Differentiation of valuable for breeding sunflower lines by resistance to Phoma rot

The purpose of the research is differentiation of valuable for breeding sunflower lines by resistance to Phoma rot. The promising constant sunflower lines were estimated by their resistance to Phoma rot using methods of artificial inoculation in the greenhouse and laboratory conditions. In greenhouse conditions, there were used two methods of artificial inoculation with a fungus Plenodomus lindquistii in the bottom of leaf petiole of a sunflower plant at a stage of the first true leaf pair. Application of the pieces of agar nutrient medium with mycelium, pycnidia and picnospores (unbroken) allowed to identify resistant and mid-resistant sunflower lines (VK 900, L 86 and L 107, L 132). At inoculation with 0.2 ml of a fungus spore suspension (concentration of 1 × 106 spores/ml), five susceptible and one very susceptible sunflower lines (L 2385, L 103, L 136, L 131, L 128, and L 82) were determined. In laboratory conditions, the quick test for estimation of susceptibility to Phoma rot (dose of infection – 12 g) allowed to reveal more than 50 % of plants with healthy root system and two constant sunflower lines L 116 and L 136 with the affection degree of one point. Estimation of susceptibility to Phoma rot infection of the different organs (stem and root system) at artificial inoculation showed a possibility to differentiate sunflower lines by resistance of roots and stems and to select the most promising for the further work in breeding.

High-pressure thermal sterilization and ε-polylysine synergistically inactivates Bacillus subtilis spores by damaging its inner membrane

The study aimed to determine the sterilization effect of a combination of high-pressure thermal sterilization (HPTS) and ε-polylysine (PL) on Bacillus subtilis spores. The spores were treated with HPTS (550 MPa at 25°C, 65°C and 75°C) combining with ε-PL at 0.1% and 0.3%. The results showed that HPTS and ε-PL synergistically inactivated the spores. The increased temperature and ε-PL concentration decreased the number of surviving spores, with the maximal inactivation of the spores in the treatment of 550 MPa, 75°C combining with 0.3% ε-PL. The increases in the temperature and ε-PL concentration significantly increased the release of the intracellular components in the spore suspension, with the maximal value for the spores treated with 550 MPa, 75°C and 0.3% ε-PL. The maximal fluidity and permeability of the cell inner membrane were observed in the treatment of 550 MPa, 75°C combining with 0.3% ε-PL. Changes in membrane lipids were detected from 3000 to 2800 cm -1 by Fourier transform infrared spectroscopy. The results may provide new insights into the mechanism by which HPTS and ε-PL synergistically sterilize Bacillus subtilis spores.

First report of a new disease caused by Fusarium tricinctum on apple tree in China

