Mycelial Mats Research Articles

Seasonal activity and phoresy rates of Nitidulid beetles (Coleoptera: Nitidulidae) captured in stands with oak wilt infections in northern Michigan, USA.

Nitidulid beetles (Coleoptera: Nitidulidae), the overland vectors of the Bretziella fagacearum fungus that causes oak wilt, were monitored in infection centers in Quercus rubra stands in northern Michigan, USA using baited, wind-oriented traps for 2 years. First nitidulid captures, accounting for <1.5% of total annual captures, occurred in late April in both years (<50 cumulative degree days [DDs]; base 10°C). A total of 1,188 and 735 beetles representing 19 and 18 species were captured in 2018 (18 traps; 3 sites) and 2019 (16 traps; 4 sites), respectively. Ground traps (1.5 m high) captured more beetles and species than traps on canopy branches of red oaks. Most nitidulids (81-86%) were captured in May and June, but frequent precipitation and cool spring temperatures extended activity into early July in 2019. In 2018, 336 beetles representing 12 species were screened for B. fagacearum spores, but only 20 beetles from 4 species bore viable spores. Mycelial mats on red oaks killed were 4-fold more abundant in 2019 than in 2018. Of the 225 beetles screened in 2019, 56 beetles representing 6 species had viable spores. Nearly all (96%) spore-bearing beetles in both years were captured in late May or June. In bimonthly xylem samples collected from healthy trees, large earlywood vessels, presumably more vulnerable to infection than latewood, were present from May to June in 2018 and until early July in 2019. Results are consistent with mid-May to mid-July high-risk periods designated in current state and regional guidelines for oak wilt management.

First Report of Mango Stem-end Rot Caused by Pestalotiopsis kenyana in Yunnan Province, China.

Stem End Rot (SER) is a devastating post-harvest disease of mango fruits causing severe losses during storage. In 22 July 2023, 31 out of 50 intact mangoes (cv. Sensation) collected from five orchards in Huaping county (26°37'N 101°15') showed typical symptoms of SER after stored for 9 d in room temperature (24-28℃). Initially, small dark brown to black spots appeared around the fruit peduncle, which rapidly expanded through the pulp tissues. The symptomatic mangoes were surface disinfected by 3% NaClO for 30 s after soaking in 75% alcohol for 3 min, and cleaned by sterile water for 3 times. Tissues were cut from the edge of lesions, dried by sterile filter paper, transferred to PDA and cultured at 28 ℃ for 5 d (Tovar-Pedraza et al., 2020). The single-spore isolation method was used to obtain pure culture. Thirty eight isolates presented four distinct kind of morphology on PDA medium. Among them, 11 isolates with same morphology were significantly distinct from common pathogens of SER. The colonies were white and pale yellow on reverse side. Mycelia grew fast and reached the edge of 90 mm Petri dish after cultured for 5d. Pycnidia were black and scattered on the mycelial mats after 15-20 d. Conidia were fusoid, straight to slightly curved, four septa, and brown. Pigmented median cells doliiform, 14.97 - 18.62(16.11 ±0.89)×5.61- 7.28 (6.61±0.51) μm. Apical cell hyaline, subcylindrical; 1-3 tubular transparent apical appendages 12.27 - 16.68 (13.65±3.78)×1.14 - 1.99 (1.59±0.36) μm. Basal cell conical with a truncate base, hyaline, and 1-2 tubulose basal appendages with 2.85 - 7.97 (5.18±1.88)×0.99 - 1.85 (1.38±0.29) μm (n=50). These fungi were described as Pestalotiopsis kenyana. based on morphological characters (Maharachchikumbura et al., 2014) which were different from isolates characterized as other common SER pathogens (Botryosphaeria, Neofusicoccum). Based on morphology, HPSX-4 was selected for further identification. ITS region, tef1-α, β-tub of HPSX-4 were amplified and sequenced (Xun et al., 2023). The sequences were deposited in GenBank (ITS:OR889126, tef1-α:OR913431, β-tub: OR913432). The ITS, tef1-α, β-tub sequence of HPSX-4 showed 100% (525/525)，99.59% (241/242), and 100% (742/742) identity to the P. kenyana CBS442.67 sequences (ITS: NR147549，tef1-α: KM199502, β-tub: KM199395), respectively. HPSX-4 clustered with P. kenyana CBS 442.67 (type strain) based on maximum likelihood method by MEGA 7.0.21(Minh et al., 2013). Pathogenicity test was performed on 12 healthy mangoes (cv. Golek) by placing mycelial plugs around the peduncle and the middle of the fruit by pin-prick method according to Feng et al.(2023). Sterile PDA were used as control (three mangoes). Every inoculated fruit was incubated at 28°C, 95% ± 3% humidity with three replicates for each treatment. The experiment was repeated three times. Typical symptoms of SER were observed. There were no symptoms in the control group. The strain was reisolated and identified as P. kenyana with the method mentioned above which fulfilled Koch's postulates. This is the first report of P. kenyana causing SER disease on Mangifera indica L.. This study expands our understanding of the pathogen range of mango SER which conducive to prevent and control the SER caused by P. kenyana.

First Report of Strawberry Anthracnose Crown Rot Caused by Colletotrichum siamense in New England.

