Mycelial Fragments Research Articles

First Report of White Mold Caused by Sclerotinia sclerotiorum on Echeveria gigantea in Mexico.

Echeveria gigantea, native of Mexico (Reyes et al. 2011), holds economic importance as it is marketed as a potted plant and cut flower due to its drought-tolerant capabilities and aesthetic appeal. In September 2023, a field sampling was conducted at the Research Center in Horticulture and Native Plants (18°55'56.6" N, 98°24'01.5" W) of UPAEP University. Echeveria gigantea cv. Quilpalli plants with white mold symptoms were found in an area of 0.5 ha, with an incidence of 40% and severity of 50% on severely affected stems. The symptoms included chlorosis of older foliage, necrosis at the base of the stem, and soft rot with abundant white to gray mycelium and abundant production of irregular sclerotia resulting in wilted plants. The fungus was isolated from 30 symptomatic plants. Sclerotia were collected, sterilized in 3% NaOCl, rinsed with sterile distilled water (SDW), and plated on Potato Dextrose Agar (PDA) with sterile forceps. Subsequently, a dissecting needle was used to place fragments of mycelium directly on PDA. Plates were incubated at 23 °C in darkness. A total of 30 isolates were obtained using the hyphal-tip method, one from each diseased plant (15 isolates from sclerotia and 15 from mycelium). After 6 days, colonies had fast-growing, dense, cottony-white aerial mycelium forming irregular sclerotia of 3.67 ± 1.13 mm (n=100). Each Petri dish produced 32.47 ± 7.5 sclerotia (n=30), after 12 days. The sclerotia were initially white and gradually turned black. The isolates were tentatively identified as Sclerotinia sclerotiorum based on morphological characteristics (Saharan and Mehta 2008). Two isolates were selected for molecular identification. Genomic DNA was extracted using the CTAB protocol. The ITS region and the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene were sequenced for two randomly selected isolates (White et al. 1990; Staats et al. 2005). The ITS and G3PDH sequences of the SsEg9 isolate were deposited in GenBank (ITS-OR816006; G3PDH-OR879212). BLAST analysis of the partial ITS (510 bp) and G3PDH (915 bp) sequences showed 100% and 99.78% similarity to S. sclerotiorum isolates (GenBank: MT101751 and MW082601). Pathogenicity was confirmed by inoculating 30 120-day-old E. gigantea cv. Quilpalli plants grown in pots with sterile soil. Ten sclerotia were deposited at the base of the stem, 10 mm below the soil surface. As control treatment, SDW was applied to 10 plants. The plants were placed in a greenhouse at 23 °C and 90% relative humidity. After 16 days, all inoculated plants displayed symptoms similar to those observed in the field. Control plants did not display any symptoms. The fungus was reisolated from the inoculated stems, fulfilling Koch's postulates. The pathogenicity tests were repeated three times. Recently S. sclerotiorum has been reported causing white mold on cabbage in the state of Puebla, Mexico (Terrones-Salgado et al. 2023). To the best of our knowledge, this is the first report of S. sclerotiorum causing white mold on E. gigantea in Mexico. Information about diseases affecting this plant is very limited, so this research is crucial for designing integrated management strategies and preventing spread to other production areas.

Response of chickpea genotypes (Cicer arietinum L.) to the fungi complex that causes wilt

Objective: The objective of the present study was to evaluate the response of 10 chickpea genotypes from INIFAP to the damage caused by the fungal complex. Design/methodology/approach: Seedlings (15-day-old) of 10 genotypes (Blanco Sinaloa '92, Blanoro, Combo 743, CUGA2054, HOGA067, CUGA3168, CUGA08-1210, CUGA09-3160, R-12-1509 and R-12-1507) were inoculated by root immersion in a suspension of mycelial fragments of two isolates (high and low virulence) of each fungi: Fusarium languescens, M. phaseolina, S. rolfsii and S. sclerotiorum. Disease severity evaluation was performed 30 days after inoculation. The entire experiment was performed twice. Results: The genotypes showed greater susceptibility to S. sclerotiorum and S. rolfsii compared to F. languescens and M. phaseolina. The highly virulent isolates caused a significant difference in the severity of the disease in the genotypes evaluated. Findings/conclusions: All chickpea genotypes showed susceptibility to the fungal complex that causes wilt.

First Report of Monosporascus cannonballus causing Root Rot and Vine Decline in Watermelon (Citrullus lanatus) in China.

In June 2023, a sudden outbreak root rot and vine decline symptoms was observed during a watermelon (Citrullus lanatus T.) variety demonstration trial located in Taizhou City, Zhejiang Province, China, with an incidence rate ranging from 75% to 100% and an affected area of nearly 2,000 square meters. The disease initially appeared with a rapid and alarming invasion of root rot and vine decline symptoms within watermelon plants. Affected plants exhibited rapid deterioration, showing symptoms of wilting, yellowing and eventual demise, predominantly during the pre-harvest stage. Notably, numerous black, spherical, erumpent perithecia were clearly visible on the watermelon's root epidermis, a characteristic trait of the disease. Symptomatic plant samples were rigorously disinfected with 75% ethanol, and plated on potato dextrose agar medium for incubation at 25°C, successfully isolate two potential strains. These isolates were inoculated in oatmeal agar and incubated in a 25℃ light incubator. After 30 days, mature perithecia, the same as those found on the watermelon's root epidermis, reached a diameter of 500 μm. Each perithecium contained several pear-shaped asci, 56 to 108.5 μm in length and 30.5 to 46.4 μm in width, typically holding 1, rarely 2 ascospores. These characteristics align precisely with the typical strains of Monosporascus cannonballus Pollack and Uecker (1974). Additionally, sequencing the internal transcribed spacer region of ribosomal DNA (ITS) gene (White et al., 1990), large subunit ribosomal RNA (LSU) gene (Rehner and Samuels 1995), and beta-tubulin (TUB) gene (Glass and Donaldson, 1995) were performed. BLAST analysis indicated the highest nucleotide sequence identity with M. cannonballus CBS 586.93 reference sequence (ITS: 100%, JQ771930; TUB: 98.99%, JQ907292). Representative sequences of isolate ZJUP0990-2 from these regions were deposited in GenBank (Accession No.: OR357656 for ITS; OR474500 for LSU; OR365762 for TUB). A multigene phylogenomic analysis (ITS-LSU-TUB) was undertaken to ascertain the exact phylogenetic position of M. cannonballus within the genus Monosporascus. The amalgamation of both morphological and molecular insights consistently reaffirmed the accurate classification of the causative agent as M. cannonballus. To validate the pathogenicity of M. cannonballus, a controlled greenhouse experiment was conducted using watermelon (cv. Nabite) as the subject. Mycelium fragments, harvested from the edge of the colony ZJUP0990-2, were inoculated into oat liquid medium and cultivated under dark conditions at a consistent temperature of 30°C for 7 days. After 20 days, the inoculated plants exhibited root rot and wilting, mirroring the symptoms observed during the field outbreak. In contrast, the control plants did not exhibit any signs of disease. M. cannonballus was successfully re-isolated from the symptomatic roots of the inoculated plants, satisfying Koch's postulates. This experiment was repeated three times. This pathogenic fungus has previously been documented as a menace to melons in various regions including Mexico (Chew-Madinaveitia et al., 2012) and Brazil (Sales et al., 2004), as well as watermelons in Brazil (Sales et al., 2010), northern Mexico (Gaytan-Mascorro et al., 2012), and Saudi Arabia (Karlatti et al., 1997). To our knowledge, this is the first reported presence of M. cannonballus on watermelons in China. This new disease poses a serious threat to watermelon production, potentially leading to severe economic losses and impacting food security.

Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis induces the death of mouse lymphocytes in a mechanism dependent on Toll-like receptor 4 and tumor necrosis factor.

Paracoccidioides fungi are thermodimorphic microorganisms that cause paracoccidioidomycosis (PCM), an autochthonous disease from Latin America, with most cases in Brazil. Humans become infected by inhaling conidia or mycelial fragments that transform into yeast at body temperature. These fungi cause chronic-granulomatous inflammation, which may promote fibrosis and parenchyma destruction in the lungs. In response to stress imposed by the host, fungi Paracoccidioides spp. increase the expression of heat shock proteins (HSP), which protect them by sustaining cellular proteostasis. Our group has studied the role of HSP60 in PCM, and previous data show that the recombinant HSP60 (rHSP60) has a deleterious effect when used in a single dose as therapy for experimental PCM. Here, we investigated the mechanism by which rHSP60 could worsen the disease. We found that rHSP60 caused the viability loss of splenic or lymph node cells from both immunized and non-immunized mice, including in splenic T lymphocytes under polyclonal stimulation with concanavalin A, probably by undergoing apoptosis. Among analyzed splenic cells, lymphocytes were indeed the main cells to die. When we investigated the death mechanisms, remarkably, we found that there was no viability loss in rHSP60-stimulated splenic cells from mice deficient in Toll-like receptor 4, TRIF adapter protein, and TNF receptor 1(TNFR1), as well as rHSP60-stimulated WT cells incubated with anti-TNF antibody. Besides, caspase-8 inhibitor IETD-CHO blocked the rHSP60 effect on splenic cells, suggesting that rHSP60 induces the extrinsic apoptosis pathway dependent on signaling via TLR4/TRIF and TNFR1.

First report of Stagonosporopsis pogostemonis causing leaf spot on cauliflower in Italy.

First report of Stagonosporopsis pogostemonis causing leaf spot on cauliflower in Italy Wassim Habib1, Mariangela Carlucci2, Roberto Fasano3, Franco Nigro1,2 † 1 Centre of Research, Experimentation and Training in Agriculture (CRSFA) - Basile Caramia, via Cisternino 281, Locorotondo, 70010 Bari, Italy 2 Department of Soil, Plant and Food Sciences, University of Bari - Aldo Moro, via Amendola 165A, 70126 Bari, Italy 3 Bioricerche, Phytopathological Lab, via Pio XI 30, Bellizzi, 84092 Salerno, Italy † Corresponding author: F. Nigro. Email: franco.nigro@uniba.it Keywords: Phoma-like, Multi-locus gene sequencing, Brassica oleracea L. var. botrytis, Campania. In September 2022, a severe foliar disease was observed on 2-3-week-old 'Corsaro' and 'Parthenon' seedlings of cauliflower (Brassica oleracea L. var. botrytis) grown under greenhouses in a nursery in Caserta province, Campania (Italy), with an incidence of 90% on about 150,000 plants. Affected plants showed numerous small, irregular, and depressed lesions bordered by a chlorotic halo on leaves that become necrotic, and later drop out giving a shot-hole effect (Fig. 1). Isolations were performed on leaf portions from 30 symptomatic samples which were surface-sterilised by dipping in 70 % ethanol for 30s, then in a sodium hypochlorite solution (1%) for 30s, and rinsed two times in distilled sterilised water. Tissue fragments were left to dry on sterile filter paper and then seeded on potato dextrose agar (PDA) amended with 0.5 g/L of streptomycin sulphate (Sigma-Aldrich S.R.L, Steinheim, Germany). After 6 to 8 days of incubation at 24 ± 1°C, colonies revealed a consistent growth of a Phoma-like fungus resembling Stagonosporopsis genus. On PDA, one-week-old colonies were dark olivaceous green to brown with white regular margin (Fig. 2B). Pycnidia (32-160 µm × 30-95 µm) were subglobose, and conidia (2.7-4.8 µm × 1-2.7 µm) ellipsoidal, with rounded ends, aseptate, with two polar guttules (Fig. 2C). All isolates had similar morphological features, therefore further analyses were performed on one representative strain (CRSFA.753.22). The multilocus phylogenetic approach using Maximum likelihood method and Tamura-Nei model on rDNA-ITS, TUB and RPB2 sequences (accession numbers: OQ318550, OQ326503, OQ326504) including reference strains of Stagonosporopsis (Dong et al. 2021) demonstrated that CRSFA.753.22 clustered with Stagonosporopsis pogostemonis Luo, Huang & Manawas holotype (ZHKUCC 21-0001) with 91% bootstrap support. ITS and RPB2 sequences had 100% BLAST match to ZHKUCC 21-0001, whereas TUB sequence had 99.69%. Pathogenicity tests were performed on 20 healthy 'Corsaro' plantlets. Leaves were surface sterilized with 70% alcohol and wounded with a sterile tip of a needle. A suspension of conidia and mycelial fragments, obtained from a 10-days-old colony of the strain CRSFA.753.22, was sprayed on the leaf surfaces at a distance of 20 cm. Five controls plants were inoculated with sterile distilled water. Plants were then covered with plastic bags and kept on the shelf in a growth chamber at 25° C, with a 12-h photoperiod. The first symptoms developed on leaves five days post-inoculation as irregular small brown spots, that were gradually expanded. Leaf tissues showed chlorosis which evolved into necrosis (Fig. 2A). Stagonosporopsis pogostemonis was consistently re-isolated from all diseased leaves, but not from control plants, thus fulfilling Koch's postulates. In Italy, Stagonosporopsis species have been reported as severe plant pathogens (Garibaldi et al. 2022; Guarnaccia et al. 2022), and this is the first report of the species S. pogostemonis in the country. References Dong, Z.Y., et al. 2021. Pathogens 10:1093. doi: 10.3390/pathogens10091093 Garibaldi, A., et al. 2022. Plant Pathol. J. 104:1157. doi: 10.1007/s42161-022-01138-7 Guarnaccia, V., et al. 2022. Plant Pathol. J. 104:1491. doi: 10.1007/s42161-022-01197-w.

