Comparison of Fungal Genera Isolated from Cucumber Plants and Rhizosphere Soil by Using Various Cultural Media.

Plant endophytic fungi and rhizosphere soil fungi are often reported as biocontrol agents against plant pathogens or with plant growth promotion potential. Four treatments were performed in field and greenhouse experiments where cucumber plants were inoculated with Trichoderma harzianum and Fusarium oxysporum in 2022. The roots, stems and leaves of cucumber plants and their rhizosphere soil were collected twice individually from the field and greenhouse for isolation of cucumber endophytic and rhizosphere soil fungi. All fungal strains were identified through sequence similarity of the ITS1-5.8s-ITS2 rDNA region. The potato dextrose agar (PDA) media yielded the highest number of genera isolated from cucumber plants, rhizosphere soil and both compared to other media. There were no significant differences among the four media for the isolation of all cucumber endophytic fungi. However, in the roots, the number of endophytic fungi isolated by MRBA was significantly higher than that isolated on malt extract agar (MEA), while in the stems, the number of fungi isolated with PDA was significantly higher than that isolated with Martin's rose bengal agar medium (MRBA). PDA had significantly higher isolation efficiency for the rhizosphere soil fungi than MRBA. The 28 fungal genera had high isolation efficiency, and the endophytic Trichoderma strains were significantly more isolated by MEA than those of MRBA. It is suggested that PDA can be used as a basic medium, and different cultural media can be considered for specific fungal genera.

Draft Genome Sequence of Alternaria longipes Causing Tobacco Brown Spot.

Alternaria is a cosmopolitan fungal genus associated with diverse hosts. Tobacco brown spot caused by Alternaria longipes is one of the most destructive diseases of tobacco. A. longipes can also infect many other plants, some animals and even humans. Here, we report a genome assembly of A. longipes CBS 540.94 using Oxford Nanopore Technologies. A total of 15 contigs were assembled, and the genome size was 37.5 Mb with contig N50 of 4.33 Mb. This genome resource will provide information for further research on comparative genomics of the genus Alternaria and be a valuable resource in investigations of the molecular interactions of pathogen and hosts.

Characterization and phytotoxicity of ophiobolins produced by Bipolaris setariae.

The Bipolaris setariae NY1 strain, isolated from a diseased green foxtail plant in Henan Province, China, showed strong pathogenicity towards green foxtail. In order to clarify the role of phytotoxic substances in the fungal pathogenicity, bioassay-directed isolation and bioactivity assays of secondary metabolites produced by the fungal strain were carried out. Five ophiobolins were obtained: 3-anhydro-ophiobolin A, 6-epi-ophiobolin A, 6-epi-ophiobolin B, 3-anhydro-6-epi-ophiobolin B and ophiobolin I. Bioassays on punctured and intact detached leaves of green foxtail indicated that 3-anhydro-ophiobolin A was the most phytotoxic, followed by 6-epi-ophiobolin A. The other three ophiobolins appeared to be inactive against green foxtail. The effects of 3-anhydro-ophiobolin A and 6-epi-ophiobolin A were synergistic. The symptoms on green foxtail caused by 3-anhydro-ophiobolin A or its mixture with 6-epi-ophiobolin A resembled those caused by the fungus. 3-Anhydro-ophiobolin A and 6-epi-ophiobolin A are likely the main pathogenic determinants of B. setariae. 6-epi-Ophiobolin A caused cytotoxicity against five kinds of human cancer cells: human colon adenocarcinoma cells (HCT-8), human liver cancer cells (Bel-7402), human gastric cancer cells (BGC-823), human lung adenocarcinoma cells (A549), and human ovarian adenocarcinoma cells (A2780). The results provide information for the development of herbicides and antitumor potential of the ophiobolin sesterterpenes.

Author Correction: Phylogeny and taxonomic revision of Kernia and Acaulium.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phylogeny and taxonomic revision of Kernia and Acaulium.