Wilting of branches and leaves was observed on 4-5 year old apple trees of the varieties Delicious and Fuji in orchards located in Wushan, Gansu Province, China in April 2018. Subsequently, the stem vascular tissue and woody xylem became discolored and necrotic. The stem dieback expanded rapidly to the entire vasculature of the branches. Finally, the epidermis of the stem bases split and was covered with light pink mold. For the pathogen isolation, 25 symptomatic stems were collected from 25 symptomatic trees in 3 individual orchards. Fragments (approximately 0.5 cm in length × 0.5 cm in width) of symptomatic stems were surface sterilized and individually transferred to Petri dishes containing potato dextrose agar (PDA), and incubated for 4 days at 25°C. Five types of isolates with distinct morphological characteristics (PJ1 to PJ5) were obtained from the 25 symptomatic stems after the single spore inoculation and sub-culture. The isolation frequency of PJ1, PJ2, PJ3, PJ4 and PJ5 types was 11%, 8%, 100%, 4% and 13%, respectively, in the 25 symptomatic stems. A spore suspension of PJ1, PJ2, PJ3, PJ4 and PJ5 types was prepared by adding 5 ml of sterile distilled water in the 14-day old culture colonies and filtered through 0.22 mm Millipore membranes, and the final concentration was adjusted to 108 per ml for inoculation. Detached healthy apple stems (15 cm in length) were surface-disinfested and used to evaluate the pathogenicity of PJ1 (7 isolates), PJ2 (5 isolates), PJ3 (32 isolates), PJ4 (2 isolates) and PJ5 (9 isolates) by dipping the stems into sterilised tubs containing the spore suspension (108 per ml) of each isolate. Apple stems dipped in sterile distilled water served as the control. Each control and treatment were repeated 3 times. At day 15 and 35, the stems infected with the spore suspension of PJ3 isolates developed symptoms that were similar to those observed in the apple orchards. However, the other four types (PJ1, PJ2, PJ4 and PJ5) exhibited either no symptoms or different symptoms from those observed in the apple orchards. There were no symptoms on the control stems. After the colony of the pathogen (PJ3 type) was re-isolated from the infected stem bases 35 days inoculation. The PJ3 type isolates with same morphological characteristics as the original PJ3 type isolates were used for further examination and identification. After 4 days of incubation on PDA, the colonies of PJ3 type isolates developed velvety aerial mycelia with white or light pink color when they were viewed from the front/top side of the PDA and orange-red color when they were viewed from the reverse/bottom side. After 14 days of incubation, the color in the centre of the colonies changed to yellow green in the front view and carmine red in the reverse view of the plates. Three types of spores (microconidia, macroconidia and chlamydospores) were observed after incubation of PJ3 type isolates for 14 days. The size (width and length) of 30 conidia in each of PJ3 type isolates was measured and averaged. The microconidia were abundant on aerial mycelia and limoniform, oval or pyriform with 0-1 septa. Their size ranged from 1.94 μm to 8.05 μm in length and 1.48 μm to 3.62 μm in width. The macroconidia were falciform and curved in shape, mostly with 3-5 septa and a size ranging from 13.52 μm to 22.43 μm in length and 2.31 μm to 3.87 μm in width. The chlamydospores were spherical, intercalary and formed in chains on PDA plates. These morphological characteristics indicate that the PJ3 type isolates could be Fusarium tricinctum (Chen et al. 2019; Aktaruzzaman et al. 2018). To confirm the morphological identification, the sequences of internal transcribed spacer (ITS), transcriptional enhancer factor-1 (TEF-lα) and ribosomal RNA large subunit gene (LSU) of the representative isolate PJ3-3 selected from the PJ3 type isolates with same morphological characteristics were sequenced and used for molecular identification (Laurence et al. 2011; Abd-Elsalam et al. 2003; Miller et al. 1996). The sequences of ITS, TEF-lα and LSU of the PJ3-3 isolate were deposited in NCBI database with the accession numbers of MZ799356, MZ820045 and MZ820044, respectively. In BLAST analyses, the obtained sequences of the PJ3-3 isolate showed 99.47%, 100% and 99.01% identity to the corresponding region of F. tricinctum ITS (JX179207.1: 3-566 Fusarium tricinctum isolate Fyx 1), TEF-lα (MK032320.1 F. tricinctum isolate ZD3) and LSU (KC311496.1 Fusarium tricinctum isolate L12), respectively. The phylogenetic analysis clustered the PJ3-3 isolate sequences within the same clade with ITS, TEF-lα and LSU sequences of F. tricinctum isolates. Thus, the PJ3-3 isolate was identified as F. tricinctum based on the pathogenicity tests, morphological characteristics and molecular analyses. Previously, the symptoms of xylem browning and dieback were observed in the twigs of wild apple trees that were collected from wild apple forests, and the species F. avenaceum, F. solani, F. tricinctum, F. proliferatum, and F. sporotrichioides were isolated from diseased wild apple trees (Chen et al. 2019). Only F. avenaceum, F. solani, F. proliferatum, and F. sporotrichioides were reported as the pathogens causing the disease symptoms of xylem browning and dieback in wild apple trees in Xinjiang, China (Chen et al. 2019). In our present study, we found that F. tricinctum can cause stem vascular and woody xylem browning, wilting, and dieback in the apple tree varieties Delicious and Fuji. These are new symptoms discovered in our present research and different from the previous paper (Chen et al. 2019). Therefore, to our knowledge, this study is the first report of F. tricinctum causing a new disease on apple trees in China following Koch’s postulates. Our findings are important for the management of apple disease and protect apple trees in the future.