In the summer of 2023, the Connecticut Agricultural Experiment Station was contacted by a farm in southern Connecticut due to reports of strawberry (Fragaria × ananassa) plants showing signs of severe wilting and crown rot across multiple fields, covering ~20 hectares. Cut crowns from diseased plants had marbled red and white lesions typically associated with anthracnose crown rot (ACR). Symptomatic plants were collected from five June-bearing cultivars (cvs. AC Valley Sunset, Lyla, Dickens, and Allstar) spanning four non-adjacent fields with incidence ranging from 5-90% and severity ranging mild wilting in low incidence fields to severe wilting/mortality in high incidence fields. Internal tissue from diseased crowns was surface sterilized in 0.6% NaOCL for 3 minutes, rinsed with sterile water, and plated on potato dextrose agar. After one-week, hyphal tips of fungi were transferred to fresh plates which formed dense mycelial mats of fluffy, greyish-white hyphae. Orange spore masses formed near the center of the colonies, each of which contained numerous cylindrical and fusiform straight conidia, matching spores within the genus Colletotrichum (De Silva et al. 2019). Average conidia (n=192) length was 15.7 ± 1.6 µm and width was 5.4 ± 0.7 µm. Fungi matching this morphology were isolated from 83% of the collected symptomatic crowns and hyphae were collected from two isolates, CT5-1 and CT23-1, for DNA extraction using the GeneJET Plant Genomic DNA Purification Kit. PCR was performed using primers targeting actin (ACT), calmodulin (CAL), β-tubulin (TUB2), GAPDH (gpdA), and ITS, followed by Sanger sequencing, which yielded identical sequences for both isolates (CT5-1 Accessions numbers: PP002078-81, OR999066)(Carbone and Kohn 1999; Hassan et al. 2018; Templeton et al. 1992). These were combined with sequences from fourteen Colletotrichum genomes, all of which were aligned, trimmed, and concatenated using Mega11 (Tamura, Stecher, and Kumar 2021). Model selection was conducted using IQ-TREE and selected parameters were used to generate maximum-likelihood trees from all five loci individually and the concatenated sequence, all of which placed the isolates in a high confidence cluster with Colletotrichum siamense (Nguyen et al. 2015). To confirm the pathogenicity of the pathogen, strawberry plants (cv. Jewel) (n=5) five weeks after bare root transplant were infected. The base of each crown was penetrated 5 mm deep with a sterile 20 µL pipette tip and then inoculated with 10 µL of spores at a concentration of 106 spores/mL. Control plants (n=5) were inoculated with 10 µL of sterile water. Plants were maintained at 30°C day (16-hour)/20°C night (8-hour) in a growth chamber and assessed after 14-days. Four of the five inoculated plants had visible wilt symptoms and bisected crowns revealed the marbled red and white lesions typifying ACR. Control plants had no clear wilting and bisected crowns were visibly healthy. C. siamense re-isolated from infected tissue presented with identical hyphal /spore morphology and ITS/Tub2 were re-amplified and sequenced, yielding identical sequences to CT5-1. Plant inoculations with the same variety were repeated, yielding identical symptom development and crown lesions. C. siamense has been a dominant source of ACR throughout the southeastern US but has not previously been a major problem in the Northeast. Given the extent of the field infection, it is likely that these isolates can survive the colder winter temperatures of New England, but further experimentation is necessary to determine the extent of the pathogen's winter hardiness.

First Report of Neopestalotiopsis spp. Causing Leaf Spot and Petiole Blight on Strawberry in New England.

In October 2023, a Connecticut grower contacted The Connecticut Agricultural Experiment Station about a field of strawberry plants (Fragaria × ananassa) (cv. Ruby June) showing symptoms of severe leaf spotting and visual wilting. Upon visiting the field, leaves had lesions with a diffuse black halo and a light brown center and wilting symptoms, which appeared driven by petiole lesions and presented as dark brown stripes with a reddish-purple halo. Symptoms were observed on 80 to 90% of plants within the block, nearly all of which (>90%) presented with both leaf spots and severe wilting. Diseased tissue was collected from 20 leaves and 25 petioles, sterilized in 0.6% NaOCL, and plated on potato dextrose agar. After hyphal tipping a morphologically identical fungus was isolated from 70% of leaves and 88% of petioles, which formed a dense white mycelial mat with moderate aerial mycelium and conidiomata that exuded dark brown conidial masses. The underside of the mycelial mat was yellowish. Conidia were fusoid, ellipsoid, straight to slightly curved, 4-septate with a single basal appendage and 2-5 apical, matching the description of species within the genus Neopestalotiopsis (Maharachchikumbura et al. 2014). The average conidia (n=74) length, not including appendages, was 29.9 ± 2.1 µm and the average width, at the widest point, was 7.5 ± 0.7 µm. Aerial hyphae were collected from two isolates, CT58-1 and CT62-2, and DNA was extracted for further molecular characterization. PCR was performed with primers targeting actin (ACT), β-tubulin (TUB2), and ITS prior to amplicon sequencing (Carbone and Kohn 1999; Hassan et al. 2018). Sequences were queried against the NCBI whole genome shotgun database, and aligned sequences from 13 species (including Neopestalotiopsis, Pestalotiopsis, and Pseudopestalotiopsis) were collected for each locus. Sequences were aligned, trimmed, and concatenated using Mega11, and IQ-TREE was employed for model selection (Nguyen et al. 2015; Tamura et al. 2021). A maximum-likelihood tree placed the isolates in a high-confidence cluster with Neopestalotiopsis rosae, confirming this placement of these isolates within the genus (CT58-1 Accession #: PP715979-89; PP707735). To confirm pathogenicity, CT58-1 was grown on autoclaved strawberry leaves to induce sporulation, and a suspension of 105 spores/ml was made. Five milliliters of this spore suspension was sprayed on six 6-week-old strawberries (cv. Jewel), and water was sprayed on the same number of control plants. Plants were at 100% humidity for two days and then kept in the greenhouse for 3 weeks to observe symptoms. Inoculated plants presented with identical leaf spot and petiole lesions to field samples and no visual symptoms were observed on control plants. New isolations were made from infected petioles, which produced morphologically identical spores to those described above, and ITS/ACT loci sequencing yielded sequences identical to those of CT58-1. Spore production and plant inoculations were repeated with this new isolate, and identical symptoms were observed. This is the first report of Neopestalotiopsis infecting strawberries in New England and given the high disease incidence in the initial infected field and relative lack of disease in a neighboring field, it is likely that this pathogen was introduced on bare root plants. As the plants were sourced from a nursery in Ontario, Canada, it is likely that the pathogen is capable of overwintering in the Northeastern United States.

