Hyphal Width Research Articles

First report of milkweed (Euphorbia geniculata) as an alternative host for Colletotrichum truncatum in soybean fields in India.

Soybean (Glycine max, L.), a major oilseed crop of India faces anthracnose disease caused by Colletotrichum truncatum (Nataraj et al. 2021). Several weeds serve as alternative hosts for Colletotrichum spp. (Hartman et al. 1986). Around 24.67% of soybean fields in the study area were infested with Euphorbia geniculata (Kutariye et al. 2021). In September 2021, milkweed plants died in the field, showing irregular circular lesions with wavy margins on the stem, change in color of veins and veinlets from brown to black and leaves exhibiting a twisted appearance at ICAR-Indian Institute of Soybean Research, India. Later on plants completely died and acervuli of average size 284 µm were visualized under stereo microscopy. Twenty milkweed samples were collected, rinsed, and surface sterilized with NaOCl (1%). Fungus isolation was done from leaf and stem and transferred to sterilized Petri plates with Potato dextrose agar (PDA). The plates were incubated at 25 ± 2°C for 48 h with dark/light (10h/14h) cycle. The fungi produced circular, raised, black to light grey colonies. Sickle shaped aseptate conidia, measuring 23.14 µm length, 3.18 µm width and hyphal width 5.49 µm were confirmed using a compound microscope with 20X magnification. The fungus was purified via hyphal tip method and pure culture was maintained on PDA at (26 ± 2°C). Milkweed seedlings in clay pots were inoculated with a conidial suspension of the fungus (106 conidia/mL) prepared from ten days old culture using serial dilution technique. Soybean variety JS 95-60 was inoculated by atomizing 20 ml of the same suspension on each plant. The negative controls for both milkweed and soybean were inoculated with sterile distilled water. Veinal necrosis and acervuli formation were observed on both milkweed and soybean, but no signs or symptoms of disease were observed in the controls. The re-isolated fungus from both the diseased hosts resembled original culture as they produced black to light grey colonies, sickle shaped aseptate conidia and ITS sequence (OR124845) exhibiting 100% resemblance to C. truncatum isolate C-17 (MN736513), thus confirming Koch's postulates. The pathogen was classified as Colletotrichum spp. based on morphological and cultural characters and the pathogenicity test (Rajput et al. 2021). To confirm identity of the pathogen infecting milkweed, DNA was extracted from the reisolated fungus using the HiPurA Fungal DNA Purification Kit (HiMedia, India). The internal transcribed spacer (ITS) region, beta-tubulin (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified (Kumar et al. 2021). The GAPDH gene was amplified under similar reaction conditions except for annealing temp 59°C. For species level identification, the ITS, TUB2 and GAPDH gene sequences were submitted to GenBank with accession numbers OR004468, OQ869780 and OQ869781, respectively. The BLAST analysis of TUB2 and GAPDH gene showed sequence homology of 100% and 98.43% respectively with C. truncatum culture-collection CBS:151.35 (GU228156, GU228254). The isolate was identified as C. truncatum on the basis of molecular analysis, corroborating the above morphological identification. This is the first report of C. truncatum infecting milkweed in India, indicating milkweed as an alternative host in soybean fields, potentially raising inoculum levels and carryover between crops.

First report of Colletotrichum shisoi causing anthracnose of Perilla frutescens in the United States.