First Report of White Mold Caused by Sclerotinia sclerotiorum on Cabbage in Mexico.

The state of Puebla is the main producer of cabbage (Brassica oleracea var. capitata) in Mexico, with an area of approximately 1,858 ha (SIAP 2023). In April 2023, a field sampling was conducted in the San Luis Ajajalpan, Tecali de Herrera (18°55.57'N, 97°55.607'W), Puebla, Mexico. The average temperature was 24°C and the relative humidity was 95% for five consecutive days. Cabbage plants cv. 'American Taki San Juan' close to harvest, with head rot symptoms were found in a commercial area of approximately 3 ha, at an estimated incidence of 35 to 45%. More than 70% of the leaves were symptomatic on severely affected plants. Typical symptoms included chlorosis of older foliage, soft rot with abundant white to gray mycelium, and abundant production of large and irregularly-shaped sclerotia. The fungus was isolated from 30 symptomatic plants. Sclerotia were collected from symptomatic heads, surface sterilized in 3% NaOCl, rinsed twice with sterile distilled water, and plated on Potato Dextrose Agar (PDA) with sterile forceps. Subsequently, a dissecting needle was used to place fragments of mycelium directly on PDA. Plates were placed in an incubator at 25°C in the dark. A total of 30 representative isolates were obtained by the hyphal-tip method, one from each diseased plant (15 isolates from sclerotia and 15 from mycelial fragments). After 8 days, colonies had fast-growing, dense, cottony-white aerial mycelium forming irregular sclerotia of 3.75 ± 0.8 mm (mean ± standard deviation, n=100). Each Petri dish produced 14-25 sclerotia (mean = 18, n = 50), after 10 days. The sclerotia were initially white and gradually turned black. The isolates were identified as Sclerotinia sclerotiorum based on morphological characteristics (Saharan and Mehta 2008). Two representative isolates were chosen for molecular identification, and genomic DNA was extracted by a CTAB protocol. The ITS region and the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene were sequenced for two isolates (White et al. 1990; Staats et al. 2005). The ITS and G3PDH sequences of a representative isolate (SsC.1) were deposited in the GenBank (ITS- OR286628; G3PDH- OR333495). BLAST analysis of the partial sequences ITS (509 bp) and G3PDH (915 bp) showed 100% similarity to S. sclerotiorum isolates (GenBank: MT436756.1 and OQ790148). Pathogenicity was confirmed by inoculating 10 detached cabbage heads of 'American Taki San Juan', using the SsC.1 isolate, according to Sanogo et al. (2015). Heads were placed on the rim of a plastic container and inserted in a moisture box with 2 cm of water on its bottom. The box was covered with a plastic sheet to maintain humidity. The control plants were inoculated with a plug of noncolonized PDA. The inoculated cabbages were covered with white to gray mycelia and abundant sclerotia within 10 days, whereas no symptoms were observed on non-inoculated controls. The fungus was re-isolated from the inoculated cabbages as described above, fulfilling Koch's postulates. The pathogenicity tests were repeated three times. White mold caused by S. sclerotiorum on Brussels sprouts was recently reported in Mexico (Ayvar-Serna et al. 2023). In 2015, S. sclerotiorum was reported on cabbage in New Mexico, causing head rot (Sanogo et al. 2015). To our knowledge, this is the first report of S. sclerotiorum causing white mold on cabbage in Mexico. This research is essential for designing management strategies and preventing spread to other production areas.

Dermatophyte antigen kit: A useful diagnostic tool for onychomycosis.

A dermatophyte antigen kit (DQT) was released in Japan as an in vitro diagnostic tool to identify tinea unguium in June 2022. From July 2022 to February 2023, we examined 75 potassium hydroxide (KOH)-negative patients (male, n = 23; female, n = 52; mean ± SD age, 63.6 ± 13.9 years) and determined the accuracy in confirming the fungal element with ZoomBlue™ staining at 400× magnification. The DQT results were classified into three categories. DQT-positive onychomycosis was detected in 27 patients with tinea unguium and two with non-dermatophyte onychomycosis. Fungal cultures were positive in 14 (51.8%) patients (Trichophyton rubrum [n = 11], T. interdigitale [n = 1], Fusarium solani [n = 1], and Talaromyces muroii [n = 1]). DQT-negative onychomycosis included ten patients with cured tinea unguium and 3 with Candida onychomycosis. Twenty-three patients had DQT-negative mimics for onychomycosis (onychauxis [n = 11], traumatic onycholysis [n = 8], yellow nail syndrome [n = 5], pincer nail deformity [n = 3], brittle nail syndrome [n = 2], contact dermatitis [n = 2], lichen planus [n = 1] and psoriasis [n = 1]). Because sparse, atrophic and/or fragmented mycelia are invisible in direct microscopy with potassium hydroxide (KOH) at 100× magnification, DQT was beneficial for diagnosing onychomycosis.