First report of Colletotrichum scovillei causing anthracnose fruit rot of pepper in Ohio.

In August 2019, the Ohio State University Vegetable Pathology laboratory received multiple bell and banana pepper fruits (Capsicum annuum, cvs. unknown) from Columbiana County, Ohio. The grower reported a disease incidence of 100% and severity of 70% in fruits across their pepper fields. Fruit lesions were brown, sunken, and covered with orange-colored sporulation. On banana peppers, the lesions mainly affected the blossom end of the fruits, while the lesions were distributed randomly on bell pepper fruits. Pieces of diseased tissue were cut from the fruit and surface sterilized in 0.5-0.6 % sodium hypochlorite, rinsed in sterile water, blotted dry, and placed on potato dextrose agar. All of the fungal cultures recovered were cottony, pale gray-green with shades of orange on the underside of the mycelial mat. Two representative isolates, SM209-19 (bell pepper) and SM210-19 (banana pepper), were grown on oatmeal agar to induce sporulation. Pink-orange concentric rings containing acervuli and conidia were present on the oatmeal agar plates after one week of growth at 22◦C (12-h dark/light). Conidia (n=29) were hyaline, aseptate, cylindrical in shape, and had an average length of 10.5 µm (std. dev. = 1.3 µm) and width of 4.1 µm (std. dev. = 0.6 µm) (Fig.1). DNA was extracted from both isolates using a DNeasy Plant Kit (Qiagen Inc, Germantown, MD), and partial sequences of the internal transcribed spacer (ITS) region, -tubulin 2 gene (TUB2), and glyceraldehyde-3-phosphate dehydrogenase gene (GDPH) were amplified by PCR with the following primers: ITS4/ITS5 (White et al. 1990), Bt1a/Bt1b (Glass et al. 1995), and GDF1/GDR1 (Guerber et al. 2003), and squenced. The ITS region of both isolates SM209-19 and SM210-19 (PP280815 and PP280816, respectively) showed 100% identity with C. scovillei (Cs) isolate LJTJ35 (KP748226). The partial sequences of GDPH, (PP320348, PP320349, respectively) showed 99% sequence identity with the Cs CBS 126528 (JQ948597) and 100% identity with Cs HP1 (MT826948) The partial sequences of TUB2 (PP472464 and PP472465, respectively) had 100% sequence similarity with the Cs HP1 and Cs CBS 126528 (MT826951, JQ949918 respectively). Pathogenicity was tested in a greenhouse experiment on blossoming bell pepper plants (cv. Carmen) by spraying 10 ml of 1 X 105 conidia/ml suspension onto flower blooms (nine plants per isolate). Control pepper plants were mock inoculated by spraying 10 mL of sterile deionized water. The plants were re-inoculated one week later. Brown, sunken lesions with orange sporulation developed on the fruits of inoculated plants 21 days after the initial inoculation (Fig. 2), while the mock-inoculated plants did not produce any symptoms. Culturing from symptomatic fruits on PDA, following the same method described above, produced fungal colonies with the same morphological traits previously described. C. scovillei causing anthracnose on pepper has been described in the US (Toporek et al. 2021), Brazil (Caires et al. 2014), China (Zhao et al. 2016), and different South Asia Countries (Khalimi et al. 2019). Open-field peppers are produced in Ohio on more than 5,400 acres, with a value of more than $53 million, with anthracnose being one of the most severe fungal diseases reducing yield. This newly reported Colletotichum species could represent a further threat for this crop. Further studies evaluating fungicide sensitivity and efficacy against this pathogen will be of crucial importance for disease management.

A new species of Xanthagaricus (Agaricaceae, Agaricales) from Lahore, Punjab, Pakistan

A new species, Xanthagaricus lahorensis, is described based on collections from the Lahore District, Pakistan. Morphological data and phylogenetic analyses based on nrITS &amp; LSU supported that the species is undescribed earlier. Xanthagaricus lahorensis is characterized by olive–yellow pileus with olive squamules at center going outward, dull yellowish-brown stipe, rough surface without rhizomorphic mycelial mat, absence of annulus, small, lacrymoid, brown walled with olivaceous guttule basidiospores (on average 4.2 × 3.2 µm), clavate, obovoid basidia, obovoid, clavate to broadly clavate cheilocystidia, and presence of clusters of cylindrical caulocystidia. With this new species, seven Xanthagaricus species have been reported from Pakistan. A key to the Pakistani Xanthagaricus species is provided in addition to the new species’ full description, illustrations, and phylogenetic analysis results.

A new foliicolous fossil-species of Asterina Lév. (Asterinaceae; Asterinales) associated with Calophyllum L. from the Siwalik of Eastern Himalaya and its implications

The present study reports and describes a new foliicolous fossil-species of Asterina Lév. (Asterinaceae: Asterinales: Dothideomycetes) namely, A. siwalika sp. nov., on a compressed leaf assigned to modern Calophyllum L. (Calophyllaceae) recovered from the Siwalik sediments (early Miocene to Pliocene: Chunabati Formation) of Darjeeling, Eastern Himalaya. This epifoliar Siwalik fungal species is characterized by a dark brown, sub-dense mycelial mat; septate, branched, superficial, oppositely arranged hyphae with one to two-celled, alternate to oppositely arranged appressoria; flattened, circular, non-ostiolate thyriothecia, scutellum with radially arranged isodiametric to cylindrical cells; and bi-celled, one-septate, conglobate to ellipsoidal, striated ascospores. Here, we reconstruct for the first time a possible sexual life cycle of A. siwalika. The in-situ occurrence of Asterina on Calophyllum leaf cuticles suggests a warm and humid tropical climate and a possible host-parasitic relationship that might have existed in the ancient forest of the Darjeeling sub-Himalaya region during the time of deposition. The conclusion is supported by published qualitative and quantitative climatic data using plant megafossils recovered from the same fossil locality.

