First report of leaf spot and stem blight on blueberry (Vaccinium corymbosum cv. Bluerain) caused by Calonectria pseudoreteaudii in China.

Blueberry has high nutritional value and is one of the five healthy fruits. In 2018, leaf spots and stem blights were observed on Vaccinium corymbosum cv. Bluerain in Guangzhou, Guangdong Province, China. Up to 80% of the plants were affected. Initial symptoms of affected leaves were red-brown, irregular, small spots, which gradually coalesced and formed larger irregular necrotic patches. The affected stems showed red-brown and irregular large lesions. Diseased tissues were surface sterilized with 75% alcohol for 15 s, followed by 2.5% NaClO for 30 s, and rinsing three times in sterile distilled water, placed on potato dextrose agar (PDA) and incubated at 25 C. Representative strains, ZHKUCC 21-0021 from diseased leaves and ZHKUCC 21-0073 from diseased stems, were selected for further studies. Colonies grew slowly at 25 C on malt extract agar (MEA) (average 5.68 mm/d), producing white aerial mycelium and red-brown color on the underside after 7 days. Macroconidiophores were hyaline, smooth, consisting of a stipe bearing fertile branches, and a stipe extension terminating in a vesicle. Each terminal branch produced 2-4 phialides, 8-13 × 3-6 µm, reniform or doliiform; Stipe extensions were septate, terminating in a narrowly clavate vesicle, 2-6 µm. Macroconidia were hyaline, straight cylindrical, round at both ends, 83-100 × 7-11 µm (average = 94 × 8 µm; n = 50), with 5 septa. These morphological characteristics were similar to the description of Calonectria pseudoreteaudii (Lombard et al., 2010). The partial calmodulin (cmdA), beta-tubulin (ß-tubulin), and translation elongation factor 1-alpha (tef1-α) genes of the two isolates were respectively amplified using primers CAL-228F/CAL-737R (Carbone et al., 1999), EF1-728F/EF2 and T1/CYLTUB1R (Lombard et al., 2015), and sequences were deposited in GenBank (cmdA: MZ516854 and MZ516855; ß-tubulin: MZ516858 and MZ516859; tef1-α: MZ516856 and MZ516857). BLAST analysis of three gene sequences showed 100% similarity to those of C. pseudoreteaudii. In the maximum likelihood (ML) tree of the concatenated sequences of the three genes, the two isolates from this study were clustered with C. pseudoreteaudii with 100% bootstrap support. Five-mm-diameter hyphal plugs of two representative isolates grown on PDA for five days were used in the pathogenicity test. Leaves were inoculated with ZHKUCC 21-0021, and stems were inoculated with ZHKUCC 21-0073 with five replicates. As controls, sterile PDA plugs were used. All inoculated plants were maintained at 25 C . After 7 days, inoculated leaves and stems developed symptoms similar to field samples, whereas the control plants remained asymptomatic. The pathogen was reisolated from inoculated plants and confirmed to be C. pseudoreteaudii by morphological characteristics. Five Calonectria species (C. canadensis, C. colhounii, C. ilicicola, C. kyotensis and C. pyrochroa), have been reported associated with blueberry (Farr and Rossman, 2022; Fei et al, 2017). Calonectria canadensis and C. ilicicola have been reported to cause stem blight and stem rot in Vaccinium spp. in China (Fei et al, 2017 and 2018). Calonectria colhounii has been reported to cause stem blight in V. angustifolium and V. corymbosum in the United States (Sadowsky et al, 2011). However, this is the first report of C. pseudoreteaudii causing leaf spot and stem blight on Vaccinium spp. worldwide. These results will provide a foundation for future research on prevention and control of this disease.

First Report of Bacterial Leaf Streak of Strelitzia reginae Caused by Burkholderia cepacia.