Determination of aspergillosis of the female genital organs in women with immunodeficiency

Background. Morphological diagnosis of aspergillosis sometimes poses great difficulties due to the fact that molds of the genus Aspergillus are very sensitive to the use of antimicrobial therapy and change under its influence. In this regard, we provide information about aspergillosis of the female genital organs, discovered accidentally during an autopsy. The purpose of the study is to conduct a complex pathomorphological analysis of biopsy, operative and sectional gynecological material of women with secondary immunodeficiency conditions, for early diagnosis of aspergillosis (which is the key to a favorable outcome of the disease), differential diagnosis and detection of invasive forms of aspergillosis. Materials & Methods. We analyzed the autopsies of 4,796 women who died in Odesa region from 2009 to 2022. Autopsies revealed 7 cases of aspergillosis with damage to the female reproductive system. All of them were found in women who had confirmed HIV infection. In 5 cases, women additionally suffered from chronic alcoholism. Results. Based on the results of these cases, it can be stated that the detection of characteristic mycelium in the preparations is a reliable sign of aspergillosis, but their absence does not deny this disease. Aspergillus hyphae change as the process develops: they can be weakly stained, and with dystrophic phenomena and the presence of necrotic fragments of mycelium, it can be almost colorless when using hematoxylin and eosin. While necrosis, tissue detritus is observed in the center of the granuloma, fibrous structures, fungal hyphae are formed perifocally. To confirm the diagnosis, it is important to detect granulomatous productive inflammation, conidial heads, inflammatory infiltration, microabscesses, foci of calcifications. However, against the background of immunodeficiency, it is not always possible to see the "classic morphological picture" of aspergillosis. In addition, detection of characteristic mycelium is possible only in a proportion of patients. Conclusions. When examining the autopsy material of deceased women with immunodeficiency states, attention should be paid to the specific etiology of inflammatory processes of the reproductive system and the nature of the inflammatory process, the presence of Aspergillus hyphae and mycelia. The presence of morphological changes characteristic of aspergillosis allows to conduct a diagnostic search in the right direction. Detection of an invasive form of aspergillosis may indicate the presence of an immunodeficient state. Keywords: aspergillosis, female reproductive system, immunodeficiency state, HIV infection, uterus, mammary gland, drug addiction.

Functional fungal pellets self-immobilized by mycelium fragments of Irpex lacteus WRF-IL for efficient degradation of sulfamethazine as the sole carbon source

In order to achieve an efficient microbial material with dual functions of self-immobilization and sulfamethazine (SMZ) degradation, this study explored the pelletization technique utilizing mycelium fragments of Irpex lacteus WRF-IL and systematically examined the pellets formation conditions and degradation capability. The Box-Behnken design results demonstrated that pure mycelium fragments, broken by frosted glass beads, could be rapidly self-immobilized to form white rot mycelial pellets (WRMPs) within 24 h, serving as the pelleting core. These WRMPs could completely remove SMZ as the sole carbon source within 20 h. The addition of sucrose expedited this process, achieving complete removal within only 14 h. Kinetic analysis showed that WRMPs could potentially remove SMZ at higher concentrations (>25 mg/L). Biodegradation was the primary pathway of SMZ removal. Seven intermediates were identified by QTOF LC/MS, and three transformation pathways initiated by SO2 overflow, molecular rearrangement, and aniline moiety oxidation were deduced.

First report of Athelia rolfsii (= Sclerotium rolfsii) causing southern blight on Pachyrhizus erosus in Mexico.

Pachyrhizus erosus, commonly named jicama, is native to Mexico and is cultivated for its tuberous roots which are edible. In November 2021, field sampling was carried out in municipality of Huaquechula (18.748640N, 98.550817W, 1,580 m above sea level), state of Puebla, México. The disease had an incidence between 20 and 30% in approximately 10 ha. Infected plants showed wilting, yellowing foliage, rotting with white mycelium, abundant sclerotia were observed in the roots and tuber. Tuber splits transversely over time. Twenty plants with symptoms of disease were carried out to isolate the fungus. The sclerotia found in the tubers were disinfected with 3% NaOCl, rinsed twice with sterile distilled water, and excess moisture was removed and, transferred on Potato Dextrose Agar (PDA) culture medium and incubated at 28°C. Mycelial fragments from symptomatic tubers, were plated directly to PDA. Twenty representative isolates were obtained by hyphal-tip method, one for each diseased plant sampled (10 isolates from sclerotia and the other 10 from fragments of mycelium). After 10 days, colonies showed fast-growing, dense, cottony-white aerial mycelium, forming globoid to irregular sclerotia, measuring 1.0-1.7 mm in diameter (mean = 1.42 mm; n=100). The number of sclerotia produced per Petri dish ranged from 54 to 542 (mean = 274, n = 50). These sclerotia were initially white and gradually turned brown. Microscopic examination showed septate hyphae with some cells having clamp connections. Based on morphological characteristics, the fungal isolates were identified as Athelia rolfsii (Curzi) CC Tu & Kimbr (Syn: Sclerotium rolfsii Sacc) (Mordue 1974). For molecular identification, a representative isolate (Sr.1), the ITS region was amplified (650 bp) using primers ITS1/ITS4 (White et al. 1990). The obtained sequence (GenBank: ON206899) was subjected to BLAST analysis, where it had 100% identity with A. rolfsii isolates (GenBank: MG836252 and MH517363). Phylogenetic analysis with the neighbor-joining method in MEGAX, grouped the Sr.1 isolate into a common clade with different A. rolfsii isolates. Pathogenicity was confirmed by inoculating 20 tubers detached from healthy P. erosus variety "Criolla de Morelos", into which a portion of mycelium from the Sr.1 isolate was inserted with a sterile wooden stick at one point per tuber. In five tubers, only a sterile wooden stick was inserted as negative controls. The tubers were placed under laboratory conditions with relative humidity close to 100% and a temperature of 28°C. Symptoms like those observed in the field were observed after five days. Control tubers showed no symptoms. Additional pathogenicity tests were performed on 50 plants of 100-day-old P. erosus of the variety "Criolla de Morelos", grown in pots with sterile soil. Ten sclerotia of 10 days old were deposited at the base of the stem, 10 mm below the soil surface; as control treatment only, sterile distilled water was deposited on 20 plants. The plants were placed in a greenhouse (Center for Technological Innovation in Protected Agriculture of the Popular Autonomous University of the State of Puebla), at 28 ± 1°C and 90% of temperature and relative humidity, respectively. After 15 days, all inoculated plants showed symptoms similar to those observed in the field. Control plants showed no symptoms. A. rolfsii was re-isolated from inoculated tubers and stem, fulfilling Koch's postulates. Previously, A. rolfsii was reported in Mexico, causing southern blight on sesame (Hernández-Morales et al. 2018). To our knowledge, this is the first report of Athelia rolfsii causing southern blight on P. erosus in Mexico (Farr and Rossman 2022). This research is important to design management strategies and prevent its spread to other P. erosus-producing areas.

