Mature Sclerotia Research Articles

Fruit rot causing by Athelia rolfsii (Sclerotium rolfsii) on jackfruit (Artocarpus heterophyllus) in China.

Artocarpus heterophyllus, known as jackfruit, was a tropical fruit and cultivated extensively as nutritional and medicinal properties in southern China in recent year. During July 2022, fruit rot was observed on the fruits at the bottom of jackfruit trees in an orchard in Zhanjiang, Guangdong (N21°9' 27" E110°17' 54") 3-4 days after typhoon. The incidence rate of fruit was about 0.3%. The initial symptom was white mycelia appearing on the surface of fruits. Mycelia with rhizomorphs spread rapidly over the fruits, formed white, often fan-shaped mats with the rapeseed size sclerotia. The infected fruits were water-soaked, quickly became rotten, and fell off. Sclerotia from disease fruits were incubated on PDA with 50 mg/L ampicillin at 25-28℃ in the dark for 2 days. Hyphae tips were transferred to get the purified isolates. Colonies with a radial growth rate of 23.2 mm/day had abundant aerial mycelia and profuse sclerotia on PDA. Hyphae of the isolates were transparent, branched, with clamp connections at septa, usually 2.9-8.3 µm (Ave. 5.8 µm) (n>30) wide. Aerial mycelia were whitish-cottony, with many narrow rhizomorphs. Spherical sclerotia developed at about 10 days after incubation, and gradually changed from white to tan-to-dark brown, and mature sclerotia were about 1.7 mm in size. The morphological characteristics was similar to those of Sclerotium rolfsii (teleomorph: Athelia rolfsii). To accurately identify the fungus, the internal transcribed spacer gene (ITS) and large subunit rRNA gene (LSU) of isolate CASS-BLM-1 were PCR amplified with primer pairs ITS1/ITS4 (White et al 1990) and V9G/LR5 (Klaubauf et al 2014). The amplicons were sequenced and deposited in GenBank with accession number OP535473 (ITS) and OP535474 (LSU). BLASTn results showed that the nucleotide sequences of ITS and LSU had high identity with corresponding sequences of A. rolfsii isolates CBS 191.62 (ITS: MH858139, 472/474(99.58%); LSU: MH869724, 882/885(99.66%)) (Vu et al 2019). Phylogenetic analysis based on ITS sequence data was obtained according to maximum likelihood method using MEGA analysis software, CASS-BLM1 was grouped in A. rolfsii clade with 100% bootstrap support value. Based on morphology and DNA sequences, the fungus was identified as A. rolfsii (anamorph: S. rolfsii). To fulfil Koch's postulates, healthy fruits on the tree and detached fruits were inoculated with 7-day-old sclerotia of isolate CASS-BLM1. Five unwound sites and five wound sites with a sterile needle were tested on each fruit and a sclerotium was put at each site. Fruits not inoculated with sclerotia were used as control the test was repeated three times. All fruit were enclosed in transparent plastic bags with sterile absorbent cotton moistened with sterile distilled water. The indoor and outdoor temperatures ranged from 25 to 30 ℃. Three days later, white mycelia were observed on all inoculation sites, and 5 days later, the inoculated fruits began to rot, while control fruits remained healthy. The same fungus with identical morphology and DNA sequences was re-isolated from the inoculated sites. Previously, A. rolfsii was reported to cause fruit rot disease on jackfruit in Bangladesh (Elahi et al 2021), this is the first report of A. rolfsii causing fruit rot on jackfruit in China. A. rolfsii is suitable for high temperature and humidity environment (Punja 1985), this report will help farmers to diagnose this disease, especially to strengthen the disease prevention during the typhoon season.

Occurrence of postharvest snow rot caused by Sclerotinia nivalis on Asian ginseng in China.

