First report of Stemphylium eturmiunum causing leaf spot and black spot on apple in Zhaotong, Yunnan, China

Apple is the largest fruit tree crop in the world, and China is the largest apple-producing County in the world. Zhaotong, Yunnan Province is a typical cold and mountainous apple-producing area in China. However, apple production is threatened by diseases during the entire growing season, and among them, apple leaf spot and fruit black spot are severe. In previous reports, the main pathogen causing apple leaf spot and fruit black spot was Alternaria sp. (Lior, et al, 2017), while different pathogens were identified. In the current study, seven red Fuji apple fruit with typical black spot samples were collected randomly in Dongda company orchard, Sujiayuan town, Zhaotong, Yunnan on March 25, 2021. The spots on the surface of these apples appear rounded, the diseased parts turn brown or black in colour and the flesh became soften and rotten. The tissues of fruit epidermis at the edge between diseased and healthy parts were cut, soaked in 75% alcohol for 30 s, washed with sterile water three times, and air-dried. Five pieces of tissue were placed on PDA medium amended with rifampicin (50 mg/ml) and incubated in the dark at 25 ℃ for 3-5 days. After colonies grew, mycelial clumps were picked out from the edges of the colonies, transferred to new PDA plates, and incubated at 25 ℃ for 6 days. The diameter of the colonies reached up to 5.7 cm. A representative isolate was retained for further work and was named P6-3-1. The hyphae were white and dense at an early stage, the culture medium on the underside became yellow and the middle parts of the colonies were darker. With maturity, hyphae were clumped, became red with other colors interspersed, and the medium became dark red. Light brown spores were produced, with more vertical septa and fewer transverse septa. Two to three transverse septa were generally observed with obvious constriction at the transverse septa. Average spore size was 22.83 µm ± 2.04 µm × 14.58 µm ± 1.97 µm. DNA was extracted from mycelium, purified and amplified with two pairs of primers, ITS1/ITS4 (White et al. 1990) and gpdF/gpdR (Marcos P. S. Câmara, et al. 2002). The PCR products were sequenced and deposited in GenBank (accession NO.OK560128 and OK627661 ). The similarity of ITS sequences between the isolate and MH843733 (Stemphylium eturmiunum strain ST14) was 100%, and that of gpd sequences between the isolate and MH843728 (Stemphylium eturmiunum strain ST20) was 100%. The maximum parsimony method of Mega7.0 was used and demonstrated that the studied isolate converged to the same branch as Stemphylium eturmiunum. Koch's postulates was applied to identify the pathogenicity of this isolate. A disc of P6-3-1-culture on PDA (5 mm in diameter) was placed on apple leaves and fruit wounds. Sterile PDA was used as a control. All plants were kept in a growth chamber at 25-30 ℃. Four days after inoculation, the disease spot was observed on the inoculated sites and fruit, and with the extension of incubation time, the diseased spots continue to grow, and the leaf spots were not limited by the veins. The pathogen was re-isolated from the inoculated leaves and fruit, satisfying Koch’s postulates. This pathogen can also cause postharvest rot of sweet cherry (Alice Spadoni, et al, 2020), postharvest rot on tomato (Prencipe Simona, et al, 2021), etc. This is the first report that Stemphylium eturmiunum can cause apple leaf spot and fruit black spot in Yunnan province, China. The apple black spot caused by Stemphylium eturmiunum was accurately identified. By distinguishing between the two similar diseases mentioned above, resistance to the host and management practices can be accrued based on the characteristics of the pathogen, its epidemiological pattern and the choice of an effective chemical fungicide.