In 2011, the bacterial leaf streak disease of the monocotyledonous flowering plant, commonly known as bird of paradise (Strelitzia reginae), occurred in a nursery in Guangzhou, Guangdong Province, China. Lesions on diseased leaves began as water-soaked leaf spots or streaks near the central and secondary veins, eventually expanded along veins and became brown necrotic streaks. Occasionally, during wet conditions, seedlings were completely blighted. The disease incidence was about 12% in the nursery. Bacteria were consistently isolated on nutrient agar (NA) (4) from surface-sterilized symptomatic lesions and purified on NA. Three bacterial strains were tested for pathogenicity on S. reginae plants. Three plants were inoculated per bacterial strain (bacterial suspensions 107 CFU/ml in nutrient broth [NB] [4]) by wounding three young, fully expanded leaves (four wounds per leaf) with needle. Plants were placed in polyethylene bags 1 day before inoculation and maintained for 7 days after inoculation. Three control plants were inoculated with NB. Water-soaked areas on leaves were observed on all inoculated plants 7 days after inoculation. Within 10 days, brown streaks were observed. All strains induced similar symptoms as those observed on the plants in the nursery. Control plants showed no symptoms. For molecular identification, a near full-length sequence of the 16S rRNA gene was amplified from strain TNT1-1 (GenBank Accession No. JX901049.1) with primers 27F and 1492R (3), obtaining a PCR product of ~1,500 bp. A BLAST search in GenBank revealed the highest similarity (99.5%) to sequences of Burkholderia cepacia (FN178432.1 and FN178432.1). BIOLOG identification showed that TTN1-1 had the highest probability index of 0.85 and highest similarity index of 0.85 to B. cepacia. For biochemical characteristics, the strain was gram negative, anaerobic growth test negative, oxidase negative, catalase positive, did not produce fluorescent pigment on KB (4), did not grow on DIM agar (4), arginine dihydrolysis negative, nitrate reduction negative, starch hydrolysis negative, gelatin liquefaction negative, citrate, D-arabinose, L-fructose, trehalose, and maltose utilization positive, didn't produce acid from glucose, and grew on Tween 80 medium at 41°C. The above characteristics were identical to that of reference isolate B. cepacia ATCC 25416. Additionally, bacteria isolated on NA from the leading edge of lesions of inoculated plants with the strain were identical to the inoculated strain based on 16S rDNA sequence analysis, but no bacteria were recovered from the wounded sites on the control plants. Therefore, bacterial leaf streak of bird of paradise is caused by B. cepacia based on Koch's postulates. In contrast, two bacterial diseases on S. reginae were previously reported to be caused by Xanthomonas campestris (1) and B. gladioli (2) in the United States and Italy, respectively. A similar leaf streak disease on S. nicolai was caused by Acidovorax avenae subsp. avenae in the United States (5). To our knowledge, this is the first report of a leaf streak disease on S. reginae caused by B. cepacia. References: (1) A. R. Chase and J. B. Jones. Plant Dis. 71:845, 1987. (2) G. Cirvilleri et al. Plant Dis. 90:1553, 2006. (3) I. M. Lee et al. Appl. Environ. Microbiol. 63:2631, 1997. (4) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001. (5) T. E. Seijo and N. A. Peres. Plant Dis. 95:1474, 2011.

Non-criteria and criteria antiphospholipid antibodies and their association with pregnancy outcomes in women with a history of miscarriage: A retrospective study.