Asian ginseng (Panax ginseng) is a valuable medicinal plant that is commercially cultivated in China. A long postharvest storage period is required before ginseng is processed. From October 2019 to May 2020, snow rot was observed on the roots of 4- and 5-year-old fresh ginseng stored in three cold storage facilities located in Tonghua and Changbai cities in northeastern China, which are the most important regions for Asian ginseng production. We sampled 1,000 ginseng roots from the three cold storage facilities, and the average disease incidence was 21%. Initially, sparse hyphae and microsclerotia appeared on the root epidermis. Lesions gradually softened and the epidermis detached easily. Multiple infected sites slowly converged, resulting in the formation of a dense complex of multiple sclerotia and thick hyphae on the surface of the ginseng root as well as internal decay. The infection eventually spread to the adjacent ginseng roots (Fig. 1). Sixteen diseased ginseng roots were collected and then sclerotia were removed from the root surface, immersed in 1% NaClO for 2 min, rinsed three times with sterile water, and placed on potato dextrose agar (PDA) containing streptomycin (40 μg/mL) in Petri dishes. After a 3-day incubation at 20 °C in darkness, 22 suspected Sclerotinia isolates were obtained. Isolates SN1 and SN2 were randomly selected for identification. On PDA, fast-growing colonies produced white, sparse, powdery, and cotton-like aerial mycelia, and the reverse side showed the same color (Fig. 2). Small and white sclerotial primordia formed 3 days later and a ring of sclerotia was detected at the plate periphery. At 7 to 10 days after incubation, the mature sclerotia were black, spherical-to-subspherical, and elongated or fused to form irregular shapes. Each Petri dish produced 55-65 sclerotia, measuring 1.1 × 1.2 to 3.2 × 3.9 mm (n = 100). The sclerotia were firmly attached to the agar surface. The isolates were initially identified as Sclerotinia sp. (Saito 1997). After sequencing the nuclear ribosomal internal transcribed spacer region (MW927134 and MW927135) and the β-tubulin gene (MW929179 and MW929180) (White et al. 1990; Glass and Donaldson 1995), BLAST searches revealed 100% homology with JX262268 and JX296007 of the published S. nivalis strain KGC-S0601, respectively. The pathogenicity of the two isolates was tested using detached ginseng roots. Briefly, healthy roots were washed, surface-disinfested with 75% alcohol, and rinsed with sterile water. Mycelial plugs (5 mm diameter) removed from the margin of actively growing colonies on PDA were placed on the ginseng roots. For each isolate, four roots were inoculated, with two plugs per root. Additionally, PDA plugs without mycelia were used as the negative control. The roots were placed in a fresh-keeping box at 20 °C in darkness and evaluated after 7 days. The pathogenicity test was repeated twice. The symptoms on the inoculated roots were the same as those observed on the roots during cold storage, whereas the control roots remained symptomless. The same fungus was reisolated consistently from all infected roots and its identity was confirmed by resequencing, thereby fulfilling Koch's postulates. To the best of our knowledge, this is the first report of S. nivalis causing postharvest snow rot on Asian ginseng in China. The occurrence of this disease threatens the postharvest storage of Asian ginseng. Hence, effective management strategies must be developed.

Effect of Different Carbon and Nitrogen Sources on Sclerotium rolfsii sacc. Mycelial Growth and Sclerotial Development

In vitro studies were conducted on Potato Dextrose Agar using different carbon (C) and nitrogen (N) sources to evaluate their effects on the mycelial growth, and the sclerotial development of three Tunisian Sclerotium rolfsii Sacc. isolates. Radial growth was optimum on basal medium supplemented with ammonium chloride (0.48 gram of nitrogen per liter (g of N.L-1)) as N source but was restricted on L-Arginine and completely inhibited on ammonium acetate amended media (0.48 g N.L-1). Sclerotial initiation occurred from the 3rd to the 12th day of incubation for all tested isolates. Potassium nitrate was the most suitable N source for sclerotial formation whereas sclerotial development was completely inhibited on ammonium acetate amended medium. Optimal sclerotial germination was recorded using L-Arginine (78-80%) followed by L-Asparagine (46-94%) and ammonium chloride (46-88%) as N sources. Nevertheless, the lowest sclerotial germination rate was noted on sodium nitrate and ammonium acetate amended media. As for C sources (16 gram of carbon per liter (g of C.L-1)), optimal radial growth occurred using D-mannitol for Sr1 and Sr2 isolates and maltose for Sr3, but no mycelial growth was recorded using sodium citrate for all isolates. All C sources tested, except sodium citrate, were suitable for sclerotial formation, production, and germination. Mature sclerotia became brownish after 6 to 12 days of incubation and sclerotial production was highest using D-mannitol, maltose, and D-glucose, depending on isolates used, as C sources. Optimal germination of sclerotia was noted using D-glucose, D-mannitol and maltose for Sr1 isolate, maltose for Sr2 and D-glucose and maltose for Sr3. It was concluded that N and C sources are both important factors for the growth of S. rolfsii and its survival.

First Report of Web Blight on Lavender Caused by Rhizoctonia solani AG-1-IB in Korea