First Report of Leaf Blight Caused by Nigrospora oryzae on Poplar in China

Populus alba L. × P. berolinensis Dipp. (a hybrid poplar, ‘PaPb poplar’) exhibits fast growth and beautiful tree shape with high drought and cold tolerance, and is widely planted in the cities of Northeast China because of its ornamental and ecological value (Wang et al. 2008). In October 2020, an unknown leaf blight symptom was observed on the seedlings of ‘PaPb poplar’ at Shenyang Agricultural University (41°49′N, 123°34′E) located in Shenyang City, Liaoning Province, China. The disease incidence was 50% in a survey of 200 seedlings on the campus. The typical symptoms were brown-to-black, irregular-shaped lesions (Fig. 1A). To investigate the disease, five symptomatic leaves were collected, and pieces were cut at the margin of diseased and healthy tissue. These pieces were surface sterilized with 2% sodium hypochlorite for 2 min, rinsed three times with sterile distilled water, air dried, placed on potato dextrose agar (PDA) and incubated at 28°C. After 5 days of incubation, three isolates with similar morphological characteristics were observed. Isolate N03 was chosen and used for pathogen identification. The fungal colonies were initially white in color, and later turned gray to black (Fig. 1D). Conidia were single-celled, black, spherical or oblate in shape measuring 9.19 to 12.78 μm × 12.61 to 14.81 μm in diameter (n=40) (Fig. 1E). These were borne on hyaline vesicles at the tip of a conidiophore. Morphologically, the isolate N03 was identified as Nigrospora oryzae (Berk. and Broome) Petch (Wang et al. 2017). The genomic DNA was extracted with a SP Fungal DNA Kit (D5542-01, OMEGA). The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1-α), and partial beta-tubulin (TUB) genes were amplified using the primers ITS5/ITS4 (White et al. 1990), EF1-728F/EF-2R (Carbone and Kohn, 1999; O’Donnell et al. 1998), and Bt-2a/Bt-2b (Glass and Donaldson, 1995) primer sets, respectively. The PCR products of ITS, TEF1-α, and TUB were amplified, sequenced, and deposited in GenBank with the following accession numbers MZ148528, MZ182080, and MZ182079, respectively. BLASTn analysis of the ITS, TEF1-α, and TUB sequences had 99.3%, 99.8%, and 99.27% nucleotide identities to MK131325, KY019328, and KY019559, respectively. A phylogenetic tree based on combined ITS, TEF1-α, and TUB sequences was constructed using a Maximum Likelihood method with 1000 bootstraps showing that N03 was grouped with other N. oryzae isolates (Fig. 2). The fungus was identified as N. oryzae based on morphological characteristics and molecular analyses. Koch’s postulates were completed to confirm the pathogenity of N. oryzae on ‘PaPb poplar’. The N03 spore suspension (105 spores/mL) was used to inoculate detached leaves and field leaves in two experiments. The two experiments were repeated three times, respectively. In the detached leaf test, 10 healthy leaves collected from 1-year-old ‘PaPb poplar’ seedlings were inoculated with N03 by spraying with the spore suspension followed by incubation at 28°C on wet filter papers in a petri dish for 7 days. 10 leaves were sprayed with sterile water to save as the controls. For field leaf test, leaves of 5 plants were spray-inoculated with the spore suspension at the 4-week-old growth stage, and an additional 5 plants were sprayed with sterile water. Seven days after inoculation, brown-to-black, irregular-shaped lesions on the margin of leaves were observed on inoculated leaves but not on the controls (Fig. 1B and C). All detached leaves inoculated with N03 were symptomatic. In the field tests, symptom appeared on 20 of the 30 inoculated leaves. N. oryzae was re-isolated from all the inoculated detached leaves and inoculated plants, but not from the controls. N. oryzae is a known pathogen of several hosts, such as Costus speciosus (Koen.) Sm. and Mentha spicata L., but has not been reported on any species of Populus. To our knowledge, this is the first report of leaf blight of ‘PaPb poplar’ caused by N. oryzae in China and the world. This disease could affect growth and development of ‘PaPb poplar’ seedlings, and may cause economic losses in the future. Appropriate strategies should be developed to manage this disease.

