Solani AG-4 HG-I Research Articles

First Report of Rhizoctonia solani AG-4 HG-I Causing Root and Basal Stem Rot on Roselle (Hibiscus sabdariffa) in Mexico.

Roselle (Hibiscus sabdariffa L.) is a crop of economic importance, refreshing drinks are prepared from its calyces, it is also attributed to antioxidant, antibacterial, and antihypertensive properties (Da-Costa-Rocha et al. 2014). In November 2022, in municipality of Iguala (18.355592N, 99.548546W, 749 m above sea level), Guerrero, México, roselle plants of approximately 1.5 months of age with basal rot were detected under greenhouse conditions. The symptoms consisted of wilting, yellowing, and root and stem rot with constriction in the base of the stem. The symptoms were detected in approximately 15% of plants at the operation. From symptomatic tissue, cuts were made into approximately 0.5 cm pieces, sterilized with 2% NaClO, washed with sterile distilled water, transferred to PDA medium amended with 50 mg/liter of Chloramphenicol, and incubated in the dark for four days at 28 °C. Rhizoctonia-like colonies were consistently obtained, and nine isolates were selected and purified by the hyphal-tip method. After four days, isolates developed a mycelium was light-white that became brown with age. Right-angled hyphal branching was also observed, in addition to a slight constriction at the base of the branches. In some older cultures, numerous dark brown sclerotia were observed. They were multinucleate cell with three to eight nuclei and measured from 1 to 2 mm in diameter. Together these characteristics were consistent with the description of Rhizoctonia solani Kühn (Parmeter 1970). The anastomosis group (AG) was confirmed by amplifying the ITS region with the primers ITS1 and ITS4 (White et al. 1990) of the RIJAM3 and RIJAM5 strains. The sequences were deposited in GenBank (Nos. OR364496 and OR364497 for RIJAM3 and RIJAM5, respectively). BLAST analysis, both isolates indicated 99.7 identity to R. solani AG-4 HG-I (GenBank: KM013470) strain ICMP 20043 (Ireland et al. 2015). The phylogenetic analysis of AGs sequences allowed assignment of isolates RIJAM3 and RIJAM5 to the AG-4 HG-1 clade. A pathogenicity test was performed on 20 one-month-old roselle plants. Mycelium of RIJAM3 isolate was inserted into the base of the stem with a sterile toothpick. As a control, a sterile toothpick with no mycelium was inserted in ten healthy plants. Additionally, 50 eight-day-old seedlings were inoculated by placing a 5-mm diameter agar plug colonized with mycelium of RIJAM3 at the base of the stem 10 mm below the soil surface. As control treatments, uncolonized PDA plugs were deposited at the base of 25 seedlings. The inoculated plants were incubated in a greenhouse with an average temperature and relative humidity of 28°C and 85%, respectively. Following inoculation, symptoms similar to those observed in the original outbreak were observed in plants after six days and only after four days in seedlings. In both experiments, the control plants and seedlings remained asymptomatic. R. solani was re-isolated from plants and seedlings, complying with Koch's postulates. The pathogenicity testing was repeated twice, with concordant results. In Nigeria and Malaysia R. solani was reported to seedling death to cause seedling dieback in roselle (Adeniji 1970; Eslaminejad and Zakaria 2011). In México R. solani AG-4 has been previously reported in crops of potato, chili and tomato (Montero-Tavera et al. 2013; Ortega-Acosta et al. 2022; Virgen-Calleros et al. 2000). To the best of our knowledge, this is the first report of R. solani AG-4 HG-I as a causing of root and basal stem rot on roselle in Mexico. This research provides information essential for informing the management of this disease, and may help design measures to prevent the spread of the pathogen to other regions.

First Report of Blight Caused by Rhizoctonia solani AG4-HGI on Pinellia ternata in Guizhou, China.

