First report of Phytopythium vexans causing root rot on Macadamia integrifolia in China.

Soil-borne plant-pathogenic Phytopythium spp. can cause root rot and damping off on important plant species, resulting in serious economic loss. A survey in October 2021 identified soil-borne diseases occurring on Macadamia integrifolia in Yunnan Province, China. Microbes were isolated from necrotic roots of 23 trees with root rot symptoms by growing on cornmeal-based oomycete-selective 3P (Haas 1964) and P5APR (Jeffers and Martin, 1986) media at 24ºC in the dark for 7 days. Of the 56 single-hyphal isolates obtained, 18 were morphologically similar to Phytopythium vexans (van der Plaats-Niterink 1981; de Cock et al. 2015). Isolates LC04 and LC051 were selected for molecular analyses. The internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit II (CoxII) gene were PCR-amplified using universal primers ITS1/ITS4 (White et al. 1990) and oomycete-specific primers Cox2-F/Cox2-RC4 (Choi et al. 2015), respectively. The PCR products were sequenced with the amplification primers and sequences were lodged in Genbank (Accession no. OM346742, OM415989 for ITS, OM453644, OM453643 for CoxII for isolates LC04 and LC051, respectively). The top BLAST hit in the Genbank nr database for all four sequences was Phytopythium vexans (>99% identity). A maximum-likelihood phylogenetic tree was constructed with analogous concatenated ITS and CoxII sequences from either type or voucher specimens of 13 Phytopythium species in the same phylogenetic clade as P. vexans (Table 1; Bala et. al 2010). Isolates LC04 and LC051 grouped most closely to P. vexans, with LC051 basal and sister to LC04 and P. vexans voucher specimen CBS119.80 with 100% support (Fig. 1). Millet seed inoculated with agar pieces colonized by P. vexans LC04 and LC51 was used to fulfill Koch's postulates (Li et al. 2015) in a completely randomized experimental design. Four 6-month-old M. integrifolia var. Keaau (660) seedlings were transplanted into pasteurized commercial potting mix containing 0.5% (w/w) inoculum. Plants were grown in free draining pots and watered once a day. At 14 days post-inoculation, roots were discolored compared to control plants inoculated with millet seed mixed with agar plugs lacking P. vexans (Fig. 2). By 30 days post-inoculation, infected roots were discolored with obvious decay and reduction in root system size. Control plants were symptomless. P. vexans was successfully re-isolated from two lesioned roots from each plant. The infection experiment was done twice, demonstrating that P. vexans LC04 and LC51 caused root disease on M. integrifolia. P. vexans causes root rot, damping-off, crown rot, stem rot or patch canker on economically important trees in many parts of the world, including seven plant species in China (Farr and Rossman 2022). This is the first report of pathogenic P. vexans on M. integrifolia in China. Reports of pathogenic P. vexans on multiple hosts in several parts of the world suggest it should be considered a quarantine risk and included in risk mitigation or pest management plans that include other species of Phytopythium, or species of Pythium or Phytophthora, to which P. vexans has many similarities (de Cock et al. 2015).

First report of Neofusicoccum parvum causing leaf spot disease on Macadamia integrifolia in China.