INTRODUCTION: The objective of this study was to investigate both antiphospholipid antibodies (aPLs) and non-criteria aPLs (NC-aPLs) in relation with pregnancy outcomes. METHODS: We retrospectively analyzed 1574 pregnant women with experienced at least one miscarriage who were tested for aPLs and NC-aPLs, and compared their clinical characteristics, immune biomarkers, and pregnancy outcomes. The χ2 test or Fisher's exact test compared pregnancy outcomes among patients negative for all aPLs, positive for NC­aPLs subtypes, and positive for criteria aPLs subtypes. RESULTS: Multivariate logistic regression analysis indicated that positive aPLs (OR = 2.216, 95 % CI 1.381-3.558), and positive NC-aPLs (OR = 1.619, 95 % CI 1.245-2.106) are linked to adverse outcomes. For fetal loss, positive aPLs (OR = 2.354, 95 % CI 1.448-3.829), NC-aPLs (OR = 1.443, 95 % CI 1.076-1.936) were significant. Premature delivery was associated with positive NC-aPLs (OR = 2.102, 95 % CI 1.452-3.043). In the NC-aPLs positive group, the rate of adverse outcomes was higher in the multiple-positive subgroup (77.8 %) compared to the double-positive (52.3 %) and single-positive (37.0 %) subgroups. The rates of fetal loss and premature delivery were also higher in the multiple-positive NC-aPLs subgroup compared to the single-positive subgroup (48.1 % vs. 22.6 % for fetal loss and 57.1 % vs. 16.5 % for premature delivery). DISCUSSION: Our findings suggest that both aPLs and NC-aPLs are associated with an increased incidence of adverse pregnancy outcomes, and patients presenting with multiple NC-aPLs positivity were found to have a higher incidence of adverse outcomes compared to their single-positive counterparts.

First Report of Leaf Spot on Tibouchina semidecandra Caused by Beltrania rhombica in China.

Tibouchina semidecandra Cogn. is a popular ornamental plant in tropical and subtropical areas (1). In August 2011, a leaf spot was observed on approximately 70% of 5,000 potted plants of T. semidecandra in a nursery in Zhongshan, Guangdong Province, China. Each leaf spot was round with a brown center surrounded by a reddish brown border, and ranged from 8 to 10 mm in diameter. A fungus was isolated consistently from the lesions by surface-sterilizing symptomatic leaf sections (each 3 cm2) with 75% alcohol for 8 s, washing the sections with sterile water, soaking the sections in 3% NaOCl for 15 s, rinsing the sections with sterile water three times, and then placing the sections on potato dextrose agar (PDA) at 28°C. Each of three single-spore isolates on PDA produced gray, floccose colonies that reached 70 mm in diameter after 5 days at 28°C. Setae were dark brown, straight, erect, distantly and inconspicuously septate, and 125 to 193 × 3.0 to 4.5 µm. Conidiophores were light brown, cylindrical, simple or sometimes branched at the base, and 105 to 202 × 3 to 5 µm. Separating cells were hyaline, oval, and 12 to 13 × 4 to 5 µm. Conidia were unequally biconic, unicellular, dark brown with a pale brown or subhyaline band just above the widest part, and 26 to 31 × 8.5 to 12 µm (mean 27.3 × 10.6 µm) with a conspicuous appendage at the apex that was 6 to 14 × 1 to 1.8 µm. These characteristics were consistent with the description of Beltrania rhombica Penz. (3). The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) of one isolate (GenBank Accession No. JN853777) was amplified using primers ITS4 and ITS5 (4) and sequenced. A BLAST search in GenBank revealed 97% similarity to the ITS sequence of an isolate of B. rhombica (GU797390.1). To confirm pathogenicity of the isolate, ten detached leaves from 3-month-old plants of T. semidecandra 'Purple Glorybush' were inoculated in vitro with 5-mm diameter, colonized mycelial plugs from the periphery of 5-day-old cultures of the isolated fungus. The agar plugs were put on the leaf surface and secured with sterile, moist cotton. Sterile PDA plugs were similarly used as the control treatment on ten detached leaves. Leaves were placed in petri dishes and incubated in a growth chamber with 12 h of light/day at 28°C. Necrotic lesions appeared on leaves after 2 to 3 days of incubation, whereas control leaves inoculated with sterile PDA plugs remained asymptomatic. B. rhombica was consistently reisolated from the lesions using the same method described above, but was not reisolated from the control leaves. Although there are approximately 77 reported hosts of B. rhombica (2), to our knowledge, this is the first report of B. rhombica causing a leaf spot on T. semidecandra. Because the disease caused foliar damage and reduced the ornamental value of the nursery plants, control measures may need to be implemented for this species in nurseries. References: (1) M. Faravani and B. H. Bakar. J. Food Agric. Env. Pap. 5:234, 2007. (4) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 30 Mar. 2012. (2) K. A. Pirozyski and S. D. Patil. Can. J. Bot. Pap. 48:567, 1970. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