Pinellia ternata (Thumb.) has been used for over 1000 years as a traditional Chinese herbal medicine (Ying et al. 2007) and is widely cultivated in Guizhou Province, China. It is cultivated over an area of 2000 hectares, and is of great value to underdeveloped regions. In April 2020, blight was observed in a field of P. ternatain Bijie County, Guizhou Province, China (27°30'N, 105°28'E). Around 20 hectares of P. ternata were surveyed and the disease incidence ranged from 10 to 12%. The disease symptoms included light brown lesions formed on the stems near the soil line. The color of the lesions became darker, and the stems became constricted around the lesions and broke, associated with the leaf blight. To identify the causal agent of this blight, 22 diseased plants (about 30 d-old） were collected, the margins of the infected parts were cut into small pieces (5 mm) and surface disinfested with 1% NaOCl for 10 min, 75% ethanol for 30 s, and rinsed three times in sterile distilled water. The pieces were blotted dry with sterile filter paper and placed on potato dextrose agar (PDA, Hopebio, China), incubated at 28℃ in darkness until fungal hyphae growth was visible. Sixteen cultures with different morphologies were recovered from the samples. When representative isolates of each culture type were inoculated onto plants, one produced similar blight symptoms. The representative isolate was called CD-1. The colony color was first white but turned light brown after grown on PDA for 6-7 d, and produced dark brown sclerotia. The hyphae were branched at right angles, with a slight constriction at the base of the branches and a septum near the junction where the branch separates from the main hyphae. Hyphal cells were stained with 0.5% Safranin O and 3% KOH and were observed to be multinucleate. These morphological features indicated that CD-1 likely is R. solani (Sneh et al. 1991). When paired with tester strains AG1 and AG4(provided by Dr. Genhua Yang, Yunnan Agricultural University). CD-1 showed anastomosis with isolate of AG4 (Fenille et al. 2002). Genomic DNA was extracted from the isolate (Thangaraj et al. 2018) using a fungal genomic DNA extraction kit (Tiangen, China). The internal transcribed spacer (ITS) regions were amplified using the primers ITS1/ITS4 (White et al. 1990). A 535 bp fragment was amplified that showed 99% coverage and 99.4% identity with an isolate of R. solani AG4-HGI (GenBank: HG934417). The gene sequence was deposited in GenBank as accession #OL518945. Pathogenicity tests were performed using 30 d-old plants planted in sterilized soil in pots. Cut mycelial discs (diameter 6 mm) from 3-day-old PDA cultures and placed beside stems of 21 healthy plants. Nine plants treated with agar plugs were control samples. Inoculated plants were maintained at 24 ± 5℃ in a green house and watered every two days with sterilized water. Typical blight symptoms developed on the inoculated plants at d 3-5 post inoculation, whereas the control plants remained healthy. The experiments were repeated three times, and the isolates was re-isolated from the inoculated plants and identified as R. solaniAG4 by morphological features and molecular method. R. solani has been reported to cause blight of many plants such as coffee (Ren et al. 2018) and sesame (Cochran et al. 2018). To the best of our knowledge, this is the first report of R. solani AG4-HGI causing disease on P. ternate, both in China and worldwide. This finding suggests that this pathogen may cause a threat to cultivation and production of P. terenata.

Effect of Oligogalacturonides on Seed Germination and Disease Resistance of Sugar Beet Seedling and Root.

Oligogalacturonides (OGs) are a bioactive carbohydrate derived from homogalacturonan. The OGs synthesized in this study significantly inhibited the mycelial growth of Rhizoctonia solani AG-4HGI in vitro, even at a low concentration (10 mg/L). The seed vigor test demonstrated that the application of 50 mg/L OGs to sugar beet seeds significantly increased average germination percentage, germination energy, germination index, and seedling vigor index. The same concentration of OGs also improved the seedling emergence percentage of sugar beet when seeds were sown in soil inoculated with D2 and D31 isolates, respectively. The lesion diameter on mature sugar beet roots caused by R. solani AG-4HGI isolates D2 and D31 also decreased by 40.60% and 39.86%, respectively, in sugar beets roots first treated with 50 mg/mL OGs in the wound site, relative to lesion size in untreated/pathogen inoculated wounds. Sugar beet roots treated with 50 mg/mL OGs prior to inoculation with the D2 isolate exhibited up-regulation of the defense-related genes glutathione peroxidase (GPX) and superoxide dismutase (SOD) by 2.4- and 1.6-fold, respectively, relative to control roots. Sugar beet roots treated with 50 mg/mL OGs prior to inoculation with D31 exhibited a 2.0- and 1.6-fold up-regulation of GPX and SOD, respectively, relative to the control. Our results indicate that OGs have the potential to be used for the protection of sugar beet against R. solani AG-4HGI.

Occurrence of Root and Stem Rot Caused by Rhizoctonia solani AG-4 HGI on Torenia fournieri in China.

Torenia fournieri (wishbone flower) is widely cultivated for ornamental and medicinal use in southern China. In July 2021, severe root and stem rot of T. fournieri were observed in approximately 70% of the 3,000 plants grown in an ornamental plant nursery under natural conditions located in Guangzhou, Guangdong Province, China. Lesions on the roots and basal stems were water-soaked and dark brown. In late infection, the whole plant wilted and died. Eventually, infected plants lose their ornamental and economic value. Fifteen diseased samples were collected, and diseased tissues (4 × 4 mm) were surface sterilized in 1% NaOCl for 45 s, rinsed with sterile water, dried, and incubated for 3 d on potato dextrose agar (PDA) medium at 25°C in the dark. Pure cultures were obtained by transferring hyphal tips to new plates of PDA and incubated at 25°C for 5 d. A fungus was consistently isolated from symptomatic root and stem samples (100% isolation rate). The colonies on PDA medium were initially white, turning light brown and with age, no sclerotia was present after 15 days. Width of hyphae was 3.78 to 11.10 μm , which with a constriction at the base of hyphal branches, septa near the branch, and right-angled branching. Multinucleate hyphal cells (3 to 12 nuclei per cell) were determined by 4'-6-diamidino-2-phenylindole (DAPI) staining (Yang et al. 2013). These characteristics matched those described for Rhizoctonia solani (Sneh et al. 1991). A representative isolate LZ1 was chosen for molecular identification, the internal transcribed spacer (ITS) region was amplified using the primers ITS1/ ITS4, and the resulting sequence (633 bp) were deposited in GenBank (accession no. MZ798228). A BLASTn search of the ITS sequence exhibited 99% identity with that of R. solani AG-4 HGI (MK430998). The phylogenetic analysis (neighbor-joining method and 1,000 bootstrap values) showed LZ1 was clearly assigned to group of R. solani AG-4 HGI. For pathogenicity testing, ten healthy T. fournieri plants (approximately 18 cm high) were planted into plastic pot filled with sterilized soil. Inoculum of R. solani was produced by adding a PDA agar plug (5 mm in diameter) from a 6-day-old colony to a flask with 150 ml of potato dextrose broth (PDB), shaken at 25°C and 150 rpm for 7 days, and poured evenly into the sterilized soil (50ml per pot ). Another 10 plants were poured with sterile PDB as controls. All the inoculated plants placed in a greenhouse at 25°C and 70-90% humidity conditions. After 14 days, rot symptoms of the roots and basal part of stems similar to those in the nursery were observed on all inoculated plants, while no visible symptoms were observed on the controls. We reisolated the fungal pathogen from the inoculated plants and identified to be R. solani AG-4 HGI by microscopy and molecular characteristics mentioned above. R. solani AG-4 HGI was previously reported causing Impatiens walleriana Stem Rot and muskmelon fruit rot in Liaoning, China (Sun et al. 2015). To the best of our knowledge, this is the first report of Rhizoctonia solani AG-4 HGI causing root and stem rot on Torenia fournieri in China as well as worldwide. This finding contributes to expand the host range known for R. solani AG-4 HGI and will assist in developing effective control strategies of this disease to minimize the losses.