Isolation, phenotypic characterization and comparative genomic analysis of 2019SD1, a polyvalent enterobacteria phage

Shigella has the remarkable capability to acquire antibiotic resistance rapidly thereby posing a significant public health challenge for the effective treatment of dysentery (Shigellosis). The phage therapy has been proven as an effective alternative strategy for controlling Shigella infections. In this study, we illustrate the isolation and detailed characterization of a polyvalent phage 2019SD1, which demonstrates lytic activity against Shigella dysenteriae, Escherichia coli, Vibrio cholerae, Enterococcus saccharolyticus and Enterococcus faecium. The newly isolated phage 2019SD1 shows adsorption time < 6 min, a latent period of 20 min and burst size of 151 PFU per bacterial cell. 2019SD1 exhibits considerable stability in a wide pH range and survives an hour at 50 °C. Under transmission electron microscope, 2019SD1 shows an icosahedral capsid (60 nm dia) and a 140 nm long tail. Further, detailed bioinformatic analyses of whole genome sequence data obtained through Oxford Nanopore platform revealed that 2019SD1 belongs to genus Hanrivervirus of subfamily Tempevirinae under the family Drexlerviridae. The concatenated protein phylogeny of 2019SD1 with the members of Drexlerviridae taking four genes (DNA Primase, ATP Dependent DNA Helicase, Large Terminase Protein, and Portal Protein) using the maximum parsimony method also suggested that 2019SD1 formed a distinct clade with the closest match of the taxa belonging to the genus Hanrivervirus. The genome analysis data indicate the occurrence of putative tail fiber proteins and DNA methylation mechanism. In addition, 2019SD1 has a well-established anti-host defence system as suggested through identification of putative anti-CRISPR and anti-restriction endonuclease systems thereby also indicating its biocontrol potential.

Identification of Vibrio cholerae O1 strains of the El Tor biovar sensitive to polymyxin B and their molecular genetic analysis

The aim of the work was the identification and genetic characterization of Vibrio cholerae O1 strains of the El Tor biovar sensitive to polymyxin B among isolates imported to Russia.Materials and methods. We used 56 toxigenic and non-toxigenic strains of V. cholerae isolated from patients and from the environmental samples on the territory of Russia in 1970-2020. Resistance to polymyxin B was determined according to MR4.2.2218-07. The ability of strains to form a biofilm on the abiotic surface was assessed by a photometric method. Nucleotide sequences of genes were determined using UGENE 1.32 and MEGA X software. Phylogenetic analysis and tree construction were performed using "maximum parsimony" method in MEGA X software.Results and discussion. Two genetically modified strains of V. cholerae O1 biovar El Tor, M1509 and 3265/80, which were imported to Russia from India in 2012 and 2014, respectively, were identified. The analysis of 12 genes responsible for the resistance of V. cholerae O1 biovar El Tor strains to polymyxin B demonstrated that these strains contain the allele of the carRS gene, which is typical for all strains of cholera vibrio sensitive to polymyxin B. Study of V. cholerae M1509 and 3265/80 phylogeny based on SNP analysis showed that they fall into the same cluster with isolates containing the carRS allele isolated in India (2015) and Bangladesh (2018). V. cholerae M1509 and 3256/8 strains had the ability to form a biofilm similar to those observed in other genetically modified strains of cholera vibrio included into analysis.Conclusion. Highly virulent strains of the cholera agent with altered diagnostically significant features are imported into Russia, which should be taken into account when identifying V. cholerae O1 biovar El Tor strains isolated from patients and environmental samples during monitoring studies.

Molecular phylogenetics of Malesian Diospyros (Ebenaceae) based trnL-F spacer sequences

Wanda IF, Djuita NR, Chikmawati T. 2021. Molecular phylogenetics of Malesian Diospyros (Ebenaceae) based trnL-F spacer sequences. Biodiversitas 22: 4106-4114. Diospyros is a genus composed of potential species as an economic commodity with high diversity. However, there is limited information on the phylogenetic relationship of this genus in the Malesian region. This study aimed to provide information on the species diversity through a DNA barcoding approach and revealing the phylogenetic information of Diospyros spp. in the Malesian region. This study used 20 Diospyros accessions from Bogor Botanical Garden collections, 40 Diospyros accessions, and four outgroup accessions obtained from the NCBI database. The DNA barcoding primer utilized comes from plastids, trnL-F intergenic spacer. The phylogenetic trees were constructed using the Maximum-Parsimony method. A total of 20 accessions of Diospyros were validated with sequence data on the genebank. The result showed that all accessions had relationships with 44 other Diospyros species globally. Here, we reported 10 new trnL-F intergenic spacer sequences of Malesian Diospyros species. A phylogenetic tree grouped 64 monophyletic Diospyros species into seven clades. The phylogenetic results supports the biogeographic hypothesis: the Malesian region, the Malesian-Caledonian Region, and Cosmopolite species in almost all bioregions.