The macadamia industry is developing rapidly in China. A brown leaf spot disease was noted in six Macadamia integrifolia plantations in Lincang, Yunnan, in October 2021. Over 60% of trees sampled had brown leaf spot symptoms, among approx.15,000 trees planted in these areas. Lesions (3 to 5 mm dia.) were small round brown spots with yellow edges. Lesions on severely infected leaves were darker and larger, with irregular shape (8 to 10 mm long, 3 to 6 mm wide). About 10% of diseased leaves had lesions characterized by a shot hole surrounded by a yellow halo. Potential pathogens were isolated from four randomly-selected symptomatic leaves from each of the six plantations by cutting lesion edges into small pieces. The pieces were surface sterilized, placed onto water agar containing 100 ppm aureomycin and incubated for 5 days at 24°C in the dark. Subculturing microbial growth on potato dextrose agar produced single-hyphal isolates with white fluffy aerial mycelia that turned pale olivaceous gray after 4 to 5 days. In four randomly-selected cultures, conidia were single celled, hyaline, spindle shaped to oval, and measured 10.9 to 16.3 µm long and 4.0 to 6.2 µm wide (n = 50). These characteristics matched those of Neofusicoccum parvum (Pavlic et al. 2009). Isolate LC013 was randomly selected as a representative individual for molecular identification. Internal transcribed spacer (ITS; ITS1/ITS4 primers; White et al. 1990), beta-tubulin gene (tub2; BT2A/BT2B primers; Glass and Donaldson 1995) and translation elongation factor 1-alpha gene (tef1-α; EF1-728F/EF2 primers; Carbone and Kohn 1999; O'Donnell et al. 1998) regions were PCR amplified from genomic DNA. Sequences of the products were used to BLAST probe the type specimen nucleotide sequences in GenBank. The LC013 sequences (GenBank accessions OM392021 (ITS); OM453641 (tub2); OM567656 (tef1-α)) had >99% sequence identity with analogous sequences from the type specimen of N. parvum CBS 138823 (accessions AY236943 (ITS); AY236917 (tub2); AY236888 (tef1-α)). Isolate LC013 was sister to N. parvum type strain in a maximum-likelihood (ML) tree constructed from analogous concatenated ITS, tef1-α, and tub2 sequences of 27 species that are phylogenetically closely-related to LC013 based on the ITS single locus ML tree. Koch's postulates were tested twice with two isolates by wounding leaves of four 14-month-old M. integrifolia seedlings with a sterile needle and placing a 5-mm-diameter agar plug containing N. parvum on the wound site. PDA plugs alone were used as uninoculated controls. Leaves were covered with sealed bags to maintain >90% humidity for 24 hours. All plants were kept in the same glasshouse under natural conditions. Leaves of inoculated plants began to discolor at 5 days post-inoculation (dpi). Brown spot symptoms were observed at 9 dpi. Control plants were symptomless. N. parvum was re-isolated from leaf lesions of the infected plants, but not from control plants, thus fulfilling Koch's postulates. N. parvum is an aggressive pathogen that causes severe disease on important tree and woody species, including M. integrifolia (Liddle et al. 2019). In China, it has been reported to cause leaf spot disease on 26 plant species (Farr and Rossman 2022), but this is the first report of N. parvum causing leaf spot disease on M. integrifolia. Further investigation is required to estimate the importance of this pathogen to the macadamia industry in China.

[Application of ultrasound-guided endoscopic retrograde appendicitis therapy in children with appendix-related chronic abdominal pain]. / è¶ å£°å¼•å¯¼å† é•œä¸‹é€†è¡Œé˜‘å°¾ç‚Žæ²»ç–—æœ¯åœ¨é˜‘å°¾ç›¸å ³æ ¢æ€§è ¹ç—›æ‚£å„¿ä¸­çš„åº”ç”¨ä»·å€¼.

OBJECTIVES: To study the clinical efficacy of ultrasound-guided endoscopic retrograde appendicitis therapy in children with appendix-related chronic abdominal pain. METHODS: A retrospective analysis was performed on the medical data of 30 children with the chief complaint of chronic abdominal pain who were admitted from August 2019 to May 2021. All the children were found to have inflammation of the appendix or intracavitary stool and fecalith by ultrasound and underwent ultrasound-guided endoscopic retrograde appendicitis therapy. The medical data for analysis included clinical manifestations, endoscopic findings, white blood cell count, neutrophil percentage, length of hospital stay, and cure rate. RESULTS: Among the 30 children with chronic abdominal pain, there were 13 boys (43%) and 17 girls (57%), with a mean age of (9±3) years (range 3-15 years) at diagnosis. The median duration of the disease was 12 months, and the median length of hospital stay was 3 days. The children had a median white blood cell count of 6.7×109/L and a neutrophil percentage of 50%±13%. Fecalith and a large amount of feces were flushed out of the appendix cavity for 21 children (70%) during surgery. The follow-up rate was 97% (29/30), and the median follow-up time was 11 months (range 5-26 months). Of the 29 children, abdominal pain completely disappeared in 27 children (93%). CONCLUSIONS: Ultrasound-guided endoscopic retrograde appendicitis therapy is effective in children with chronic abdominal pain caused by feces or fecalith in the appendix cavity.

Effect of Timing and Duration of Soil Saturation on Soilborne Pythium Diseases of Common Bean (Phaseolus vulgaris).