Lavender (Lavandula officinalis L.) is an annual or short-lived perennial herb belonging to the mint family (Lamiaceae) that originates from eastern Africa, the Mediterranean, southwest Asia, to southeast India. Lavenders are cultivated extensively as ornamental plants for garden and landscape uses, for usage as culinary herbs, and commercially for the extraction of essential oils. In June 2017, approximately 5 to 10% of plants showed web blight symptoms in a private garden located near Gangneung, Gangwon Province, Korea. Specifically, leaf tissues were blighted, and white mycelial growth was seen on the stems. Symptomatic tissues were surface sterilized with 1% NaOCl, washed twice in sterilized water, blotted on sterile filter paper, and plated on 2% water agar at 25°C. After 2 days, hyphal tips were transferred to potato dextrose agar (PDA) to obtain pure cultures. A total of five morphologically similar fungal isolates (LWB001 to LWB005) were obtained from diseased samples; isolate LWB002 was selected for identification. The fungus produced a white mycelium when young but produced a brown mycelium with dark brown sclerotia on PDA at 20 ± 2°C for 10 days. Mature sclerotia (n = 20) were dark, spheroidal, with diameters ranging from 0.2 to 2.2 mm (average 0.7 mm). The young hyphae had acute angular branching near the distal septum of the multinucleated cells, and mature hyphal branches showed approximately 90° angles. Morphological characteristics were consistent with those of Rhizoctonia solani (Sneh et al. 1991). A representative isolate (LWB002) was deposited in the Korean Agricultural Culture Collection (KACC), National Institute of Agricultural Science, Rural Development Administration, Wanju, South Korea (KACC48671), to be used for further studies. The identification of the anastomosis group of the R. solani isolate was carried out on sterilized glass slides coated with 2% water agar medium placed on Petri dishes. The representative isolate was paired with tester strains of R. solani (AG-1-IA, AG-1-IB, AG-1-IC, AG 2-2-IIIB, AG 4, AG 7, and AG 11), which were collected from KACC (Ogoshi 1987) and examined microscopically. The fungal isolate anastomized only with tester strain AG-1-IB. To obtain a DNA sequence-based identification, a total DNA was extracted directly from the mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Following DNA extraction, the internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and sequenced (White et al. 1990). BLASTn analysis of the 706 bp showed a 100, 99.71, and 99.86% similarity with sequences of R. solani AG-1-IB (teleomorph Thanatephorus cucumeris) in GenBank (JQ692293.1, GU585667.1, and KF907717.1, respectively). The nucleotide sequence has been assigned GenBank number MH122653. To confirm pathogenicity, three fungal mycelial discs (14 days old, 6.5 mm) were placed onto three wounded young plant stems on the soil line. Another three plants served as controls, for which only an agar plug was used on wounded plant stems. Then all the plants were incubated in a growth chamber (at 28 ± 2°C, 90% relative humidity, with 12/12-h light/dark). After 10 days, blight symptoms were similar to the original symptoms developed on inoculated plant leaves and stems, whereas no symptoms were observed on control plants. Five fungal isolates were reisolated from disease lesions of inoculated plants on PDA with the same morphological characters as before, and the identity was confirmed by microscopic studies, which fulfilled Koch’s postulates. Lavender web blight caused by R. solani AG-1-IB has been recorded in Italy (Garibaldi et al. 2013). To our knowledge, this is the first disease report of web blight on lavender caused by R. solani AG-1-IB in Korea, which poses a potential threat to lavender production, and our findings will be useful for the development of effective control strategies and further research.

Rice stem rot Sclerotium hydrophilum Sace isolated and characterized in Southern Karnataka

In the summer season of 2016 and 2017, in some fields of Cauvery command area of Karnataka the rice crop showed stunted growth with brownish to black lesions without distinct margin on stem at the water level during tillering and reproductive phase . The causal organism was tissue isolated on potato dextrose agar, colonies of pure cultures showed white mycelium at first and after two days white coloured scelerotial formation started which later changed to chocolate brown and after five days became black colour. The hyphal width was measured to be within a range of 6 to 10 mm and binucleate hyphae were observed between the septa. Small, globose and reddish brown to black coloured mature sclerotia measuring 0·24-0·43 mm were observed in culture. Koch's postulates was proved by inoculating five days old culture disc containing sclerotia on the stem of 6 week-old healthy rice plants. After seven days symptoms were observed on the inoculated seedlings but not on control plants. The fungus was re-isolated and similar cultural characteristics were observed as of the original culture. DNA fingerprinting of the rDNA-ITS sequence of the representative isolate MNDSR01 (Gene Accession MH393598.1) matched with that of Sclerotium hydrophilum reference (MH393598) with 100% identity. To our knowledge, this is the first report of S. hydrophilum in the Cauvery command area of Karnataka.

Comparative transcriptomics reveals potential genes involved in the vegetative growth of Morchella importuna.

True morels (Morchella spp.) are edible, medicinal mushrooms which have recently been artificially cultivated in China but stable production remains a problem. Here, we describe complete and comprehensive transcriptome of Morchella importuna at the stages of vegetative mycelium (VM), initial sclerotium (IS) and mature sclerotium (MS) by deep transcriptional sequencing and de novo assembly for the first time and which will potentially provide useful information for improving its cultivation. A total of 26,496 genes were identified with a contig N50 length of 1763bp and an average length of over 1064bp. Additionally, 11,957 open reading frames (ORFs) were predicted and 9676 of them (80.9%) were annotated. The 2605 differentially expressed genes (DEGs) identified by gene expression clustering were mainly involved with energy metabolism and could be divided into three broad clusters, of which genes in cluster_1 and cluster_2 were involved in the metabolic process of carbohydrate, polysaccharide, hydrolase, caprolactam, beta-galactosidase, and disaccharide, respectively. Genes in cluster_3 were the largest category, mainly identified with the catalytic activity and transporter activity. Overall, the enzymes involved in the carbohydrate metabolism were highly expressed, and the CAZyme (carbohydrate-active enzyme) genes were significantly expressed within cluster_3. For expression verification, 16 CAZYme genes were selected for qRT-PCR, and the results suggested that the catabolism of carbohydrates occurs mainly in the vegetative mycelium stage, and the anabolism of the energy-rich substances is the main event of mycelial growth and sclerotial morphogenesis of M. importuna.