First report of the new Neopestalotiopsis species causing strawberry leaf spot and fruit rot in Georgia.

In April 2023, symptomatic strawberry (Fragaria × ananassa) plants (cv. 'Camarosa' and 'Florida Brilliance') were observed at a commercial farm in Worth County, GA (USA). Symptoms included foliar, irregularly distributed, and different-sized spots (dark brown with light brown centers) and dark brown V-shaped necrotic areas starting at the leaf edge. By the time of sample collection, ~50% incidence was reported in the field. Leaf samples were collected and shipped overnight to the laboratory. Black acervuli were observed readily on old necrotic foliar lesions. Conidial morphology was consistent with that observed with Neopestalotiopsis species (Maharachchikumbura et al. 2014). Conidia were ellipsoid to fusiform, five-celled, with three light brown median cells and one hyaline apical and basal cell. Apical cells had two-to-four flexuous appendages, and the basal cell had one non-flexuous appendage (Fig 1). The average (n = 20) conidia length, not including the appendages, was 26.6 µm (SD: 2.8), and width was 6.3 µm (SD: 0.94). Fungal isolation was conducted on acidified PDA and incubated at 25°C for 6 days. Dense, white mycelia were observed on the upper plate surface, while a pale pink/orange coloration was observed on the underside (Fig 1). Black acervuli formed on the surface of the white mycelial mat. Six isolates were purified and selected to confirm the species identity. DNA was extracted from 6-day-old cultures and PCR was conducted following Kaur et al. (2023). Amplified DNA was digested with the restriction enzyme BsaWI and two bands were clearly visualized (~130 and ~290 bp), along with a faint band of 20-bp (Fig 2). Four of the six isolates were selected for sequencing of the β-tubulin gene. BLAST queries using the consensus sequence showed that all isolates had 100% identity to strain N21002 from Florida (FL), characterized as Neopestalotiopsis sp. (Kaur et al. 2023). One representative isolate (AJ07-2023) was deposited in GenBank (accession No. PP316103). Pathogenicity tests were performed on 27-day-old transplants of Sensation 'Florida127' provided by Natalia Peres from the UF. Plants were grown in 10.5 cm pots in the greenhouse. Isolate AJ07-2023 was grown on PDA for 30 days at 25°C, and the spore suspension was adjusted at 106 spore/ml. Five strawberry plants were sprayed with 5 ml of inoculum using a Preval sprayer with a CO2 canister, and 5 plants were sprayed with sterile distilled water. Plants were placed in a growth chamber for 6 days and covered with plastic bags after the sixth day to maintain ~85% relative humidity and 25°C. Foliar symptoms, including dark-brown circular lesions occurring towards the edge of leaves with light-brown center and light-yellow halo, developed 13 days after inoculation. No symptoms were observed on control plants. Neopestalotiopsis sp. was reisolated from inoculated plants as described above. Colony, conidial morphology, and PCR results were consistent with the original isolates. Neopestalotiopsis disease has been reported on strawberry in FL (Baggio et al. 2021), OH (Rotondo et al. 2022) and IN (Guan et al. 2023). Although the disease has been observed sporadically in GA since 2020 (Brannen, personal communication), to our knowledge, this is the first official report of the new Neopestalotiopsis sp. in GA. It has been reported that this new strain is more aggressive on fruits and leaves than other Neopestalotiopsis spp. (Baggio et al. 2021), therefore, accurate identification is critical for proper management.

The chemotrophic behaviour of Aspergillus niger: Mapping hyphal filaments during chemo-sensing; the first step towards directed materials formation

In the development of fungal based materials for applications in construction through to biomedical materials and fashion, understanding how to regulate and direct growth is key for gaining control over the form of material generated. Here, we show how simple ‘chemical food’ cues can be used to manipulate the growth of fungal networks by taking Aspergillus niger as an exemplar species. Chemotrophic responses towards a range of nitrogen and carbon containing biomolecules including amino acids, sugars and sugar alcohols were quantified in terms of chemotrophic index (CI) under a range of basal media compositions (low and high concentrations of N and C sources). Growth of filamentous networks was followed using fluorescence microscopy at single time points and during growth by an AI analytical approach to explore chemo sensing behaviour of the fungus when exposed to pairs (C-C, C-N, N-N) of biomolecules simultaneously. Data suggests that the directive growth of A. niger can be controlled towards simple biomolecules with CI values giving a good approximation for expected growth under a range of growth conditions. This is a first step towards identifying conditions for researcher-led directed growth of hyphae to make mycelial mats with tuneable morphological, physicochemical, and mechanical characteristics.

First report of Bretziella fagacearum infecting chestnut in Michigan.

