Exploring the Diversity and Aromatic Hydrocarbon Degrading Potential of Epiphytic Fungi on Hornbeams from Chronically Polluted Areas

Plants can ‘catch’ and mitigate airborne pollutants and are assisted by fungi inhabiting their leaves. The structure and function of the fungal communities inhabiting the phyllosphere of hornbeam trees growing in two chronically polluted areas, the oilfield of Bóbrka and the city center of Warsaw, were compared to the ones growing in one nature reserve, the Białowieża National Park. Fungi were isolated and characterized both phylogenetically and functionally for their potential role in air pollution mitigation. Both culture-dependent (e.g., enzyme assays and tolerance tests) and culture-independent methods (e.g., ITS and shotgun sequencings) were used. Furthermore, the degradation potential of the fungi was assessed by gas chromatography mass spectrometry (GC-MS). Shotgun sequencing showed that the phyllosphere fungal communities were dominated by fungi belonging to the phylum Ascomycota. Aureobasidium was the only genus detected at the three locations with a relative abundance ≥1.0%. Among the cultivated epiphytic fungi from Bóbrka, Fusarium sporotrichioides AT11, Phoma herbarum AT15, and Lophiostoma sp. AT37 showed in vitro aromatic hydrocarbon degradation potential with laccase activities of 1.24, 3.62, and 7.2 μU L−1, respectively, and peroxidase enzymes with activities of 3.46, 2.28, and 7.49 μU L−1, respectively. Furthermore, Fusarium sporotrichioides AT11 and Phoma herbarum AT15 tolerated exposure to airborne naphthalene and benzene. Lophiostoma sp. AT37 was the most tolerant to exposure to these pollutants, in line with being the best potential aromatic hydrocarbon degrader isolated in this study.

First Report of Phoma herbarum Causing Leaf Spot on Rhapis humilis in China

Rhapis humilis Blume is an ornamental plant for landscaping that is widely distributed in China. In February 2020, a leaf spot disease was observed on R. humilis in a nursery shed in Zhanjiang (21.17 N, 110.18 E), Guangdong, China. The disease incidence was more than 90%. The early symptom was small water-soaked lesions, which then turned into black necrotic spots. Eventually, the individual lesions coalesced into larger ones, leading to the death of diseased leaves. Ten diseased leaves were collected from the nursery. The diseased tissues were cut into 2 × 2 mm pieces, surface disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s, and then rinsed three times with sterile water before pathogen isolation. The tissues were plated on potato dextrose agar (PDA) medium and incubated at 28°C in the dark for 4 days. Pure cultures were produced by transferring hyphal tips to new PDA plates. Three isolates (RHPH-1, RHPH-2, and RHPH-3) were obtained. The colonies of the isolates were approximately 5 cm in diameter after 7 days. They were initially whitish and later became grayish white. The NaOH testing on MEA cultures was negative. No sporulation was detected after 30 days. The fertile structures of the specimens collected in the nursery were examined. Pycnidia were globose, measured 68 to 265 × 72 to 360 µm (n = 20), and mostly embedded. Conidia were aseptate, hyaline, and ellipsoid, measuring 3.6 to 6.5 × 2.2 to 2.7 µm (n = 30). Based on the morphological characteristics, the fungus was identified as in genus Phoma (Boerema et al. 2004). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), partial RNA polymerase II largest subunit (RPB2), and beta-tubulin (β-tub) loci of three isolates using primer pairs ITS4/ITS5, RPB2-6F/RPB2-7R, and BT2a/BT2b, respectively (Chen et al, 2015; White et al, 1990). The sequences were deposited in GenBank (ITS, MZ419364-MZ419366; RPB2, MZ562293-MZ562295; and β-tub, MZ562296-MZ562298). Based on BLAST analysis, the sequences of the ITS, RPB2, and β-tub all showed 100% similarity to Phoma herbarum Westend. (CBS 377.92, accession nos. KT389536 for ITS; KT389663 for RPB2; and KT389837 for β-tub). Pathogenicity testing was performed in a greenhouse with 80% relative humidity at 25 to 30°C. Ten healthy plants of R. humilis were grown in pots, with one plant in each pot. The leaves were pinpricked with sterile needles before inoculation. They were inoculated with mycelial plugs of the isolates or sterile agar plugs (as control), with four plugs for each leaf. Five plants were used in each treatment. Disease symptoms similar to those in the nursery were observed on the inoculated plants 2 weeks after inoculation, whereas the control plants remained healthy. The fungus was reisolated from the symptomatic leaves and confirmed as P. herbarum by morphology and ITS analysis. P. herbarum was reported to cause leaf spot on Atractylodes lancea, Camellia sinensis, Elaeis guineensis, Lilium brownii, and Vetiveria zizanioides in China; Bituminaria bituminosa, Glycine max, Medicago sativa, and Pisum sativum in Australia; and Salvia nemorosa in Italy (Li et al. 2011; Li et al. 2012; Thangaraj et al. 2018). To our knowledge, the present study was the first to report P. herbarum causing leaf spot on R. humilis in China. P. herbarum seriously affects the supply of seedlings in R. humilis, and its epidemiology on R. humilis should be further studied.