First Report of Anthracnose Caused by Colletotrichum gloeosporioides on Cymbidium sinense in China.

Cymbidium sinense are among the most important commercial orchids cultivated in China for flower production. In April of 2010, a leaf spot was sporadically observed on C. sinense in fields located in Guangzhou, China. Symptoms first appeared as yellow to brown, irregular-shaped lesions on the leaf margin or tip. As the infection continued on the tissues, the spot expanded and became dark brown along the margins and developed gray brown centers. At later stages, numerous epidermal acervuli developed on the lesions and mucilaginous conidial masses appeared on the lesions under moist conditions. Ten samples from tissue along the margins of lesions were collected and surface sterilized by washing in 70% ethanol for 30 s, followed by washing in 1% sodium hypochlorite for 30 s, and rinsing in sterile distilled water. These samples were plated onto potato dextrose agar (PDA) and incubated at 25°C with a 12-h alternating light and dark cycle. After 5 days, fungal colonies that grew from the tissue were subcultured onto PDA and pure cultures were obtained using the single-spore method. The fungus was identified as Colletotrichum gloeosporioides based on typical cultural characteristics and conidial morphology (1). PDA cultures were white at first and subsequently became grayish white to gray and pink to reddish brown. Conidia were straight, one-celled, hyaline, oblong, or cylindrical, slightly curved with truncate base and rounded apex and measured 14.0 to 19.5 × 4.0 to 6.0 µm. Chlamydospores, sclerotia, and a teleomorph were not found. Genomic DNA was extracted from one isolate and the internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1-5.8S-ITS2) was amplified using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-CCTCCGCTTATTGATATGC-3') primers. The ITS region was further cloned and sequenced and showed 100% homology with many GenBank sequences (e.g., HQ333546.1) of C. gloeosporioides as the closest match. Pathogenicity tests were done by transferring one 4-mm-diameter disk of PDA that was colonized by the test isolates to wounds (4 × 4 mm) made with a needle in the leaves of 1-year-old C. sinense plants. Control plants received a sterile agar plug in wound. Ten inoculated plants were covered with plastic bags to maintain a high relative humidity and maintained in a greenhouse at 25 ± 2°C for 72 h. Five days after inoculation, no symptoms developed on the control plants. Foliar lesions closely resembled those observed in the field. C. gloeosporioides was reisolated consistently from symptomatic tissue collected from greenhouse experiments. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on C. sinense in China. Reference: (1) B. C. Sutton. Colletotrichum Biology, Pathology and Control. CAB International, Wallingford, UK, 1992.

First Report of Leaf Spot on Gerbera jamesonii Caused by Corynespora cassiicola in China.