In the summer of 2021, a 20-year-old 'Colossal' (Castanea sativa × C. crenata hybrid) tree in a commercial chestnut orchard in northwest Michigan suddenly declined. Until 2023, an additional 26 adjacent trees declined, suggesting the occurrence of root-graft transmission of the pathogen. The initial wilting of leaves progressed to complete tree death in about 10 days. Symptoms included wilting, and bronzing, followed by tanning starting at leaf apex and margins, with significant defoliation. Sometimes black-to-brown streaks of discoloration appear in the sapwood, with no signs of mycelial mat production on dead trees. Branches from symptomatic trees in two different areas of the orchard were submitted to Plant and Pest Diagnostics at Michigan State University. Bretziella fagacearum (Bretz) Z.W. de Beer, Marinc., T.A. Duong & M.J. Wingf. was detected in both samples using nested PCR (Wu et al. 2011) and qPCR (Bourgault et al. 2022). The products of the nested PCR were sequenced (GenBank accession nos. OR522695-OR522696) and BLASTn search results showed 100% identity to an ex-type strain of B. fagacearum (MH865866). Surface-sterilized discolored sapwood chips were plated on acidified potato dextrose agar (aPDA). Bretziella fagacearum was consistently recovered; colony and endoconidia morphology aligned with the description of the pathogen (De Beer et al. 2017). A pure culture (BF277) was obtained for inoculation experiments. To confirm pathogenicity, 10 'Colossal' chestnut seedlings (average stem diameter of 9 mm) were inoculated in the greenhouse with a 14-day old culture of BF277. Using a conical drill bit, two 0.4 mm diameter holes were drilled, one was 5 cm above the soil line at a 45° angle and the other was on the opposite side of the stem at least 10 cm above the soil line. A 50-µl conidial suspension (1 × 107 conidia per ml) was applied and the holes were sealed with Parafilm. Five 'Colossal' seedlings were inoculated with sterile water. Leaf epinasty with bent petioles was observed 14 days later. Leaf wilting and necrosis similar to natural infection in the orchard were observed at 24 and 34 days after inoculation, respectively. Water-inoculated control plants showed no symptoms. Bretziella fagacearum was reisolated from symptomatic plants by surface sterilizing leaf petioles with 75% ethanol (30 s), followed by 10% (v/v) bleach (1 min), and two rinses with sterile deionized water (>1 min). Petiole pieces (~1 cm) were plated on aPDA. The pathogen was reisolated from six symptomatic plants and detected using qPCR in the remaining four seedlings. Bretziella fagacearum was not detected in control plants. The identity of the recovered fungus was confirmed following the amplification of the internal transcribed spacer (ITS) from extracted genomic DNA, as described in Chahal et al. 2022. The resulting PCR product was sequenced and assembled into a consensus sequence using Geneious Prime. The consensus sequence (accession no. OR515809) revealed 100% identity to the ex-type of B. fagacearum (KU042044). This is the first record of B. fagacearum infecting chestnut trees in Michigan. Previously, B. fagacearum has been reported infecting Chinese chestnut (C. mollissima) in Missouri (Bretz and Long, 1950). Oak wilt is widely distributed in Michigan and is the predominant disease afflicting red oaks in the Midwestern U.S. Consequently, constant vigilance and monitoring are essential in chestnut orchards to promptly detect and effectively manage potential infections.

Analysis of Molecular Diversity in Castanopsis sieboldii with Felt Disease Caused by Septobasidium sp.

In 2020, within the Dongbaekdongsan area in Jeju Island, a Septobasidium sp. associated with a felt disease in &lt;i&gt;Castanopsis sieboldii&lt;/i&gt; (Makino) Hatus. ex T. Yamaz. &amp; Mashiba was identified. The symptom included the presence of brown, thin, and silk-like mycelial mats attached to the tree's bark, displaying variations in size from large to small. To induce hyphal growth, the samples collected were incubated in a moist chamber, and the newly formed hyphae were subjected to genomic DNA extractions. The nucleotide sequences of the internal transcribed spacer and small subunit rDNA genes were determined, and molecular characteristics among the isolates were investigated through polymerase chain reaction-based restriction fragment length polymorphism analysis. This &lt;i&gt;Septobasidium&lt;/i&gt; sp. exhibited distinct morphological and phylogenetic features compared to those that were previously reported in South Korea. Consequently, this strain is taxonomically classified as a provisionally novel species of &lt;i&gt;Septobasidium&lt;/i&gt;. Furthermore, the observed felt disease exhibited a high degree of host specificity, as it was exclusively identified in &lt;i&gt;C. sieboldii&lt;/i&gt; without occurrence in other tree species at the time of observation.

Invitro analysis of antifungal effects of botanicals on sclerotinia sclerotiorum causing white mold disease

White mold, Sclerotinia sclerotiorum, is a devastating fungal plant pathogen that has affected many crop species worldwide. Using chemicals to control the disease has been practiced over the years, whose prolonged application has negatively impacted the environment, thus finding an organic solution is crucial. The analysis quantifies the effect of 5 different local plants that have been proven to have fungicidal properties; Artemisia vulgaris L., Azadirachta indica L., Zingiber officinale Roscoe, Allium sativum L., and Lantana camara L. Poisoned food technique was used to study the inhibition effect, carried out by inoculating and growing the fungus on PDA media infused with botanical extracts. The data of mycelial mat diameter was recorded till the control plates were fully occupied. The growth-inhibiting capacity was found as 100% by Allium sativum, 28% &amp; 43% by Azadirachta indica, 22.44% &amp; 44% by Zingiber officinale, and 16.33% &amp; 8.55% by Lantana camara at 10% and 20% concentrations respectively. Only a slight difference between the overall inhibition effect of the two concentrations was found with 37.22% inhibition by 20% concentration and 33.38% inhibition by 10% concentration. No inhibition effect was observed from Artemisia vulgaris which could be due to heat neutralization of the active constituent during sterilization. Further research needs to be conducted using the botanical with different sterilization techniques. This in-vitro study identified garlic as a critical antifungal alternative to conventional fungicides. Field experiments need to be done to prove its effectiveness.