Perilla mint (Perilla frutescens (L.) Britt.) is an annual plant native to Asia and considered invasive in North America where it has escaped cultivation as an ornamental (Miller 1947; Swearingen et al. 2010). In August 2021, an anthracnose disease was observed on invasive perilla found along the disturbed margins of a forest in Frederick County, Maryland, United States. Symptoms included necrotic lesions with chlorotic halos, were concentrated in the lower canopy, and caused premature defoliation of lower leaves (Figure S1). Leaves from four plants were surface sterilized by rinsing for 30 s in 70% ethanol, 60 s in 0.8% NaClO, and 60 s in sterile water and then incubated on 2% water agar under ambient laboratory conditions to permit sporulation. After three days, spores that exuded from individual lesions were streaked onto acidified potato dextrose agar. Two single-conidium isolates were recovered from each plant. All eight isolates were identified to species using DNA sequences. A single isolate (21-067) was selected at random for morphological characterization and completion of Koch's postulates. Morphological features were recorded after seven days of growth on synthetic low-nutrient agar (SNA) and potato dextrose agar (PDA) incubated at 22°C under 12 hr UV-B and white fluorescent lighting. Measurements were based on a minimum of 20 observations per structure. Cultures on SNA were flat, hyaline to pale salmon, lacked sporodochia and grew at a rate of 1.3 mm day-1 (n = 3). Vegetative hyphal width was (minimum-maximum) 1.5-4.0 μm, (average ± standard deviation) 2.7 ± 0.9 μm. Conidiophores arose directly from hyphae, were hyaline, smooth, unbranched and measured 40.0-180.0 x 1.5-4.5 μm, 102.3 ± 33.9 x 2.7 ± 0.8 μm. Conidia were single celled, straight, hyaline, glabrous, rounded at both ends and measured 10.0-20.0 x 3.8-6.3 μm, 15.4 ± 2.5 x 4.9 ± 0.7 μm. Setae, observed only on PDA, were pale brown, 1-2 septate, straight, blunt tipped and measured 42.5-97.5 μm, 70.8 ± 13.4 μm. Appressoria formed on PDA were single-celled, pigmented, smooth, and obovoid with entire margins, measuring 5.2-7.7 x 8.6-13.8 μm, 6.8 ± 0.6 x 10.8 ± 1.4 μm. These characteristics were consistent with members of the Colletotrichum destructivum species complex. Partial DNA sequences from five loci were amplified following the procedures of Damm et al. 2014: internal transcribed spacer region of rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), and beta-tubulin (TUB2). Pairwise sequence comparisons to references using the Blastn algorithm found 99.8% similarity between isolate 21-067 and ex-type C. shisoi isolate JCM 31818: MH660930 (ITS, 545/546 bp), MH660931 (GAPDH,192/192 bp), MH660929 (CHS-1, 278/280 bp), MH660928 (ACT, 262/262 bp), and MH660932 (TUB2, 511/511 bp) (Altschul et al. 1990; Gan et al. 2019). Subsequently, sequences from all eight isolates were aligned with Clustal Omega (Sievers and Higgins 2018), concatenated, and used in a Bayesian phylogenetic reconstruction (Huelsenbeck and Ronquist 2001) of the C. destructivum species complex (Figure S2), confirming the species identity as C. shisoi. Sequences were deposited in NCBI GenBank under accession numbers OM865277-OM865284 and OM885059-OM885090. Koch's postulates were fulfilled using perilla grown in a greenhouse until second true leaves emerged. Inoculum washed from two-week-old fungal cultures grown on potato dextrose agar was adjusted to 4 x 104 conidia mL-1 and applied to three replicate plants with an aspirator until runoff. Three plants were sprayed with sterile water as a negative control. Plants were covered with plastic bags for 72 hrs and maintained in a growth chamber at 20°C and 80% RH for 14 days. Inoculated plants displayed disease symptoms similar to those observed under field conditions, and control plants did not develop symptoms. Colletotrichum shisoi was reisolated from symptomatic tissue and reidentified based on morphology. The experiment was completed twice. To the authors' knowledge, this is the first report of C. shisoi on P. frutescens in the United States. Colletotrichum shisoi has not been reported as a pathogen on other plants in the United States and may have potential use as a biological control agent for invasive perilla.

Effect of agar concentration on structure and physiology of fungal hyphal systems

Hyphae are the filamentous branches that form a mycelial network, acting as the constitutive structure of mushroom-forming Fungi. Hyphae grow on many substrates and in multitudes of environmental conditions. Due to this versatility, hyphae are of increasing interest in the area of bio-constructed materials derived from sustainable and non-petroleum-based inputs. By understanding the effects of external factors on the growth and physical properties of the hyphae, we can manipulate the physical properties of a cultivated mycelial sheet. This study demonstrates how altering the agar concentration during in vitro culturing can affect the physical growth and structure of the mycelial sheet without altering its permeability or chemical makeup. When the agar concentration was increased from 1.5% agar to 6.0% agar, the mycelium density and hyphal width increased by 32.3% and 63.6%, respectively. The implications of these findings will allow for the advancement and tuning of fungi-based materials, particularly for the application of sustainable textiles and fine particulate filters.

First Report of Powdery Mildew of American Ginseng Caused by Erysiphe heraclei in Tennessee and the United States.