Gerbera (Gerbera jamesonii Bolus ex. Hook f.) is a popular cut flower and flowering potted plant. In August 2011, a new leaf spot disease was observed on double-type Gerbera growing in outdoor ground beds in Guangzhou, Guangdong Province, China. Approximately 30% of about 20,000 Gerbera plants in the Guangzhou ground beds were affected. Leaf spots were round or irregular with grayish centers surrounded by dark brown borders and ranged from 5 to 15 mm in diameter. Leaves with multiple lesions became blighted. A fungus was isolated from the lesions and single-spore isolates plated on potato dextrose agar (PDA) produced gray, floccose colonies, which reached 65 mm on PDA after 7 days at 28°C. Conidiophores were brown or olivaceous, cylindrical, straight and unbranched, two to seven septations, and 25 to 83 × 4 to 7 µm. Conidiogenous cells were olivaceous or brown, cylindrical, and 11 to 21 × 4 to 6 µm. Conidia were borne singly or in chains of two to five, brown, cylindrical, straight to slightly curved, two to eight pseudosepta, and 30 to 90 × 5.5 to 11.5 µm (mean 70.4 × 7.3 µm), with a conspicuous hilum. These characteristics were consistent with the description of Corynespora cassiicola (Berk. & M.A. Curtis.) C.T. Wei (1). The internal transcribed spacer region (ITS) of one isolate (GenBank Accession No. JN853778) was amplified using primers ITS4 and ITS5 (3) and sequenced. A BLAST search in GenBank revealed highest similarity (99%) to sequences of C. cassiicola (AY238606.1 and FJ852715.1). Pathogenicity tests were conducted on 10 potted double-type Gerbera plants. Five wounded and five unwounded leaves on each plant were inoculated with 5-mm mycelial plugs from the periphery of 5-day-old cultures of the isolated fungus. The plugs were put on the leaf surface and secured with sterile wet cotton. Sterile PDA plugs were used as the control treatment on different leaves of the same plants that were inoculated. Plants were covered with plastic bags and incubated in a growth chamber with 12 h of light at 28°C. Necrotic lesions appeared on wounded leaves after 2 to 3 days of incubation and on unwounded leaves 5 to 7 days after incubation. Symptoms on wounded and unwounded leaves were similar to those observed in the field, whereas control leaves inoculated with sterile PDA plugs remained symptomless. C. cassiicola was consistently reisolated from these lesions. Although there are approximately 644 reported hosts of C. cassiicola (2), to our knowledge, this is the first report of C. cassiicola leaf spot on G. jamesonii. Because the disease caused damage to the foliage and affected the flowering of the plants, control measures may need to be implemented for the production of Gerbera in cut flower nurseries. References: (1) M. B. Ellis. CMI Mycol. Pap. 65:15, 1957. (2) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 21 November 2011. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

First Report of Myrothecium Leaf Spot of Hemionitis arifolia Caused by Myrothecium roridum in China.

Hemionitis arifolia (Burm.) Moore. was grown commercially as an ornamental plant in China. In January 2010, a new foliar disease with typical leaf spot symptoms was observed on H. arifolia in Dongguan City, Guangdong Province. Approximately 10% of the plants in the Dongguan nursery were affected. Leaf spots were circular to subcircular, dark brown, with distinct concentric zones, and ranged from 10 to 20 mm in diameter. Lesions developed mostly on the lower leaves and black sporodochia with white mycelial tufts were produced mostly in older lesions under high humidity. Single-spore isolates from lesions plated on potato dextrose agar (PDA) produced white, floccose colonies and dark green-to-black sporodochia. Colonies reached 60 mm on PDA at 25°C after 14 days. Conidiophores branched repeatedly. Conidiogenous cells in whorls of two to six on ultimate branches were hyaline, cylindrical, and 13 to 20 × 1.2 to 1.8 µm. Conidia were hyaline, cylindrical, mostly with both rounded ends, occasionally one blunt end, and mean size was 6.1 (4.5 to 7.0) × 2.3 (1.8 to 3.0) µm. These characteristics were consistent with the descriptions of Myrothecium roridum Tode ex Fr. (2-4). The internal transcribed spacer regions including ITS1, ITS2, and the 5.8S rRNA of one isolate were PCR amplified and sequenced. A BLAST search in GenBank revealed highest similarity (99%) to M. roridum sequences from isolates collected from Germany (Accession Nos. AJ302001.1 and AJ301995.1). The M. roridum sequence from the Chinese isolate was submitted to GenBank (Accession No. JF343832). To confirm pathogenicity, five leaves on five H. arifolia plants were inoculated with 5-mm mycelial plugs from the periphery of 7-day-old cultures; sterile PDA plugs were used as the control treatment. Plants were covered with plastic bags and incubated in a growth chamber at 28°C. Necrotic lesions appeared 2 to 3 days after inoculation and the symptoms described above were observed after 7 days on all inoculated leaves, whereas sterile PDA plugs did not produce any leaf lesion. The pathogen was reisolated from the inoculated leaves and confirmed to be M. roridum on the basis of morphological characteristics. There are approximately 271 hosts of M. roridum (1), including many ornamental plants such as salvia (2) and garden hydrangea (3). To our knowledge, this is the first report of Myrothecium leaf spot on H. arifolia. Because the disease caused damage to the foliage and reduced the ornamental value of H. arifolia plants, control measures may need to be implemented for production of this species in ornamental nurseries. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrived from http://nt.ars-grin.gov/fungaldatabases/ , 6 February 2011. (2) J. A. Mangandi et al. Plant Dis. 91:772, 2007. (3) M. T. Mmbaga et al. Plant Dis. 94:1266, 2010. (4) M. Tulloch. Mycol. Pap. No. 130. CMI, Wallingford, UK, 1972.