RNA-seq Analysis of Rhizoctonia solani AG-4HGI Strain BJ-1H Infected by a New Viral Strain of Rhizoctonia solani Partitivirus 2 Reveals a Potential Mechanism for Hypovirulence.

Rhizoctonia solani partitivirus 2 (RsPV2), in the genus Alphapartitivirus, confers hypovirulence on R. solani AG-1-IA, the causal agent of rice sheath blight. In this study, a new strain of RsPV2 obtained from R. solani AG-4HGI strain BJ-1H, the causal agent of black scurf on potato, wasidentified and designated as Rhizoctonia solani partitivirus 2 strain BJ-1H (RsPV2-BJ). An RNA sequencing analysis of strain BJ-1H and the virus RsPV2-BJ-free strain BJ-1H-VF derived from strain BJ-1H was conducted to investigate the potential molecular mechanism of hypovirulence induced by RsPV2-BJ. In total, 14,319 unigenes were obtained, and 1,341 unigenes were identified as differentially expressed genes (DEGs), with 570 DEGs being down-regulated and 771 being up-regulated. Notably, several up-regulated DEGs were annotated to cell wall degrading enzymes, including β-1,3-glucanases. Strain BJ-1H exhibited increased expression of β-1,3-glucanase after RsPV2-BJ infection, suggesting that cell wall autolysis activity in R. solani AG-4HGI strain BJ-1H might be promoted by RsPV2-BJ, inducing hypovirulence in its host fungus R. solani AG-4HGI. To the best of our knowledge, this is the first report on the potential mechanism of hypovirulence induced by a mycovirus in R. solani.

Identification of Leaf Spot and Stem Rot on Plectranthus verticillatus Caused by Rhizoctonia solani AG-4 HGI in China.

Plectranthus verticillatus (Swedish ivy) is a plant in the family Lamiaceae (Labiatae), native to South-east Africa, that is commonly grown as a houseplant in China. In June 2018, the initial symptoms water-soaked spots were found on the stem base of sixteen P. verticillatus plants(1-year-old plants) in a commercial greenhouse, situating at Daxing district, Beijing, China (116°22'E, 39°42'N), where there were 150 potted P. verticillatus plants obtained by cuttage propagation and grown in conventional nutrient soils. Almost amonthlater, the disease was more severe and leaves appeared symptoms. Infected leaves displayed water-soaked, darkbrown lesions and fell off easily. Symptoms on the stems were dark brown, rotted and lodging. In the survey, the incidence of affected plants was 70%. To isolate the causal agent, the infected leaf and stem tissues were cut into 3 mm segments and surface disinfected in 1% NaClO for 1 min, then rinsed three times with sterile distilled water and cultured on potato dextrose agar (PDA) at 25±1 °C with a 12-h photoperiod. After 2 days, hyphal tips were transferred to new PDA plates to obtain pure cultures. Six morphologically similar isolates (CC1 to CC6) were obtained from the symptomatic samples. All the fungal colonies were white initially, turned brown gradually, and formed irregular shaped sclerotia after 9 days of incubation on PDA. Observationofmicrostructures indicated that all the isolates resembled Rhizoctonia sp.(Sneh et al. 1991), including septate hyphal with right-angled branching, a slight constriction at the base of hyphal branches and 3 to 8 nuclei per cell. Total genomic DNA was extracted from pure isolates using a Fungal DNA Kit (GBCBIO, Guangzhou, China) and the internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and sequenced (White et al. 1990 and Garibaldi et al.2019).The results showed that sequences of 6 isolates were identical and had a 100% identity with those of R. solani AG-4 HGI (GenBank accession no. MH172679 and MH172664). The phylogenetic analysis performed by the neighbour-joining method (MEGA 7 software) showed that the representative isolate (accession no. MZ723940) was assigned to group ofR. solaniAG-4 HG-I. The pathogenicity of these six isolates to the leaves and stems of P. verticillatus were conducted. Healthy P. verticillatus plants(1-year-old) were grown in 2-liter pots with sterile substrate in a greenhouse at 14-19°C (night)/23-28°C (day). After 7 days, five P. verticillatus plantsfor each isolate were inoculated with a 6-mm-diameter PDA plug taken from a 10-day-old PDA culture, which was placed onto leaf blade and the base of the stem. Five control pots were inoculated with sterile PDA plugs. All the inoculated plants were incubated in a glass cabinet under 90% humidity and 25°C for 2 days and were then moved back to the greenhouse with natural daylight conditions. The test was repeated three times. Ten days later, all inoculated leaves and stems appeared dark brown and water-soaked lesions similar to those observed on naturally infected plants, whereas control plants remained asymptomatic. The fungus that was reisolated from diseased tissues had the same morphological characters as the original isolates and confirmed to beR. solaniAG-4 HG-I by molecular identification. R. solani AG-4 HGI has an extensive host range, which had previously been recorded to cause leaf, root and stem rot on Plectranthus sp. in Florida in 1984 (Alfieri Jr. et al. 1984). To our knowledge, this is the first report ofR. solani AG-4 HGI infecting P. verticillatus in China. Its confirmation is a significant step toward management recommendations for growers.