Experimental Outcrossing in Agaricus bisporus Revealed a Major and Unexpected Involvement of Airborne Mycelium Fragments.

Agaricus bisporus var. bisporus, the button mushroom, has a predominantly pseudohomothallic life cycle. Most of its spores are heterokaryotic and give rise to fertile heterokaryons. However, previous studies have suggested that outcrossing should not be rare in wild populations. In order to discover how outcrossing occurs, we experimentally favored it between aerial propagules of a fruiting donor mycelium and a delayed receiver mycelium that only invaded culture trays. Two donor/receiver pairs were studied, and potentially hybrid basidiomata collected on the receiver trays were analyzed with a mitochondrial marker, two unlinked nuclear CAPS markers, then haplotype markers based on DNA sequences obtained after PCR cloning of the rDNA ITS region and the fruk gene. For one of the two pairs, most basidiomata were hybrids between the donor and the receiver. Genotyping of the hybrids revealed only two genotypes consistent with outcrossing involving airborne mycelium fragments rather than basidiospores. The resident receiver heterokaryon that provided its mitochondria to the hybrid basidiomata is suspected to have had a trophic contribution to their growth and successful fruiting. The high level of heterozygosity and the cultivar introgression previously revealed in wild populations of this pseudohomothallic species may result from outcrossing involving airborne pieces of mycelium.

First report of southern blight caused by Athelia rolfsii on Spotted laurel (Aucuba japonica) in China.

Aucuba japonica, also known as spotted laurel, is a woody, broadleaf, evergreen shrub with variegated leaves in the Garryaceae family, widely used in urban parks, green spaces and landscaping. In October 2019, an outbreak of a disease with southern blight symptoms was observed on A. japonica planted as a green barrier in Kunshan city, Jiangsu province of China (N31°32'37", E120°00'41"). The disease incidence was estimated up to 30%. The infected plants showed symptoms including brown to black necrotic stems, white mycelium and white to dark reddish brown sclerotia at the base of the stem and decayed tissues. Fifteen samples (10 sclerotia and 5 mycelial fragments) were collected from symptomatic plants for causal agent isolation. The sclerotia were disinfected with 70% ethanol for 2 to 3 s and 5% NaClO for 2 min, rinsed three times with sterile water, then cultivated on potato dextrose agar (PDA) plate at 25°C. Mycelial fragments were transferred to PDA plates by an inoculation needle directly. In total 15 fungal strains were obtained and purified by transferring single hyphal tips to fresh media. All the strains showed consistent phenotype, white mycelia on PDA, with an average growth rate of 13.6 to 16.9 mm/day (n=30), and mycelia with clamp connections were observed under the microscope. Globose sclerotia formed at 4 days post inoculation (dpi), initially whitish, turning to beige and eventually dark reddish brown. The number of sclerotia produced per plate ranged from 280 to 486 (mean = 378; n = 30), and the diameter of mature sclerotia ranged from 0.8- to 1.6-mm (mean = 1.24; n = 150). Three strains YKY2020.02, YKY2020.03, and YKY2020.07 were selected for further molecular identification. Genomic DNA was extracted from these strains using a CTAB method (Mahadevakumar et al. 2018). ITS primer pair ITS1/ITS4 was used to amplify the internal transcribed spacer region (White et al. 1990). PCR products were then sequenced by Sangon Biotech (Shanghai, China), and subsequently, the ITS sequences (686 bp) were deposited in GenBank under accession number OM647806, OP279917 and OP279918, respectively. All sequences showed 99-100% similarity with Athelia rolfsii sequences from GenBank by BLAST analysis in NCBI. The phylogenetic tree of ITS sequences generated by the neighbor-joining analysis in MEGA-X also shows that all selected strains clustered with different strains of A. rolfsii into one big branch, indicating that these strains are the same. Based on morphological and molecular characteristics, these strains were identified as A. rolfsii (Curzi) C.C. Tu & Kimbr. (syn. Sclerotium rolfsii) (Stevens 1931; Paul et al. 2017). Pathogenicity tests were conducted on healthy plants of A. japonica (n = 15). Five-day-old mycelial discs (5 mm) were inoculated at the basal part of the plants with mycelial side inward and secured with wet absorbent cotton, while plants inoculated with sterile water were used as a control (n = 5). All plants were kept in a greenhouse with a temperature of 26 to 33°C and an average relative humidity higher than 65%. At 5 dpi, all inoculated plants showed symptoms similar to those observed in fields. Control plants remained asymptomatic. To fulfill Koch's postulates, identities of all the causal pathogens were confirmed by reisolation in PDA and identification by morphology. To our knowledge, this is the first report of A. rolfsii causing southern blight on A. japonica worldwide. Our findings are important for future disease control strategy development.