DETERMINATION OF FUNGI RESISTANCE OF SEVERAL SAMPLES OF NATURAL LEATHER IN CONDITIONS OF ARMENIA

In this work we studied the effect of microscopic soil fungi on some lather samples. The species composition of micromycetes inhabiting the samples under study was revealed, and an assessment of the fungal resistance of the materials under study was given. In the process of work, species of microscopic soil fungi were isolated and identified from the soil. In order to determine the degree of resistance to fungi for the infection of samples, a water-spore suspension was obtained. The leather materials were partially destroyed by microscopic fungi, and mold resistance ranged from 2 to 3 on a 5-point scale.

First Report of Gray Leaf Spot Caused by Pyricularia oryzae (synonym: Magnaporthe oryzae) in Oat (Avena sativa) in Georgia, USA.

In southeastern U.S., oat (Avena sativa L.) is predominantly grown as a grain or forage crop due to its exceptional palatability (Buntin et al. 2009). In November 2020, leaf spot symptoms were observed in an oat field (cv. Horizon 720) in Screven County, Georgia (GPS: 32°38'57.6"N 81°31'32.178"W). Lesions were oblong, whitish to gray in color, and surrounded by dark brown borders. Symptomatic oat leaves were sampled from the field and cut into 1 cm2 sections that were surface sterilized, plated onto Potato Dextrose Agar (PDA) media and incubated in the dark at 23°C. To obtain pure cultures, fungal hyphal tips were transferred onto fresh PDA plates 3 times. The pathogen was identified as Pyricularia (Magnaporthe) based on typical conidial morphology (Ellis 1971). Conidia were hyaline, pyriform, 2-septate, and displayed a basal hilum. Conidia measured 5.32 to 10.64 μm (average 8.24 μm) wide by 15.96 to 29.26 μm (average 25.40 μm) long. The identification of Pyricularia was further confirmed genetically via PCR amplification followed by sequencing. Genomic DNA was extracted from a 14-day old pure culture using a CTAB method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (CaM) gene, and -tubulin (TUB) gene were amplified using ITS5-ITS4 (White et al. 1990), CMD5-CMD6 (Hong et al. 2005), and Bt2a- Bt2b (Glass and Donaldson 1995) primer sets, respectively. Amplicons were Sanger sequenced and blasted against the NCBI database. Results exhibited 100% (ITS), 100% (CaM), and 99.61% (TUB) homology with Pyricularia oryzae Cavara (GenBank accession no. LC554423.1, CP050920.1, and CP050924.1, respectively). The ITS, CaM, and TUB sequences of the isolate were deposited in GenBank as MZ295207, MZ342893, and MZ342894, respectively. In a greenhouse (23°C, 80% RH), Koch’s postulates were carried out by using oat seedlings cv. Horizon 270 grown in Kord sheet pots filled with Sun Gro professional growing mix, and a P. oryzae spore suspension containing 104 conidia ml−1. The spore suspension (10 ml) was sprayed with an air sprayer onto 7 pots of oat seedlings at the two-leaf stage. Seven supplementary pots of oat seedlings of the same cultivar were sprayed with sterile water to act as controls. After inoculation, plants were covered with black plastic bags that had been sprayed with sterile water to maintain high humidity and incubated overnight in the greenhouse. The bags were removed the next day, and plants were evaluated for symptoms in the following days. Seven days after inoculation, plants displayed symptoms similar to those found in the original field sample. Control plants showed no symptoms. Pyricularia oryzae was consistently re-isolated from inoculated symptomatic oat tissues. To our knowledge, this is the first report of gray leaf spot caused by P. oryzae on oat in the state of Georgia and in the continental United States. Pyricularia oryzae can infect several graminaceous plants, including agronomically important crops such as rice (Oryza sativa) and wheat (Triticum spp.) (Chung et al. 2020). Phylogenetic analysis on the ITS region using 6 different host lineages was performed and revealed that this oat isolate was most closely related to the Lolium lineage. This outbreak could have economic implications in oat production.