Removal of perfluorooctanoic acid (PFOA) in the liquid culture of Phanerochaete chrysosporium

Perfluorooctanoic acid (PFOA) is becoming a concern due to its persistence, bioaccumulation, and potential harmful effects on humans and the environment. In this study, the fungus Phanerochaete chrysosporium (P. chrysosporium) was used to remove the PFOA in liquid culture system. The results showed that the average activities of laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP) enzymes secreted by P. chrysosporium were 0.0003 U/mL, 0.013 U/mL, and 0.0059 U/mL, respectively, during the incubation times of 0–75 days. The pH of 3 and incubation time of 45–55 days were the optimum parameters for the three enzymes activities. The enzyme activities in P. chrysosporium incubation system were firstly inhibited by adding PFOA and then they were enhanced after 14 days. The maximum removal efficiency of PFOA (69.23%) was achieved after 35 days in P. chrysosporium incubation system with an initial PFOA concentration of 0.002 mM and no veratryl alcohol (VA). Adsorption was not a main pathway for PFOA removal and the PFOA adsorbed in fungi mycelial mat accounted for merely 1.91%. The possible products of PFOA contained partially fluorinated aldehyde, alcohol, and aromatic ring. These partially fluorinated compounds might result from PFOA degradation via a combination of cross-coupling and rearrangement of free radicals.

Genome-wide identification of Sclerotinia sclerotiorum small RNAs and their endogenous targets

BackgroundSeveral phytopathogens produce small non-coding RNAs of approximately 18–30 nucleotides (nt) which post-transcriptionally regulate gene expression. Commonly called small RNAs (sRNAs), these small molecules were also reported to be present in the necrotrophic pathogen Sclerotinia sclerotiorum. S. sclerotiorum causes diseases in more than 400 plant species, including the important oilseed crop Brassica napus. sRNAs can further be classified as microRNAs (miRNAs) and short interfering RNAs (siRNAs). Certain miRNAs can activate loci that produce further sRNAs; these secondary sRNA-producing loci are called ‘phased siRNA’ (PHAS) loci and have only been described in plants. To date, very few studies have characterized sRNAs and their endogenous targets in S. sclerotiorum.ResultsWe used Illumina sequencing to characterize sRNAs from fungal mycelial mats of S. sclerotiorum spread over B. napus leaves. In total, eight sRNA libraries were prepared from in vitro, 12 h post-inoculation (HPI), and 24 HPI mycelial mat samples. Cluster analysis identified 354 abundant sRNA clusters with reads of more than 100 Reads Per Million (RPM). Differential expression analysis revealed upregulation of 34 and 57 loci at 12 and 24 HPI, respectively, in comparison to in vitro samples. Among these, 25 loci were commonly upregulated. Altogether, 343 endogenous targets were identified from the major RNAs of 25 loci. Almost 88% of these targets were annotated as repeat element genes, while the remaining targets were non-repeat element genes. Fungal degradome reads confirmed cleavage of two transposable elements by one upregulated sRNA. Altogether, 24 milRNA loci were predicted with both mature and milRNA* (star) sequences; these are both criteria associated previously with experimentally verified miRNAs. Degradome sequencing data confirmed the cleavage of 14 targets. These targets were related to repeat element genes, phosphate acetyltransferases, RNA-binding factor, and exchange factor. A PHAS gene prediction tool identified 26 possible phased interfering loci with 147 phasiRNAs from the S. sclerotiorum genome, suggesting this pathogen might produce sRNAs that function similarly to miRNAs in higher eukaryotes.ConclusionsOur results provide new insights into sRNA populations and add a new resource for the study of sRNAs in S. sclerotiorum.

Phase-Dependent Differential In Vitro and Ex Vivo Susceptibility of Aspergillus flavus and Fusarium keratoplasticum to Azole Antifungals.

Fungal keratitis (FK) is an invasive infection of the cornea primarily associated with Aspergillus and Fusarium species. FK is treated empirically with a limited selection of topical antifungals with varying levels of success. Though clinical infections are typically characterized by a dense network of mature mycelium, traditional models used to test antifungal susceptibility of FK isolates exclusively evaluate susceptibility in fungal cultures derived from asexual spores known as conidia. The purpose of this study was to characterize differences in fungal response when topical antifungal treatment is initiated at progressive phases of fungal development. We compared the efficacy of voriconazole and luliconazole against in vitro cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. A porcine cadaver corneal model was used to compare antifungal efficacy of voriconazole and luliconazole in ex vivo tissue cultures of A. flavus and F. keratoplasticum at 0, 24, and 48 h of fungal development. Our results demonstrate phase-dependent susceptibility of both A. flavus and F. keratoplasticum to both azoles in vitro as well as ex vivo. We conclude that traditional antifungal susceptibility testing with conidial suspensions does not correlate with fungal susceptibility in cultures of a more advanced developmental phase. A revised method of antifungal susceptibility testing that evaluates hyphal susceptibility may better predict fungal response in the clinical setting where treatment is often delayed until days after the initial insult.

First report of Athelia rolfsii associated with southern blight disease of Amorphophallus muelleri in China.

