Occurrence of Phoma macdonaldii, the Causal Agent of Sunflower Black Stem Disease, in Sunflower Fields in China.

Phoma macdonaldii Boerma is the pathogen of sunflower black stem disease, causing dark black, oval to long lesions on stems of sunflower plants. Infection during early growth stages can reduce yield by 10 to 30% (3). This fungal disease is distributed mainly in North and South America and Europe. In China, the first case was reported in Xinjiang in 2008 (1), and was believed to be introduced as a result of hybrid sunflower seeds being imported from abroad. The Chinese government included this fungus into its quarantine pests list in 2010 (2). Since China imports a great number of sunflower seeds to grow in its Northern provinces from epidemic areas such as the United States, Argentina, and France, monitoring the disease occurrence in planting areas became crucial. During 2010 and 2011 growing seasons, surveys were conducted in 37 commercial farms or individual households in 12 counties of five areas (Xinjiang, Inner Mongolia, Ningxia, Hebei, and Beijing). A total of 185 suspicious samples of sunflower black stem disease were collected and all were found from imported hybrid seed fields. The presence of P. macdonaldii was confirmed as following: 4 mm2 tissue pieces cut from lesion margins were disinfected with 1% NaOCl, plated on APDA (acid potato dextrose agar, 4.5 to 5.0 pH adjusted with lactic acid), and incubated at 25°C with 12L:12D photoperiod. After 3 days of incubation, colonies were opalescent or ivory in color, and fluffy or flocculent in appearance. After 4 to 6 days, a large number of spherical or oblate black-brown pycnidia were formed separately or in clusters with thin wall and papillate ostiole in diameter of 135 to 324 µm (average 178 µm). A light pink or opalescent gelatinous substance (pycnidiospores) exuded from the ostiole. Pycnidiospores were single celled, oval or kidney-shaped and hyaline both with and without oil balls, and 1.5 to 3.0 µm × 3.0 to 6.5 µm (average 2.0 × 4.7 µm). Sequences of ITS1-5.8S- ITS2 rDNA fragment of all isolates (GenBank Accession No. JQ979487, JQ979488) were identical and had 100% homology with P. macdonaldii isolates from Xinjiang (HM003206) and Australia (DQ351823, DQ351825) and 99% homology with isolates from the former Yugoslavia (DQ351821, DQ351822) in GenBank. Pathogenicity studies of the isolate were performed by injecting 10 × 106/ml spore suspension into the hypocotyl of four true leaves of sunflower seedlings with a syringe. Sterile water was injected as control. After being inoculated in a plastic bag in the shade at room temperature for 48 h, the plastic bag was removed and the seedlings were grown under natural light. Symptoms of black stem disease were observed in all P. macdonaldii inoculated seedlings and the fungus was reisolated from the lesions for confirmation. The current survey found that 105 of 185 suspicious samples were P. macdonaldii positive and were all from four counties in Xinjiang, suggesting that the disease has not spread to other areas since its introduction. The monitoring of sunflower black stem disease is continuing, as is the research for measuring P. macdonaldii adaptability in China and the development of rapid molecular detection technology. References: (1) W. M. Chen et al. J. Yunnan Agric. Univ. 23:609, 2008. (2) J. Luo et al. Australas. Plant Pathol. 40:504, 2011. (3) E. Miric et al. Aust. J. Agr. Res. 50:325, 1999.

Embryo transfer and sex determination following superovulated hinds inseminated with frozen-thawed sex-sorted Y sperm or unsorted semen in Wapiti (Cervus elaphus songaricus).