First Report of Anthracnose on Camellia japonica Caused by Colletotrichum siamense in Zhejiang Province, China

Camellia japonica is an attractive flowering woody plant with great ornamental and medicinal value in China. However, typical anthracnose lesions on the leaves are usually observed in summer in Zhejiang province. A number of 100 trees have been investigated with over 70% of leaf disease incidence. The symptom initially develops from the tip or edge of the leaf and dark green infected spots appear. The diseased spots expand and become yellow brown. The lesions are covered with abundant, small and black acervuli at the center with yellow edges. The diseased leaves become brittle, cracked, and finally fall off. Sixty leaves with typical anthracnose symptoms were sampled from gardens in Lin’an, Zhejiang province. The diseased tissues were cut into pieces and incubated in moist chambers at 25°C. The spore mass was collected using a sterile needle under dissection microscope and put on 2% malt extract agar (MEA). The cultures were incubated at 25°C in the dark for one week. Thirty single spore cultures were obtained and grown on 2% MEA at 25°C for morphological characterization. White aerial mycelia and black conidiomata with orange masses of conidia developed seven days later. Conidia are cylindrical in shape, 12-19 μm, mean lengths ranging from 15.5 ± 1.0 to 16.0 ± 1.2 μm. The morphological characteristics are consistent with those of Colletotrichum species. DNA was extracted from three selected isolates (HT-71, J-5, J-20) for sequencing. The partial regions of ribosomal internal transcribed spacers (ITS), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin gene (ACT), beta-tubulin (TUB2), Apn2-Mat1-2 intergenic spacer and partial mating type gene (ApMat), and glutamine synthetase (GS) were amplified as described by Liu et al. (2015). Sequences of the above seven loci for the selected isolates were obtained, and deposited in the GenBank database (MZ014901 to MZ014905, MZ514915 to MZ514922, MZ514925 to MZ514930, MZ497332 and MZ497333). BLAST results indicate they represent Colletotricum siamense. Multi-locus phylogenetic analysis including ex-type of C. siamense (ICMP18578=CBS130417) and related species was conducted using Maximum Likelihood method, and C. acutatum (CBS 112996) served as the outgroup. The three obtained isolates clustered with the ex-type isolate of C. siamense. Eight leaves on two Camellia plants were inoculated to confirm the pathogenicity in the field. The leaves were surface sprayed with 75% ethanol and dried with sterilized filter paper. The leaves were inoculated using the wound/drop inoculation method: an aliquot of 10 μL of spore suspension (1.0 × 106 conidia per mL) was dropped on the left side of a leaf after wounding once by pin-pricking with a sterilized needle. The sterile water was dropped on the right side of the same leaf in parallel as control. The initial symptoms were observed seven days later, all inoculated leaves developed lesions similar to those observed in the field, and no symptoms observed in the control. The fungus was successfully re-isolated only from lesions inoculated with spore suspension exhibiting morphological characteristics resembling those in C. siamense, and further confirmed with sequence data. To our knowledge, this represents the first report of anthracnose on C. japonica caused by C. siamense worldwide. Confirmation of this pathogen in the region will be helpful for the disease management on C. japonica, considering previous report of C. camelliae-japonicae on the same host. References Fu, M., et al. 2019. Persoonia. 42: 1. https://doi.org/10.3767/persoonia.2019.42.01 Guarnaccia, V., et al. 2017. Persoonia. 39: 32. https://doi.org/10.3767/persoonia.2017.39.02 Hou, L. W., et al. 2016. Mycosphere. 7: 1111. Doi 10.5943/mycosphere/si/2c/4 Liu, F., et al. 2015. Persoonia. 35: 63. http://dx.doi.org/10.3767/003158515X687597 Vieira, A. D. S., et al. 2019. Mol. Phylogenet. Evol. https://doi.org/10.1016/j.ympev.2019.106694.