Effects of pH and Aeration on Sclerotium rolfsii sacc. Mycelial Growth, Sclerotial Production and Germination

Sclerotium rolfsii is one of the devastating soilborne fungus responsible for significant plant losses. The effects of pH and aeration on pathogen mycelial growth, sclerotial production and germination were investigated for three Tunisian isolates. Optimal mycelial growth occurred at pH 6 for Sr2 and Sr3 isolates and at pH 6-7 for Sr1. Dry mycelial growth was optimum at pH values ranging between 4 and 7. Sclerotial initiation started on the 3rd day of incubation at all pH values tested and mature sclerotia were formed after 6 to 12 days. Optimal sclerotial production was noted at pH 5. The dry weight of 100 sclerotia varied depending on isolates and pH and occurred at pH range 4-7. At pH 9, mycelial growth, sclerotial production and dry weight of 100 sclerotia were restricted. The optimum sclerotial germination, noted after 24 h of incubation, varied depending on isolates and pH and occurred at pH 4-9. Mycelial growth was optimum in aerated plates with a significant isolates x aeration treatments interaction. Sclerotial initiation occurred at the 3rd day of incubation and mature sclerotia were observed after 6-9 days. Sclerotial development was very slow in completely sealed plates and dark sclerotia were produced only after 15 days of incubation. The highest sclerotial yields were noted in aerated plates. The highest dry weight of 100 sclerotia for Sr1 isolate was recorded in ½ sealed, no sealed and completely sealed plates, while for Sr2, it was noted in ½ and ⅔ sealed plates. For Sr3, the maximum dry weight of 100 sclerotia was recorded in ½, ⅔ and completely sealed plates. Germination of S. rolfsii sclerotia, after 24 h of incubation, did not vary significantly depending on aeration treatments and ranged from 90 to 100% for all isolates.

Effect of Temperatures and Culture Media on Sclerotium rolfsii Mycelial Growth, Sclerotial Formation and Germination

Sclerotium rolfsii is a serious soilborne phytopathogenic fungus causing serious yield loss in crops of high economic importance. In this study, the effects of temperatures and culture media were evaluated on S. rolfsii mycelial growth, sclerotial production and germination. The three isolates tested grew over a range of temperatures from 10°C to 35°C but not at 5°C and 40°C. Radial growth and dry mycelium production were highest at 30°C. Sclerotial initiation started on the 3rd day after incubation at 30°C and 35°C, and mature sclerotia were observed after 15 and 6 days of incubation, respectively. Optimal sclerotial production and the dry weight of 100 sclerotia varied depending on isolates and temperatures and occurred at 25°C-35°C. No mature sclerotia were produced at 5°C, 10°C, 15°C and 40°C. Sclerotial germination, noted after 24 h of incubation, was 96%-100% at 25°C-35°C, declined at 15°C-20°C, and was totally inhibited at 5°C, 10°C and 40°C even after 72 h of incubation. For all S. rolfsii isolates, optimal radial growth occurred on OAT medium followed by CZA, MYEA and PDA. When grown on PDA and OAT media, S. rolfsii developed cottony colonies with abundant mycelium whereas on WA and CZA pathogen growth was thin and scanty. Colonies formed on the other media developed sparse and flat mycelia. For sclerotial formation, WA and CZA were the most suitable culture media but no dark brown sclerotia were observed on NA and YDA. The highest sclerotial formation was observed on PDA and CZA. The highest sclerotium weight was produced by Sr3 isolate on PDA whereas for Sr1 and Sr2 isolates, sclerotial weights were significantly comparable on PDA, MYEA and CZA. Sclerotial germination was optimal on all tested media except NA. The significance of these findings on S. rolfsii biology is discussed.

Structure, biogenesis and nutricional dependence of <em>Paxillus ammoniavirescens</em> sclerotia (Boletales, Paxillaceae)

The objective of this work was to study the structure, composition, and biogenesis of Paxillus ammoniavirescens sclerotia, as well as their possible use as inoculum for birch mycorrhizae. By means of histochemical stains, a change of the composition and internal organization of these structures during their maturation was observed. The formation of sclerotia begins with a clustering of hyphae that gives rise to a structure with pseudoparenchyma in the central part; these immature sclerotia develop to form, at maturity, a pseudoparenchymatic outer cortex of 6 or 7 layers of hyphae around an inner medulla. The hyphae of mature sclerotia have a large number of protein bodies in contrast to scarce lipid accumulations. The formation of sclerotia is influenced by the characteristics of the culture medium and the temperature. The greatest number of sclerotia was obtained by reducing the concentration of the diammonium phosphate to 0.94 mM in the MMN medium, and maintaining the cultures at 8 °C for 2 weeks. Finally it was shown that the sclerotia of this species can be used directly as inoculum, both to generate mycelium in the culture media and to initiate mycorrhizae in birch plants in vitro.

