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Melon (Cucumis melo L.) is a protected crop in China with high economic value. Agrobacterium-mediated genetic transformation is a powerful tool to improve agronomic traits and obtain elite germplasm. However, current transformation protocols in melons are inefficient and highly genotype-dependent. To improve transformation in melon, we tested different infiltration methods for Agrobacterium-mediated transformation. Among these methods, micro-brushing and sonication for 20 s, followed by vacuum infiltration at −1.0 kPa for 90 s, resulted in the strongest green fluorescent protein signal and increased the proportion of infected explants. We transformed melon with developmental regulatory genes AtGRF5, AtPLT5, AtBBM, AtWUS, AtWOX5, and AtWIND1 from Arabidopsis and estimated regeneration frequencies as the number of regenerating shoots/total number of inoculated explants in the selection medium. The overexpression of AtGRF5 and AtPLT5 in melon resulted in transformation efficiencies of 42.3% and 33% in ZHF and 45.6% and 32.9% in Z12, respectively, which were significantly higher than those of the control. AtGRF5 and AtPLT5 expression cassettes were added to CRISPR/Cas9 genome-editing vectors to obtain transgenic phytoene desaturase CmPDS knockout mutants. Using AtGRF5 or AtPLT5, multi-allelic mutations were observed at CmPDS target sites in recalcitrant melon genotypes. This strategy enables genotype-flexible transformation and promotes precise genome modification technologies in melons. [ABSTRACT FROM AUTHOR]

As an endemic fruit tree in China, the Chinese dwarf cherry (Cerasus humilis (Bge.) Sok.) has substantial ecological and economic value. In 2018, characteristic symptoms of the crown gall of the Chinese dwarf cherry were observed in a nursery base located in Ningxia Hui Autonomous Region, China. The occurrence of the disease among seedlings was observed to be 9.48%. Diseased plants were collected and brought to the laboratory for the isolation of the pathogens. In this study, the isolates were identified by a combination of morphological, biochemical, pathogenicity-related, as well as molecular methods. The isolated strains were white, circular, domed, and glistening. Using 16S rRNA gene sequence analysis and biochemical tests, five strains were identified as Agrobacterium tumefaciens/ biovar 1. Polymerase chain reaction (PCR) was used to detect pathogenicity-associated genes in isolates through the amplification of the partial nucleotide sequences of tms2, virD2 and ipt genes, which proved that the isolates contained the Ti plasmid. In the pathogenicity assay, the crown gall symptom developed on the young stem of tomato and sunflower after inoculation. Based on its morphological, biochemical, pathogenicity and genetic characteristics, the pathogen of the Chinese dwarf cherry crown gall was identified to be A. tumefaciens/ biovar 1. [ABSTRACT FROM AUTHOR]

Apple replant disease (ARD) occurs frequently in old orchards and is an important disease that limits apple production in China. Fusarium oxysporum is one of the causal agents of ARD and its pathogenic mechanism is still unclear. To explore the potential pathogenesis-related genes of this pathogen, a T-DNA insertion library of F. oxysporum HS2 was constructed via optimized Agrobacterium tumefaciens-mediated transformation (ATMT). The ATMT system yielded 160–200 transformants/106 conidia, indicating a relatively high conversion efficiency. The genetically stable transformants were tested through five generations of successive subculture on hygromycin-free media, and the alien gene was detected via PCR. A total of 3500 transformants were obtained to produce a T-DNA insertion library of F. oxysporum HS2, and 223 of these mutants were randomly selected for sporulation and pathogenicity tests. Most of the tested mutants presented weakened virulence, and one of them displayed obviously attenuated sporulation and reduced pathogenicity. The T-DNA in six pathogenicity-deficient mutants existed as a single copy, as confirmed by Southern blotting. The T-DNA flanking sequence was obtained rapidly by successive chromosome walking via the high-efficiency thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR) method. The T-DNA right flanking sequences of five transformants were identified further, and the obtained sequences were aligned to the genomic sequence, revealing novel loci related to pathogenesis. This study provided a large number of various pathogenesis-related mutants and pathogenesis-related genes, which will be valuable resources for characterizing the molecular mechanisms of pathogenesis-related genes. [ABSTRACT FROM AUTHOR]