Fusarium pallidoroseum: A potential entomopathogenic agent for the biological management of Aphis gossypii

With rising need of switching over to sustainable agricultural practices, utilization of entomopathogenic fungi (EPF) as biocontrol agents, provides better substitute against chemical pesticides- having several side-effects. Therefore, an attempt have been made to explore the potential EPF fungi that could be incorporated into IPM practices for control of Helicoverpa armigera Hubner and Aphis gossypii Glover. Regarding this, an entomopathogenic fungus, Fusarium pallidoroseum (Cooke) Sacc, was isolated from natural population of H. armigera infesting chickpea (Cicer arietinum L.) and explored efficacy under in-vitro & field conditions. The findings of present investigation shows efficacy of F. pallidoroseum as potential biocontrol agent against okra aphid (A. gossypii), as it inflicted initial mortality of 43.33% nymphs on 2nd day and lead to complete annihilation (93.33%) of nymph population on 8th day of spore suspension application at 1x1010 spores/mL concentration. The observations against adult okra aphid clearly demonstrated that spraying of 1 x 1010 spores/mL of F. pallidoroseum resulted 66.67% mortality after 8th days of spraying. Increased mortality was recorded with increase in spore suspension concentrations. The LC50 & LC90 value for F. pallidoroseum against nymphs of A. gossypii was recorded 3.79 x 105 and 2.74 x 108, respectively. The findings were used to develop formulations (1 × 104 to 1 × 1010 spore suspension/mL conc), and tested at field-level. The results showed that formulation at 1 x 1010 spores/mL conc was most effective against A. gossypii, recorded 93.33% mortality of nymphs & 66.67% mortality of adults; could be used under IPM practices.

First Report of Cobweb Disease in Oudemansiella raphanipes Caused by Cladobotryum varium in Beijing, China