American ginseng (Panax quinquefolius L.), native to the forested regions of northeast U.S is a perennial herb valued as traditional Chinese medicine. It has been cultivated in North America for several decades due to high global demand. Powdery mildew symptoms were observed on 8-year-old cultivated American ginseng leaves (Fig. 1a, b) on a residential property in Rutherford Co., TN in May 2022. Disease severity was 40 to 60% of leaf area and incidence was 33% out of 30 plants. Affected plants exhibited white fungal colonies on the leaves. Under severe infection, the leaves were chlorotic and senescing. Microscopic observation revealed masses of conidia and mycelia on symptomatic tissue. Conidiophores were cylindrical and unbranched (2- or, rarely, 3-septate), measuring 66.7 ± 12.5 μm (n=78) with a range of 24.3 to 90.7 μm. Conidia produced singly or in pseudo-chains (Fig. 1c). Conidiophore foot cells measured 23.2 ± 4.3 μm long (n=54) and the width at the foot cell septum was 5.1 ± 0.6 μm (n=54). Hyphal width was 3.3 ± 0.6 um (n=59). Fresh vacuolated spores were oblong-elliptical to oblong (Fig. 1d) and measured 31.5 × 11.9 μm (n=55), lacked fibrosin bodies. The length-to-width ratio of conidia was 1.9 to 4.4 (avg. 2.7). Superficial mycelia and germinating spores displayed lobed appressorium (Fig. 1e). Detached spore surfaces were wrinkled (Fig. 1f). Morphological characteristics of the fungus matched the description of Erysiphe heraclei (Braun and Cook, 2012) and Erysiphe sp. (Cho et al. 2016) except for conidiophore length, which was shorter in our sample. To confirm pathogen identity, total DNA was extracted directly from single spore cultures (isolates FBG1668 and FBG1728). The ribosomal internal transcribed spacer (ITS) region was amplified using ITS4 and ITS5 primers (White et al. 1990). The sequences (GenBank accession nos. OP458196 and OP469994) showed 100% identity and 100% query coverage to E. heraclei (KY073878 and LC270862). The sequences were also 100% identical to the ITS sequences of E. betae and E. malvae. Solano-Báez et al. (2022) noted that the species in the E. malvae/E. heraclei/E. betae species complex are phylogenetically undistinguishable. E. betae and E. malvae infect plants in Chenopodiaceae and Malvaceae, respectively (Braun and Cook, 2012). However, E. heraclei has been reported to infect plants in Apiaceae. American ginseng belongs to Araliaceae which is a close family to Apiaceae and both belong to Apiales. Based on morphological and molecular identification, both isolates were identified as E. heraclei. Pathogenicity was confirmed by inoculating the adaxial leaf surface of six 2-year-old American ginseng plants. Spores from detached symptomatic leaves were tapped onto the adaxial surface of healthy leaves. Six non-inoculated and inoculated plants were maintained in a greenhouse at 21 to 23°C, 70%RH, with 16-h photoperiod. After 2 weeks, powdery mildew symptoms developed on the inoculated plants. The microscopy and molecular analysis confirmed infection and all control plants remained asymptomatic. Cho et al. (2016) reported powdery mildew on Korean ginseng (P. ginseng C.A. Mey) caused by Erysiphe sp., and Sholberg et al. (1996) reported Erysiphe sp. on P. quinquefolius in Canada, but to our knowledge, this is the first report of powdery mildew caused by E. heraclei on American ginseng in Tennessee and the U.S. Identification and timely management of powdery mildew on American ginseng will be necessary to control this disease in affected growing sites.

Lithium bioaccumulation in Lentinus crinitus mycelia grown in media with different lithium sources and pH values.

Lentinus crinitus bioaccumulates lithium in mycelia, but bioaccumulation may be affected by pH of the culture medium. Lithium is used in clinical practice as a mood stabilizer and antidepressant. This study aimed to assess the effect of culture medium pH and lithium source (LiCl or Li2CO3) on lithium bioaccumulation in vegetative mycelia of L. crinitus grown in malt extract broth. Lentinus crinitus U9-1 was cultured in malt extract broth supplemented with Li2CO3 or LiCl (50mg L-1 lithium) in the pH range of 3.0 to 6.0. The pH was adjusted using HCl solution. The results showed that medium pH affected mycelial biomass production, lithium bioaccumulation in mycelial biomass, and lithium transfer from the culture medium to mycelial biomass. The effect of lithium source on the bioaccumulation capacity of mycelial biomass varied according to pH. At pH 4.0, both lithium sources stimulated mycelial biomass production compared to the control without the addition of lithium. At pH 5.5, Li2CO3 provided the highest lithium bioaccumulation in mycelial biomass. Lithium transfer from the culture medium to mycelia was highest in Li2CO3-supplemented cultures at pH 4.5. LiCl reduced hyphal width compared with Li2CO3 and the control. However, pH and lithium sources did not affect the formation of clamp connections in hyphae. For the first time, the influence of the pH of the culture medium on lithium bioaccumulation by Lentinus crinitus is reported. Finally, we conclude that the culture medium pH affected lithium transfer and bioaccumulation in mycelial biomass differently depending on the lithium source. Additionally, we report the presence of clamp connections in the hyphae of L. crinitus as an indicator of even growth.