Morphological and Molecular Characterization of Paragonimus skrjabini Complex from Yunnan, China: A Brief Report

Purpose To perform environmental sampling and molecular identification of Paragonimus in endemic regions, which may help in minimizing transmission among humans. Methods Mountain crabs from the genus Potamiscus were collected and the encysted metacercariae were extracted and subjected to morphological identification, followed by animal inoculation in Sprague–Dawley (SD) rats. After 112 days of infection, animals were killed and adult worms were extracted from lungs and muscles. The morphology of adult worms was characterized by microscopy and molecular identification was done by polymerase chain reaction, followed by sequencing of cox1 and ITS2 genes. Phylogenetic analysis was done by maximum parsimony method. Results A total of 447 crabs were captured from the streams of Tongchang Town, Jinping County, Yunnan Province, China. The infection rate was found to be 41% (186 out of 447 crabs). The metacercariae of Paragonimus skrjabini was identified by the characteristics round or spherical encysted form measuring 410 to 460 × 400 to 460 µm. After animal infection in SD rats, adults were presumptively confirmed to be P. skrjabini, which was also confirmed by gene amplification and sequence analysis of cox1 and ITS2 regions. Paragonimus skrjabini clustered with previously reported P. skrjabini from Yunnan and Vietnam. The confidence values of their branches were > 95%. Phylogenetic analysis of the ITS2 region revealed two distinct clusters with distinct geographical grouping. Phylogenetic analysis with the combined data sets reiterated the geographical grouping with P. skrjabini from Yunnan clustering with strains from Vietnam. Conclusion Metacercariae of P. skrjabini was discovered in freshwater crabs in Yunnan province, China, and the strains were phylogenetically related to P. skrjabini from Vietnam.

First Report of Leaf Spot Disease Caused by Alternaria brassicae on Orychophragmus violaceus in China

Orychophragmus violaceus, belonging to the Brassicaceae family, is widely grown in many provinces of China as an ornamental plant and also as a green manure crop. In December 2019, field investigations showed that a leaf spot disease occurred on O.violaceus with 50% to 80% incidence in Huize City, Yunnan Province of China. Infected leaves showed symptoms of small black point spots in the early stage of onset. The lesions are distributed throughout the leaves and finaly expand to 10-15 mm in diameter after 10-15 days of onset. At this time, the lesions are gray to black, and some have round patterns, and gray-white mildew layers can be seen on the front and back of the lesions in a humid environment. The leaves with typical lesion symptoms were sampled and photographed, and then subjected to isolate and characterize the pathogen. Six pure cultures (HEYA2; HEYA4; HEYC6; HEYD7; HEYD8; HEYD10) were obtained by single-hyphae isolation. On PCA medium, colony can reach 27 mm after 7d, at 25°C in darkness. Aerial hypha is cottony with white to pale gray color, while the colony reverse is fawn to dark. on V8 medium, conidiophore solitary or clustered, erect or knee-curved, occasionally branched, pare brown, separated, 82–130 × 5–9 µm. Conidia are solitary, straight or slightly curved, inverted rod-shaped, pare brown to brown, with 6-10 transverse septa, 0-5 oblique and longitudinal septa, columnar beak, conidial bodies (47.7-)69.3-103.8(-119.6)(11.2-)16.6-23.6(-27.8) µm. Beak septum, pare brown, (29.2-)34.4-72.4(-101.3)(4.2-)6.6-9.5(-11.3) µm. Morphologically these isolates resembled species belonging to genus Alternaria (Simmons, 2007). Genomic DNA of each culture was quickly extracted from mycelia using QS method (Chi et al. 2009). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2) and translation elongation factor -1α (TEF -1α) genes were amplified according described procedures (White et al. 1990; Berbee et al. 1999; Liu et al. 1999；Sung et al. 2007; Carbone & Kohn 1999). The sequences obtained in this study were deposited in GenBank with accession numbers: MW867245, MW867246, MW867247, MW867248, MW867249, MW867250, MW882913, MW882914, MW882915, MW882916, MW882917, MW882918, MW882919, MW882920, MW882921, MW882922, MW882923, MW882924, MW882925, MW882926, MW882927, MW882928, MW882929, MW882930. Phylogenetic analysis was conducted with combined sequences of the four loci, using the maximum likelihood method and the maximum parsimony method. In the phylogenetic tree, the six isolates and Alternaria brassicae (CBS 116528) clustered together with high bootstrap support values (MLBS=100; MPBS= 100). Based on both morphological characters and phylogenetic results, the isolates were identified as Alternaria brassicae. Pathogenicity test of isolate HEYA2 was carried out on the detached leaves in a dark thermostat incubator at 25°C. Five pots per leaf were inoculated with mycelia plugs (5 mm in diameter), another five pots were inoculated with pure agar plugs and used as the negative control. In addition, conidia suspension (105 conidia/ml) of isolate HEYD8 were sprayed on 3-month-old healthy plants grown in a greenhouse at 22 °C–28 °C. The plants sprayed with sterilized water were used as negative controls. The test was conducted three times. After 5-7 days, the leaves inoculated with other the conidia suspension or the mycelium plugs showed brown necrotic lesions that are similar to the symptoms observed in the field, but the controls remained healthy. The pathogen was reisolated and confirmed to be A. brassicae, completing Koch’s postulates. To our knowledge, this is the first report of leaf spot disease caused by A. brassicae on Orychophragmus violaceus in China.