First Report of Rhizoctonia solani AG-4 HG-I Causing Fruit Rot on Tomato in Mexico.

The culture of tomato (Solanum lycopersicum L.) is an important food, fruits are source of vitamins and minerals, they also prevent chronic diseases such as cancer and vascular diseases (Willcox, 2003); in Mexico 47,372 ha are destined to tomato cultivation (SIAP, 2019). In February 2021, in a tomato plantation located in Rancho el Progreso (17.743639N, 99.243250W, 1554 masl), municipality of Zitlala, Guerrero, Mexico, the presence of rot of tomato fruits hybrid DRD-8551 (seed company, Seminis) was detected in tomatoes grown in greenhouses. Disease incidence was 8% in a sample of 250 plants. The infected fruits initially exhibited circular to irregular lesions, with a soft rot of infected tissue, which in advanced stages infected more than 50% of the fruit surface and a white to light brown mycelium cover was observed. It was observed that fruits were those that were closer to the soil were heavily affected. From infected fruits, cuts of approximately 0.5 cm were made, disinfested with 1% NaOCl and transferred to PDA medium, using this technique for all samples that were processed. Eight representative isolates were purified; developed colonies were initially light-white, after seven days they turned light brown. Right-angle hyphae were observed, with a slight constriction at the base of the branches, and produced dark brown sclerotia; morphological characteristics were consistent with those described for Rhizoctonia solani Kühn (Parmeter 1970). To confirm the anastomosis group (AG), the ITS region was amplified with the ITS1 and ITS4 primers (White et al. 1990) from the ESCJZ9 and ESCJZ10 strains. A search with BLAST tool indicated that both strains were 99.4% identity with R. solani AG-4 HG-I strain ICMP 20043 (GenBank: KM013470) (Ireland et al. 2015). Phylogenetic analysis compared with different sequences of AGs, allowed to group ESCJZ9 and ESCJZ10 strains with the clade AG-4 HG-I. The sequences were deposited in GenBank (MZ274468 for ESCJZ9 and, MZ274469 for ESCJZ10). Pathogenicity was confirmed by inoculating 15 healthy tomato fruits (hybrid DRD-8551), in which a portion of mycelium from the ESCJZ9 strain was inserted with a sterile toothpick in two points / fruit. In eight fruits only a sterile toothpick (two points / fruit) was inserted. The fruits were housed in laboratory conditions at average temperature of 27 °C. After five days, fruit rot symptoms were observed in all inoculated fruits, similar to those observed in the field. Control fruits remained asymptomatic. Additional pathogenicity tests were performed on 50 tomato plants (hybrid DRD-8551) of 15-day-old. Two 5 mm diameter mycelial discs from strain ESCJZ9 were deposited at the base of the stem 10 mm below the soil surface. As control treatments, sterile PDA was deposited in 20 plants. The plants were housed in a greenhouse at an average temperature and relative humidity of 28 ° C and 85%, respectively. After four days, all the inoculated plants showed root and stem rot and leaf flaccidity. Control plants remained asymptomatic. R. solani was re-isolated from the infected fruits and plants, fulfilling Koch's postulates. Previously, in Iran and Brasil R. solani AG-4 HG-I has been reported that caused stem and fruit rot on tomato (Rahimian 1988; Kuramae et al. 2003). In Mexico R. solani AG-4 has been reported in other solanaceae species such as potato and chili (Virgen-Calleros et al. 2000; Montero-Tavera et al. 2013). To our knowledge, this is the first report of R. solani AG-4 HG-I causing tomato fruit rot in Mexico. This research provides information in order to establish disease management strategies. It is important to establish measures to minimize the spread of this pathogen to other tomato-producing areas of this country.