First Report of Binucleate Rhizoctonia AG-L Causing Root and Stem Rot of Wishbone Flower (Torenia fournieri) in Taiwan.

Wishbone flower (Torenia fournieri L.) is a common ornamental plant for flower bed in Taiwan. In August 2018, root and stem rot of wishbone flower occurred on the flower bed in the campus of National Chung Hsing University, Taichung city, with 100% incidence. Symptoms were dark brown discoloration of basal stems and brown necrosis of roots. Lesions from base of stems were excised into 5 mm long fragments, which were then surface sterilized in 1% sodium hydrochloride for 1 min, rinsed in sterile distilled water, dried on filter paper and thereafter placed onto 2% water agar. After 24 h, hyphae characteristic of Rhizoctonia (Sneh et al. 1991) appeared and dominated in every isolation. Hyphal tips were transferred to potato dextrose agar (PDA). After 5 days of incubation at 28°C, characteristic brown colonies of Rhizoctonia (Sneh et al. 1991) were observed. Hyphal width was 4.29±0.52 μm. No sclerotia were visibly present after 21 days of growth on PDA at 28°C. Hyphae were stained by 0.3% safranin-O and 1% KOH. There were two nuclei in each hyphal compartment, suggesting a binucleate Rhizoctonia fungus. ITS sequence has been used as the best tool to identify specific anastomosis group (AG) of Rhizoctonia as shown by Sharon et al. (2006, 2008). ITS sequence was amplified using the primers Bd1a and ITS4 (Goka et al. 2009; White et al. 1990). Blast search analysis of this acquired sequence (acc. no. LC498494) revealed the highest similarity (98.75 to 99.83%) with the reference sequences (acc. nos. AB286934, AB286933, and AB196653) of binucleate Rhizoctonia AG-L, namely Ceratobasidium sp. AG-L. Pathogenicity test was carried out using seedlings of 4-week-old wishbone flower each grown in a pot of 6.35 cm diameter. To prepare the inoculum, a PDA agar block (6 mm in diameter) excised from the growing front of 5-day-old colony was transferred into a flask with 200 ml of potato dextrose broth (PDB) incubated in a shaker at 26°C and 120 rpm for 6 days. The PDB broth was then blended into slurry. Ten pots each with a seedling were inoculated by pouring 50 ml of slurry onto the potting medium. Five pots were served as the controls by pouring PDB only. Pots were maintained in a greenhouse (26 to 33°C). Three days after inoculation, all inoculated plants exhibited symptom of root and stem rot. The same fungus was reisolated and confirmed by sequencing rDNA-ITS. This is the first report of root and stem rot of wishbone flower caused by binucleate Rhizoctonia AG-L in Taiwan and in the world. Although this is the second cases, since Wang and Hsieh (1993), for binucleate Rhizoctonia AG-L to be pathogenic, this study shows that this fungus has the potential to cause damages and is worth of further investigations.

Salicylic acid affects mycorrhizal features, antioxidant enzyme activities and seed yield of linseed under water-deficit stress in open-field conditions.

