Invasive cardiac aspergillosis as contiguous infiltration from mediastinum: Imaging evaluation using cardiac MRI

Invasive cardiac aspergillosis is a rare, potentially fatal disease which poses a significant diagnostic challenge. Combination of clinical details, imaging features, fungal markers, serology and fungal isolation with demonstration of invasion is usually necessary to establish the diagnosis. High index of suspicion is the key for early diagnosis with potential role of cardiac MRI in its early detection, delineation of extent of involvement and guidance to the appropriate site for tissue sampling, thereby allowing for improved prognosis with early institution of appropriate therapy.

Detection of the Phoma pathogens Plenodomus biglobosus subclades ‘brassicae’ and ‘canadensis’ on wasabi, and ‘canadensis’ in Europe

Phoma stem canker / blackleg is an internationally important disease of Brassicas including B. napus (oilseed rape, OSR), caused by multiple genetic subclades of the fungi Plenodomus lingam (formerly Leptosphaeria maculans) and P. biglobosus (L. biglobosa). In Spring 2021, Phoma-like disease symptoms were observed on leaves and stems of Eutrema japonicum (wasabi) crops at three UK sites (Northern Ireland, Southern England and the West Midlands). Fungal isolation from wasabi leaf spots yielded colonies with two distinct phenotypes on potato dextrose agar (PDA). Isolates from the Northern Ireland and Southern England sites had white colonies with abundant pink cirri that were confirmed (based on ITS rDNA, beta tubulin and actin sequences) as P. biglobosus subclade ‘canadensis’ (Pbc). Those from the West Midlands site, however, had yellow pigmented colonies and were confirmed by sequencing as P. biglobosus subclade ‘brassicae’ (Pbb). Greenhouse pathogenicity testing showed that Pbb and Pbc wasabi isolates were pathogenic not only to this host but also OSR, B. oleracea (cabbage), and B. rapa (pak choi). Re-isolation of the fungi was attempted and confirmed from lesions that developed on inoculated OSR and wasabi, thus completing Koch’s postulates. These findings represent new discoveries for both Pbb and Pbc on wasabi, plus for Pbc in Europe. The crop health implications of these results are briefly considered.

New record of Gymnoascus udagawae associated with Clathria sp. sponge from Indonesia and the potency as anti-Candida

Marine sponges are associated with marine fungi. The associated fungi produce secondary metabolites for sponge survival in extreme habitats. Despite the important role of the associated fungi on their host, the research on marine fungi however has not been studied well. This study aimed to isolate sponge-associated marine fungi and analyze the potency of fungal secondary metabolites against Candida albicans. The sponge used was Clathria sp. collected from Pramuka Island, Indonesia. Fungal isolation used the direct inoculation method. Fungal identification was done by morphological and molecular characteristics of ITS rDNA region. Detection of anti-Candida used the well diffusion method. The isolate has typical morphological characteristics of the genus Gymnoascus with noduled chlamydospore and arthroconidia. The isolated fungus was identified as Gymnoascus udagawae based on morphological and molecular analysis. This is the first record of marine fungi G. udagawae from Indonesia. The ethyl acetate extract of fungal filtrate showed 1.4 cm inhibition diameter of 500 mg/μL extract. The inhibition is moderate category compared to that of clotrimazole a drug commonly used for candidiasis as the positive control with showed 2.8 cm inhibition diameter at 100 mg/μL. The fungus is a potential source of the secondary metabolite active against C. albicans.

Flower endophytic fungi of Geodorum densiflorum endangered orchid

Geodorum densiflorum is an ornamental and medicinally important orchid. The medicinal metabolites are produced by endophytic fungi associated with orchid tissue, particularly flowers. However, there is no report of the endophytic fungi from the G. densiflorum flowers. This research aimed to study the endophytic fungi from different parts of G. densiflorum flower. Fungal isolation was carried out from sepal, petal, labium, stigma, and anther using surface sterilization method. The fungi were identified by combining morphological and molecular characteristics of ITS rDNA region. The results showed that each flower organ had different species of endophytic fungi. A total of seven species of endophytic fungi were obtained; four species were successfully identified by molecular identification and three species based on morphology. The four species, namely Hypomontagnella barbarensis, Aspergillus oryzae, Curvularia pseudobranchyspora, and Nigrospora chinensis, while the three species, namely Gonatobotrys sp., Humicola sp., Aspergillus section Nigri. The labium inhabited by Curvularia pseudobranchyspora, Nigrospora chinensis, Aspergillus section Nigri. Three species isolated from petals, namely Hypomontagnella barbarensis, Gonatobotrys sp., and Aspergillus oryzae. The sepal is inhabited by Humicola sp. There were no endophytic fungi in stigma and anther. This indicates that each flower part is a unique habitat of endophytic fungi.