Isolation and identification of mycorrhizal fungus from an epiphytic orchid (Rhynchostylis retusa L. Bl.)

Isolation and identification of mycorrhizal fungi from the roots of Rhynchostylis retusa indicated that the cultural and microscopic features, namely colony appearance, colony colour, diameter of vegetative hyphae, presence of monilioid cells, right angle branching pattern of the fungal endophyte corroborated the identity of the fungus Rhizoctonia like anamorphs of Ceratobasidium species. Fungal identity was further confirmed through sequencing and analysis of internal transcribed spacer (ITS) sequences of the nuclear ribosomal DNA (nrDNA). ITS sequence of the isolate RhY10A6 (accession no. MN120903.1) showed > 99% similarity with several isolates of the teleomorphic fungus Ceratobasidium sp. in NCBI mega blast search. The phylogenetic analysis based on maximum parsimony method, this orchid mycorrhizal fungus clustered with several isolates of Ceratobasidium sp. or Rhizoctonia like fungi. It showed near distant relation with Ceratobasidium ramicola (GeneBank accession no. NR_138368.1) which is an orchid mycorrhizal fungus. Therefore, molecular characterization validated the morphological data. The techniques established in this orchid will facilitate to isolate and accurate identification of mycorrhizal fungus.

Genetic Diversity of Natural Morchella spp. from the Southern Gansu Province Based on rDNA-ITS, China

Background: China is a mainland country rich in natural Morchella spp. resources. At present, about half of the natural Morchella spp. in the world has been recorded in China. The current study was aimed at the classification and cultivation of Morchella spp. This study provided a more accurate molecular trait for the systematic classification of Morchella spp. in southern Gansu Province, China.Methods: From April to May 2019. Based on the molecular biological analysis of 16 natural Morchella spp. strains collected from the southern Gansu province, China. the ITS fragments between transcripts of the rDNA gene were amplified by PCR and sequenced.Result: The results of BLAST comparison according to GenBank showed that the 16 tested strains of Morchella spp. were classified into 5 species, which were M.angusticeps, M.esculenta, M.elata, M.crapssipes and M.conica. According to the molecular evolutionary trees constructed by the Maximum-Parsimony method (MP) and the Neighbour-Joining method (NJ), the topological structures of the two molecular evolutionary trees were similar and the Bootstrap verification had a high support rate, indicating that the phylogenetic relationship had high credibility.