Understanding combined abiotic (waterlogging) and biotic (Pythium spp.) stress resistance remains an important challenge to improving common bean (Phaseolus vulgaris) productivity in disease-prone regions with irregular but intensive rainfall patterns. This study documented the effects of timing (1, 3, 5, 7, and 9 days after sowing) and duration (3, 6, 12, and 24 h) of soil saturation (waterlogging) on damping-off, as well as hypocotyl and root diseases of common bean caused by Pythium irregulare. There were significant effects of timing of waterlogging as well as the presence or absence of the pathogen on emergence of the three bean varieties tested; namely, 'Gourmet Delight', 'Brown Beauty', and 'Pioneer'. The interaction between time of waterlogging and variety was significant for both root and hypocotyl disease severities. In the presence of P. irregulare, waterlogging 1 day after sowing resulted in the least emergence (55.2 ± 5.6%), although plants that survived after 5 weeks had less hypocotyl and root disease (percent hypocotyl disease index [%HDI] ± standard deviation [SD] = 42.0 ± 2.1% and percent root disease index [%RDI] ± SD = 42.4 ± 2.1%, respectively) than nonwaterlogged plants (%HDI = 50.8 ± 2.1% and %RDI = 48.0 ± 2.1%, respectively). The most severe disease assessed 5 weeks after sowing occurred when plants were subjected to waterlogging 9 days after sowing (%HDI = 61.3 ± 2.1% and %RDI = 56.0 ± 2.1%). In general, both hypocotyl and root disease severity increased as the duration of waterlogging increased from 1 to 24 h, with %HDI increasing from 53.9 ± 3.2% to 70.9 ± 3.2%, while %RDI increased from 57.2 ± 1.5% to 73.7 ± 1.5%. Varieties responded differentially in terms of disease development after waterlogging, with the least hypocotyl and root disease on Gourmet Delight (%HDI = 51.4 ± 3.2 and %RDI = 60.1 ± 1.5, respectively) and greatest on Pioneer (%HDI = 66.2 ± 3.2 and %RDI = 64.9 ± 1.5, respectively). Despite being susceptible to hypocotyl and root disease, Pioneer had the greatest emergence and shoot dry weight overall among the three varieties, suggesting that this variety has a degree of tolerance to waterlogging, P. irregulare infection, and the combination of these two stresses. Although the resistance of Gourmet Delight could be exploited to breed bean varieties that exhibit less hypocotyl and root disease when waterlogging occurs, the tolerance to both P. irregulare infection and waterlogging observed for Pioneer could also be exploited to breed varieties that incur less damage from hypocotyl or root disease or waterlogging. Furthermore, this study demonstrated what appears to be independent resistance to hypocotyl versus root infection by P. irregulare, which offers an opportunity to combine resistance to both stresses to reduce the impact of damping-off and root rot in conditions conducive for P. irregulare.

Species of Pythium Associated with Seedling Root and Hypocotyl Disease on Common Bean (Phaseolus vulgaris) in Western Australia.

The occurrence and distribution of Pythium spp. were determined by collecting isolates of Pythium from common bean (Phaseolus vulgaris) plants showing root or hypocotyl disease symptoms from different areas of Western Australia in 2012. Eight different Pythium species (Pythium conidiophorum, P. diclinum, P. intermedium, P. irregulare, P. lutarium, P. mamillatum, P. pachycaule, and P. perplexum) were isolated and identified according to molecular sequences. P. irregulare was the most widespread Pythium sp. All species, except P. perplexum, were pathogenic to the hypocotyl and root of common bean. We believe this is the first report of P. intermedium as a pathogen on common bean worldwide. This is also the first report of P. conidiophorum, P. intermedium, P. lutarium, P. mamillatum, P. pachycaule, and P. diclinum as pathogens on common bean in Australia and the first report of P. irregulare as a pathogen on common bean in Western Australia. P. intermedium was the most pathogenic species, causing the most severe disease on 'Gourmet Delight' (percent root disease index [%RDI] 75 ± 2.9 and percent hypocotyl disease index [%HDI] 59.2 ± 3.2) and 'Pioneer' (%RDI 75 ± 2.9 and %HDI 65.8 ± 3.2). That the relative susceptibility or resistance (the ability of a plant to reduce the extent of invasion by the pathogen) of a given bean variety to one Pythium sp. was, in general, similar across the other Pythium spp. was an important finding, because this opens up opportunities to utilize a single virulent isolate of one Pythium sp. to identify general resistance to a wider spectrum of Pythium spp.

Temporal and Spatial Changes in the Pea Black Spot Disease Complex in Western Australia.