Amorphophallus muelleri, known as konjac, is widely used in the biomedicine and food processing due to its richness in glucomannan. Between the years of 2019 to 2022, severe outbreaks of southern blight on Am. muelleri were observed during August and September in the main planting region of Mile city. The average disease incidence was 20%, resulted in 15.3% of economic losses in approximately 10,000 m2. Infected plants showed wilting and rotting and were covered with white dense mats of mycelia and sclerotia on both petiole base and tubers. Am. muelleri petiole base covered with mycelial mats were collected for pathogen isolation. The infected tissues (n=20) were washed with sterile water and surface disinfected with 75% alcohol for 60 seconds, rinsed three times with sterile water, cultured on rose bengal agar (RBA) and incubated at 27 ℃ for two days (Adre et al. 2022). Individual hyphae were transferred to new RBA plates and incubated at 27 ℃ for 15 days to obtain purified cultures. Five representative isolates were subsequently obtained and exhibited identical morphological characteristics. All isolates produced dense, cotton-white aerial mycelia and had a daily growth rate of 1.6 ± 0.2 mm (n=5). After 10 days, all isolates formed sclerotia in spherical (diameter range 1.1 to 3.5 mm, aver. 2.0 ± 0.5 mm; n=30) and irregular shapes. The number of sclerotia per plate ranged from 58 to 113 (aver=82; n=5). These sclerotia were initially white and gradually turned brown as they matured. A representative isolate (17B-1) was selected for molecular identification and the translation elongation factor (TEF, 480 nt.), internal transcribed spacer (ITS, 629 nt.), large subunit (LSU, 922 nt.), and small subunit (SSU, 1016 nt.) regions were amplified with the primers EF595F/EF1160R (Wendland and Kothe 1997), ITS1/ITS4 (Utama et al. 2022), NS1/NS4, and LROR/LR5 (Moncalvo et al. 2000), respectively. The ITS (GenBank accession no. OP658949), LSU (OP658955), SSU (OP658952), and TEF (OP679794) sequences were 99.19%, 99.78%, 99.31%, and 99.58% similar to those of At. rolfsii isolates (MT634388, MT225781, MT103059, and MN106270, respectively). Thus, the fungus represented by isolate 17B-1 was identified as At. rolfsii, corroborating the identification of Sclerotium rolfsii Sacc., the anamorph, based on cultural and morphological features. Pathogenicity tests were performed on six-month-old asymptomatic Am. muelleri plants (n=30) grown in pots with sterile soil in a greenhouse at 27 °C and 80% relative humidity. The petiole base was scratched with a sterile blade and 20 plants were inoculated by placing a 5 mm2 mycelial plug of five-day-old isolate 17B-1 on the wound. Sterile RBA plugs were used on 10 wounded control plants. After 12 days, all inoculated plants exhibited symptoms similar to those observed in the field, while the control plants showed no symptoms. The morphological and molecular identification of the fungus reisolated from inoculated petioles confirmed its identity as At. Rolfsii, fulfilling Koch's postulates. S. rolfsii was first reported on Am. campanulatus in India (Sarma et al. 2002). As At. rolfsii causes konjac diseases in all Amorphophallus growing areas (Pravi et al. 2014), the importance of At. rolfsii as an endemic pathogen of Am. muelleri in China needs to be recognized, and its prevalence should be determined as a first step to managing this disease.

Ganoderma zonatum causes butt rot of areca (Dypsis lutescens) and robellini (Phoenix roebelenii) palms in Florida.

Ganoderma butt rot is a lethal disease of palms (Arecaceae) prevalent in palm-growing regions in the US that infects at least 58 species of palms (Elliott and Broschat 2001). Early symptoms appear as wilting of older fronds in the lower part of the canopy, and as disease progresses, wilting advances to younger leaves higher in the canopy towards the unopened spear leaf eventually killing the palm. A characteristic sign of the disease is the appearance of fruiting bodies (basidiomata) at the base of the palm trunk close to soil line. Ganoderma butt rot disease was detected on clustering palm species, areca palms, with 9 (82%) clusters showing Ganoderma basidiocarps and dead stumps, and mortality was observed in 5 (45%) clusters. A sterile scalpel was used to transfer the context tissue from Ganoderma basidiomata to full-strength potato dextrose agar selective media supplemented with streptomycin (100 mg/l), lactic acid (2 ml/l) and benomyl (4 mg/l). The pure culture for isolate GAN-33 was grown at 28°C in complete darkness for 10 days. The fungal colony was ivory white in color that grew radially as a dense mycelial mat without any sporulation. To establish the identity of the fungus, DNA was extracted using the Qiagen DNeasy PowerSoil kit (Cat. #12888). Three barcoding genes, nuclear ribosomal DNA internal transcribed spacer (ITS) region, RNA polymerase II subunit 2 (rpb2) and translation elongation factor 1α (tef1α) were amplified using primers ITS1/ITS4 (White et al 1990), bRPB2-6f/bRPB2-b7.1R (Matheny et al 2007) and EF1-983F/EF1-2212R (Matheny et al 2007), respectively. The sequences were deposited in GenBank, accession numbers KX853442, KX853466 and KX853491 for ITS, rpb2 and tef1α, respectively (Elliott et al 2018). Comparison to the NCBI nucleotide sequence database identified isolate GAN-33 as Ganoderma zonatum based on 100, 99 and 99% similarity to ITS, rpb2 and tef1α sequences, respectively. Pathogenicity of G. zonatum isolate GAN-33 was determined on 1-year old seedlings of areca palm (Dypsis lutescens) and pygmy date palm (Phoenix roebelenii). Ganoderma zonatum inoculum was prepared by transferring two-week old cultures to autoclaved wheat berries and allowed to colonize for two weeks. Seedlings were gently removed from the pot and the roots were trimmed before placing them back in the pot ensuring that the roots were in contact with the G. zonatum colonized wheat berries. The inoculated and control seedlings were maintained in a growth chamber at 28°C 60% RH (daytime) and 24°C 50% RH (night time), 12h:8h light:dark period, and watered twice a week. Initial wilting symptoms started appearing approximately one month after inoculation and mortality was observed for a total of four seedlings at three months after inoculation i.e., 2 out of 3 G. zonatum inoculated seedlings died for both areca and robellini palms, whereas the non-inoculated areca and robellini palm control seedlings remained healthy and alive. The pathogen was re-isolated from inoculated roots, and its identity was confirmed by colony morphology and PCR using G. zonatum specific primers (Chakrabarti et al 2022). To the best of our knowledge this is the first report establishing G. zonatum as the pathogen responsible for Ganoderma butt rot of palms.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Stevia rebaudiana in Arizona, USA.