The genera Kernia and Acaulium comprise species commonly isolated from dung, soil, decaying meat and skin of animal. The taxonomy of these fungi has been controversial and relies mainly on morphological criteria. With the aim to clarify the taxonomy and phylogeny of these fungi, we studied all the available ex-type strains of a large set of species by means of morphological and molecular phylogenetic analyses. Phylogenetic analysis of the partial internal transcribed spacer region (ITS) and the partial 28S rDNA (LSU) showed that the genera Kernia and Acaulium were found to be separated in two distinct lineages in Microascaceae. Based on morphological characters and multilocus phylogenetic analysis of the ITS, LSU, translation elongation factor 1α and ß-tubulin genes, the species in Kernia and Acaulium were well separated and two new combinations are introduced, i.e. Acaulium peruvianum and Acaulium retardatum, a new species of Kernia is described, namely Kernia anthracina. Descriptions of the phenotypic features and molecular phylogeny for identification are discussed for accepted species in two genera in this study.

The potential of endophytic fungi isolated from cucurbit plants for biocontrol of soilborne fungal diseases of cucumber.

The ability of endophytic fungi isolated from cucurbit plants to suppress soilborne diseases and the relationship between antagonism and disease suppression were studied. In dual culture tests of 1044 strains of 90 genera and three pathogenic fungi, 47.1 % of the endophytic fungal strains showed antagonistic effects on at least one pathogen; 186 strains against Rhizoctonia solani, 371 strains against Sclerotinia sclerotiorum, and 403 strains against Fusarium oxysporum f. sp. cucumerinum. The main antagonistic type of the strains of one genus generally was identical to one pathogen. In the pot experiment of cucumber inoculated with R. solani and endophytic fungi, 74.3 % and 33.3 % of 288 strains showed control efficacy of more than 50 % and more than 80 % on cucumber Rhizoctonia root rot respectively. These strains were mostly distributed in Fusarium, Chaetomium, Colletotrichum and Acrocalymma. There were some differences in the proportion of strains with better disease suppressive effects between strain sources. No significant correlation existed between the disease suppression of a strain in vivo and its antagonism against the pathogen in vitro. Most growth-promoting strains had good suppressive effects on cucumber Rhizoctonia root rot. In this study, 82 endophytic fungal strains had good disease suppressive effects and no obvious adverse effects on cucumber growth, and 35 of them showed obvious growth-promoting effects, which suggested that endophytic fungi from cucurbit plants have excellent potential for plant disease control.

Multilocus phylogenetic analysis of Talaromyces species isolated from cucurbit plants in China and description of two new species, T. cucurbitiradicus and T. endophyticus.

During a survey of endophytic fungi from cucurbit plants in China, 21 Talaromyces strains were isolated from ten symptomless plants. Phylogenetic analysis of the partial RNA polymerase II largest subunit gene (RPB2) showed that the strains belong to Talaromyces sections Talaromyces and Islandici. Based on morphological characters and multilocus phylogenetic analysis of the nuc rDNA internal transcribed spacer region (ITS1-5.8S-ITS2 = ITS), calmodulin (CaM), and ß-tubulin (TUB) genes, the strains were identified as four known species, T. cnidii, T. pinophilus, T. radicus, and T. wortmannii, and two new species. Two new species, T. cucurbitiradicus from pumpkin roots and T. endophyticus from cucumber stems, are described in this study. Talaromyces cucurbitiradicus is morphologically similar to T. funiculosus but differs in the number of phialides per metula and by the production of chlamydospores. Talaromyces endophyticus is morphologically similar to T. cerinus and T. chlamydosporus but differs by producing yellowish colonies and by lacking chlamydospores. Further analyses of polymorphisms in ITS and TUB sequences supported the distinctions among T. cucurbitiradicus, T. endophyticus, and similar species.

[Suppression of three soil-borne diseases of cucumber by a rhizosphere fungal strain].

