First Report of Nigrospora hainanensis Causing Leaf Blight on Brachiaria Griseb in China.

Brachiaria Griseb is an important gramineous forage grown in tropical regions, and also a main grass species uses to restore grasslands in tropical and subtropical regions of China. In August 2022, symptoms of leaf blight were observed on nearly 30% of the Brachiaria forage grass in the base of the Chinese Academy of Tropical Agricultural Sciences, Hainan, China. Symptomatic leaves initially exhibited small, reddish-brown, round or oval spots on their tips, subsequently expanding in size along the leaf margin, and gradually becoming wilted and dry. Twenty leaves showing typical symptoms were randomly collected and pieces (5×5 mm) from the junction of diseased and healthy region were cut, sterilized with 75% alcohol for 30 s, followed by 5% sodium hypochlorite for 30 s. Rinsed three times with sterile water and dried with sterile filter paper. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 28℃. The colonies were white on the surface and gray on the reverse side. The conidiogenous cells were monoblastic, hyaline, globose or ampulliform, and 6 to 8.7(13.1) ×5 to 7.2 (9) m (n=200). Conidia is single celled, smooth, black, spherical, or ellipsoidal, and (11)13 to 16.5 × (8.2) 10.3 to16.1 m (n=100). Setae were not observed. The morphological characteristics of the isolates were consistent with Nigrospora species. A representative isolate (LNH-5) was selected for genomic DNA extraction. Sequences of the transcribed spacer region of rDNA (ITS), partial translation elongation factor (TEF1), and beta-tubulin fragment (TUB) were amplified using primer pairs ITS1/ITS4(White et al. 1990), EF-728F and EF-986R (Carbone et al. 1999) and Bt2a and Bt2b (Glass et al. 1995), respectively. The sequences of ITS (OQ473493), TEF1 (OQ506059) and TUB gene (OQ506055) were submitted to GenBank. They were 99 to 100% identical to the Nigrospora hainanensis ITS(OM283581.1)(538 out of 519 bp),TEF1(YK019415.1)(274 out of 276 bp),and TUB (OK086377.1)(405 out of 405 bp) sequences. The phylogenetic maximum likelihood analysis using the combined ITS, TEF1 and TUB sequences indicated that the isolate was part of the N. hainanensis clade (100% bootstrap value) that also contained the type isolate LC6979 of this species. Pathogenicity was tested on 15 healthy Brachiaria plants. Fungal conidia were harvested by flooding two-week-old single conidial cultures with sterile water, centrifuging, and adjusting the concentration to 107 spores/mL. Then 10 µL of conidial suspension was dropped onto the surfaces of leaves wounded with a sterile needle. Sterile distilled water was used for control treatment. The test was repeated three times. After inoculation, the plants were kept at 90~100% relative humidity at 25 to 28°C in a greenhouse for two weeks, and monitored daily for lesion development. Seven days post inoculation, all the inoculated leaves presented symptoms similar to those observed under natural conditions, while the control leaves showed no symptoms. The fungus was re-isolated from the diseased tissues by the single spore isolation method (Choi et al. 1999) to complete Koch's postulates. This pathogen has been reported on sugarcane in China (Raza et al., 2019; Zheng et al., 2022). To our knowledge, this is the first report of N. hainanensis causing leaf blight on Brachiaria plants in China.

Comparative karyotype analysis of eight Cucurbitaceae crops using fluorochrome banding and 45S rDNA-FISH.

To have an insight into the karyotype variation of eight Cucurbitaceae crops including Cucumissativus Linnaeus, 1753, Cucumismelo Linnaeus, 1753, Citrulluslanatus (Thunberg, 1794) Matsumura et Nakai, 1916, Benincasahispida (Thunberg, 1784) Cogniaux, 1881, Momordicacharantia Linnaeus, 1753, Luffacylindrica (Linnaeus, 1753) Roemer, 1846, Lagenariasicerariavar.hispida (Thunberg, 1783) Hara, 1948 and Cucurbitamoschata Duchesne ex Poiret, 1819, well morphologically differentiated mitotic metaphase chromosomes were prepared using the enzymatic maceration and flame-drying method, and the chromosomal distribution of heterochromatin and 18S-5.8S-26S rRNA genes (45S rDNA) was investigated using sequential combined PI and DAPI (CPD) staining and fluorescence in situ hybridization (FISH) with 45S rDNA probe. Detailed karyotypes were established using the dataset of chromosome measurements, fluorochrome bands and rDNA FISH signals. Four karyotype asymmetry indices, CVCI, CVCL, MCA and Stebbins' category, were measured to elucidate the karyological relationships among species. All the species studied had symmetrical karyotypes composed of metacentric and submetacentric or only metacentric chromosomes, but their karyotype structure can be discriminated by the scatter plot of MCA vs. CVCL. The karyological relationships among these species revealed by PCoA based on x, 2n, TCL, MCA, CVCL and CVCI was basically in agreement with the phylogenetic relationships revealed by DNA sequences. CPD staining revealed all 45S rDNA sites in all species, (peri)centromeric GC-rich heterochromatin in C.sativus, C.melo, C.lanatus, M.charantia and L.cylindrica, terminal GC-rich heterochromatin in C.sativus. DAPI counterstaining after FISH revealed pericentromeric DAPI+ heterochromatin in C.moschata. rDNA FISH detected two 45S loci in five species and five 45S loci in three species. Among these 45S loci, most were located at the terminals of chromosome arms, and a few in the proximal regions. In C.sativus, individual chromosomes can be precisely distinguished by the CPD band and 45S rDNA signal patterns, providing an easy method for chromosome identification of cucumber. The genome differentiation among these species was discussed in terms of genome size, heterochromatin, 45S rDNA site, and karyotype asymmetry based on the data of this study and previous reports.