Aspergillus flavus aswA, a gene homolog of Aspergillus nidulans oefC, regulates sclerotial development and biosynthesis of sclerotium-associated secondary metabolites

Aspergillus flavus aswA (AFLA_085170) is a gene encoding a Zn(II)2Cys6 DNA-binding domain and a transcriptional activation domain, DUF3468. Disruption of aswA yielded strains that made a truncated gene transcript and generated a fungus that produced a greatly increased number of sclerotia. These sclerotia were odd-shaped and non-pigmented (white) and different from oval and pigmented (dark brown to black) mature sclerotia. Transcriptomic analysis of the ΔaswA strain grown on potato dextrose agar plates and Wickerham agar plates showed that expression of clustering genes involved in the biosynthesis of three sclerotium-associated secondary metabolites was down-regulated. These included gene clusters of asparasone, aflatrem, and aflavarin. In contrast, those of aflatoxin, cyclopiazonic acid and kojic acid were not affected. Metabolite analyses confirmed that the non-pigmented sclerotia contained aflatoxin and cyclopiazonic acid but not other aforementioned metabolites, three asparasone analogs and dihydroxyaflavinine commonly present in mature sclerotia. Impairment in aswA gene function stalls normal sclerotial development, which in turn prevents biosynthesis and accumulation of sclerotium-specific metabolites.

First Report of Web Blight on Rose Campion (Lychnis coronaria) Caused by Rhizoctonia solani AG 1-IB in Italy.

Lychnis coronaria (syn. Silene coronaria), rose campion, is a perennial in the Caryophyllaceae used in gardens. In the summer of 2014, a web blight was observed in a private garden located near Biella (northern Italy), approximately 45°39'N 8°00'E, on 40% of 100 5-month-old plants grown in sandy soil. In the days preceding the outbreak of the disease, daytime temperatures ranged from 18 to 24°C and relative humidity from 45 to 83%. Affected plants showed pale brown discoloration of stems, starting from the base, and eventually collapsed. Under conditions of high relative humidity, a scant amount of whitish mycelium developed on leaves of about 50% of diseased plants. Eventually, infected plants died about 10 days after symptoms appeared. Symptomatic tissues of stems and leaves were disinfected for 10 s in 1% NaOCl, rinsed in sterile water, and plated on potato dextrose agar (PDA). A fungus with the morphological characters of Rhizoctonia solani (3) was consistently recovered. Three representative isolates were paired with tester strains of R. solani (AG 1, AG 2-2-IIIB, AG 4, AG 7, and AG 11) (2) and examined microscopically. The Lychnis isolates anastomosed only with the AG 1 tester strain, with low fusion frequency. The anastomosis point was obvious: the hyphal diameter at the point of anastomosis was reduced and death of adjacent cells was observed, indicating an anastomosis reaction (1). Mycelium maintained on PDA at 23 ± 1°C was coarse and reddish brown. After 5 days of growth, mycelium started differentiating numerous sclerotia, often aggregated. Mature sclerotia were dark, spheroidal, with diameters ranging from 0.2 to 1.6 (average 0.6) mm. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLASTn analysis of the 609-bp amplicon (GenBank Accession No. KM596899) showed a 98% homology with the sequence of the R. solani isolate FJ746937 obtained from Zoysiagrass. On the basis of molecular and cultural characteristics and anastomosis tests, the isolates from L. coronaria were identified as R. solani AG 1-IB (4). For pathogenicity tests performed in August, mycelial disks (8 mm diam.) from 10-day-old PDA cultures of an isolate of the fungus were placed on four healthy 6-month-old L. coronaria plants (four stem and six leaf disks per plant). Four plants inoculated with disks of PDA served as controls. Plants were covered with plastic bags for 4 days and maintained in a garden located in the same area in which the disease appeared, at field temperatures ranging from 15 to 28°C. The first symptoms developed 4 days after inoculation, and 15 days after the artificial inoculation, all inoculated plants were dead. R. solani was re-isolated from the stem of symptomatic plants, whereas no colonies developed from controls, which all remained healthy. This is the first report of blight of L. coronaria caused by R. solani in Italy or anywhere else in the world. The impact of this disease may become a significant problem for L. coronaria, a very common species in Italian gardens.

First Report of Sclerotinia minor on Brassica rapa subsp. pekinensis in Central China.