Assessment of fungi and viruses in Artichoke (Cynara scolymus L.) in Da Lat, Lam Dong province

Artichoke (Cynara scolymus L.), a high economic value crop, was brought to Vietnam by the French in the late 19th century. The artichoke was mainly planted in Lam Dong, Lao Cai, Vinh Phuc provinces, etc. At present, the disease situation of Artichoke plants and the lack of disease-free seedlings result in insufficient source of Artichoke for producers. Artichoke plants are mainly vegetative propagation and pathogens easily transferred from mother to daughter plants. Therefore, low propagation rate and fungal infection are two main factors hindering the expansion and development of Artichoke cultivation (in Lam Dong). Therefore, studying and evaluating the situation of fungi and viruses as well as establishing the in vitro propagation procedures in order to produce high number of disease-free seedlings are urgent for the current Artichoke shortage. In this study, samples of purple and white Artichoke varieties, which suspected fungal manifestations, were collected to assessment of fungi and viruses in artichoke. In addition, in vitro propagation by cultivating apical meristem was applied to produce disease-free seedlings. The recorded results showed that, 19 strains of mold were identified on purple and white Artichoke belonged to nine genera including Mucor (M. sp., M. circinelloides, M. fragilis, M. irregularis, and M. racemosus), Alternaria (Alternaria sp., A. alterinata, A. gaisen, A. tenuissima, and A. tillandsiae), Fusarium (F. acuminatum and F. solani), Cylindrobasidium (Cylindrobasidium sp1 and Cylindrobasidium sp2), Actinomucor elegans, Curvalaria clavata, Plectosphaerella oligotrophica, Phoma herbarum, Rhizomucor variabillis; meanwhile, the Tomato mosaic virus (ToMV) was isolated only purple Artichoke. Shoot explants obtained from apical meristem culture were completely disease-free and used for micropropagation at the next stage.

A HARNESSING THE POTENTIAL OF NOVEL BIOACTIVE COMPOUNDS PRODUCED BY ENDOPHYTIC Phoma spp.: BIOMEDICAL AND AGRICULTURAL APPLICATIONS

Endophytes are those inhabiting in plants without causing any apparent loss to the host plant. Phoma is a ubiquitously found genus of fungi in soil, water and air. Endophytic Phoma spp. are distributed with high specific diversity, those occur in plants and are mainly responsible for the production of a vast range of secondary metabolites. These secondary metabolites or the bioactive compounds have demonstrated a wide range of activity ranging from antibacterial, antifungal, antiviral, algicidal, cytotoxic, antitubercular and plant growth promoting, etc. Bioactive compounds are produced by Phoma herbarum, P. sorghina, P. exigua, P. macrostoma, P. medicaginis, P. betae, P. tropica and others. The present review emphasizes on different species of endophytic Phoma as novel source of bioactive compounds, and their applications in medicine and agriculture are documented.

Swim bladder mycosis in farmed rainbow trout Oncorhynchus mykiss caused by Phoma herbarum and experimental verification of pathogenicity

In this study, spontaneous swim bladder mycosis was documented in a farmed fingerling rainbow trout from a raceway culture system. At necropsy, the gross lesions included a thickened swim bladder wall, and the posterior portion of the swim bladder was enlarged due to massive hyperplasia of muscle. A microscopic wet mount examination of the swim bladder contents revealed abundant septate hyphae, and histopathological examination showed periodic acid-Schiff-positive mycelia in the lumen and wall of the swim bladder. Histopathological examination of the thickened posterior swim bladder revealed muscle hyperplasia with expansion by inflammatory cells. The causative agent was identified as Phoma herbarum through morphological analysis and DNA sequencing. The disease was reproduced in rainbow trout fingerlings using intraperitoneal injection of a spore suspension. Necropsy in dead and moribund fish revealed extensive congestion and haemorrhages in the serosa of visceral organs and in liver and abdominal serosanguinous fluid. Histopathological examination showed severe hepatic congestion, sinusoidal dilatation, Kupffer cell reactivity, leukostasis and degenerative changes. Fungi were disseminated to the liver, pyloric caeca, kidney, spleen and heart. Although infections caused by Phoma spp. have been repeatedly reported in fish, species identification has been hampered by extensive taxonomic changes. The results of this study confirmed the pathogenicity of P. herbarum in salmonids by using a reliably identified strain during experimental fish infection and provides new knowledge regarding the course of infection.