The aim of this study was to evaluate embryo production in superovulated wapiti hinds inseminated with either Y-sorted or unsorted semen. Eighteen hinds were allocated to three treatment groups: AI following multiple ovulation (CIDR/FSH) with 10×10(6) Y-sorted frozen-thawed semen (Y group, n=6), or 10×10(6) and 100×10(6) unsorted frozen-thawed semen for the unsorted (n=6) and the control group (n=6). The embryos from the sixth day following insemination were collected and classified. Fifteen embryos from the unsorted or the control group, and four embryos from the Y group were sex determinated based on DNA analysis of the amelogenin gene. Twenty-one embryos from the Y group and 42 embryos from the unsorted or the control group were transferred into 21 and 42 synchronized recipients via standard procedures on 6th day post estrus, respectively. There were no significant differences in the number of recovered eggs, transferable embryos, degenerated embryos or unfertilized oocytes per hind among the three groups of the control (9.2±3.6, 4.7±1.9, 3.0±2.0, 1.5±1.4), the unsorted (8.2±1.9, 4.8±0.7, 1.7±1.0, 1.7±1.0) and the Y group (8.8±4.2, 4.2±1.8, 2.2±1.2, 2.5±2.1), respectively (P>0.05). The sex ratio of embryos from the Y group (4M/0F) was significantly (P<0.05) distinct from that of the unsorted and control group (8M/7F). The sex ratio of the offspring from sexed embryos (8M/0F) was deviated significantly (P<0.05) from that of the non-sexed embryos (11M/9F). In conclusion, the results suggested that the male embryos of predicted sex can be achieved with AI of sex-sorted cryopreserved sperm. PCR amplification using the amelogenin gene primers can be applied to DNA analysis of micro samples from wapiti embryo biopsies for sex identification. The male offspring can be produced after transferred with the male embryos of predicted sex.

First Report of Cochliobolus sativus Causing Leaf Spot on Paper Mulberry in China.

Paper mulberry, Broussonetia papyrifera (L.) Vent., is a highly adaptable, fast-growing tree that is native to eastern Asia. Its ability to absorb pollutants makes it ideal for ornamental landscapes, especially in industrial and mining areas. During the summer of 2010, brown lesions were observed on leaves of paper mulberry in Baiwangshan Forest Park, Beijing, China. These lesions were ovoid to fusiform and 4 to 9 × 2 to 4 mm with dark brown centers and light brown irregular edges. Spots on severely infected leaves sometimes coalesced to form long stripes with gray centers. To isolate the causal agent of the lesions, 4-mm2 pieces of diseased leaf tissue from 12 leaves were collected at the lesion margins and surface disinfected in 0.5% NaOCl for 3 min, rinsed three times with sterile water, plated on water agar, and incubated at 25°C with a 12-h photoperiod. After 5 days, the cultures, which became dark brown to black, were observed. Conidiophores (120 to 220 × 4 to 7 µm) were solitary or in groups of two to five, straight or flexuous with swollen bases, and light or dark brown. Conidia were dark olive brown, spindle- or oval-shaped with truncated ends (60 to 120 × 15 to 30 µm), slightly curved, and containing 3 to 12 distoseptate (mostly 6 to 10). Pseudothecia, produced after 14 days in culture, were dark brown to black and flask shaped (420 to 530 µm in diameter with 85 to 100 × 75 to 90 µm ostiolar beaks). Asci were cylindrical (100 to 220 × 30 to 40 µm) and contained eight ascospores. Ascospores were filiform, (150 to 360 × 6 to 9 µm), hyaline, with 6 to 11 septations. Isolates were identified as Cochliobolus sativus (Ito & Kurib.) Drechsler & Dastur (anamorph Bipolaris sorokiniana (Sacc. & Sorok.) Shoem.) on the basis of culture color and dimensions and colors of pseudothecia, asci, ascospores, conidiophores, and conidia (2,3). The identity of one isolate was confirmed by ITS1-5.8S-ITS2 rDNA sequence (GenBank Accession No. HQ 654781) analysis that showed 100% homology to C. sativus listed in Berbee et al. (1). Koch's postulates were performed with six potted 3-month-old paper mulberry plants. An isolate was grown on potato dextrose agar for 14 days to obtain conidia for a conidial suspension (3 × 104 conidia/ml). Three of the potted plants were sprayed with the conidial suspension and three were sprayed with sterile water as controls. Each plant was covered with a plastic bag for 24 h to maintain high humidity and incubated at 25°C with a 12-h photoperiod. After 7 days, the inoculated plants showed leaf symptoms identical to those previously observed on paper mulberry trees in the Baiwangshan Forest Park, while control trees remained symptom free. Reisolation of the fungus from the inoculated plants confirmed that the causal agent was C. sativus. C. sativus is widely distributed worldwide causing a variety of cereal diseases. Wheat and barley are the most economically important hosts. To our knowledge, this is the first report of C. sativus as a pathogen causing leaf spot of paper mulberry in China. References: (1) M. L. Berbee et al. Mycologia 91:964, 1999. (2) M. B. Ellis. Dematiaceous Hyphomycetes. CABI, Oxon, UK, 1971. (3) A. Sivanesan et al. No.701 in: Descriptions of Pathogenic Fungi and Bacteria. CAB, Kew, Surrey, U.K., 1981.