Chinese cabbage (Brassica rapa subsp. pekinensis Hanelt) is a leafy vegetable widely grown in China. In December 2012 to March 2013, a leaf rot disease was observed on the lower part of cabbage leaves in a field in Xianning, Hubei Province, China, with the incidence of 6.3% in that field. The diseased leaves showed water-soaked rot and brown symptoms at the top surface. White fluffy mycelia and small black sclerotia were produced on the lesion surface. Cabbage leaf tissues from the disease/healthy-bordering areas and the sclerotia from the lesions were separately surface-sterilized in 75% ethanol (v/v) for 30 s, followed by rinsing three times in sterilized water. The surface-sterilized tissues and sclerotia were placed on potato dextrose agar (PDA) and incubated at 22°C. Individual emerging fungal colonies from the leaf tissue pieces and the sclerotia were transferred to new 9-cm-diameter PDA plates and incubated for 15 days. A total of 40 isolates (20 from diseased tissue and 20 from sclerotia) were obtained. All the isolates grew rapidly on PDA with an average growth rate of 2.2 cm/day and produced abundant sclerotia on the colony surface (1,179 sclerotia/plate on average). None of the isolates produced conidia and any other spores in the PDA cultures. Mature sclerotia were black, irregular, spherical or elliptical, had a diameter of 0.5 to 1 mm, and easily detached from the colonies. The cultural and morphological characteristics of the isolates matched the description for Sclerotinia minor Jagger (3). Two isolates, A1 (from leaf tissue) and S2 (from a sclerotium), were further identified by analysis of the ITS region (ITS1-5.8S rDNA-ITS2) using the primer pair ITS1/ITS4. The resulting 540-bp DNA sequences (GenBank Accession Nos. KC836493 for A1 and KC836494 for S2) shared 100% identity S. minor isolate 62907 (JF279880). Pathogenicity of the isolates A1 and S2 was tested by inoculating detached cabbage leaves with mycelial agar plugs removed from the colony margin of the 3-day-old cultures. Isolates A1 and S2 were each inoculated on three leaves with three plugs per leaf. Three cabbage leaves inoculated with PDA plugs were treated as a control. The treated leaves were covered with plastic films to maintain high humidity (>90% RH) and incubated at 22°C for 72 h under the regime of 12 h light/12 h dark. Results showed that while the control leaves remained healthy, brown and water-soaked lesions appeared around the mycelial agar plugs of each isolate. Average lesion diameters were 47.5 mm for A1 and 47.8 mm for S2. Abundant small sclerotia were produced on necrotic leaf lesions after 7 days. The fungus in diseased leaf tissues was re-isolated and the morphological characteristics of the resulting fungus were the same as S. minor isolated from infected field-grown cabbages. Therefore, S. minor is the causal agent for the leaf rot disease on Chinese cabbage. S. minor has been reported to infect a few plant species in the genus Brassica, including B. rapa subsp. oleifera (3), B. oleracea var. gemmifera (3), B. napus (2), B. oleracea var. capitata (3), B. oleracea var. botrytis (3), and B. rapa (3). It was found on B. rapa subsp. pekinensis in Korea (1). To our knowledge, this is the first report of S. minor on B. rapa subsp. pekinensis in China.

Transcriptomic profiles of Aspergillus flavus CA42, a strain that produces small sclerotia, by decanal treatment and after recovery

Aspergillus flavus is a ubiquitous saprophyte and is capable of producing many secondary metabolites including the carcinogenic aflatoxins. The A. flavus population that produces small sclerotia (S strain) has been implicated as the culprit for persistent aflatoxin contamination in field crops. We investigated how the plant volatile decanal, a C10 fatty aldehyde, affected the growth and development of the S strain A. flavus. Decanal treatment yielded fluffy variants lacking sclerotia and conidia and exhibiting a dosage-dependent radial colony growth. We used RNA-Seq analysis to examine transcriptomic changes caused by decanal and after removal of decanal. Mature sclerotia contained only 80% of the total transcripts detected in all samples in comparison to 94% for the decanal treated culture. Gene ontology (GO) analysis showed that decanal treatment increased expression of genes involved in oxidoreductase activity, cellular carbohydrate metabolism, alcohol metabolism and aflatoxin biosynthesis. The treatment affected cellular components associated with cell wall, and gene expression of glucanases, α-amylases, pectinesterase and peptidase required for its biosynthesis was increased. After decanal was removed, the culture resumed sclerotial production. Moreover, its GO terms significantly overlapped with those of the untreated culture; five of the enriched molecular functions, oxidoreductase activity, monooxygenase activity, electron carrier activity, heme binding, and iron binding were found in the untreated culture. The GO term of cellular component enriched was mainly integral protein constituents of the membrane. The results suggested that decanal halted development at the vegetative state rendering the fungus unable to produce conidia and sclerotia. The induced fluffy phenotype could be related to lower transcript abundance of flbB, flbD, and flbE but not to veA expression. Increased abundance of the laeA transcript in the treated culture correlated with early transcriptional activation of aflatoxin and kojic acid biosynthesis gene clusters. Expression profiles revealed subtle differences in timing of activation of the respective 55 secondary metabolite gene clusters.

First Report of Garlic Leaf Blight Caused by Botrytis porri in China.