To understand the effect of rhizosphere fungi on soil-borne diseases of cucumber, 16 fungal, strains from rhizosphere soil were investigated for the antagonistic activity to three soilborne pathogenic fungi with dual culture method and for suppression of cucumber diseases caused by the pathogens in pot experiments. Four strains showed antagonism to one or more pathogenic fungi tested. The strain JCL143, identified as Aspergillus terreus, showed strong antagonistic activity to the three pathogenic fungi Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani and Sclerotinia sclerotiorum. In greenhouse pot experiments, inoculation with strain JCL143 provided 74% or more of relative control effect to all the three diseases of cucumber seedling caused by the above three pathogenic fungi, and provided 85% or more of relative control effect to Rhizoctonia root rot and Sclerotinia root and stem rot in pot experiment with non-sterilized substrate. In pot experiment with natural soil as substrate, inoculation with strain JCL143 provided average 84.1% of relative control effect to Fusarium wilt of cucumber at vine elongation stage. The fermentation broth of strain JCL143 showed inhibitory effect in different degrees on the colonial growth of the three pathogenic fungi tested, and reached 63.3% of inhibitory rate of colonial growth to S. sclerotiorum. The inhibitory activity of the fermentation broth decreased with increasing treatment temperature, was liable to decrease to alkaline pH than acid pH, and stable to protease treatment. The results indicated that A. terreus is an important factor in suppression of plant soil-borne diseases, and strain JCL143 with stable disease suppression is potential in biocontrol application.

Effect of Trichoderma longbrachiatum T2 on functional diversity of cucumber rhizomicrobes.

After biocontrol Trichoderma longbrachiatum T2 was applied in the rhizosphere of cucumber, the functional diversity of the rhizomicrobes utilizing carbon sources was investigated by using Biolog Eco-microplates technique. The result showed that T longbrachiatum T2 had a significant effect on the rhizomicrobes in terms of their metabolic activity and their capacity to utilize carbon sources. The ability of the rhizomicrobes to utilize carbon sources was enhanced at seedling stage. The significant change of the richness and the degrees of the rhizomicrobes occured at seedling stage, flowering stage, and final stage, while no obvious difference was observed at fruiting stage. Principal Component Analysis (PCA) indicated that the ability of the rhizomicrobes to utilize carbon sources was enhanced at seedling stage and final stage. At flowering stage, the ability to utilize carbohydrates, amino acids, carboxylic acids, amines and phenolic compounds was reduced, but the ability to untilize polymers was slightly enhanced. The ability to utilize carbohydrates, amino acids, carboxylic acids, polymers and amines was enhanced, but the ability to utilize phenolic compounds was reduced at fruiting stage.

[Isolation and identification of a Bacillus amyloliquefaciens YB-3 against Rhizoctonia solani].

OBJECTIVE: An antagonistic bacterial strain YB-3 against Rhizoctonia solani was isolated from soils. METHODS: Antagonistic strains were isolated by a reporter strain method. YB-3 was identified based on morphology observation, physiological and biochemical characterizations, Biolog, G + C content and 16S rDNA sequence analysis. The antagonistic spectrum and the properties of the inhibitor produced by Bacillus amyloliquefaciens YB-3 against plant pathogenic fungi and bacteria were investigated by means of plate two-way cultivation and disc diffusion method. RESULTS: The strain YB-3 against Rhizoctonia solani was identified as Bacillus amyloliquefaciens. The antagonistic results showed that it had distinctively inhibitive effects on 14 pathogenic fungi and 7 bacteria. In addition, it also had inhibitive effects on strains from genus Bacillus to which YB-3 belongs. Antagonistic properties of B. amyloliquefaciens YB-3 was thermostable, acid resistant, and protease sensitive. CONCLUSION: Bacillus amyloliquefaciens YB-3 was isolated and characterized which had distinctively inhibitive effects on Rhizoctonia solani and had broad-spectrum, highly efficient to plant pathogens.

[Construction and analysis of the SSH library with the resistant wheat near-isogenic line and its susceptible parent infected by Puccinia striiformis Westend. f. sp. tritici].

To analyze the differentially expressed genes between resistant and susceptible wheat near-isogenic lines infected by Puccinia striiformis Westend. f. sp. tritici, a subtractive library containing about 1300 clones was constructed using suppression subtractive hybridization (SSH) in which the cDNA from resistant Yr4/6 × Taichung 29 seedlings inoculated with race CY26 was used as the tester, and the corresponding cDNA from susceptible Taichung 29 as the driver. Six hundred clones from the library were analyzed with reverse Northern blot. The positive clones were further tested by Northern blotting analysis. Twelve clones were verified and showed significant difference. By means of sequencing and BlastX analysis, six function-known differentially expressed sequences were detected, and their putative products were leucine-rich repeat protein, catalase, thioredoxin H-type, RNA binding protein, ascorbate peroxidase, and heat shock protein, respectively. Among them, leucine-rich repeat protein belongs to signal transduction protein, and others belong to defense response protein.