Molecular Mechanisms Associated with the Resistance of Rhizoctonia solani AG-4 Isolates to the Succinate Dehydrogenase Inhibitor Thifluzamide

Thifluzamide, a succinate dehydrogenase (SDH) inhibitor, possesses high activity against Rhizoctonia. In this study, 144 Rhizoctonia solani AG-4 (4HGI, 4HGII, and 4HGIII) isolates, the predominate pathogen associated with sugar beet seedling damping-off, were demonstrated to be sensitive to thifluzamide with a calculated mean median effective concentration of 0.0682 ± 0.0025 μg/ml. Thifluzamide-resistant isolates were generated using fungicide-amended media, resulting in four AG-4HGI isolates and eight AG-4HGII isolates with stable resistance and almost no loss in fitness. Evaluation of cross-resistance of the 12 thifluzamide-resistant isolates and their corresponding parental-sensitive isolates revealed a moderately positive correlation between thifluzamide resistance and the level of resistance to eight other fungicides from three groups, the exception being fludioxonil. An active efflux of fungicide through ATP-binding cassette and major facilitator superfamily transporters was found to be correlated to the resistance of R. solani AG-4HGII isolates to thifluzamide based on RNA-sequencing and quantitative reverse transcription-PCR analyses. Sequence analysis of sdhA, sdhB, sdhC, and sdhD revealed replacement of isoleucine by phenylalanine at position 61 in SDHC in 9 of the 12 generated thifluzamide-resistant isolates. No other mutations were found in any of the other genes. Collectively, the data indicate that the active efflux of fungicide and a point mutation in sdhC may contribute to the resistance of R. solani AG-4HGI and AG-4HGII isolates to thifluzamide invitro. This is the first characterization of the potential molecular mechanism associated with the resistance of R. solani AG-4 isolates to thifluzamide and provides practical guidance for the use of this fungicide.

First Report of Multinucleate Rhizoctonia solani AG4 HG-I Causing Crown and Root Rot on Strawberry in Italy

First Report of Multinucleate <i>Rhizoctonia solani</i> AG4 HG-I Causing Crown and Root Rot on Strawberry in Italy

First report of Rhizoctonia solani AG-4 HGI Causing Stem Canker on Fagopyrum tataricum (Tartary buckwheat) in China.

Tartary buckwheat (Fagopyrum tataricum) is an ideal functional food source, which is well known to be gluten-free and rich in proteins, fats, vitamins, minerals, and flavonoids of various pharmaceutical uses, such as rutin, quercitin and epicatechin (Zhou, M et al. 2018). The Rhizoctonia solani AG-4 HGIII causing severe canker disease was first isolated from common buckwheat (F. esculentum) in Inner Mongolia of China (Zhou, H et al. 2015). In 2018, sunken lesion and dark brown symptoms were observed on the root and stem of ten days old Tartary buckwheat in Liangshan (28°21'N, 103°19''E), Sichuan Province and Fenghuang (28°19' N, 109°48' E), Hunan Province in China. In the beginning, water soaked spots appeared on the stem base, where gradually became rotten and necrotic, finally resulting in the damping-off and death of buckwheat seedlings. This disease had over 40% incidence and lead to serious losses to the buckwheat production in 2018. To isolate the pathogens on Tartary buckwheat, ten plants with typical symptoms were collected from each location. The infected tissue was taken and cut into 3mm pieces from the margin between healthy and diseased tissue, surface sterilized with 1% sodium hypochlorite solution for 4 min, washed three times with sterile distilled water, dried on sterilized filter paper and then placed on potato dextrose agar (PDA) with 100 mg/ml streptomycin sulfate. After incubation at 28℃ in the dark for 2 days, mycelial tips of four fungal cultures were transferred to PDA plates for purification. Initially, colonies were pale white, and then turned brown after 2 days incubation. The mycelium was hairy and concentrically whorled in the culture medium. Microscopic observation showed that the hyphae characteristically branched at right angles and had constriction at the base of hyphal branches. Nuclear staining showed that the hyphae cells were multinuclear. These morphological features revealed that the isolates belonged to R. solani (Sneh et al. 1991). Subsequently, the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of one isolate was amplified by PCR (White et al. 1990) and sequenced (GeneBank accession no. MT078642) by Shanghai Majorbio Bio-pharm Technology Co.,Ltd. DNA was extracted by Fungal genomic DNA Extraction Kit, D3390-02, OMEGA. The BLAST similarity analysis showed a 99.96% match with R. solani AG-4 HGI (GenBank accession no. JQ343830) and 99.85% identity to R. solani AG-4 HGI isolate SX-8 (GenBank accession no. KJ170346) (Suli, Sun et al. 2015). Furthermore, the phylogenetic analysis performed by the neighbour-joining method (MEGA 7 software) showed that the isolate was clearly clustered with the group of R. solani AG-4 HGI (Ireland et al. 2015). Pathogenicity was tested in the greenhouse condition to satisfy Koch's postulates. Tartary buckwheat plants of seven days old and fifty days old were respectively inoculated near the base of the stem neck with one mycelial plug contacted directly. Ten plants in a pot were inoculated as one treatment, four pots were used for replicates. Control plants were inoculated with PDA medium plugs without fungi. All the plants were kept at 26℃ with 14 h light, 10 h dark and 96% humidity. After five days (Suli, Sun et al. 2015), over 90% of the inoculated plants exhibited necrotic brown lesions on stems that was similar to those symptoms observed in the field, whereas control plants remained asymptomatic. The visible characteristics and ITS sequence of the pathogen re-isolated from symptomatic plants were in accordance with the original isolate (R. solani AG-4 HGI). Based on disease symptoms in the fields, morphological characteristics, ITS sequence analysis, and pathogenicity assay, we concluded that R. solani AG-4 HGI was the principle cause of Tartary buckwheat blight in Liangshan, Sichuan Province and Fenghuang, Hunan Province in China. Previously, R. solani AG-4 HGI has been identified as a Chinese chive pathogen (Shi, Y et al. 2017). To the best of our knowledge, this is the first report of the natural occurrence of Rhizoctonia solani AG-4 HGI affecting Tartary Buckwheat in China. This finding is helpful for the early diagnosis and identification of the disease, which will be the guiding of effective control methods to the devastating disease at the early stage.