Antifungal Activity of Streptomyces spectabilis SP-O2 against Aflatoxin Producing Mold, Aspergillus flavus

Among soil isolated species of actinomycetes bacteria that affected the growth of aflatoxin producing mold; Aspergillus flavus TISTR 3041, the isolate SP-O2 showed a promising inhibitory activity on the dual culture assay. This isolated bacterium was characterized on various International Streptomyces Project media. The macroscopic morphological analysis showed mycelium, sporogenesis, and orangish pink of colonial pigment. The scanning electron microscope (SEM) photograph showed that the sporogenesis has occurred by shrinking of both sides of aerial mycelium. After the sporogenesis has been completed, the fragmentation of altered mycelium started from the tips of aerial mycelium to produce 0.8-1 micrometer fragmented spores. The 16S rRNA sequences analysis revealed that isolate SP-O2 shared a 99.93 % (1 mismatch/1436 nt) similarity with Streptomyces spectabilis NBRC 13424 AB184393. Co-culture liquid assay was analyzed for the aflatoxin produced by A. flavus in the presence of Streptomyces spectabilis SP-O2. The result showed that UV-visualized band of aflatoxin B1 was not detected by co-cultivation of Streptomyces spectabilis SP-O2 with A. flavus by thin layer chromatography compared to the control. Indeed, fungal growth of A. flavus was destroyed by the antifungal substance produced by Streptomyces spectabilis SP-O2 in the liquid culture. Furthermore, biocontrol of contaminated A. flavus was conducted on peanut kernels and the results showed that Streptomyces spectabilis SP-O2 has antifungal activity against the presence of A. flavus. Together, these results suggested that further investigation on the development of biocontrol use of Streptomyces spectabilis SP-O2 to overcome the contamination of aflatoxin producing mold in the feedstock and agricultural-based industry is needed. HIGHLIGHTS The isolated actinobacteria in this study was identified as Streptomyces spectabilis isolate SP-O2 based on its morphological characteristics, physiological properties and molecular identification SEM showed the sporogenesis of 0.8-1 micrometer fragmented spores Streptomyces spectabilis isolate SP-O2 exhibited the promising fungicidal activity against flavus in the dual culture agar assay, co-culture liquid assay and peanut kernels assay with consistency results The secreted metabolites play a crucial role on the fungal killing against Aspergillus flavus rather than the aflatoxin degradation GRAPHICAL ABSTRACT

Antifungal Potential of Acetone and Ethyl Acetate Extracts of Thevetia peruviana on Development of Phytophthora colocasiae, Causal Agent of Late Blight of Taro (Colocasia esculenta (L.) Schott) from Three Agro-Ecological Zones of Cameroon

Aims: This study was aimed to evaluate the antifungal activities of acetone and ethyl acetate extracts of Thevetia peruviana seeds on the in vitro growth of the fungus.
 Study Design: A randomized sample block design containing four treatments (T- = Negative control; T2= Ethyl acetate extract; T3= Acetone extract; T+=Callomil Plus) with three repetitions was used. Plant extracts were used at three concentrations: C1: 12.5 µl/ml; C2: 25 µl/ml and C3: 50 µl/ml; the chemical fungicide at the dose of 12.5 μL/ml.
 Place and Duration of Study: The study was conducted in the University of Yaoundé 1, Faculty of Sciences, Department of Plant Biology, Laboratory of Phytopathology and Crop Protection, and in the Institute of Agricultural Research for Development (IARD) of Yaoundé, Laboratory of Phytopathology, during the year 2019-2020.
 Methodology: acetone and ethyl acetate extracts of T. peruviana were prepared and used at concentrations of 12.5, 25 and 50 µl/ml. P. colocasiae was isolated from infected taro leaf cultivars "Macumba or Ibo coco" located in three different regions: west, Littoral and Centre. The various explants were were put in V8 agar medium and maintained in pure culture. Mycelial fragments of P. colocasiae of about 0.8 cm in diameter were cut and placed in sterile Petri dishes containing Potato Dextrose Agar (PDA) medium supplemented with different concentrations of plant extracts and incubated at 23±1°C for seven days for the evaluation of the radial growth.
 Results: The results obtained showed that the acetone and ethyl acetate extracts have completely inhibited the growth of the strain of West at 25 μ/ml while total inhibition of the pathogen was not obtained with strain of Centre region. The lowest inhibition was obtained with the strain of Littoral region: 93.88 % for acetone extract and 90.78 % for ethyl acetate extract compare to 100 % for west and Centre region at highest concentration.
 Conclusion: The acetone and ethyl acetate extracts at the concentration of 25 μ/ml totally inhibited the in vitro radial growth of some strains of P. colocasiae. These extracts, which are effective against P. colocasiae, may substitute fungicides in the fight against taro leaf blight.

P469 Feline aspergillosis caused by Aspergillus candidus: A case report

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMAspergillosis is an emerging mycosis that can occur in apparently healthy animals under environmental stress or immune-compromised condition. In cats, the clinical forms described are sinonasal, synoorbital, and, less frequently, invasive and disseminated forms.Feline aspergillosis is caused mainly by cryptic species of Aspergillus closely related to Aspergillus fumigatus. It is rare to recover species such as A. candidus, an endophytic fungus, opportunistic pathogen, and contaminant of cereals, nuts, and flour. It is described to cause allergic alveolitis or asthma, mycotoxicosis, and other related diseases. Mycelial fragments, inhaled spores, and substances such as terpenoids, flavones, and indoles in the lungs could be involved in the underlying pathogenesis.This presentation aims to describe a clinical case of feline pneumonia caused by A. candidus.Clinical caseWe present the clinical case of a common European breed feline of ∼ 7 years of age. The cat was rescued from the streets of an urban area in Buenos Aires and examined in April 2021. The feline suffered from mild hyperthermia, 39.4°C, had pale to slightly purple mucous membranes, cachexia, dehydration, dyspnea (abdominal respiration), tachycardia, and a large number of parasites. There were no nasal discharge, cough, or skin lesions. A hemogram with a blood biochemistry profile showed no significant alterations. Chest X-rays showed a pattern compatible with severe miliary pneumonia. A bronchoalveolar lavage (BAL) was performed for cytology and microbiological culture; direct examination showed nucleated superficial epithelial cells without atypia, few macrophages, no leukocytes, and gram-positive cocci, while ZN and mycological direct examination were negative. Treatment with doxycycline 10 mg/k every 12 h and dexamethasone was indicated. Flea treatment and control were conducted every 72 h. During the following consultations, the cat continued tachypnoeic and showed increased vesicular murmur with rales and cough. Corticosteroids (Fluticasone propionate 125 mg) were given every 12 h intranasal. Oral parasitization was performed due to the presence of Dipylidium caninum in feces. After 7 days of incubation, white cottony colonies phenotypically identified as Aspergillus grew on Sabouraud dextrose agar. Even though serum galactomannan GM (Platelia Aspergillus EIA BIORAD®) is useful as a biomarker in humans with OD >0.5 positive for invasive aspergillosis, it was used in this case and the result was OD 0.75. Antifungal treatment was started with itraconazole orally at 5 mg/kg/d. A mycobacterial culture was negative.The isolate was identified by partial sequencing of the calmodulin gene CaM as A. candidus.The feline showed a remarkable recovery. After 35 days from the start of the treatment, the final consultation showed a decrease in the pattern of military pneumonia through radiological images and an absence of any evident respiratory symptoms.ConclusionWe describe a fungal infection in a cat caused by an unusual species of Aspergillus. Moreover, this clinical case was proved to be pneumonia. Given this, it is important to consider this species as an emerging pathogen in cats.

