were initially white with abundant aerial hyphae, and white-gray after three days at 28°C on PDA. Isolate SL2-1 eventually turned greenish-grey after 14 days, while the center of C. horii isolates turned iron-gray with white-gray marginal. Both species of Colletotrichum had hyaline conidia that were terete, smooth, apex obtuse, base truncate, and there were no significant differences (P>0.05) in conidial size between C. horii (10.5 to 33.6 × 3.6 to 9.3 μm) (n=300) and C. fructicola (13.1 to 16.2 × 4.7 to 7.1 μm) (n=100). Pathogenicity tests were conducted in the greenhouse on 1-year-old star anise seedlings, and performed with a conidial suspension (10 µL of 106 conidia/mL) containing 0.1% Tween 20 placed onto lightly wounded sites on healthy leaves. Light cross-shaped wounds were made with sterilized toothpicks, gently scratching the surface without piercing the leaf. Each isolate was inoculated onto three seedlings, with at least eight leaves per seedling inoculated in two spots after light wounding. Control seedlings were inoculated with water containing 0.1% Tween 20. All inoculated seedlings were maintained in the greenhouse (12 h/12 h light/dark, 25±2°C), and covered with plastic bags to maintain high humidity throughout. The wounded sites inoculated with C. horii darkened to greenish-brown after 24 h, and C. fructicola gave similar symptoms after 36 h. Then the wounds turned to light brown round spots, and after 5 days, the spots expanded to water-stained spots with dots of acervuli arranged in a circular pattern. No symptoms were observed for the non-inoculated control. Each fungal isolate was consistently re-isolated from inoculated leaves, thus fulfilling Koch's postulates. There were significant differences (P<0.05) in aggressiveness between the two species, with C. horii showing larger diameter lesions (averaging 10.2 mm) than C. fructicola (averaging 8.4 mm). Anthracnose of star anise caused by C. horii (Liao et al. 2017) and C. coccdes (Wu et al. 2003) has been previously reported in China; however, to our knowledge, this is the first report of C. fructicola infecting star anise in China. This study may provide reference for further epidemiological study and prevention of anthracnose on star anise.

Testicular Torsion: Preliminary Results of In Vitro Cell Stimulation Using Chorionic Gonadotropin

Testicular torsion is a pathology that occurs in young males generally before the age of 25. Despite surgery representing the only effective approach, there is still a need to identify a marker that can predict whether a preserved testicle will be functional. In addition, no therapeutic approach is currently considered in the post-operative phase. Through an approach based on the in vitro culture of a tissue strictly linked to the testicle, the gubernaculum, we defined the healthy state of the organ and the possible responsiveness to a therapy used in the andrology field, chorionic gonadotropin (hCG). Firstly, we optimized a protocol to obtain viable cells starting from a small piece of gubernacular tissue harvested during surgery with the aim to amplify cells in vitro. Intriguingly, only for a patient whose testicle had been removed during surgery due to an excessive necrotic area, gubernacular cells were not able to grow in culture. These data support the possibility of exploiting the gubernaculum to evaluate the healthy state of the testicle. Then, as we demonstrate that gubernacular cells express a luteinizing hormone receptor, to which hCG is specific, we analyzed the cellular response to hCG treatment on in vitro cultured cells derived from patients affected by testicular torsion. Our study opens the way for the possibility of evaluating testicle wellbeing after derotation through in vitro culture of a small piece of gubernaculum together with predicting the response to the treatment with hCG, which can have a positive effect on cell proliferation and vascularization.