Association between genetic variants in ZNF365 and inflammatory bowel disease risk in Caucasians: a meta-analysis and trial sequential analysis.

OBJECTIVE: The published studies regarding the relationships between zinc finger 365 (ZNF365) polymorphisms and inflammatory bowel disease (IBD) risk in Caucasians have yielded conflicting results. Therefore, we performed a meta-analysis to clarify this issue. METHODS: The Electronic databases of PubMed, Web of Science, Wiley Online Library, and EMBASE were searched for eligible studies up to 31 November 2020. The quality of eligible studies was evaluated using the Newcastle-Ottawa Scale. The pooled odds ratios (ORs) with 95% confidence intervals (CIs) under different genetic models were calculated to assess the strength of associations. RESULTS: A total of 22 relevant case-control studies with 9542 ulcerative colitis (UC) patients and 13,886 controls, as well as 13,651 Crohn's disease (CD) patients and 15,256 controls, were involved in our meta-analysis. rs10761659 polymorphism significantly decreased CD and UC risk (except for the heterozygous model and the dominant model in UC), and rs10995271 polymorphism was significantly associated with UC (except for the heterozygous model and dominant model) rather than CD. CONCLUSIONS: The meta-analysis demonstrated that the rs10761659 polymorphism might be a protective factor for both UC and CD in Caucasians, while the rs10995271 polymorphism might be a risk factor for UC rather than CD in Caucasians.

First Report of Paramyrothecium breviseta Causing Leaf Spot Disease of Coffea canephora in China.

Coffee is a tropical plant with two widely cultivated species, namely Coffea arabica and Coffea canephora. A leaf spot disease causing brownish and necrotic lesions was broken out on the C. canephora coffee seedlings in a nursery in Ruili County, Yunnan Province, China, during 2018 to 2019. The incidence of the disease was 15% ~ 20%. Ten diseased leaf samples from five diseased plants were collected for pathogen isolation by tissue separation method. Leaf pieces were cut from the margin of the necrotic lesions (4 × 6 mm), surface-sterilized for 30 s in 75% ethanol, followed by 0.1% arsenic mercury solution for 15 s, then washed 3~4 times with sterilized distilled water and transferred onto potato dextrose agar (PDA) medium in petri plates. Four morphologically similar isolates were obtained from lesions and cultivated on PDA at 25°C. Initial colonies of isolates were round, neat edge, white, floccose mycelium and developed dark green-to-black concentric rings that were sporodochia bearing viscid spore masses after 5~7 days. Conidia were acetates, hyaline and cylindrical with both rounded ends and 4.8 to 6.4 µm long × 1.6 to 2.6 µm wide. Koch's test were conducted on three healthy plants leaves of original source variety C. canephora No.2 and C.arabica Catimor CIFC7963 (control plants) with spore suspension (1 × 106/mL), respectively. Meanwhile, equal numbers of healthy plants were inoculated with water as controls. After inoculation, the plants were transferred into an incubator at 25℃ with saturated humidity. After 10 days of inoculation, all the tested plants presented similar typical symptoms with the diseased leaves under natural conditions; whereas the controls remained healthy. Koch's postulates were performed by re-isolating the fungus from the inoculated leaves and verifying its colony and morphological characters. Two single spore isolates cultured on PDA medium were selected for DNA extraction. The ribosomal internal transcribed spacer (ITS) was PCR amplified by using primers ITS1 and ITS4 (White et al., 1990), ß-tubulin gene by Bt2a and Bt2b (Glass and Donaldson, 1995), the RNA polymerase II second largest subunit (rpb2) by RPB2-5F2 and RPB2-7cR (O'Donnell et al, 2007), calmodulin (cmda) gene by CAL-228F and CAL2Rd (Groenewald et al., 2013). The sequences of ITS (MT853067 ~ MT853068), ß-tubulin (MT897899 ~ MT897900), rpb2 (MW256264~ MW286265) and cmda (MT897897~ MT897898) were deposited in GenBank databases. BLAST analysis revealed that the representative isolates sequences shared 99.31%~99.65% similarities to the ITS sequence of Paramyrothecium breviseta (Accession Nos. NR_155670.1), 99.43% similarities to the ß-tubulin sequence of P. breviseta (Accession Nos. KU846406.1), 98.98% similarities to the rpb2 sequence of P. breviseta (Accession Nos. KU846351.1), and 98.54%~98.71% similarities to the cmda sequence of P. breviseta (Accession Nos. KU846262.1). As it shown in the phylogenetic tree derived from combined ITS, ß-tubulin, rpb2, and cmda gene sequences, the two representative isolates were clustered together with P. breviseta CBS 544.75 with 98% strong bootstrap support, which confirmed that P. breviseta is the causal agent of leaf spot of Coffea canephora. To our knowledge, this is the first report of a leaf spot disease caused by P. breviseta on C. canephora in China, which raised the caution that P. breviseta is also pathogenic to Coffea Arabica.