Stevia (Stevia rebaudiana Bertoni) is an important medicinal crop grown worldwide. Leaves of stevia contain a non-caloric sweetener, stevioside, which is used as a substitute to artificial sweeteners. In August 2022, symptoms of chlorosis, wilting, and root rot were observed in about 30 % of stevia plants growing at the Agricultural Station at Yuma Agricultural Center, Yuma, AZ, USA (32.7125° N, 114.7067° W). Infected plants initially showed chlorosis and wilting, and the plants eventually died with foliage remaining intact to the plant. Cross sections of the crown tissue of affected stevia plants showed necrotic tissue and a dark brown discoloration in areas of the vascular and cortical tissues. Dark brown microsclerotia were observed on stem bases and necrotic roots of the infected plants. Five symptomatic plants were sampled to isolate the pathogen. Root and crown tissues (0.5 to 1 cm) were surface disinfested with 1% sodium hypochlorite for 2 min, rinsed three times with sterile water, and plated onto potato dextrose agar (PDA). All the five isolates displayed rapid mycelial growth on PDA at 28°C with a 12-h photoperiod. The mycelia were initially hyaline and turned from gray to black after 7 days. Masses of dark spherical to oblong microsclerotia were observed after 3 days on PDA, measuring an average of 75 µm width × 114 µm length (n=30). For molecular identification, genomic DNA was extracted from mycelia and microsclerotia of a representative isolate (Yuma) using the DNeasy Plant Pro kit (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS), translation elongation factor-1α (TEF-1α), calmodulin (CAL), and β-tubulin (β-TUB) regions were amplified using the primer sets, ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), MpCalF/MpCalR (Santos et al. 2020), and T1/T22 (O'Donnell and Cigelink 1997), respectively. A BLAST search of sequences revealed 98.7 to 100% identity to Macrophomina phaseolina sequences (MK757624, KT261797, MK447823, MK447918). Both morphological and molecular characteristics confirmed the fungus as M. phaseolina (Holliday and Punithaligam 1970). Sequences were submitted in the GenBank under accession numbers OP599770 (ITS), OP690156 (TEF-1α), OP612814 (CAL), and OP690157 (β-TUB). Pathogenicity assay was performed on 9-week-old stevia plants (var. SW2267), grown in 4-inch planters in the greenhouse. The inoculum was made from a 14-day-old culture of M. phaseolina grown in conical flasks (250 ml) in potato dextrose broth at 28°C. Mycelial mats of the fungus were blended in 250 ml of sterile distilled water, filtered through four layers of cheesecloth, and then calibrated to 105 microsclerotia/ml using a hemocytometer. Twenty healthy plants were inoculated by soil drenching 50 ml of the inoculum per pot. Soil drenching using sterile distilled water was done on 5 non-inoculated control plants. Plants were maintained in the greenhouse at 28 ± 3°C with 12 h photoperiod. After 6 weeks, necrosis at the base of petioles and chlorosis of the leaves, followed by wilting were noticed on all 20 inoculated plants, whereas all the 5 control plants remained healthy. The fungus was reisolated and identified as M. phaseolina based on the morphology and sequences of ITS, TEF-1α, CAL and β-TUB regions. Although M. phaseolina has been reported earlier on stevia in NC, USA (Koehler and Shew 2018), this is a first report from AZ, USA. M. phaseolina is known to be favored by high soil temperatures (Zveibil et al. 2011), thus represents a potential threat to stevia production in AZ, USA in coming years.

A Rapid Method for Measuring In Vitro Growth in Entomopathogenic Fungi.

Quantifying the growth of entomopathogenic fungi is crucial for understanding their virulence and pathogenic potential. Traditional methods for determining growth, such as biomass determination or colony growth area, are time-consuming and quantitatively and spatially limited in scope. In this study, we introduce a high-throughput method for rapidly measuring fungal growth using spectrophotometry in small-volume, liquid media cultures in 96-well microplates. Optical density (OD) changes were directly correlated with dry weight of samples for six isolates from three species of the genus Metarhizium to validate spectrophotometric growth measurements, and investigate species- and isolate-specific effects. We quantified fungal biomass from the microcultures by extracting, drying, and weighing mycelial mats. From the relationship established between OD and biomass, we generated standard curves for predicting biomass based on the OD values. The OD measurements clearly distinguished growth patterns among six isolates from three Metarhizium species. The logistic growth phase, as captured by the OD measurements, could be accurately assessed within a span of 80 h. Using isolates of M. acridum, M. brunneum, and M. guizhouense, this technique was demonstrated to be an effective, reproducible, and simple method for rapidly measuring filamentous fungal growth with high precision. This technique offers a valuable tool for studying the growth dynamics of entomopathogenic fungi and investigating the factors that influence their growth.

A layered solid-state culture system for investigating the fungal growth and decay behaviour on the cellulosic substrate

Mycelium-based materials have attracted growing interest, facilitating their development for various new applications. Finding for suitable fungal species and strains enables further technical breakthroughs in their development and quality control. Here, we developed a model solid-state culture system to screen fungal strains efficiently for mycelium-based materials production. A piece of silicone-coated paper set on the general agar plate allows for isolation of the mycelial mat from the substrate. The mycelial growth and density can be evaluated by weighing the mycelial mat. We used the paper substrate after fungal incubation to investigate the relationship between substrate degradation and the contact time with hyphae. It yielded further insights into the fungal decay. Ten basidiomycetes were assessed for their fungal growth and degradation behaviour of the substrate using this method. Pleurotus floridanus FBCC375 showed a dense and elastic mycelial mat and mild degradation of the substrate. A unique decay behaviour was found in Hypsizygus ulmarius FBCC573 and Trametes versicolor FBCC564. They indicated a positional imbalance in the decay activity within the colony. This simple method is helpful for screening fungal strains and facilitates the further development of mycelium-based materials.