The research aims were to study salicylic acid (SA) effects on mycorrhiza [hyphal width (HW), vesicle diameter (VD) and mycorrhizal colonization (MC)] and interaction between them on greenness index (GI), drought tolerance index (DTI), antioxidant enzymes activities, and seed yield of linseed under drought. A factorial experiment was conducted in an open-field place with mycorrhiza [non-inoculation, Funneliformis mosseae (FM), and Rhizoglomus intraradices (RI)], SA (250μM and non-SA), and irrigation levels [100%, 70%, and 40% field capacity (FC)] as treatments. Severe drought increased VD, MC, superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase activities while decreased GI, DTI, and yield. The RI-linseed had higher MC, GI, SOD, and glutathione reductase (GR) activities, but FM-linseed had greater VD and yield under drought. Inoculated linseed with both mycorrhiza showed a reduction in DTI and yield under SA than non-SA. In RI-linseed, SA increased GI, MC, HW, VD, catalase and GR, but decreased in FM-plants. Mycorrhiza (particularly RI) alleviated drought (40% FC)-caused negative effects onlinseed via the improvement of SOD, APX, and GI. Regardless of other treatments, SA had negative effects on HW and VD, but SA effects varied depending on mycorrhizal species so that SA increased HW, VD, and MC in RI. Due to the positive correlation between MC and HW, SA reduces FM colonization by reducing the HW of FM. Totally, SA along with RI species can mitigate the harmful effects of drought and improve tolerance via increasing MC, HW, VD, catalase, peroxidase, and GR activities.

Surveillance of Rhizoctonia solani in Meerut region of Uttar Pradesh

The potato (Solanum tuberosum L.) is widely grown in the world and contributes significantly not only to food security but to nutritional security. It is a vegetable crop characterized by a shallow and superficial root system and has been shown to be a heavier nutrient feeder taking up nutrition until maturity. The underground portion of the stem has buds which give rise to lateral slender underground stems called Stolons. It is the Stolons which enlarge to form tubers.Four hundred seventy Rhizoctonia solani isolates from different leguminous hosts originating from 16 agro-ecological regions of India covering 21 states and 72 districts were collected. The disease incidence caused by R. solani varied from 6.8 to 22.2% in the areas surveyed. Deccan plateau and central highlands, hot sub-humid eco-region followed by northern plain and central highlands and hot semi-arid eco-region showed the highest disease incidence. R. solani isolates were highly variable in growth diameter, number, size and pattern of sclerotia formation as well as hyphal width.

Trait-Based Models of Decomposition with Interspecific and Environmental Interactions

Most saprotrophic fungi are dominant decomposers, and they play a vital role in the carbon cycle and stability of ecosystem. Previous researchers examined that how traits and environments influence the decomposition rate(DR), but few studies last long enough to scale short-term results up to long-term trends. In this paper, we build several trait-based models of decomposition with interspecific and environmental interactions to describe both short- and long-term trends. First, we build the model of decomposition ratio versus time with covariates – hyphal extension ratio and moisture tolerance for each species. Second, we define Interspecific Interference Ratio (IIR) to reflect interactions between fungi species and rank the relative competitiveness based on Grey System Theory with four evaluation indices: hyphal extension rate, moisture niche width, the hyphal density and thermal niche width. Then, we predict the relative advantages and disadvantages for different species by IIR and analyze the impact of biodiversity. Finally, we perform sensitivity and robustness analysis of our models, which exhibits the wide prospects and a deeper understanding of the role of fungi in the ecosystem.

First Report of Sclerotium hydrophilum Sace Causing Stem Rot Disease of Rice in North Eastern Plain Zone of India.

High incidence of stem rot in rice (Oryza sativa L.) caused by Sclerotium hydrophilum was observed in Eastern Gangetic plains of India including eastern Uttar Pradesh and Bihar states in rice-wheat irrigated ecosystem in the months of August and September of the years 2016-17 at the maximum tillering stage of the crop plants. A survey was conducted for the prevalence of rice diseases in Eastern Uttar Pradesh and Bihar. Stem rot incidence (12-14%) was observed in widely cultivated rice varieties viz., Swarna, Sabour Surbhit, Samba Mahsuri and Bhagalpur katarani. Brownish to black water-soaked lesions without distinct margins were visually observed on rice leaf sheaths in Pusa, Bihar and Allahabad, Uttar Pradesh (India). To isolate the causal pathogen, pieces of symptomatic leaf sheaths were treated with 1% sodium hypochlorite solution for 1 min, rinsed with sterile distilled water for 2 min, and then transferred to potato dextrose agar (PDA) medium for incubation at 280C. The cultivated mycelium was transferred to fresh PDA medium. Colonies of pure cultures on PDA medium were initially white and turned brown about 2 weeks later. The hyphal width was measured with a range of 4.0 to 6.0 μm. Large numbers of small globose sclerotia were observed on surface of the colonies at 5 days after sub-culturing. The sclerotia were white at first and then turned black over time with maturity. The diameters of sclerotia ranged from 0.32 to 0.51 mm with an average of 0.41 mm (n = 50). DNA of a representative isolate named SH1 was extracted, and the ITS region was amplified by PCR with universal primer pair ITS1/ITS4 (White et al. 1990). Sequence analysis showed 99.66% identity with Sclerotium hydrophilum isolate VC228 (accession no. KT362098) and accession no. obtained (KX181457). Phylogenetic analysis based onneighbour-joining method grouped the isolatesalongwith other isolates from Asia. The pathogen was identified as Sclerotium hydrophilum on the basis of cultural, morphological, pathogenic and molecular characteristics. Further, culture was deposited to Indian Type Culture Collection (ITCC) at the Indian Agricultural Research Institute, New Delhi for the identification where it was identified as Sclerotium hydrophilum with identification number 10039.15. Another culture specimen of the pathogen was also deposited at the ITCC-IARI, New Delhi with accession no. 19160.25. Koch's postulates were completed by inoculating rice plants with PDA disks of5 mm in size bearing both mycelium and sclerotia of S. hydrophilum in 45 days old plants of rice genotype 'Samba Mahsuri' under glass house conditions. After 4th days leaf sheath necrosis was observed around the inoculation plugsunder field conditions.The same fungus was re isolated from the symptomatic lesions of inoculated plants, fulfilling Koch's postulates. Pathogen was reported with high disease incidence in Northern Karnataka (Pramesh et al. 2017). However, based on literature and fungus-host distribution records this is the first report of S. hydrophilum on leaf sheath of rice from North-Eastern plain zone of India.