Black spot (also referred to as Ascochyta blight, Ascochyta foot rot and black stem, and Ascochyta leaf and pod spot) is a devastating disease of pea (Pisum sativum) caused by one or more pathogenic fungi, including Didymella pinodes, Ascochyta pisi, and Phoma pinodella. Surveys were conducted across pea-growing regions of Western Australia in 1984, 1987, 1989, 1996, 2010, and 2012. In total, 1,872 fungal isolates were collected in association with pea black spot disease symptoms. Internal transcribed spacer regions from representative isolates, chosen based on morphology, were sequenced to aid in identification. In most years and locations, D. pinodes was the predominant pathogen in the black spot complex. From 1984 to 2012, four new pathogens associated with black spot symptoms on leaves or stems (P. koolunga, P. herbarum, Boeremia exigua var. exigua, and P. glomerata) were confirmed. This study is the first to confirm P. koolunga in association with pea black spot symptoms in field pea in Western Australia and show that, by 2012, it was widely present in new regions. In 2012, P. koolunga was more prevalent than D. pinodes in Northam and P. pinodella in Esperance. P. herbarum and B. exigua var. exigua were only recorded in 2010. Although A. pisi was reported in Western Australia in 1912 and again in 1968 and is commonly associated with pea black spot in other states of Australia and elsewhere, it was not recorded in Western Australia from 1984 to 2012. It is clear that the pathogen population associated with the pea black spot complex in Western Australia has been dynamic across time and geographic location. This poses a particular challenge to development of effective resistance against the black spot complex, because breeding programs are focused almost exclusively on resistance to D. pinodes, largely ignoring other major pathogens in the disease complex. Furthermore, development and deployment of effective host resistance or fungicides against just one or two of the pathogens in the disease complex could radically shift the make-up of the population toward pathogen species that are least challenged by the host resistance or fungicides, creating an evolving black spot complex that remains ahead of breeding and other management efforts.

[Expression and Activity Regulation of Indoleamine 2,3-Dioxygenase in Acute Myeloid Leukemia Cells].

OBJECTIVE: To investigate the expression and activity regulation of indoleamine 2,3-dioxygenase(IDO) in acute myeloid leukemia cells. METHODS: Expression of IDO and TLR9 in HL-60 and K562 cells cocultured with or without IFN-γ,Tα1,IFN-γ+Tα1 and chloroquine were determined by reverse transcription-polymerase chain reaction(RT-PCR). Then, the IDO activity in HL-60 and K562 cells cocultured with or without IFN-γ,Tα1,IFN-γ+ Tα1 was assayed by coomassie brilliant blue staining and modified colorimetric method. RESULTS: Both IDO and TLR9 mRNA were expressed in HL-60 and K562 cells; IFN-γ increased the expression and activity of IDO in a concentration-dependent manner; Tα1 decreased the expression and activity of IDO in a concentration-dependent manner; the up-regulation of IFN-γ on IDO induced expression and activity had been weakened by Tα1(P<0.01); Chloroquine had no effect on the expression of IDO. The expression of TLR9 in HL-60 cells and K562 cells cocultured with IFN-γ,Tα1,IFN-γ+Tα1 and chloroquine was not significantly changed. CONCLUSION: IDO can be expressed in acute myeloid leukemia cells and possesses the activity. IDO may play an important role in immune tolerance induced by leukemia cells, and become a new predictor of AML prognosis. Tα1 decreases the expression and activity of IDO, which can weaken the induction of IFN-γ on IDO expression and activity, thus Tα1 as an immune modulator may be a new agent for AML immunotherapy.

Activity and expression of nitric oxide synthase in pork skeletal muscles.

The objective of this study was to investigate the biochemical changes of nitric oxide synthase (NOS) in pork skeletal muscles during postmortem storage. Longissimus thoracis (LT), psoas major (PM) and semimembranosus (SM) muscles of pork were removed immediately after slaughter and stored under vacuum condition at 4°C for 0, 1 and 3d. Results showed that all three muscles exhibited NOS activity until 1d while SM muscle retained NOS activity after 3d of storage. The content of nNOS in SM muscle was stable across 3d of storage while decreased intensity of nNOS was detected at 1 and 3d of aging in PM and LT muscles due to the degradation of calpain. Immunostaining showed that nNOS was located at not only sarcolemma but also cytoplasm at 0 and 1d of storage. Our data suggest that postmortem muscles possess NOS activity and nNOS expression depends on muscle type.

Effect of nitric oxide on µ-calpain activation, protein proteolysis, and protein oxidation of pork during post-mortem aging.

The aim of the current research was to examine the influence of nitric oxide (NO) on calpain activation, protein proteolysis, and oxidation in post-mortem pork. Five longissimus muscles were removed from carcass after slaughter, and samples were incubated with water, nitric oxide synthase (NOS) inhibitor, or NO donor for 24 h at 4 °C. The samples were taken out and then stored under 4 °C for 1, 4, and 7 d. Results showed that autolysis of µ-calpain increased by incubation with NOS inhibitor after storage for 1 d (P<0.05). Degradation of titin and nebulin increased by treatment of NOS inhibitor among three treatments (P<0.05). Higher levels of protein oxidation were observed after samples incubated with NO donor than treatment of NOS inhibitor (P<0.05). These data indicated that NO could participate in regulating calpain activation and its proteolysis activity during post-mortem aging.