In the spring of each year from 2007 to 2009, a leaf blight of garlic (Allium sativum L.) was observed in more than 50 fields in Zhushan County of Hubei Province, China. Gray mold was observed on many of the blighted garlic leaves. The percentage of garlic plants with blight and gray mold symptoms ranged from 10 to 50% with one to three blighted leaves on each plant, which severely reduced the yield of young garlic plants (produced as a green vegetable). Ten strains of a Botrytis sp. were isolated from symptomatic garlic leaves collected from 10 different fields. These strains were inoculated onto potato dextrose agar (PDA) in petri dishes and incubated at 20°C for 3 to 15 days for observation of colony characteristics and morphology of sclerotia and conidia. All 10 Botrytis strains formed flat and "ropy" mycelia (mycelial strands) on PDA. Abundant sporulation with a gray powdery appearance was observed on the colonies after 6 days. Conidiophores were erect with alternate branches at the top and ranged from 907 to 1,256 μm high. Conidia were borne in botryose clusters on conidiophores, obovate, and 10.4 to 17.6 × 7.6 to 13.1 μm with an average length/width ratio of 1.36. Discrete sclerotia were produced on each colony after 15 days. Mature sclerotia were black, cerebriform and convoluted, and 1.9 to 9.1 × 1.6 to 6.5 mm. Morphological characteristics of the colonies, conidia, and sclerotia of these Botrytis strains were similar to Botrytis porri Buchwald (1,2). Strain GarlicBC-16 was selected as a representative for molecular identification. Genomic DNA was extracted from mycelia of this strain and used as a template for amplification of the internal transcribed spacer (ITS) region of rDNA using primer pair ITS1/ITS4. A 539-bp amplicon was obtained and sequenced (GenBank Accession No. EU519206). Excluding the flanking regions, the amplicon contained a 453-bp ITS sequence (ITS1 + 5.8S rDNA + ITS2) 100% identical to the ITS sequence of strain MUCL3234 of B. porri (GenBank Accession No. AJ716292). Pathogenicity of strain GarlicBC-16 was tested by inoculation of 10 young and fully expanded garlic leaves taken from 100-day-old garlic plants with mycelial agar plugs (three plugs per leaf and spaced by 5 cm). Ten garlic leaves inoculated with agar plugs of PDA alone served as controls. Inoculated garlic leaves were covered with a plastic film (0.1 mm thick; Gold Mine Plastic Industrial Ltd. Jiangmen, China) and incubated at 20°C with 12-h light/12-h dark. Control leaves remained healthy after 48 to 120 h, but gray, water-soaked lesions appeared on leaves inoculated with strain GarlicBC-16 after 48 h. The average lesion length reached 27.3 mm after 90 h and abundant sporulation was produced on necrotic leaf lesions after 120 h. Microscopic examination showed the shape and size of conidia that formed on garlic leaf lesions were similar to those formed by strain GarlicBC-16 on PDA. On the basis of the isolation, identification, and pathogenicity tests, B. porri was determined to be the causal agent of garlic leaf blight in Zhushan County. B. porri has been reported to cause neck rot of leek (A. porrum) (1) and clove rot of garlic (2), and has been isolated from asymptomatic foliage and seeds of A. cepa (3). To our knowledge, this is the first report of garlic leaf blight caused by B. porri in China.

The development-specific protein (Ssp1) from Sclerotinia sclerotiorum is encoded by a novel gene expressed exclusively in sclerotium tissues

The gene encoding a development-specific protein (Ssp1) was identified; it previously was described as the major protein present in mature sclerotia of Sclerotinia sclerotiorum. To determine the developmental specificity of ssp1 gene expression in relation to protein accumulation we examined transcript and protein accumulation during various growth and development stages of the lifecycle. We found that ssp1 transcript accumulated exclusively within developing sclerotium tissue and not in any other examined stage of growth or development. In contrast high levels of Ssp1 protein were detectable by western blot and tandem mass spectrometry analyses in all stages of sclerotium as well as apothecium development. Immunolocalization further indicated that Ssp1 protein bodies were depleted from the sclerotium tissue surrounding the site of apothecium germination, but by this method Ssp1 was not detected in the apothecium. Together these findings suggest that Ssp1 is not metabolized during carpogenic germination, instead it is translocated from the sclerotium to the apothecium in an antigenically novel form. Outside the Sclerotiniaceae ssp1 homologs were found only from the sclerotium-forming Aspergillus species A. flavus and A. oryzae. Further studies concerning the regulation and function of this gene and its occurrence in other species have the potential to inform our understanding of sclerotium development and the evolution of sclerotia and other forms of fungal stroma.

Symptomatology and morphology of Claviceps cyperi on yellow nut sedge in South Africa

Symptoms of ergot on yellow nut sedge, germination of sclerotia of the causal organism, Claviceps cyperi, and morphology of fresh specimens of the pathogen are described for the first time. The initial symptom of infection was a black sooty layer on inflorescences of infected plants due to colonization of the ergot honeydew by Cladosporium cladospor-ioides. Sclerotia of C. cyperi started to develop in March and April and could be discerned as small protuberances on inflorescences in the place of seed. Mature sclerotia were purplish-black. They generally remained viable for less than a year and germinated without prior cold treatment, although exposure for 21 d to 5 C before incubation significantly increased the germination rate. Under moist conditions at 24 C in the laboratory, germination commenced within 4–8 wk. Stromata took about 12 d to mature. Mature capitula were distinctly lobulate with a perithecium embedded in each lobe and a collar-like appendage around the base. Although dimensions of sclerotia, stipes, capitula, asci and ascospores were larger than in the original description, the general morphology supports treatment of C. cyperi as a distinct species.