Molecular Cytogenetic Characterization of a New T2BL·1RS Wheat-Rye Chromosome Translocation Line Resistant to Stripe Rust and Powdery Mildew.

Wheat (Triticum aestivum) genotypes with rye (Secale cereale) 1RS chromosomal translocations are widely used in wheat breeding programs because 1RS carries genes for resistance to several diseases. However, some of the pathogens have evolved into new races that overcome the resistance due to extensive use of cultivars with the resistance genes from rye. Therefore, identification and deployment of new resistance sources with desirable agronomic characteristics are important and urgent. We have used winter rye cultivar German White as a source of genes for desirable traits in wheat improvement. A new genotype named WR04-32 was produced through hybridization and chromosome manipulation between common winter wheat cultivar Xiaoyan 6 and German White. This genotype was highly resistant to a wide spectrum of the wheat stripe rust (Puccinia striiformis f. sp. tritici) and powdery mildew (Blumeria graminis f. sp. tritici) pathotypes prevalent in China. The polymerase chain reaction (PCR) result using EST-STS (expressed sequence tag-site tagged sequence) marker STSWE126 specific to 1RS confirmed 1RS in WR04-32, and it was further proved to be a wheat-rye T2BL·1RS translocation line using sequential genomic in situ hybridization (GISH) and multicolor fluorescence in situ hybridization (FISH) with probes pAs1 and pSc119.2 (or pHvG38). In addition to its resistance to stripe rust and powdery mildew, WR04-32 was genetically stable and had desirable agronomic traits, making it a desirable germplasm for wheat breeding.

[Analysis and follow-up study on 8 children with combined congenital heart disease treated with simultaneous trans-catheter therapy].

OBJECTIVE: Interventional treatment for childhood combined congenital heart disease (CHD) has developed very quickly and more new types of occluders have emerged in recent years. The aim of this study is to investigate the efficiency and safety of interventional treatment for combined CHD in children. METHODS: Eight children with combined CHD (4 boys and 4 girls), aged 6.1+/-2.9 years, underwent simultaneous transcatheter therapy. Of the 8 children with CHD, 1 case had atrial septal defect (ASD), ventricular septal defect (VSD) and patent ductus arteriosus (PDA), 1 case had ASD, PDA and pulmonary stenosis (PS), 1 case had ASD and PDA, 1 case had patent foramen ovale (PFO) and PS, and 4 cases had ASD and PS. The methods of transcatheter intervention for these patients were as follows: in patients with ASD,VSD and PDA, the occlusion of VSD was performed first, followed by PDA and ASD occlusions; in patients with ASD, PDA and PS, the occlusion of percutaneous balloon pulmonary valvuloplasty (PBPV) was performed first, followed by PDA and ASD occlusions; in patients with PFO and PS, the occlusion of PBPV was performed first, and PFO occlusion followed; in patients with ASD and PS, the occlusion of PBPV was performed first, and ASD occlusion followed. RESULTS: The intervention operation was successfully performed in all of the 8 patients. No serious adverse events occurred during the operation. No residual shunt was found and all the occlusion devices were in the suitable sites shown by transthoracic echocardiography (TTE) and X-ray right after the operation. In the 6 patients with PS, the systolic pressure across the pulmonary valve decreased from 75.3+/-15.6 mmHg (before operation) to 14.0+/-5.6 mmHg after operation (P<0.05).A 3.4+/-1.2 years follow-up demonstrated that no residual shunt occurred and gradients across valve or coarctation sites were within the limit of satisfactory results. No complications were observed during the follow-up. CONCLUSIONS: Transcatheter interventional therapy for childhood combined CHD can obtain satisfactory results by proper procedures.

Mapping of a major stripe rust resistance gene in Chinese native wheat variety Chike using microsatellite markers.