Phoebe bournei, a plant species endemic to China, is a precious timber tree and widely used in landscaping. This tree contains numerous secondary metabolites, underscoring its potential economic value. However, studies on this species, including molecular genetic research, remain limited. In this study, both a somatic embryogenesis (SE) technical system and Agrobacterium-mediated genetic transformation were successfully employed in P. bournei for the first time. The SE technical system was constructed using immature embryos as original material. The primary embryo and embryonic callus induction rates were 30.66% and 41.67%, respectively. The highest rate of embryonic callus proliferation was 3.84. The maximum maturity coefficient and germination rate were 53.44/g and 39%, respectively. Agrobacterium-mediated genetic transformation was performed using the SE technical system, and the highest transformation rate was 11.24%. The results presented here are the first to demonstrate an efficient approach to achieve numerous P. bournei plantlets, which serves as the basis for artificial cultivation and resource conservation. Furthermore, the genetic transformation platform constructed in this study will facilitate assessment of gene function and molecular regulation. [ABSTRACT FROM AUTHOR]

Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae . Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5′ terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana ; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant–virus–vector interactions, and especially for discerning the differences among the three genotypes. [ABSTRACT FROM AUTHOR]

The article discusses a study that investigates the effects of overexpressing purple acid phosphatase 2 encoded by Arabidopsis (AtPAP2) in Camelina sativa. It is given that agrobacterium transformation was examined to see multiple independent overexpression lines of Camelina sativa with AtPAP2 protein expression. It is mentioned that plants were transferred immediately after first flowering to greenhouse conditions under Hong Kong, China's natural day/light photoperiod. It is found that under controlled environmental conditions, overexpression of AtPAP2 in Camelina sativa resulted in longer hypocotyls.

Abstract: Carbonaceous sorbents including black carbon (BC) and carbon nanotubes have attracted research attention around the world because of their effects on bioavailability of hydrophobic organic compounds (HOCs) in sediments and soils. In this research, 14C-labeled and unlabeled phenanthrene were spiked into three artificial sediments: (i) a sediment sample without amorphous organic carbon (OC) and with BC collected from the Yangtze River (BC-YR), (ii) a sediment without OC and with multi-walled carbon nanotubes (MWCNTs), and (iii) a sediment without OC and with fresh wood char. Biodegradation and mineralization of adsorbed phenanthrene by Agrobacterium and XAD-2 assisted abiotic desorption of adsorbed phenanthrene were studied. The results showed that microbes could utilize a fraction of adsorbed phenanthrene by BC and MWCNTs after aging for 21–40d. With aging for 28d, the biodegradation efficiencies of phenanthrene after incubation for 21d were 83.8%, 73.5% and 54.2% for BC-YR, char and MWCNTs, respectively; with aging for 40d, the mineralization rates of 14C-labeled phenanthrene after incubation for 25d were 38.3%, 25.1% and 24.6%, respectively. The desorption and biodegradation processes showed similar residual concentration of phenanthrene. However, the biodegradation rates were higher than the desorption rates during the fast biodegradation stage, suggesting that bacteria could promote desorption or access and utilize the sorbed phenanthrene. The biodegradation and mineralization efficiencies of phenanthrene associated with MWCNTs were significantly lower than with BC (p <0.01), implying adsorption by MWCNTs may lead to a greater decrease of HOCs bioavailability in the environment. [Copyright &y& Elsevier]

A copper-resistant bacteria CCNWSX2332 was isolated from root nodules of Lespedeza cuneata growing in a gold mining tailing region in northwest of China. The specific growth rate of the strain was 0.62 μh−1 in the presence of 2.0 mM Cu2+ in TY liquid media, and the maximum copper accumulation of whole cell reached 147.03 μM Cu2+ per gram (dry weight) after 4 h incubation. A partial sequence of the copper resistance gene copA was amplified from the strain, and the phylogenetic analysis based on 16S rDNA sequence showed that CCNWSX2332 belonged to Agrobacterium, and it had 100% similarity with Agrobacterium tumefaciens type strain IAM13129T. [ABSTRACT FROM AUTHOR]

