AG-4 HG-I Research Articles

Biogenic Synthesis of Silver and Iron Oxide Nanoparticles Using Aronia prunifolia Leaf Extract and Its Inhibitory Action Against Pathogenic Fungi

Because of the environment and abundant renewable resources, exploiting plant extracts to form metallic nanoparticles has become a promising alternative to chemical and physical methods. Numerous studies have shown that nanoparticles of silver (AgNPs) and iron oxide (Fe2O3NPs) have inhibitory effects against pathogenic fungi. In this study, we used the leaf extract of Aronia prunifolia to generate biogenic AgNPs and FeNPs, aiming to demonstrate the impact of nanoparticles on pathogenic fungi. Nanoparticles are characterized by UV-Vis, X-ray diffraction, EDX spectrum, and SEM techniques. Leaf extracts used for nanosynthesis yielded silver and iron oxide nanoparticles with distinct color changes and absorption peaks, showcasing tetragonal, pentagonal, and hexagonal shapes (15-50 nm) for silver and spherical morphology (16-60 nm) for iron oxide. The antifungal effectiveness of nanoparticles against Aspergillus fumigatus, Rhizoctonia solani Ag4 HgII, and Aspergillus flavus was investigated using a well diffusion method. Inhibition zones, ranging from 12.5 to 35.0 mm for AgNPs and 7.1 to 17.1 mm for FeNPs at concentrations of 10 to 30 µg/ml respectively, demonstrated the superior inhibitory potential of AgNPs over FeNPs. This study aims to address a gap in the literature by examining the inhibitory effects of green AgNPs and FeNPs on pathogenic fungi. Encased nanoparticles can be very useful in treating fungal infections; this will be the first investigation into the production of nanoparticles from A. prunifolia leaves.

Genetic and pathogenic characterization of Rhizoctonia solani AG-4 isolates obtained from common bean

Common bean (Phaseolus vulgaris L.) is the most important grain legume. Crop production of common bean is affected by a number of diseases, such as Rhizoctonia root rot, being detected worldwide. Herein, we aimed to genetically and pathologically characterize Rhizoctonia solani isolates obtained from different provinces in Turkey. Anastomosis groups (AG) and subgroups of isolates were identified based on the sequence analysis of the rDNA-ITS region. The most prevalent subgroup was AG-4 HGIII, according to BLAST analysis and the phylogeny of resultant sequences, followed by AG-4 HGI and AG-4 HGII, respectively. iPBS retrotransposons highly supported the phylogenetic tree and provided a high level of genetic variability among isolates to discriminate AG subgroups. The results indicated that the iPBS DNA marker system based on retrotransposons could be used to discriminate Rhizoctonia isolates relying on AG grouping. The virulence of the pathogen isolates changed from 3.67 to 5 based on the agar-plate assay on the susceptible cv. Gina. The reactions of thirty common bean cultivars were also evaluated against the most aggressive isolate in each subgroup. The reaction assay showed significant differences among both isolates and cultivars; the highest disease severity among cultivars was observed to AG-4 HGI, followed by AG-4 HGIII and AG-4 HGII, respectively. None of the cultivars showed resistance to all AG subgroups. Screening of AGs among the isolates and selecting cultivars are critical to managing Rhizoctonia disease due to the range of host suitability according to AG subgroups.

Binucleate Rhizoctonia induced tomato resistance against Rhizoctonia solani via affecting antioxidants and cell wall reinforcement

Isolates of Rhizoctonia solani (AG-3 PT, AG-4 HG-I, AG-4 HG-II) and one binucleate Rhizoctonia sp. (BNR) belonging to AG-Bb were investigated for pathogenicity on tomato cultivar Mobil. The BNR isolate revealed the lowest virulence and it was used as biocontrol agent against R. solani AG-4 HG-II, which showed the highest virulence on tomato. Inoculation of tomato plants with the hypovirulent BNR isolate reduced the disease symptoms of R. solani and induced resistance. Resistance induction was observed not only on the plants simultaneously inoculated with BNR and R. solani, but also when the plants were inoculated by the BNR and R. solani with time intervals. The peroxidase (POX), superoxide dismutase (SOD) and catalase (CAT) activities and expression levels of the corresponding genes in tomato plants increased after R. solani or BNR inoculation. The highest level of antioxidant activities and expression of their genes, lignin and callose formation were observed in the plants inoculated with the BNR and R. solani, simultaneously. The BNR inoculation reduced H2O2 accumulation. The highest level of priming was observed for the POX among other antioxidants tested via application of the BNR. Treatment with potassium cyanide (as a POX inhibitor) reduced basal resistance and BNR-induced resistance (BNR-IR) via reduction of lignification and callose deposition in tomato plants. These findings demonstrated the role of antioxidant enzymes, mainly the POX, in both basal resistance and BNR-IR. Therefore, redox state and antioxidants are involved in cell wall strengthening via lignin and callose formation, as important defense components which decrease the pathogen progress in plant tissues.