First Report of Black Spot Caused by Colletotrichum gloeosporioides on Paper Mulberry in China.

Paper mulberry, Broussonetia papyrifera (L.) Venten. (family Moraceae), is a fast-growing tree with luxuriant branches and leaves. Because of strong adaptability and tolerance to unfavorable environmental conditions, it is an important tree species for shade or shelter and reforestation in mined areas and on hillsides. During the summer of 2010, brown-to-black spots were observed on leaves of paper mulberry in Baiwangshan Forest Park in Beijing, China. Early symptoms were round or elliptic, light brown, small lesions that later extended to round or irregular spots (4 to 6 × 4 to 8 mm) that were dark brown or black in the center with brown or light brown margins. Several dozen spots were found on severely infected leaves. Leaf tissues (2 × 2 mm), cut from the margins of lesions, were surface disinfected in 0.5% NaOCl solution for 3 min, rinsed three times with sterile water, plated on potato dextrose agar (PDA) and incubated at 25°C with a 12-h light and 12-h dark period. Numerous waxy subepidermal acervuli with setae were observed after 3 days. Acervuli were brown or black, round or elongate, and 100 to 250 µm in diameter. Setae were dark brown, erect straight or slightly curved, and 60 to 74 × 4 to 8 µm with one to two septa. Conidiophores were hyaline or light brown, short with no branches, and cylindrical with dimensions of 12 to 21 × 4 to 5 µm. Conidia were 11 to 21 × 3 to 6 µm, hyaline, aseptate, and cylindrical. Mycelia on PDA were off white-to-dark gray on the reverse side of the colony. Six isolates (BP21-1 to BP21-6) were obtained from different infected leaves and identified as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (teleomorph Glomerella cingulata (Stonem.) Spaulding & Schrenk) on the basis of reverse colony color, dimensions and colors of acervuli, conidiophores, and conidia (3). ITS1-5.8S-ITS2 rDNA sequence analysis was performed on all six isolates. The resultant sequences were identical (GenBank Accession No. HQ 654780) and revealed 99% similarity (100% coverage) with C. gloeosporioides isolates in the GenBank (Accession Nos. EU371022.1 and AY376532.1) (2). Pathogenicity was demonstrated using six potted 3-month-old paper mulberry trees. Isolate BP21-2 was grown on PDA for 14 days and conidia were harvested to prepare a suspension of 106 conidia/ml. Three plants were sprayed with the conidial suspension and three were sprayed with sterile water. All trees were covered with plastic bags for 24 h to maintain high humidity and incubated at 25°C for 6 days. All conidia-inoculated trees showed identical symptoms as the infected leaves in the park, while the control trees remained symptom free. Reisolation of the fungus confirmed that the causal agent was C. gloeosporioides. C. gloeosporioides is distributed worldwide causing anthracnose on a wide variety of plants including members of mulberry family Moraceae, e.g., mortality of stem cuttings and death of saplings on mulberry (Morus alba L.) in India (1). To our knowledge, this is the first report of C. gloeosporioides causing black spots on paper mulberry in China. References: (1) V. P. Gupta et al. Indian Phytopathol. 50:402, 1997. (2) K. D. Hyde et al. Fungal Divers. 39:147, 2009. (3) J. E. M. Mordue. No. 315 in: Descriptions of Pathogenic Fungi and Bacteria. CMI. Kew, Surrey, UK, 1971.