Gene profiling involved in immature CD4+ T lymphocyte responsible for systemic lupus erythematosus.

We attempted to characterize the genes expression of CD4+ T lymphocytes for the pathogenesis of systemic lupus erythematosus (SLE). Genomewide gene expression profiles of CD4+ T cells, which were isolated from the disease severe activity (T4-1s) and nonactivity (T4-2s) with an SLE patient by using long serial analysis of gene expression (LongSAGE). We picked out 289 genes matching to Unigene cluster with different expression more than four copies between T4-1s and T4-2s libraries and analyzed their roles from the collectedly published articles of PubMed by genes functional clustering. The genes functions were related to a diverse cellular process including: (1) most of these genes were associated with CD4+ T cells functions, particularly related to cellular developments; (2) Ras pathway genes as RANBP10, GMIP, RASGRP2 and ARL5 might be responsible for the abnormal development of CD4+ T cells of SLE; (3) HIG2, TCF7, KHSRP, WWP1, SMAD3, TLK2, AES, CCNI and PIM2 belong to Wnt/beta-catenin way, they could play roles in modulating proliferation and differentiation of T lymphocytes; (4) uncertain viral infections may initiate autoimmunity because high levels expression genes were detected in T4-1s such as TRIM22, IER2, ABCE1, DUT, G1P2, G1P3, HNRPUL1, EVER2, IFNAR1, TNFSF14, TMP21 and PVRL2; and (5) apoptosis relating genes as EIF3S8, SH3BGRL3, GPX4, TOSO, PFDN5, BIN1, XIAPAF1, TEGT and CUGBP2 may contribute to over uploading of selfantigens in SLE cells. Abnormalities findings of multiple genes expression involving with a variety of CD4+ T cells process might be meaningful to understanding the pathogenesis of SLE, and immature CD4+ T cells may be responsible for SLE.

Translocation of the zinc finger protein basonuclin from the mouse germ cell nucleus to the midpiece of the spermatozoon during spermiogenesis.

Basonuclin was first described as a human keratinocyte zinc finger protein present in the nuclei of proliferative basal keratinocytes in the epidermis. It disappears from keratinocytes that have lost their proliferative ability and have entered terminal differentiation. We now report that basonuclin is present also in the germ cells of the mouse testis and ovary. Immunocytochemical staining detected basonuclin in the nuclei of spermatogonia and spermatocytes at various developmental stages. During spermiogenesis, it relocated from the nucleus to the midpiece of the flagellum of the spermatozoa. In the ovary, basonuclin was found mainly in the nuclei of developing oocytes. The dual presence of basonuclin in differentiated spermatozoa and oocytes suggests that it may play a role in their differentiation and the early development of an embryo.