Chike (accession number Su1900), a Chinese native wheat (Triticum aestivum L.) variety, is resistant to the currently prevailing physiological races of Puccinia striiformis Westend. f. sp. tritici in China. Genetic analysis indicated that resistance to the physiological race CY32 of the pathogen in the variety was controlled by one dominant gene. In this study, BSA (bulked segregant analysis) methods and SSRs (simple sequence repeats) marker polymorphic analysis are used to map the gene. The resistant and susceptible DNA bulks were prepared from the segregating F2 population of the cross between Taichung 29, a susceptible variety as maternal parent, and Chike as paternal parent. Over 400 SSR primers were screened, and five SSR markers Xwmc44, Xgwm259, Xwmc367, Xcfa2292, and Xbarc80 on the chromosome arm 1BL were found to be polymorphic between the resistant and the susceptible DNA bulks as well as their parents. Genetic linkage was tested on segregating F2 population with 200 plants, including 140 resistant and 60 susceptible plants. All the five SSR markers were linked to the stripe rust resistance gene in Chike. The genetic distances for the markers Xwmc44, Xgwm259, Xwmc367, Xcfa2292, and Xbarc80 to the target gene were 8.3 cM, 9.1 cM, 17.2 cM, 20.6 cM, and 31.6 cM, respectively. Analysis using 21 nulli-tetrasomic Chinese Spring lines further confirmed that all the five markers were located on chromosome 1B. On the basis of the above results, it is reasonable to assume that the major stripe rust resistance gene YrChk in Chike was located on the chromosome arm 1BL, and its comparison with the other stripe rust resistance genes located on 1B suggested that YrChk may be a novel gene that provides the resistance against stripe rust in Chike. Exploration and utilization of resources of disease resistance genes in native wheat varieties will be helpful both to diversify the resistance genes and to amend the situation of resistance gene simplification in the commercial wheat cultivars in China.

[Identification of RAPD markers linked to the resistance gene Yr5 against wheat stripe rust with denaturing PAGE-silver staining].

RAPD analysis was performed between a near-isogenic line (NIL) Yr5/6 x Avocet S carrying the resistance gene Yr5 against wheat stripe rust and its susceptible parent Avocet S, using the Yr5 gene donor parent Triticum spelta album as control. Amplified DNA fragments were separated on 4% denaturing PAGE (polyacrylamide gel electrophoresis) and displayed by silver staining. Fifty to 100 bands were detected, 5 folds more than those revealed on agarose gels. A total of 240 random primers were screened, and 23 reproducible polymorphic DNA fragments were found, out of which 6 polymorphic bands appeared to be linked to Yr5 gene. Genetic linkage was tested on 121 segregating F2 plants derived from a cross between Avocet S and Yr5/6 x Avocet S. It was showed that the polymorphic DNA fragment S1320(207) was completely linked to Yr5 gene, and S1348(363) closely linked to Yr5 gene. The results suggested that using denaturing PAGE-silver staining could increase the level of DNA polymorphisms detected in wheat and also improve the repeatability of RAPD analysis.

[Inheritance of stripe rust resistance in the native wheat variety Dazicao from China].

Dazicao, a native wheat variety with stripe rust resistance from Henan, China, was crossed with susceptible cultivar Mingxian 169 as the female parent. The F(1) progeny was selfed to produce F(2) progeny and backcrossed with Mingxian 169 to produce BC(1) progeny. In air-conditioned greenhouse,seedlings of the F(1), F(2), BC1 progenies and their parents were inoculated with the prevalent races CY28 and CY32 of Puccinia striiformis respectively. The phenotypes of the F(1), F(2) and BC(1) plants were analyzed for resistance to the two races. The results indicated that the resistance in the Dazicao to race CY32 was controlled by one recessive gene,and the resistance to race CY28 by complementary action of one dominant gene and one recessive gene.

[RAPD markers linked to the stripe rust resistance gene Yr5 in the wheat variety Triticum spelta album].

A total of 520 10-mer random primers were used to identify the RAPD markers linked to the Yr5 gene between the near-isogenic line Yr5/6 x Avocet S and recurrent parent Avocet S. Three polymorphic DNA fragments, S1496(761), S1453(880) and S1418(1950), were found linked to the Yr5 gene. In which the genetic distance between S1496(761) and Yr5 gene was 2.7 cM. The fragment S1496(761) was recovered from the gel and cloned and sequenced. A pair of specific PCR primers was designed based on the sequence. The specific primers amplified the same fragment about 761bp as the random primer S1496 did. Because the primers could amplify another non-specific fragment, the PCR products must be analyzed by electrophoresis on polyacrylamide gels.