Invasive growth of Aspergillus oryzae in rice koji and increase of nuclear number

Background‘Rice koji’ is a solid culture of Aspergillus oryzae on steamed rice grains. Multiple parallel fermentation, wherein saccharification of rice by A. oryzae and alcohol fermentation by the budding yeast occur simultaneously, leads to the formation of a variety of ingredients of Japanese sake. In sake brewing, the degree of mycelial invasive growth into the steamed rice, called ‘haze-komi’, highly correlates with the digestibility and quality of rice koji, since the hyphae growing into the rice secrete amylases and digest starch.ResultsIn this study, we investigated mycelial distribution of GFP-tagged A. oryzae in rice koji made with different types of rice, such as sake rice and eating rice, with 50 or 90% polishing rate to remove abundant proteins and lipids near the surface. In addition, we compared transcriptomes of A. oryzae in the different types of rice koji. Finally, we found that A. oryzae increases the nuclear number and hyphal width in the course of 1–3 days cultivation.ConclusionsOur imaging analyses indicate that A. oryzae hyphae grew more deeply into 50% polished rice than 90% polished rice. The increases of nuclear number may be a selectively acquired characteristic for the high secretory capacity during the long history of cultivation of this species.

The cultural and morphological variability among Rhizoctonia solani isolates causing banded leaf and sheath blight of maize in Indonesia

Banded leaf and sheath blight caused by Rhizoctonia solani Kuhn has become a constraint on maize production in Indonesia. Forty-one isolates from 11 provinces in Indonesia were characterized based on cultural and morphological characteristics. Most isolates were characterized with sparse fluffy hyphae and pale yellow mycelium. They were categorized into two, three and four groups based on hyphal width, their growth rate and sclerotia production, respectively. The number of sclerotia was highly variable across different isolates. They were arranged in subcentral-peripheral, irregular, scattered, and peripheral manner on the Petri dish. The UPGMA analysis showed four major clusters, designated as cluster I, II, III, and IV at 0.75 similarity coefficient according to cultural and morphological characters. Cluster III was the largest cluster comprising 34 isolates; while cluster I, II, and IV comprised of four, one, and two isolate, respectively. Our results indicated that the cultural and morphological variability among R. solani isolates in Indonesia was not related to geographic origins.

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.

Oxalis purpurea sclerotium rot caused by Athelia rolfsii

Over a 3-year period (2015–2017), sclerotium rot was observed on purple wood sorrel (Oxalis purpurea) in the exhibition field of Gyeongsangnam-do Agricultural Research and Extension Services, Jinju, South Korea. Infected plants exhibited blight and rot symptoms. White mycelial mats spread over lesions, and numerous sclerotia formed on the petiole near the soil line. Sclerotia were globoid in shape, 1–3 mm in size, and white to brown in color. The optimum temperature for mycelial growth and sclerotium formation on potato dextrose agar (PDA) was 30 °C and the hyphal width was 4–8 μm. Typical clamp connections were observed in the hyphae of fungi grown on PDA. Molecular identification was conducted by sequencing and analysis of the complete internal transcribed spacer (ITS) rDNA sequence of the causal fungus. On the basis of mycological characteristics, molecular identification, and pathogenicity to host plants, this fungus was identified as Athelia rolfsii. This is the first report of sclerotium rot on purple wood sorrel caused by A. rolfsii.