First report of dragon fruit (Hylocereus undatus) stem rot caused by Diaporthe ueckerae in Taiwan

Dragon fruit (Hylocereus polyrhizus & H. undatus) is a rapidly growing commodity in Taiwan. The production acreage has been tripled since 2011, with an estimation of over 2,800 ha in 2019. From disease survey conducted in July 2020, reddish orange to blackish brown lesions similar to stem canker caused by Neoscytalidium dimidiatum on dragon fruit cladodes (Supplementary Fig. S1, Q) were observed from two orchards in Central Taiwan. Diseased cladodes were brought back to the lab, surface disinfested with 70% ethanol for 15 to 30 sec, and then blotted dried with a paper towel. Small pieces (about 3x3 mm) of necrotic spots were excised, placed on 2% water agar (WA) plates, and incubated with 12 h photoperiod at 28 ± 2 ℃ for 3 days. Among the necrotic spots that were used for fungal isolation, some were detected to have N. dimidiatum accounting for 21 isolates, while three isolates detected in other spots were unknown. Single hyphal tips of the three unknown fungal colonies with similar morphology were transferred on potato dextrose agar (PDA). Brownish- to grayish-white colonies with fluffy aerial mycelium were observed on PDA (Supplementary Fig. S1, A, B, E, F, I and J) after 8 days of incubation. To induce the sporulation, all the fungal isolates were cultivated on autoclaved cowpea pods on 2% WA plates with 12 h photoperiod at 25 ± 2 ℃ for 3 weeks. Black pycnidia embedded in cowpea tissues and creamy yellowish exudates with pycnidiospores extruding from the ostiole were observed (Supplementary Fig. S1, C, G and K). Alpha-conidia were characterized as aseptate, hyaline, smooth, ellipsoidal or fusiform, often bi-guttulate and measured about 6.0 to 6.5 μm × 2.0 to 2.3 μm (n = 50 for each isolate) (Supplementary Fig. S1, D, H and L). Beta-conidia were not observed. Morphological characteristics of these isolates were similar to Diaporthe spp. described by Udayanga et al. (2015). To further identify the fungal isolates, the internal transcribed spacer (ITS), β-tubulin (TUB) and translation elongation factor 1-α (EF1-α) regions were amplified using primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass & Donaldson 1995) and EF1-728F/EF1-986R (Carbone & Kohn 1999), respectively. BLAST analysis of isolates CH0720-010 (ITS: OK067377; TUB: OK149767; EF1-α: OK149764), CH0720-013 (ITS: OK067378; TUB: OK149768; EF1-α: OK149765) and TC0720-016 (ITS: OK067379; TUB: OK149769; EF1-α: OK149766) showed 99.78 to 100% of ITS identity, 98.8 to 99.2% of TUB identity, and 100% of EF1-α identity with Diaporthe ueckerae (ITS: KY565426; TUB: KY569384; EF1-α: KY569388). Phylogenetic trees were constructed using concatenated ITS, TUB, and EF1-α sequences based on maximum likelihood with HKY+G model, maximum parsimony, and Bayesian inference method in MEGA X and Geneious Prime 2020.2.4. All isolates were clustered in D. ueckerae with similar topology based on aforementioned methods, hence the phylogram of maximum likelihood was presented (Supplementary Fig. S2). To confirm the pathogenicity, detached dragon fruit (H. polyrhizus and H. undatus) cladodes (20 to 30 cm in length) were surface disinfested, wounded with sterilized syringe (about 2 mm in depth), and inoculated with mycelial plugs (6 mm in diam.) from 5-day-old colonies on PDA. Each isolate had three mycelial plugs and the PDA plugs without mycelium were inoculated as negative control. Inoculated cladodes were placed in a moisture chamber and incubated at 30 ± 2 ℃ with 12 h photoperiod. Two days after inoculation (DAI), the agar plugs were removed and symptom development on the cladodes was photo recorded every other day. The inoculation experiment was repeated twice. At 6 DAI, round to irregular, dark-brown, and water-soaking lesions were observed on the cladodes of both species inoculated with the three D. ueckerae isolates whereas all negative controls remained asymptomatic (Supplementary Fig. S1, M-P). Morphologically identical fungi were re-isolated from inoculated cladodes, fulfilling Koch’s postulates. Several Diaporthe species have been reported infecting dragon fruit in the southeastern Asian countries such as Thailand, Bangladesh and Malaysia (Udayanga et al. 2012; Karim et al. 2019; Huda-Shakirah et al. 2021). To our knowledge, this is the first report of stem rot caused by D. ueckerae in Taiwan. Since the field symptoms may be easily confused with those caused by N. dimidiatum, the potential threat of Diaporthe species complex on dragon fruit should be aware and may warrant further study.