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.

Diversity and pathogenicity of anastomosis groups of Rhizoctonia associated with brassicas in Brazil

Seventy-eight Rhizoctonia isolates were obtained from brassicas in five Brazilian states between 2015 and 2017. The isolates were identified by phylogenetic analysis of the rDNA internal transcribed spacer region (ITS) and tested for pathogenicity and aggressiveness. Sixty-eight isolates were identified as R. solani and 10 isolates as binucleate Rhizoctonia. The most prevalent anastomosis group (AG) was AG-4 HG-I (83.3% of isolates), followed by AG-A (7.7%), AG-1 IB (2.6%), AG-G (2.6%), AG-R (2.6%) and AG-2-2 IIIB (1.3%). AG-4 HG-I isolates were widely distributed and recovered from all Brazilian states sampled, including 12 brassicas-growing fields. All isolates were pathogenic to broccoli, cauliflower and kale, causing necrotic lesions on subterraneous stems. There was no significant difference in aggressiveness among the different AGs in broccoli, but AG-1 IB isolates were more aggressive to cauliflower and AG-4 HG-I isolates were more aggressive to kale. This study is the first report of AG-4 HG-I, AG-2-2 IIIB, AG-A, AG-G and AG-R infecting brassica plants in Brazil. To our knowledge, this is the first detailed research of the AG composition of Rhizoctonia populations causing brassica diseases in Brazil.

Rhizoctonia solani AG1-IB, AG1-IC, and AG4-HGII cause bottom rot of field lettuce in Vermont.

Members of Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) species complex cause bottom rot on lettuce (Latuca sativa) and yield losses up to 70% (Subbarao et al. 2017). Severe symptoms include necrosis, stem rot, and/or discoloration especially on the leaf midrib. In Vermont, vegetable farms are small (0.5-30 acres) and grow lettuce concurrently with other vegetable crops in the same field but the AG(s) that causes the disease in Vermont has not been determined. Isolates (n = 157) were collected from 31 fields with reported history of bottom rot between July 10 and October 8, 2019, across Addison, Caledonia, Chittenden, Franklin, Lamoille, and Orleans counties. Isolates were collected from lettuce tissue or potato (Solanum tuberosum), a common rotation crop, or uncropped soil baited using radish (Raphanus sativus). Pieces of tissue (5-10 mm) were cut from the leading margin of lesions, surface disinfested with 0.1% NaClO for 1 min followed by 2 rinses with sterile water, blotted dry, and plated onto acidified 2% water agar (0.085% lactic acid, pH 4.8). After incubation for 48 to 72 h, mycelia resembling Rhizoctonia were examined for morphological characteristics including hyphal branching at ca. 90o angles, a septum near the branching point, multiple nuclei per cell, and lack of both clamp connections and conidia (Sneh et al. 1991). Colonies were white to dark brown, and some produced small sclerotia. Koch's postulates were performed by inoculating nine 8-week-old (9 leaf pairs) romaine lettuce plants (Johnny's Seeds, Winslow, ME, cv. Monte Carlo) per isolate. Isolates were grown on 2% potato dextrose agar for 1 week, from which a 5-mm agar plug was placed on the adaxial leaf surface at the base of a petiole. Plants were enclosed in a plastic bag to maintain high humidity and grown under a 16-hour photoperiod at 24 °C. Disease severity was rated 4 days after inoculation (0: healthy, 1: isolated lesions, 2: lesions across multiple petioles, and 3: systemic disease). Putative AG were determined by Sanger sequencing of the internal transcribed spacer (ITS) region using the ITS1F and ITS4B primer pair (758 bp) (Gardes and Bruns 1993). Contigs were assembled using CAP3 software (Huang and Madan 1999). Taxonomy was assigned to each OTU via the NCBI BLASTn database with criteria as 0.0 E and nucleotide match of at least 97%. Of the 10 isolates sequenced with sufficient coverage (735 to 784 bp alignment length) and definitive resolution (96.7 to 99.9% identity), 5 were putative AG 1-IB (Genbank Accession HG934430.1), 2 AG 1-IC (Genbank Accession AF354058.1), 2 AG 3 (Genbank Accession AF354064.1), and 1 AG 4-HGII (Genbank Accession AF354074.1). Fasta files and metadata are archived at 10.6084/m9.figshare.20301324, 10.6084/m9.figshare.20301375. Putative AG 1-IB was highly virulent on lettuce plants whether it originated from potato (mean 2.6) or lettuce (mean 1.3 to 3). AG 4-HGII and AG 1-IC isolated from lettuce and radish, respectively, were moderately severe (mean 1.4 to 2.2) on lettuce with identical symptoms. The two potato isolates (AG3) were not pathogenic on lettuce. Similarly, higher incidence of AG 1-IB is reported on lettuce in Quebec (Wallon et al. 2021), Ohio (Herr 1993), and Germany (Grosch et al. 2004). Because AG vary in their host range (Sneh et al. 1991), knowing the AG will inform management decisions such as crop rotation and weed control. This is the first report of the causal agent of bottom rot of lettuce or any AG of R. solani in Vermont.