Symptomatology and morphology of Claviceps cyperi on yellow nut sedge in South Africa

Symptoms of ergot on yellow nut sedge, germination of sclerotia of the causal organism, Claviceps cyperi, and morphology of fresh specimens of the pathogen are described for the first time. The initial symptom of infection was a black sooty layer on inflorescences of infected plants due to colonization of the ergot honeydew by Cladosporium cladosporioides. Sclerotia of C. cyperi started to develop in March and April and could be discerned as small protuberances on inflorescences in the place of seed. Mature sclerotia were purplish-black. They generally remained viable for less than a year and germinated without prior cold treatment, although exposure for 21 d to 5 C before incubation significantly increased the germination rate. Under moist conditions at 24 C in the laboratory, germination commenced within 4-8 wk. Stromata took about 12 d to mature. Mature capitula were distinctly lobulate with a perithecium embedded in each lobe and a collar-like appendage around the base. Although dimensions of sclerotia, stipes, capitula, asci and ascospores were larger than in the original description, the general morphology supports treatment of C. cyperi as a distinct species.

Morphological characteristics of sclerotia formed from hyphae of Grifola umbellata under artificial conditions

The morphological characteristics of sclerotia were induced in cultures of the fungus Grifola umbellata by introducing an unidentified companion fungus were studied by light microscope, scanning and transmission electron microscope (SEM and TEM). Light microscope and SEM investigations of developing sclerotia revealed that aerial mycelial hyphae diminished with age, and mature sclerotia had two tissue layers, the rind and medulla. The medulla was comprised of thin and thick-walled hyphae of varying diameter. The thick-walled cells always formed below the hyphal tips. Retraction of the cytoplasm was accompanied by the thickening of cell wall. There were crystalline initials in the newly formed sclerotium. Crystalline initials were always formed in the tip of medullary hyphae and were not of regular shape. A series of changes occurred in the cells in which the crystalline initials would be formed, such as enlargement of size, formation of one or several large vacuoles. Crystalline initials developed via amorphous materials in the cytoplasm deposited in the vacuoles. At last crystalline initials was released by degradation of the cell wall.

First Report of False Smut of Corn (Zea mays) in the Mississippi Delta.

False smut, caused by Ustilaginoidea virens, occurs on rice, corn, and other Gramineae, but is of little economic importance. False or green smut of corn resembles most closely false smut of rice (1). During surveys in 2000 at Mississippi State University, Delta Experiment Station, Stoneville, 32 (2.5%) of 1,280 commercial and inbred corn hybrids had false smut on the tassel. The percentage of plants infected with false smut ranged from 5 to 15% based on a sample of 20 corn plants from a single 4.9-m row plot. Galls (sclerotia), which are similar to ergot, replaced flowers on tassels of infected plants. Disease severity ranged from 2 to 50% infected flowers per inbred. Mature sclerotia were spherical, 6 to 17 mm in diameter, olive green to black, and velvety with white interiors. The fungus could not be isolated on potato dextrose agar. Only the tassels were infected, which resulted in little damage to the plant, except for a decrease in pollen production. Although the disease has been reported to infect other parts of the corn plant, the injury will likely be less on corn than rice. In 2001 and 2002, the disease was not observed. Disease incidence was more prevalent in 2000 because of hot, wet weather immediately after the crop was planted. The observation of the disease in one growing season suggests the pathogen may be common in the Mississippi Delta and the disease may occur under favorable environmental conditions and susceptible corn hybrids. Reference: (1) J. K. Pataky. False smut. Page 35 in: Compendium of Corn Diseases, 3rd ed. D. G. White, ed. American Phytopathological Society, St. Paul, MN, 1999.

An improved inoculation technique for sheath blight of rice caused by Rhizoctonia solani

To develop a simple and reliable inoculation technique allowing to differentiate minor differences in susceptibility of rice cultivars to sheath blight caused by Rhizoctonia solani Kühn, the effects of different pathogen and host factors on disease development were investigated under greenhouse conditions. The most critical factor affecting uniform development of sheath blight was the amount of inoculum. Approximately 0.2 mg of inoculum placed inside the leaf sheath with a few drops of sterile water invariably induced single discrete, uniform-sized lesions, irrespective of the type of inoculum used (i.e., mature sclerotium, immature sclerotium, or mycelium). Considering the ease of maintaining uniformity of inoculum, the use of immature sclerotia is recommended. This inoculation method is not only simple, rapid, and highly reproducible, but it also allows to distinguish minor differences in susceptibility under greenhouse conditions.