First report of root rot caused by Fusarium armeniacum on soybean in Korea

Fusarium wilt samples were collected in 2017 and 2019 from two soybean (Glycine max) fields, Yesan (36°73′N, 126°81′E) and Gimje (35°76′N, 126°80′E), in Korea. The disease incidence rate in each field was approximately 1%. For fungal isolation, root lesion fragments were surface-sterilized in 1% sodium hypochlorite for 2 min, rinsed thrice with sterile distilled water, and then incubated on water agar (WA) plates at 28 °C in an incubator for 5 days. Two isolates (YS37231 and GJ3050) were obtained using the hyphal tip method. Colonies of the isolates on potato dextrose agar (PDA) produced white aerial mycelia, which later turned pinkish yellow. The isolates on PDA formed abundant chlamydospores and macroconidia, but microconidia were absent. Macroconidia were 3–5 septate and prominently curved, measuring 12.4 to 41.2 × 3.3 to 4.3 µm (Leslie and Summerell, 2006). For the identification of the isolates, translation elongation factor 1 alpha (EF-1α) and RNA polymerase second largest subunit (RPB2) regions were amplified and sequenced using EF1, EF2, RPB2-5f2, and RPB2-7cr primers, respectively (O’Donnell et al. 2010). EF-1α sequences of YS37231 (MT445439) and GJ3050 (MT445440) showed 99.01 and 99.67% identity with F. armeniacum (FD_01843 and FD_01305; FUSARIUM-ID database), respectively. The RPB sequences of YS37231 (MT445442) and GJ3050 (MT445441) showed 100 and 98.48% identity with that of F. armeniacum (FD_01869; FUSARIUM-ID database), respectively. The sequences MT445439, MT445440, MT445441, and MT445442 were deposited in NCBI GenBank. The pathogenicity of the two isolates on the soybean cultivar Daewonkong was determined using two inoculation methods. In the first method, a pathogenicity assay was performed on seedlings using WA plates (Cruz Jimenez et al. 2018). Eight surface-sterilized seeds were transferred to WA plates, with or without actively growing cultures, for 3 days; and then incubated at 25 °C in a growth chamber (12 h photoperiod) for 7 days. After 7 days, brown lesions were observed on the roots in inoculated plates; however, no symptoms were observed in the control. -In the second method, 10-day old soybean seedlings were inoculated by cutting and soaking the roots in the spore suspension (1 × 106 conidia/mL) for 2 h. The inoculum was prepared by incubating isolates on PDA for 10 days, then adding sterile distilled water, scraping the growth medium, and filtering the suspension. The seedlings were then transplanted into 18 cm plastic pots (20 cm height) and grown under greenhouse conditions (26 °C ± 3 °C, 13 h photoperiod) for 2 weeks. After 7 days, the inoculated plants showed wilting symptoms, developed brown lesions in the roots, and eventually died within 2 weeks after inoculation. No such symptoms were observed in the control (inoculated with sterile distilled water). The isolates were re-isolated from the inoculated seedlings for confirmation. Although the fungus and associated mycotoxins have been reported in the rice produced in Korea (Hong et al. 2015), to the best of our knowledge, this is the first report of F. armeniacum causing Fusarium wilt on soybean in Korea. In the US, it was first reported by Ellis et al. (2012). Fusarium wilt is a soilborne disease of growing concern in soybean cultivation worldwide. Our findings will help increase awareness about the global spread of this disease.