Anastomosis group and subgroup identification of Rhizoctonia solani strains deposited in NARO Genebank, Japan

The Rhizoctonia solani species complex is classified by anastomosis groups (AGs)-1–13 based on the hyphal fusion reaction. Most AGs are further divided into subgroups. However, the AG and subgroup designations of many strains of Japanese R. solani, including pathogens isolated from important crops, have not been determined. We thus identified AGs and subgroups of 45 R. solani strains (38 AG unknown and seven subgroup unknown strains), which had been isolated from 30 crops and identified by depositors in the NARO Genebank, Japan, based on the number of nuclei, culture morphology, PCR amplification with specific primers, and sequences of the rDNA-ITS region. Of the 45 strains, 37 were multinucleate, however, eight were binucleate. Of the multinucleate strains, 18 were identified as R. solani AG-4 HG-I, the rest as AG-1 IA (one strain), AG-1 IB (one strain), AG-2-1 (one strain), AG-2-2 IIIB (one strain), AG-2-2 LP (five strains), AG-2-2 hybrid subgroup between IIIB and IV (newly designated in this study; one strain), AG-3 PT (one strain), AG-4 hybrid subgroup between HG-I and HG-II (one strain), AG-4 HG-III (two strains), AG-5 (two strains), and AG-6 (three strains). Six of the eight binucleate strains were identified as binucleate Rhizoctonia AG-R (one strain) and AG-U (five strains). The rDNA-ITS sequences of the other two binucleate strains did not match any from known binucleate Rhizoctonia AGs-A–W. This study revealed new findings of Rhizoctonia diseases in Japan in 28 strains obtained from 21 crops.

Culture media to detect and criteria to evaluate and report the activity of extracellular enzymes produced by phytopathogenic fungi

ABSTRACT: Extracellular enzymes are involved in the fungal pathogenesis in plants. Currently, culture media, data analyses, and data report related to extracellular enzymes produced in vitro conditions are different and therefore, lack standardization. This work aimed to compare the culture media cited on the literature (normal) with the potato-dextrose-agar (PDA) medium combined with a specific compound to produce extracellular enzymes through three soilborne phytopathogenic fungi (F. solani f. sp. passiflorae, S. rolfsii, and R. solani AG-4 HGI), as well as to analyze and report enzyme data based on five different criteria. The assay was randomized, with three factors (culture media, isolates, and enzymes) and six repetitions. The studied enzymes were amylase (AM), carboxymethylcellulase (CMCase), lipase (LP), laccase (LC), catalase (CT), and gelatinase (GT). The normal media detected more enzymes and was more precise compared to the PDA medium plus specific compound. The criteria that calculated the area of the circular crown of AM, CMCase, LP, and LC and measured the intensity (0 = absence, up to 4 = intense) of CT and GT adopting note scale were the best to evaluate and report the results of the enzymes. We suggest the normal media culture to study enzyme production, as well as the criteria mentioned to assess and report the data related to enzyme activities.

Phylogenetic diversity and antagonistic traits of root and rhizosphere pseudomonads of bean from Iran for controlling Rhizoctonia solani

Fluorescent pseudomonads from bean root and rhizosphere in Iran were investigated for biocontrol of the fungal pathogen Rhizoctonia solani. Phylogenetic analysis of concatenated 16S rRNA, gyrB and rpoD sequences for 33 Pseudomonas isolates showed that 15 belonged to four clusters within the ‘P. fluorescens’ group, i.e. one corresponding to P. thivervalensis, two others including P. moraviensis or P. baetica, and the last one without closely-related established species. The 18 other isolates belonged to five clusters within the ‘P. putida’ group, one including P. mosselii and P. entomophila, another including strains currently described as P. putida, and three without closely-related species described. Ten isolates were selected based on in vitro inhibition of R. solani. Cellulase activity was identified in three pseudomonads, chitinase activity in two pseudomonads, extracellular protease activity in nine pseudomonads and hydrogen cyanide production in two pseudomonads. Genes coding for production of phenazine, pyoluteorin, pyrrolnitrin and 2,4-diacetylphloroglucinol were not found, whereas the 1-aminocyclopropane-1-carboxylate deamination gene acdS was present in three pseudomonads. The antagonistic acdS+ strain VKh13 from the ‘P. putida’ group effectively protected soil-grown bean from R. solani AG 4-HGI. Results show that pseudomonads from uncharacterized taxa were readily obtained from Iranian soils and displayed biocontrol potential against R. solani.