Kiwifruit is moderately sweet and sour and quite popular among consumers; it has been widely planted in some areas of the world. In 2019, the crown gall disease of kiwifruit was discovered in the main kiwifruit-producing area of Guizhou Province, China. This disease can weaken and eventually cause the death of the tree. The phylogeny, morphological and biological characteristics of the bacteria were described, and were related to diseases. The pathogenicity of this species follows the Koch hypothesis, confirming that A. fabacearum is the pathogen of crown gall disease of kiwifruit in China. In this study, Loop-mediated isothermal amplification (LAMP) analysis for genome-specific gene sequences was developed for the specific detection of A. fabacearum. The detection limit of the LAMP method is 5 × 10−7 ng/μL, which has high sensitivity. At the same time, the amplified product is stained with SYBR Green I after the reaction is completed, so that the amplification can be detected with the naked eye. LAMP analysis detected the presence of A. fabacearum in the roots and soil samples of the infected kiwifruit plant. The proposed LAMP detection technology in this study offers the advantages of ease of operation, visibility of results, rapidity, accuracy and high sensitivity, making it suitable for the early diagnosis of crown gall disease of kiwifruit. [ABSTRACT FROM AUTHOR]

In the present study, a total of 154 bacterial strains isolated from nodules of eighteen Vicia species mainly grown in the temperate Chinese provinces were characterized by ARDRA, ITS PCR–RFLP, BOX-PCR, sequencing of 16S rDNA, nodC, nifH, atpD and glnII, and nodulation tests. The results demonstrated that most of the R. leguminosarum strains were effective microsymbionts of the wild Vicia species, while genomic species related to Rhizobium gallicum, Mesorhizobium huakuii, Ensifer meliloti and Bradyrhizobium spp. were symbiotic bacteria occasionally nodulating with Vicia species. In addition, fourteen strains related to Agrobacterium, Phyllobacterium, Ensifer, Shinella and R. tropici, as well as 22 strains of R. leguminosarum might be nodule endophytes without symbiotic genes. Diverse symbiotic gene lineages were found among the test strains and a strong association was found among the symbiotic gene types and genomic species, indicating the absence of lateral gene transfer. These results greatly enlarged the rhizobial spectrum of Vicia species. [ABSTRACT FROM AUTHOR]

Crown gall induced by Agrobacterium vitis is a worldwide plant disease in grape-growing regions. Rahnella aquatilis HX2, a new isolate from vineyard soil in Beijing, showed a significant inhibition effect on the development of crown galls in grapevines. In field trials, immersion of the basal ends of grape cuttings with HX2 cell suspension inhibited or completely prevented crown gall formation caused by A. vitis K308 in the roots of the plants from the cuttings. The 3-year average disease incidence in grape plants treated with HX2 was 30.8% compared to 93.5% in plants without HX2. The culture supernatant of HX2 exhibited a stronger inhibition effect on disease development than did the cell suspension. HX2 could be found in the grape rhizosphere, grown under field conditions, for up to 90 days after inoculation. There was no significant difference in the mean population sizes of root microflora between plants treated and not treated with HX2. The inhibition effect of HX2 on crown gall in sunflower, caused by different agrobacterial strains, varied between 30.7 and 100%, depending on strains. Our results showed that Rahnella aquatilis HX2 may be used as a biological control agent for crown gall disease of grapes. [ABSTRACT FROM AUTHOR]