Novel Symbiotic Association Between Euwallacea Ambrosia Beetle and Fusarium Fungus on Fig Trees in Japan

Ficus carica plantations in Japan were first reported to be infested by an ambrosia beetle species, identified as Euwallacea interjectus, in 1996. The purpose of this study was to determine the symbiotic fungi of female adults of E. interjectus emerging from F. carica trees infected with fig wilt disease (FWD). Dispersal adults (51 females) of E. interjectus, which were collected from logs of an infested fig tree in Hiroshima Prefecture, Western Japan, were separated into three respective body parts (head, thorax, and abdomen) and used for fungal isolation. Isolated fungi were identified based on the morphological characteristics and DNA sequence data. Over 13 species of associated fungi were detected, of which a specific fungus, Fusarium kuroshium, was dominant in female head (including oral mycangia). The plant-pathogenic fungus of FWD, Ceratocystis ficicola, was not observed within any body parts of E. interjectus. We further discussed the relationship among E. interjectus and its associated fungi in fig tree.

Fungal Endophytes: A Promising Frontier for Discovery of Novel Bioactive Compounds

For years, fungi have served as repositories of bioactive secondary metabolites that form the backbone of many existing drugs. With the global rise in infections associated with antimicrobial resistance, in addition to the growing burden of non-communicable disease, such as cancer, diabetes and cardiovascular ailments, the demand for new drugs that can provide an improved therapeutic outcome has become the utmost priority. The exploration of microbes from understudied and specialized niches is one of the promising ways of discovering promising lead molecules for drug discovery. In recent years, a special class of plant-associated fungi, namely, fungal endophytes, have emerged as an important source of bioactive compounds with unique chemistry and interesting biological activities. The present review focuses on endophytic fungi and their classification, rationale for selection and prioritization of host plants for fungal isolation and examples of strategies that have been adopted to induce the activation of cryptic biosynthetic gene clusters to enhance the biosynthetic potential of fungal endophytes.

Prevalence of Pulmonary Mycotic Lesions and Antifungal Susceptibility Pattern of Fungi Isolated from Nigerian Trade Cattle

Pulmonary mycosis is a major cause of deaths in many livestock herds, especially in immune-compromised animals. A total of 138 lung samples (6mm in size) were collected from cattle slaughtered for meat at Nsukka abattoir. Swab sticks were dipped into each lung sample and streaked on freshly prepared Sabouraud Dextrose Agar (SDA) for fungal isolation. Isolated fungi were identified using morphological and microscopic features. Disc diffusion method was used to test the antifungal susceptibility pattern of a randomly selected isolates with voriconazole (1μg), fluconazole (25μg) and methanol extract of Garcinia kola, using disc diffusion method. The lung samples were examined for lesions using histopathological procedures. Out of the 138 lung samples, 23 had fungal growths while 115 had no growth of any sort. A total of 34 fungi were isolated as pure cultures. Aspergillus spp. had the highest frequency of isolation, 44.92%, followed by Penicillium spp. 11.76%, Trichophyton spp. 5.88%, Conidiobolus and Sporothrix species had 2.94% isolation rate each. About 29.6% of the isolated fungi could not be identified. Fungal positive lungs showed acute to sub-acute inflammatory responses with bronchiolar septal, alveolar interstitium and peribronchial inflammatory cell infiltration and thickening of the lung tissues. All the screened fungal isolates were sensitive to voriconazole with the zone of inhibition ranging from 12-27mm. The fungal isolates were all resistant to fluconazole and G. kola extract, except one that showed sensitivity to the extract. Voriconazole showed strong antifungal effect on all the fungi; but the efficacy of the G. kola on the fungal isolates was very low compared to voriconazole.

First report of Diplodia fraxini and Diplodia subglobosa causing canker and dieback of Fraxinus excelsior in Slovenia

Over the last decades the vitality and productivity of European ash trees in Slovenia has been seriously impacted by the onset of canker and dieback disease symptoms on young and old trees, primarily identified as ash dieback caused by Hymenoscyphus fraxineus. Given the limited information available about the aetiology of this emerging disease, a study was carried out to isolate, identify and characterize the fungal species involved in the observed ash symptoms. Field surveys were conducted in five forest sites where 50 symptomatic branch samples were collected. All samples were inspected and used for fungal isolation. Based on morphology, colony appearance and DNA sequence data of the internal transcribed spacer region (ITS), 125 fungal colonies belonging to five species were isolated and identified. Only a few symptomatic ash samples yielded colonies of H. fraxineus, whereas Botryosphaeriaceae species were isolated with a high frequency, with Diplodia fraxini as the dominant species. A pathogenicity test proved that all isolated species were pathogenic on European ash, causing bark lesions and wood discoloration. All Botryosphaeriaceae species isolated in this study are reported for the first time on European ash in Slovenia.