Occurrence and characterisation of Rhizoctonia species causing diseases of ornamental plants in Italy

During surveys conducted in 2010–2012 Rhizoctonia symptoms were observed on 30 ornamental species in different nurseries located in eastern Sicily (Southern Italy). Eighty-eight isolates of Rhizoctonia spp. were obtained from symptomatic leaves, roots and stems. Fifty-six of the isolates were binucleate and 32 were multinucleate Rhizoctonia. Characterisation of anastomosis groups (AGs) was performed using morphological characteristics and sequence analysis of the internal transcribed spacer of ribosomal DNA ( rDNA-ITS) region. Most isolates collected were Rhizoctonia solani AG-4 HG-I (35.2% of all isolates) and one isolate was AG-2-2 IIIB. The binucleate isolates belonged to AG-R (27.3%), AG-A (21.6%), AG-G (12.5%), AG-V (1.1%) and AG-Fb (1.1%). The pathogenicity of 38 representative isolates collected from each host was tested on seedlings or cuttings grown in a growth chamber. All R. solani AG-4 HG-I isolates, most of the binucleate AG-R, AG-A and AG-G and AG-V were pathogenic and reproduced symptoms identical to that observed in nurseries, while binucleate AG-Fb and R. solani AG-2-2 IIIB isolates were nonpathogenic. This is the first report of the occurrence of Rhizoctonia species on some ornamental plants and the first report of binucleate Rhizoctonia AG-R and AG-V in Europe.

Biosynthesis, Chemical Structure, and Structure-Activity Relationship of Orfamide Lipopeptides Produced by Pseudomonas protegens and Related Species.

Orfamide-type cyclic lipopeptides (CLPs) are biosurfactants produced by Pseudomonas and involved in lysis of oomycete zoospores, biocontrol of Rhizoctonia and insecticidal activity against aphids. In this study, we compared the biosynthesis, structural diversity, in vitro and in planta activities of orfamides produced by rhizosphere-derived Pseudomonas protegens and related Pseudomonas species. Genetic characterization together with chemical identification revealed that the main orfamide compound produced by the P. protegens group is orfamide A, while the related strains Pseudomonas sp. CMR5c and CMR12a produce orfamide B. Comparison of orfamide fingerprints led to the discovery of two new orfamide homologs (orfamide F and orfamide G) in Pseudomonas sp. CMR5c. The structures of these two CLPs were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. Mutagenesis and complementation showed that orfamides determine the swarming motility of parental Pseudomonas sp. strain CMR5c and their production was regulated by luxR type regulators. Orfamide A and orfamide B differ only in the identity of a single amino acid, while orfamide B and orfamide G share the same amino acid sequence but differ in length of the fatty acid part. The biological activities of orfamide A, orfamide B, and orfamide G were compared in further bioassays. The three compounds were equally active against Magnaporthe oryzae on rice, against Rhizoctonia solani AG 4-HGI in in vitro assays, and caused zoospore lysis of Phytophthora and Pythium. Furthermore, we could show that orfamides decrease blast severity in rice plants by blocking appressorium formation in M. oryzae. Taken all together, our study shows that orfamides produced by P. protegens and related species have potential in biological control of a broad spectrum of fungal plant pathogens.

Interplay between orfamides, sessilins and phenazines in the control of Rhizoctonia diseases by Pseudomonas sp. CMR12a.

We investigated the role of phenazines and cyclic lipopeptides (CLPs) (orfamides and sessilins), antagonistic metabolites produced by Pseudomonas sp. CMR12a, in the biological control of damping-off disease on Chinese cabbage (Brassica chinensis) caused by Rhizoctonia solani AG 2-1 and root rot disease on bean (Phaseolus vulgaris L.) caused by R. solani AG 4-HGI. A Pseudomonas mutant that only produced phenazines suppressed damping-off disease on Chinese cabbage to the same extent as CMR12a, while its efficacy to reduce root rot on bean was strongly impaired. In both pathosystems, the phenazine mutant that produced both CLPs was equally effective, but mutants that produced only one CLP lost biocontrol activity. In vitro microscopic assays revealed that mutants that only produced sessilins or orfamides inhibited mycelial growth of R. solani when applied together, while they were ineffective on their own. Phenazine-1-carboxamide suppressed mycelial growth of R. solani AG 2-1 but had no effect on AG 4-HGI. Orfamide B suppressed mycelial growth of both R. solani anastomosis groups in a dose-dependent way. Our results point to an additive interaction between both CLPs. Moreover, phenazines alone are sufficient to suppress Rhizoctonia disease on Chinese cabbage, while they need to work in tandem with the CLPs on bean.