Successful low dose insemination of flow cytometrically sorted Sika (Cervus nippon) sperm in Wapiti (Cervus elaphus).

The purpose of this study was to determine a practical method in Wapiti (Cervus elaphus) of using predetermined sexed Sika (Cervus nippon) semen. Semen was collected by electro-ejaculation from one stag of proven fertility and transported to the laboratory where it was retained as unsorted (control) or was separated into X- and Y-chromosome-bearing sperm using a modified high-speed cell sorter. Wapiti hinds (n=81) were inseminated into the uterus by rectum manipulation with 1 x 10(6) (X1 and Y1 group, respectively) or 2 x 10(6) (X2 and Y2 group, respectively) of sorted frozen-thawed and 1 x 10(7) non-sorted frozen-thawed (a commercial dose control) Sika motile sperm 60-66h after removal of intra-vaginal progesterone-impregnated CIDR devices and administration of 700IU of PMSG at the time of CIDR removal. The percentage of hinds calving after insemination was similar for X1 (38.5%), X2 (41.7%), Y1 (44.4%), Y2 (38.9%) groups (P>0.05), but higher for control (75%) treatment (P<0.05). Ultimately 15 out of the 16 Sika and Wapiti-hybrid calves produced by Wapiti hinds inseminated with Y-sorted sperm were male (93.7%) and 10/10 (100%) Sika and Wapiti-hybrid calves from hinds inseminated with X-sorted sperm were female. The sex ratio of the Sika and Wapiti-hybrid calves born to hinds inseminated with sex-sorted sperm deviated significantly (P<0.05) from 50% and 50.0% in the control group. All Sika and Wapiti-hybrid calves were born between 237 and 250d of gestation. Male and female calves in the control group had similar birth weights and weaning weights as calves from hinds inseminated with X- or Y-sorted sperm. In conclusion it can be said that normal Sika and Wapiti-hybrid calves of predicted sex can be produced after artificial insemination of Wapiti does with low numbers of sex-sorted cryopreserved Sika sperm.

Catalytic wet air oxidation of coke-plant wastewater on ruthenium-based eggshell catalysts in a bubbling bed reactor.

Catalytic wet air of coke-plant wastewater was studied in a bubbling bed reactor. Two types of supported Ru-based catalysts, eggshell and uniform catalysts, were employed. Compared with the results in the wet air oxidation of coke-plant wastewater, supported Ru uniform catalysts showed high activity for chemical oxygen demand (COD) and ammonia/ammonium compounds (NH3-N) removal at temperature of 250 degrees C and pressure of 4.8 MPa, and it has been demonstrated that the catalytic activity of uniform catalyst depended strongly on the distribution of active sites of Ru on catalyst. Compared to the corresponding uniform catalysts with the same Ru loading (0.25 wt.% and 0.1 wt.%, respectively), the eggshell catalysts showed higher activities for CODcr removal and much higher activities for NH3-N degradation. The high activity of eggshell catalyst for treatment of coke-plant wastewater can be attributed to the higher density of active Ru sites in the shell layer than that of the corresponding uniform catalyst with the same Ru loading. It has been also evidenced that the active Ru sites in the internal core of uniform catalyst have very little or no contribution to CODcr and NH3-N removal in the total oxidation of coke-plant wastewater.