First Report of Southern Blight on Aloe vera Caused by Athelia rolfsii in China.

Aloe vera (L.) Burm f., which belongs to the family Aloaceae, is a perennial succulent plant and cultivated for its medicinal, cosmetic, vegetable and ornamental uses. In summer of 2021, about 15% (60 infected among 400 surveyed plants) of A. vera (A. barbadensis) plants in two gardens in Lishui, Zhejiang Province of China showed symptoms of southern blight disease. Symptomatic plants primarily exhibited slightly sunken water-soaked, dark brown lesions on taproot and basal part of the stems. As the disease progressed, leaves in the basal part of stems and subsequently the whole plant rotted and withered, with white mycelial mats occurring on infected stems and leaves. Numerous brown, spherical sclerotia were observed on the colonized tissues and soil surfaces around the infected plants. Mycelial fragments and sclerotia from symptomatic leaves were plated directly to potato dextrose agar (PDA) amended with 100 μg/ml streptomycin and incubated at 26°C in the dark. By hyphal-tip method, a total of five pure isolates were obtained from five diseased leaf samples. When cultured on PDA at 26°C for three days, colonies showed white and thick aerial mycelium, with a radial growth rate of 23.7 mm/day. Typical clamp connection structures were observed microscopically after three days and numerous globoid, rapeseed shape sclerotia, measuring 1 to 2 mm in diameter (n=50) formed after six days. These sclerotia were initially white and gradually turned dark brown with age. On the basis of morphological characteristics, the fungal isolates were identified as Athelia rolfsii (Curzi) C.C. Tu & Kimbr (anamorph Sclerotium rolfsii Sacc) (Mordue 1974). The internal transcribed spacer (ITS) and translation elongation factor 1-α gene (TEF1) regions of a representative isolate LHBJ2-4 were amplified and sequenced using the primers ITS4/ITS5 (White et al. 1990) and EF1/EF2, respectively (accession no. MZ956758 and OL365370). BLASTn search showed that the amplified ITS and TEF1 sequences had 99.71% (680/682 bp) and 99.80% (498/499 bp) identity with the A. rolfsii isolates CBS 115.22 (MH854711.1) and Sr_286 (JF267815), respectively. Neighbor-joining phylogenetic tree based on the ITS sequences revealed that LHBJ2-4 clustered with A. rolfsii isolates. For pathogenicity test, three potted A. vera plants (~30 cm tall) were inoculated by placing a 0.5 cm mycelial plug of isolate LHBJ2-4 (three-day old) at the base of each A. vera plant. Three A. vera plants inoculated with sterile PDA plugs served as controls. All the inoculated plants were placed in a growth chamber at 27°C under a 12/12 h light/dark cycle. The inoculation assays were carried out twice. After 5 to 7 days, stem bases of the inoculated plants showed brown lesions that were similar to those observed in the field. However, control plants remained symptomless. Athelia rolfsii was re-isolated from all the inoculated plants and identified using morphological and molecular method described above, thus confirming Koch's postulates. Although A. rolfsii has been reported to cause disease on A. vera in India (Dubey and Pandey 2009), to the best of our knowledge, this is the first report of A. rolfsii causing southern blight on A. vera in China. Because A. rolfsii has a wide host range and is difficult to control (Punja 1985), occurrence of southern blight in China might be a serious threat for A. vera production and appropriate management strategies should be developed to control this disease.

First Report of Botrytis Blight Caused byBotrytis cinereaonHelleborus nigerin Italy.