Phenotypic Diversity and Molecular Identification of the Most Prevalent Anastomosis Group of Rhizoctonia solani Isolated from Diseased Faba Bean Plants

One hundred and thirty one isolates of Rhizoctonia spp. were isolated from faba bean plants showing root rot and stem canker collected from different fields in Delta region of Egypt. Forty six isolates were found to be polynucleate and the remaining isolates were binucleates. According to morphological features of isolates, they were classified into 12 groups. Polynucleate isolates were identified as Rhizoctonia solani (Kuhn). The most aggressive isolate of R. solani was identified according to sequences of ITS1-5.8S rDNA-ITS4 and the sequence was compared with Thanatephorus cucumeris (teleomorphic phase) and other R. solani (NCBI GenBank). Sequence and comparison revealed that this isolate is R. solani AG4-HGI. Anastomosis test carried out between molecular identified isolate and 11 randomly chosen isolates resembles all groups of polynucleate R. solani. All tested isolates were completely fused between each other indicating that the prevalent AG of R. solani on faba bean is AG4-HG1.

First report of white leaf rot on Chinese chives caused by Rhizoctonia solani AG-2-1

Delayed sprouting and white rot of leaf tips were found on Chinese chives in a greenhouse in Hokkaido, Japan, in the spring of 2006 and 2007. The causal fungus was identified as Rhizoctonia solani anastomosis group (AG)-2-1 and discriminated from the leaf rot pathogen R. solani AG-4 HG-I in terms of pathogenicity. Symptoms and the time of year that the disease occurs also apparently differ for the two pathogens. This is the first report of white leaf rot on Chinese chive caused by R. solani AG-2-1.

Efeitos da simulação da solarização do solo com materiais vegetais sobre o crescimento micelial de fungos fitopatogênicos habitantes do solo

A incorporação de materiais vegetais específicos associados à solarização do solo tem sido um avanço promissor no controle de fungos fitopatogênicos habitantes do solo. O objetivo do trabalho foi avaliar determinados efeitos da incorporação e decomposição de brócolis, mamona, mandioca brava e mansa, no solo, em condições de microcosmo mantido em BOD (37±2ºC), sobre o micélio de Fusarium oxysporum f. sp. lycopersici Raça 2, Macrophomina phaseolina, Rhizoctonia solani AG-4 HGI e de Sclerotium rolfsii. Assim, quatro ensaios idênticos foram instalados em conjunto de microcosmos, com cinco tratamentos e quatro períodos de tempo diferentes e independentes (7, 14, 21 e 28 dias). O parâmetro avaliado foi os efeitos inócuo, fungistático e fungicida dos tratamentos sobre o micélio dos fungos. Verificou-se efeito fungistático e fungicida no crescimento micelial de F. oxysporum f. sp. lycopersici Raça 2, R. solani AG-4 HGI e de S. rolfsii. Os fungos que apresentaram efeito fungistático apresentaram uma velocidade média de crescimento micelial inferior ao controle geral, que consistiu na incubação dos fungos em temperatura de 25±2ºC. O efeito fungicida ocorreu aos 21 dias de incubação para F. oxysporum e R. solani e aos 28 dias para S. rolfsii. Para M. phaseolina, observou-se apenas efeito inócuo. Associação da temperatura de 37±2ºC mais o período de tempo dos tratamentos foi o fator responsável pelos efeitos fungistático e fungicida no micélio dos fitopatógenos estudados. Essa associação também interferiu na velocidade do crescimento micelial dos fungos que apresentaram efeito fungistático.

Influence of soil type and indigenous pathogenic fungi on bean hypocotyl rot caused by Rhizoctonia solani AG4 HGI in Cuba

Calcisol, ferralsol and vertisol soils, representative of different bean production areas of Villa Clara province in Cuba, were selected to determine the impact of soil type on bean hypocotyl rot severity caused by Rhizoctonia solani AG4 HGI (isolate CuVC-Rs7). In inoculated autoclaved soil, hypocotyl rot was most severe in calcisol soil, followed by ferralsol soils and then vertisol soils. In inoculated natural soils, disease severity was lower in vertisol and calcisol soils and higher in ferralsol soil, indicating that biological factors are suppressing or stimulating the pathogenic efficiency of R. solani. Native binucleate Rhizoctonia AGF, Sclerotium rolfsii and R. solani AG 4 HGI were isolated from bean plants grown in natural calcisol, vertisol and ferralsol soils, respectively. Subsequent studies about the interaction between these fungi and R. solani indicated that they were involved in the variability of disease severity caused by R. solani. The addition of R. solani AG4 HGI (isolate CuVC-Rs7) into each autoclaved soil inoculated with binucleate Rhizoctonia or S. rolfsii resulted in a reduction of disease severity caused by this pathogen while in soils inoculated with native R. solani AG4 HGI, disease severity increased. Irrespective of fungal interactions, calcisol was always the most disease conducive soil and vertisol the most disease repressive soil. The mechanisms by which native pathogenic fungi could influence disease severity caused by R. solani are discussed.