To analyze the diversity and relationships of rhizobia in the subtropical and tropical zones of China, we characterized 67 bacterial strains isolated from root nodules of five legume species in the genera Trifolium, Crotalaria and Mimosa . PCR-amplified 16S rDNA RFLP, numerical taxonomy, SDS-PAGE of whole cell proteins, sequencing of 16S rDNA and DNA–DNA hybridization grouped the isolates into 17 lineages belonging to Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Burkholderia, as well as a non-symbiotic group of Agrobacterium. The Rhizobium group contained twenty strains isolated from Mimosa pudica, Crotalaria pallida and two species of Trifolium. Fifteen of them were R. leguminosarum. Twenty-one strains isolated from four species of Trifolium, Crotalaria and Mimosa were classified into five groups of Bradyrhizobium, including B. japonicum. Agrobacterium group composed of 20 isolates from Mimosa pudica, C. pallida and Trifolium fragiferum. In addition, several strains of Sinorhizobium and Mesorhizobium associated with Trifolium and Burkholderia associated with Mimosa pudica were also identified. The predominance of Bradyrhizobium in the nodules of Trifolium was a novel finding and it demonstrated that the nodule microsymbionts might be selected by both the geographic factors and the legume hosts. [ABSTRACT FROM AUTHOR]

A bi-directional promoter of Tomato yellow leaf curl China virus (TYLCCNV) was obtained with the total DNA from TYLCCNV isolate Y10 infected tobacco leaves as a template. Plant expression vectors were constructed by fusing the amplified DNA fragment with the gus gene and nopaline terminator in different orientations. The vectors containing promoter fragments were transferred into leaf celIs and plant stems of Nicotiana benthamiana by Agrobacterium-mediated method. Transient expression results showed that both the complementary and virion-sense promoters could drive the gus gene to express, and the GUS activity of the complementary-sense promoter was stronger than that of the virion-sense. Co-expression of the vector containing βC1 gene of TYLCCNV DNAβ with the vector containing a bi-directional promoter revealed that the βC1 protein has no impact on expression of either the virion- or the complementary-sense promoter. [ABSTRACT FROM AUTHOR]

Chromoblastomycosis (CBM) is a chronic cutaneous or subcutaneous mycosis that is prevalent worldwide. Though CBM tends not to be fatal, it is difficult to treat and complications can include chronic, marked lesions, lymphatic damage, and neoplastic transformation. Fonsecaea monophora, as a new species segregated from Fonsecaea pedrosoi , is the predominant causative pathogen of CBM in southern China. However, research about F. monophora has been limited, which may be due to a lack of an effective genetic manipulation system for F. monophora. In this study, we successfully established a random insertional mutagenesis system by Agrobacterium tumefaciens -mediated transformation (ATMT) in F. monophora for the first time. In order to improve the efficiency of ATMT, various co-culture conditions were optimized, including: acetosyringone (AS) concentrations, co-culture duration, ratio of bacteria to conidia, and the A. tumefaciens strains. In addition, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to identify the transferred DNA (T-DNA) flanking sequences of the F. monophora transformants. The valuable transformants obtained in this study will be used for research in the future. • ATMT is an efficient tool for random insertional mutagenesis in Fonsecaea monophora. • Several transformation parameters were optimized. • TAIL-PCR was performed to identify T-DNA flanking sequences of the transformants. [ABSTRACT FROM AUTHOR]

A 3-hydroxypyridine degrading bacterium, designated strain DW-1, was isolated from petroleum contaminated soil in Liao River China. 16S rRNA-based phylogenetic analysis indicates that strain DW-1 belongs to genus Agrobacterium. The optimal cultivation temperature and pH for strain DW-1 with 3-hydroxypyridine were 30 °C and 8.0, respectively. Under optimal conditions, strain DW-1 could completely degrade up to 1500 mg/L of 3-hydroxypyridine in 66 h. The 3-hydroxypyridine degradation pathway of strain DW-1 was suggested by HPLC and LC–MS analysis. The first reaction of 3-hydroxypyridine degradation in strain DW-1 was α-hydroxylation so that the major metabolite 2,5-dihydroxypyridine was produced, and then 2,5-dihydroxypyridine was transformed by a Fe2+-dependent dioxygenase to form N-formylmaleamic acid. N-Formylmaleamic acid will be transformed to maleic acid and fumaric acid through maleamic acid. This is the first report of the 3-hydroxypyridine degradation pathway and the utilization of 3-hydroxypyridine by a Agrobacterium sp. It may be potentially used for the bioremediation of environments polluted with 3-hydroxypyridine. [ABSTRACT FROM AUTHOR]