Molecular Characterization of the Alfalfa mosaic virus Infecting Solanum melongena in Egypt and the Control of Its Deleterious Effects with Melatonin and Salicylic Acid

During the spring of 2019, distinct virus-like symptoms were observed in the Kafr El-Sheikh Governorate in Egypt in naturally infected eggplants. Leaves of affected plants showed interveinal leaf chlorosis, net yellow, chlorotic sectors, mottling, blisters, vein enation, necrotic intervention, and narrowing symptoms. The Alfalfa mosaic virus (AMV) was suspected of to be involved in this disease. Forty plant samples from symptomatic eggplants and 10 leaf samples with no symptoms were collected. The samples were tested by double antibody sandwich ELISA (DAS-ELISA) using AMV-IgG. Six of the 40 symptomatic leaf samples tested positive for AMV, while, DAS-ELISA found no AMV in the 10 leaf samples without symptoms. The AMV Egyptian isolate (AMV-Eggplant-EG) was biologically isolated from the six positive samples tested by DAS-ELISA and from the similar local lesions induced on Chenopodium amaranticolor and then re-inoculated in healthy Solanum melongena as a source of AMV-Eggplant-EG and confirmed by DAS-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) assay with a pair of primers specific for coat protein (CP) encoding RNA 3 of AMV yielded an amplicon of 666 bp from infected plants of Solanum melongena with AMV-Eggplant-EG. The amplified PCR product was cloned and sequenced. Analysis of the AMV-Eggplant-EG sequence revealed 666 nucleotides (nt) of the complete CP gene (translating 221 amino acid (aa) residues). Analysis of phylogeny for nt and deduced aa sequences of the CP gene using the maximum parsimony method clustered AMV-Eggplant-EG in the lineage of Egyptian isolates (shark-EG, mans-EG, CP2-EG, and FRE-EG) with a high bootstrap value of 88% and 92%, respectively. In addition to molecular studies, melatonin (MTL) and salicylic acid (SA) (100 μM) were used to increase the resistance of eggplant to AMV- infection. Foliar spray with MLT and SA caused a significant increase in the morphological criteria (shoot, root length, number of leaves, leaf area, and leaf biomass), chlorophyll and carotenoid content, antioxidant enzymes, and gene expression of some enzymes compared to the infected plants. On the other hand, treatment with MLT and SA reduced the oxidative damage caused by AMV through the reduction of hydrogen peroxide, superoxide anions, hydroxyl radicals, and malondialdehyde. In conclusion, MLT and SA are eco-friendly compounds and can be used as antiviral compounds.

Molecular record for the first authentication of Isaria cicadae from Vietnam

The entomopathogenic fungus T011, parasitizing on nymph of Cicada, collected in the coffee garden in Dak Lak Province, Vietnam, was preliminarily morphologically identified as Isaria cicadae, belonged to order Hypocreales and family Clavicipitaceae. To ensure the authenticity of T011, phylogenetic analysis of the concatenated set of multiple genes including ITS, nrLSU, nrSSU, Rpb1, and Tef1 was applied to support the identification. Genomic DNA was isolated from dried sample T011. The PCR assay sequencing was applied to amplify ITS, nrLSU, nrSSU, Rpb1, and Tef1 gene. For phylogenetic analysis, the concatenated data of both target gens were constructed with MEGAX with a 1,000 replicate bootstrap based on the neighbor-joining, maximum likelihood, maximum parsimony method. As the result, the concatenated data containing 62 sequences belonged to order Hypocreales, families Clavicipitaceae, and 2 outgroup sequences belonged to order Hypocreales, genus Verticillium. The phylogenetic analysis results indicated that T011 was accepted at subclade Cordyceps and significantly formed the monophyletic group with referent Cordyceps cicadae (Telemorph of Isaria cicadae) with high bootstrap value. The phylogenetically analyzed result was strongly supported by our morphological analysis described as the Isaria cicadae. In summary, phylogenetic analyses based on the concatenated dataset were successfully applied to strengthen the identification of T011 as Isaria cicadae.