First Report of Sclerotium hydrophilum Causing Stem Rot of Rice in Northeast China

In August 2016, rice plants with brown to black lesions with irregular shape and water-soaked margins in leaf sheaths were observed in Baoqing county in Heilongjiang province, Northeast China. To isolate the causal pathogen, pieces of symptomatic leaf sheaths were treated with 1% NaClO for 1 min, rinsed with sterile distilled water for 2 min, and then transferred to potato dextrose agar (PDA) medium for incubation at 28°C. The cultivated mycelium was transferred to new PDA medium. Colonies of pure cultures on new PDA medium were initially white and turned brown about 3 weeks later. The hyphal width was measured with a range of 4.5 to 6.2 μm. Large numbers of small and globose sclerotia were observed on surface of the colonies at 3 days after subculturing. The sclerotia were white at first and then turned black over time. The diameters of sclerotia ranged from 0.31 to 0.54 mm with an average of 0.41 mm (n = 50). DNA of a representative isolate named Hbq001 was extracted, and the 18S rDNA region was amplified by PCR with universal primer pair NS1/NS6 (White et al. 1990). The 18S rDNA sequence of isolate Hbq001 (GenBank accession no. KY995575) shared 99.7% nucleotide identities with those of Sclerotium hydrophilum (GenBank accession no. KC354147). Therefore, based on these cultural characteristics and rDNA sequence, isolate Hbq001 was identified as S. hydrophilum (Demirci et al. 2009). To complete Koch’s postulates, 4 mm diameter PDA disks containing mycelium and sclerotia of S. hydrophilum isolate Hbq001 were placed on the stem of 4-week-old healthy rice seedlings and wrapped with Parafilm. Sterile PDA disks were used as controls. After 4 days at 28°C, symptoms similar to those in the field were observed on the inoculated seedlings but not on control plants. Finally, the fungus S. hydrophilum, which showed the same cultural characteristics as described above, was reisolated from the inoculated seedlings. In China, S. hydrophilum was first identified in Yunnan province and was found to be distributed in southern China (Hu et al. 2008; Ruan and Chen 2002; Wang et al. 2013). Northeast China is a major rice-producing region. Differing from the rice-producing areas of southern China, this region has specific climatic conditions with cold temperature and abounds in high-quality japonica rice. To our knowledge, this is the first report of S. hydrophilum in the cold northern region of China.

Sclerotium rolfsii에 의한 동부 흰비단병

Sclerotium rot disease on cowpea (Vigna sinensis King) was observed in the exhibition field of Gyeongsangnam-do Agricultural Research and Extension Services in September 2015. Lesions were covered by white mycelial mats, and numerous sclerotia were formed on the stem near the soil line. The sclerotia were globoid in shape, 1~3 mm in size and white to brown in color. The optimum temperature for mycelial growth and sclerotia formation on potato dextrose agar (PDA) was 30oC, with the hyphal width of 4~8 μm. For molecular identification, the complete internal transcribed spacer (ITS) rDNA region of the causal fungus was sequenced and analyzed. Based on the mycological characteristics, ITS rDNA sequence analysis, and pathogenicity test, this fungus was identified as Sclerotium rolfsii. This is the first report of sclerotium rot on cowpea caused by S. rolfsii in Korea.

Morphological and Molecular Variability of Rhizoctonia solani Isolates Causing Banded Leaf and Sheath Blight in Maize