HomePlant DiseaseVol. 106, No. 1First Report of Botrytis Blight Caused by Botrytis cinerea on Helleborus niger in Italy. PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Botrytis Blight Caused by Botrytis cinerea on Helleborus niger in Italy.A. Garibaldi, D. Bertetti, G. Tabone, I. Luongo, and M. L. GullinoA. GaribaldiCentre of Competence AGROINNOVA, University of Torino, 10095 Grugliasco, ItalySearch for more papers by this author, D. BertettiCentre of Competence AGROINNOVA, University of Torino, 10095 Grugliasco, ItalySearch for more papers by this author, G. TaboneCentre of Competence AGROINNOVA, University of Torino, 10095 Grugliasco, ItalySearch for more papers by this author, I. LuongoDISAFA, University of Torino, 10095 Grugliasco, ItalySearch for more papers by this author, and M. L. Gullino†Corresponding author: M. L. Gullino; E-mail Address: [email protected]https://orcid.org/0000-0002-7706-1915Centre of Competence AGROINNOVA, University of Torino, 10095 Grugliasco, ItalySearch for more papers by this authorAffiliationsAuthors and Affiliations A. Garibaldi1 D. Bertetti1 G. Tabone1 I. Luongo2 M. L. Gullino1 † 1Centre of Competence AGROINNOVA, University of Torino, 10095 Grugliasco, Italy 2DISAFA, University of Torino, 10095 Grugliasco, Italy Published Online:9 Jan 2022https://doi.org/10.1094/PDIS-06-21-1290-PDNAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Helleborus niger (Christmas Carol) is a perennial herbaceous plant belonging to the Ranunculaceae family, appreciated for the abundant production of winter flowers. In Italy, it is marketed as potted plant. In January 2021, symptoms of an unknown blight were observed on all the 2-year-old potted plants of H. niger grown in a private garden located in Santa Margherita Ligure (Genova Province, northern Italy). Symptoms appeared on stems and petioles that turned brown, rotted, and collapsed starting from the base. The tepals also were affected and desiccated. A soft, gray mycelium appeared on affected tissues and plants totally lost their aesthetic value. Several affected stems and tepals were immersed in a solution of sodium hypochlorite (NaOCl, 1%) for 30 s. Successively, they were washed in sterilized water. Small pieces were excised from the margin of affected tissues and cultured on potato dextrose agar (PDA, Merck KGaA, Darmstadt, Germany) medium. Plates were incubated at temperatures ranging from 20 to 25°C. Gray fungal colonies developed consistently and produced dark, spheroidal to elongated sclerotia that measured 1.0 to 1.5 × 0.5 to 1.3 (mean: 1.1 × 0.9) mm. After 10 days, colonies produced branched conidiophores that supported unicellular, ovoid conidia measuring 7.9 to 12.1 × 5.2 to 9.2 (mean: 10.3 × 7.8) µm. These characteristics permitted us to identify the fungus isolated from H. niger as Botrytis cinerea Pers.: Fr. (Ellis 1971). The DNA of the isolate 21-1-O1 was extracted with the E.Z.N.A. Plant DNA Kit (Omega Bio-Tek, Norcross, GA, U.S.A.) from a pure culture grown on PDA. Successively, the product obtained from a PCR reaction with primers ITS1/ITS4 (White et al. 1990) was purified and sequenced. A 516-bp sequence (GenBank accession no. MZ417544) was achieved. BLASTn analysis (Altschul et al. 1997) of this sequence had 100% nucleotide identity with the ex-type CBS 156.71 of B. cinerea (GenBank accession no. MH860044). The pathogenicity of the isolate 21-1-O1 was confirmed by inoculating three plants of H. niger with a conidial and mycelial suspension obtained from 20-day-old cultures grown on PDA at 20 to 24°C. Each plant was sprayed with 20 ml of suspension at the concentration of 5 × 104 CFU (conidia and mycelial fragments)/ml. Three control plants were sprayed with sterile water. All plants were closed in a moistened chamber for 8 days and maintained outdoors, under the shade, at temperatures ranging from 17 to 26°C. First symptoms of browning appeared on tepals of inoculated plants about 6 days after inoculation. Later, symptoms developed on stems and petioles, affected tissues rotted and were covered by a gray soft mycelium. The pathogenicity test was repeated once. B. cinerea was reisolated from the affected tissues. Controls remained healthy. B. cinerea has been reported on H. niger in the United States (Anonymous 1960). To authors’ knowledge, this is the first report of B. cinerea on H. niger in Italy as well as in Europe. The economic importance of this disease is nowadays limited in Italy due to the reduced production of H. niger. However, the cultivation of this species might increase, following the selection of genotypes more suitable as potted plants and for cut flower production under the Mediterranean climate.The author(s) declare no conflict of interest.

English

Histoplasma capsulatum is a dimorphic fungus usually found in the few regions of West Bengal and Assam, the Gangetic Plains, western parts of India, and sporadically from southern and few northern Indian states.1 Humans are infected by inhalation of microconidia and mycelial fragments of H. capsulatum. 2 It commonly infects immunocompromised patients (HIV/AIDS, transplant patients and patients receiving immunosuppressive agents). In immunocompromised patients, it presents as nonspecific symptoms such as fever of unknown origin, weight loss, oral and aphthous ulcers, lymphadenopathy and hepatosplenomegaly.3 Here, we are reporting a case of disseminated histoplasmosis in an immunocompromised patient.

Antimicrobial activity of the edible mushroom Pleurotus eryngii (DC.) Quél grown in liquid medium

Edible mushrooms have a number of medicinal properties and this study aimed to investigate the antimicrobial activity of Pleurotus eryngii DPUA1816 in metabolic broths after being grown in submerged cultivation. Mycelial fragments of pure P. eryngii culture was inoculated in sweet potato culture medium and incubated at 150 rpm for 15 days at 25°C. Pleurotus eryngii was also cultivated for 18 days under the same conditions, the mycelial biomass was separated by filtration for quantification. The supernatant was used in the diffusion test in agar and performed against Escherichia coli CCCD-E005, Staphylococcus aureus CCCD-S009, Pseudomonas aeruginosa CCCD-P004, Candida albicans CCCD-CC001, Candida parapsilosis CCCD-CC004 and Candida tropicalis CCCD-CC002. The samples showed no inhibitory activity against bacteria, however they showed some activity against C. albicans (12.17 mm), C. parapsilosis (27.67 mm) and C. tropicalis (13.67 mm). After being cultivated for 18 days, P. eryngii was able to inhibit all yeasts after 12 days of culture, with an inhibition halo of 29.33 mm at 16 days against C. parapsilosis. This study demonstrates the antifungal potential filtered liquids from P. eryngii cultivated in purple-skinned sweet potato culture medium, which suggests the possibility of the use of this species by the pharmaceutical industry as a natural source of biological action.

Curing two predominant viruses occurring in Lentinula edodes by chemotherapy and mycelial fragmentation methods

Previous research has established that Lentinula edodes mycovirus HKB (LeV-HKB) and L. edodes partitivirus 1(LePV1) are major mycoviruses identified in L.edodes germplasm. In this paper, two different methods for curing these two dsRNA mycoviruses, ribavirin treatment and mycelial fragmentation, were evaluated for the first time. Mycelial fragmentation was found to resulted in LeV-HKB- and LePV1-cured fungal strains, whereas ribavirin treatment could eliminate LeV-HKB only. Although no LePV1-cured strain was obtained via ribavirin treatment by the end of the experiment, the relative LePV1 concentration in the eighth successive subcultures was lower than that of the untreated control. The culture features of several virus-cured strains had faster mycelial growth rate and higher colony density than the infected ones. It was also suggested that LeV-HKB infection may affect the pigmentation in plate- and bag-cultivated mycelia of L. edodes strain L135.