Oudemansiella raphanipes is an edible mushroom with medicinal properties,which has been recently cultivated throughout China (Hao et al. 2016). In October 2019, a disease with symptoms similar to that of cobweb disease (Carrasco et al. 2017) was observed in O. raphanipes in the Tongzhou District, Beijing, China, infecting 25% of the fruiting bodies (Fig. 1A, B). White cotton-like net of hyphae were present typically on the casing soil or on the stipe of the fruiting bodies; they gradually spread to the pileus, covering the fruiting body, which eventually wilted and died (Fig. 1C, D), resulting in yield reduction and economic loss. Cultures were obtained by aseptically transferring the diseased fruiting bodies onto potato dextrose agar (PDA) at 25 °C; they were deposited in the culture collection (ID: JZBQA1) of the Beijing Academy of Agricultural and Forestry Sciences, China. The colonies were pale white/white, with an occasional formation of yellow diffusing pigments on the reverse side (Fig. 1E–G). Conidiophores were Cladobotryum-like, phialides were solitary or commonly divergent in whorls of 2–3 (–4), lageniform to subulate, 20–63.5 (–66) × (3.8–) 4–5.3 (–9) μm (n = 40) (Fig. 1H, I); conidia were hyaline, oval to ellipsoidal, with one or two septa, (10.4–) 11.4–20 (–22) × 6.6–9.5 (–10) μm (n = 40) (Fig. 1J); chlamydospores were globose or ellipsoidal (Fig. 1K). The morphological characteristics were consistent with that of Cladobotryum varium (Back et al. 2012a, b; Sun et al. 2019). For species-level fungal identification, genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, USA). The internal transcribed spacer (ITS) regions, translation elongation factor 1 alpha exon (TEF1-α), RNA polymerase II subunit b (RPB2), and RNA polymerase I largest subunit (RPB1) genes were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-983F/2218R (Rehner and Buckley 2005), RPB2-5F/7cR (Liu et al. 1999), and RPB1F1 (5'-GCCGATGAAGTTGGTCTA-3')/RPB1R1 (5'-TATGTTGCGGTGAGCCTT-3'), respectively. A BLAST nucleotide search showed 99.34% (449/452 bp), 99.24% (914/921 bp), 98.08% (1,022/1,042 bp), and 99.66% (588/590 bp) homology, respectively, with those of the ex-type culture of Hypomyces aurantius TFC 95-171 (FN859425.1, FN868743.1, FN868679.1, and FN868805.1). The four sequences were deposited in GenBank (accession numbers: MW534093, MW560066, MW560064, and MW560065). Phylogenetic trees based on the assessed gene loci revealed that the JZBQA1 strain was closely related to C. varium (Fig. 2). A in vivo pathogenicity test was performed using the fruiting bodies (Fig. 1L, O). Spore suspension (108 spores/mL) of the JZBQA1 strain or sterile distilled water was sprayed on six healthy fruiting bodies, maintained in an artificial climate chamber at 24-26°C. Cobweb-like features were observed on the fruiting bodies treated with the spore suspension 2-3 days post-inoculation; while those treated with water did not exhibit such features (Fig. 1L, O). The same pathogen was re-isolated and confirmed from the infected fruiting bodies by integrated analysis of morphological characteristics and gene sequencing data. Cladobotryum spp. infects different varieties of cultivated edible mushrooms, resulting in the development of cobweb diseases (Cao et al. 2020; Carrasco et al. 2017). Cladobotryum varium is the causal agent of cobweb disease in Flammulina velutipes and Hypsizygus marmoreus (Back et al. 2012a, b). To our knowledge, this is the first report of cobweb disease caused by C. varium in O. raphanipes. This finding is a valuable contribution to the knowledge of cobweb disease development in edible fungi.

Optimization of Mycoremediation Potential of A Fungi: Aspergillus Ochraceus Strain

Mycoremediation is an important process that targets the removal of petroleum hydrocarbons by fungi. Accordingly, colorimetric method was used in the preliminary investigation of petroleum degradation with ten fungal strains as Aspergillus ochraceus, Aspergillus parasiticus, Aspergillus niger, Fusarium acuminatum, Fusarium graminearum, Fusarium equiseti, Fusarium oxysporum, Paecilomyces lilac, Penicillium crustosum, and Penicillium chrysogenum. Petroleum degradation of spore suspension, live biomass (fungal pellet and disc) and cell-free culture supernatant of the potent A. ochraceus strain were determined by gravimetric analysis. It was obtained that the fungal disc (94%) was more successful than the spore suspension (87%) in petroleum degradation under optimized conditions as pH:5.0, 1% of petroleum concentration, %5 (v/v) of inocum concentration, 1 g/100mL of inoculum amount and 7 days of incubation period. The degradation rate constant and half-life period of spore suspension were calculated as 0.291 day− 1 and t1/2 = 0.340 and of fungal disc were 0.401 day− 1 and t1/2 = 0.247. 7.5% and 10% (v/v) concentration of cell-free culture supernatant were achieved more than 80% of petroleum removal. However, the cell-free culture supernatant was not as effective as fungal disc. According to GC/MS analysis, the fungal disc of A. ochraceus strain degraded long chain n-alkanes such as C35 and C36 more effectively than n-alkanes in the range of C22-C34. Drop-collapse and oil-spreading methods showed that A. ochraceus is a good biosurfactant producer. This study clearly pointed out that Aspergillus ochraceus NRRL 3174 strain with high its removal capacity can be used as an effective agent in petroleum bioremediation process.