Twenty seven isolates of Rhizoctonia solani causing Banded Leaf and Sheath Blight in maize were collected from different maize varieties and one from rice grown in various regions of Telengana State, India for easy breeding task. The isolates were analysed for their morphological and genetic characterization. The hyphal width varied from 5.00 (RS2, RS20) to 7.97 μm (RS10) of rice isolate (7.34 μm). The isolates RS7, RS 8, RS9 (Karimnagar), RS10, RS11, RS12 (Khammam) RS16 (Krishna) and RS26 (Kurnool) and one rice isolate RS28 produced barrel shaped monilioid cells. The clamp connection was present only in RS1 isolate from Pragnyapur mandal of Medak district. The three isolates RS16, RS17, RS18 from Krishna district and rice isolate RS28 recorded maximum colony growth of 90.00 mm within 48 h of incubation. The colour of most isolates varied from light grey to brown while the rice isolate showed a typical yellowish brown shade. The sclerotial number per 5.00 mm culture disc of test isolates ranged from between (7.00) to (17.67). Their size varied between 0.21 μm (RS 24) and 1.57 μm (RS11). The rice isolate RS28 recorded highest (19.00) sclerotial count compared to that of maize isolates. Sclerotial colour in all isolates except for RS 24 was yellowish brown to dark reddish brown whereas the rice isolate was dark yellowish brown. Molecular characterization of the test isolates using RAPD markers with UPGMA exhibited two major clusters with 66 to 90% genetic similarity. Art ic le History

English

Sheath blight (Rhizoctonia solani) causes severe loss in the production of rice in India. Twenty five (25) isolates were collected from different parts of India. Morphological and cultural characters were investigated, on the basis of colony colour, growth pattern, hyphal width, colony growth, formation of sclerotia, size of sclerotia, diameter and number of sclerotia. Pathogenic variability was studied on highly susceptible cultivar Pusa Basmati-1 and isolates were classified into two major groups that is, highly virulent and virulent. Genetic variability of R. solani was also analyzed using 10 RAPD markers and on the basis of Jaccard’s similarity coefficient, 4 major clusters were formed. The range of genetic similarity varied from 17 - 77%. Maximum similarity (77%) was found between two isolates (RS-14 and RS-15) both from Kerala whereas, lowest similarity (17%) was observed between RS-22 (Delhi) and RS-4 (Punjab). Isolates from same geographical regions showed similarity in DNA profiles except few isolates from Uttar Pradesh and Punjab. Isolates were classified based on morphological, cultural characters and some isolates were identified as highly virulent and virulent. Key words: Rice, sheath blight, Rhizoctonia solani, cultural, pathogenic, molecular, variability.

Diversity of Rhizoctonia solani associated with pulse crops in different agro-ecological regions of India

Four hundred seventy Rhizoctonia solani isolates from different leguminous hosts originating from 16 agro-ecological regions of India covering 21 states and 72 districts were collected. The disease incidence caused by R. solani varied from 6.8 to 22.2% in the areas surveyed. Deccan plateau and central highlands, hot sub-humid ecoregion followed by northern plain and central highlands and hot semi-arid ecoregion showed the highest disease incidence. R. solani isolates were highly variable in growth diameter, number, size and pattern of sclerotia formation as well as hyphal width. The isolates obtained from aerial part of the infected plants showing web blight symptoms produced sclerotia of 1-2mm in size whereas, the isolates obtained from infected root of the plants showing wet root rot symptoms produced microsclerotia (<1mm). Majority of R. solani isolates showed <8μm hyphal diameter. Based on morphological characters the isolates were categorized into 49 groups. Seven anastomosis groups (AGs) were identified among the populations of R. solani associated with the pulse crops. The frequency (25.6%) of AG3 was the highest followed by AG2-3 (20.9%) and AG5 (17.4%). The cropping sequence of rice/sorghum/wheat-chickpea/mungbean/urdbean/cowpea/ricebean influenced the dominance of AG1 (16.3%). Phylogenetic analysis utilizing ITS-5.8S rDNA gene sequences indicated high level of genetic similarity among isolates representing different AGs, crops and regions. ITS groups did not correspond to the morphological characters. The sequence data from this article has been deposited with NCBI data libraries with JF701707 to JF701795 accession numbers.

サカキに発生した白絹病（新称）

Blight-like symptoms on Cleyera japonica Thunb. were first found on plants growing on the shrine in Omura, Nagasaki, Japan on June 19, 2009. White mycelia and brown, spherical, rapeseed-sized sclerotia were observed on the diseased plants. A fungus similar to Sclerotium rolfsii Saccardo was isolated from infected plants. The optimum growth temperature of these isolates, on potato dextrose agar, was 25℃for mycelial growth and 30℃for sclerotium formation. The mycelial tufts were white to grayish brown, and the main hyphal width ranged from 4.5-7.8μm（avg. = 6.6μm）with clamp connections. These mycological characters are identical to those of S. rolfsii reported in other plants. The isolates were found to be pathogenic to C. japonica in artificial inoculation test, and could be re-isolated from infected tissue. It is proposed that the disease should be referred to as southern blight of C. japonica caused by S. rolfsii.