Comparative molecular cytogenetic characterization of five wild Vigna species (Fabaceae).

To extend our knowledge on karyotype variation of the genus Vigna Savi, 1824, the chromosomal organization of rRNA genes and fluorochrome banding patterns of five wild Vigna species were studied. Sequential combined PI (propidium iodide) and DAPI (4',6-diamidino-2-phenylindole) (CPD) staining and fluorescence in situ hybridization (FISH) with 5S and 45S rDNA probes were used to analyze the karyotypes of V. luteola (Jacquin, 1771) Bentham, 1959, V. vexillata (Linnaeus, 1753) A. Richard, 1845, V. minima (Roxburgh, 1832) Ohwi & H. Ohashi, 1969, V. trilobata (Linnaeus, 1753) Verdcourt, 1968, and V. caracalla (Linnaeus, 1753) Verdcourt,1970. For further phylogenetic analysis, genomic in situ hybridization (GISH) with the genomic DNA of V. umbellata (Thunberg, 1794) Ohwi & H.Ohashi, 1969 onto the chromosomes of five wild Vigna species was also performed. Detailed karyotypes were established for the first time using chromosome measurements, fluorochrome bands, and rDNA-FISH signals. All species had chromosome number 2n = 2x = 22, and symmetrical karyotypes that composed of only metacentric or metacentric and submetacentric chromosomes. CPD staining revealed all 45S rDNA sites in the five species analyzed, (peri)centromeric GC-rich heterochromatin in V. luteola, V. trilobata and V. caracalla, interstitial GC-rich and pericentromeric AT-rich heterochromatin in V. caracalla. rDNA-FISH revealed two 5S loci in V. caracalla and one 5S locus in the other four species; one 45S locus in V. luteola and V. caracalla, two 45S loci in V. vexillata and V. trilobata, and five 45S loci in V. minima. The karyotypes of the studied species could be clearly distinguished by the karyotypic parameters, and the patterns of the fluorochrome bands and the rDNA sites, which revealed high interspecific variation among the five species. The V. umbellata genomic DNA probe produced weak signals in all proximal regions of V. luteola and all (peri)centromeric regions of V. trilobata. The combined data demonstrate that distinct genome differentiation has occurred among the five species during evolution. The phylogenetic relationships between the five wild species and related cultivated species of Vigna are discussed based on our present and previous molecular cytogenetic data.

[Spotting the positions of T-DNA on the genome for Magnaporthe grisea transformants and study on the mode of T-DNA insertion].

Using thermal asymmetric interlaced PCR (TAIL-PCR), 39 specifical fragments of Magnaporthe grisea genomic DNA flanked on the T-DNA were successfully amplified from 37 M. grisea transformants randomly selected from the mutants induced by T-DNA insertion in our laboratory. These fragments were sequenced and then compared by BLAST with the sequences of M. grisea genomic DNA published in netwwork. T-DNA insertion positions for 17 transformants were spotted on the genome of M. grisea. Of all the 39 amplified specifical fragments, 19 were M. grisea genomic DNA and 20 contained the vector backbone sequences. Of the 19 M. grisea genomic DNA fragments, 10 flanked on the right and 9 on the left border of T-DNA inserted. Of the 10 M. grisea genomic DNA fragments flanked on the right border of T-DNA, 9 had a 102bp identical sequence. However, the 7 fragments flanked on the left border of T-DNA did not have this regularity. The above results demonstrated that T-DNA right nick positions were relatively fixed on the M. grisea genomic DNA, similar to the ones on plant genomic DNA. The spotted positions of T-DNA on the genome for 17 M. grisea transformants established a solid foundation for further functional genomic research of M. grisea.