First Report of Target Spot Caused by Rhizoctonia solani AG-5 on Tobacco in China.

Tobacco is one of the vital economic crops in China. Nevertheless, tobacco diseases cause substantial economic losses each year. Tobacco target spot is a fungal disease which commonly found on the leaves. While both sexual and asexual reproduction can occur, asexual reproduction is much more common in tobacco. In June 2022, target spot was found on tobacco leaf samples from Yibin, Sichuan Province and Meitan, Guizhou Province, China. The typical symptoms were light brown tissue with concentric ring marks, and the necrotic part of the disease spot was fragile and forming perforation after falling off. The diseased tissue in the sample was cut off and sterilized in 75% ethanol for 1 min, and rinsed three times in sterilized distilled water. Finally, the tissues were placed on potato glucose agar (PDA) medium with kanamycin (0.1 mg/mL). After incubation at 28 °C in darkness for 3 days,the culture of the isolate grew in the form of radial mycelium on PDA dishes, the mycelium was white initially, turned brown generally at the later stage, and finally thickened and separated with the growth of the culture. Nine pathogenic strains were isolated, including four isolates from Yibin and five from Meitan. They were all used for pathogen identification. Genomic DNA of each isolate was extracted using the CATB method, and PCR analysis was performed with primers specifically designed to detect individual fusion groups or fusion subgroups of solani: AG-1 IA, IB, and IC; AG-3 PT; AG-4 HG-I, HG-II and HG-III; AGs-5-6 and P-21-22. Among the 11 specific primer pairs, only AG-5-specific primer amplified the fungal DNA, indicating that the nine isolates tested all belonged to the R. solani AG-5 fusion group. BLASTn search was performed on the gene sequences obtained from these strains and they deposited in GenBank under accession no. OP647851-OP647859. These gene sequences were aligned with the voucher specimen R. solani AG-5, with more than 99% similarity . The nine isolates were then tested for mycelial anastomosis reactions using the R. solani AG-5 standard strain following the method described by Ogoshi (1987). A decrease in the diameter of the mycelia at the anastomosis site and death of adjacent cells were observed, indicating their anastomosis response. Therefore, these nine strains were identified as R. solani AG-5 based on morphological and genetic analysis. Subsequently, one pathogenic strain from Meitan and another one from Yibin were selected for pathogenicity verification. Mycelial PDA blocks (6 mm in diameter) of the two isolates were inoculated on healthy tobacco plants, while leaves containing only PDA blocks were used as controls. A total of 6 replicates were conducted. After inoculation, they were incubated at 85% relative humidity and 15 to 25 °C. Koch's hypothesis was confirmed by reisolating pathogens from diseased leaves 5 days after inoculation. Typical symptoms were observed on tobacco plants inoculated with the pathogen strains but not on control tobacco plants. To the best of our knowledge, tobacco target spot has been reported caused by R. solani AG-3, AG-6 and AG-2.1 groups in the field in China and in Argentina. Up until now, this is the first report of R. solani AG-5 causing tobacco target spot on tobacco in the field in China. It was also found to be highly virulent to chickpea in Turkey. Due to serious damages caused by this disease in the last five years in China, more attention should be paid in disease control measures to avoid economic losses. In addition, it also provides some theoretical help for the damage caused by this pathogen on other hosts and helps people to better understand Rhizoctonia solani AG-5.

Determination of Disease Severity of Rhizoctonia solani Kühn (Telemorph: Thanatephorus cucumeris (Frank) Donk) Isolates from Bean, Sugar Beet and Potato Planting Areas in Konya

This study was carried out to determine the disease severity of Rhizoctonia solani isolates isolated from plant samples collected from bean, sugar beet and potato cultivation areas in Konya in 2020 and the anastomosis groups of the most virulent isolates. A total of 40 R. solani isolates were obtained as a result of the isolations made from 86 plant samples (36 beans, 25 sugar beets and 25 potatoes) showing root rot symptoms in general appearance. The number of R. solani isolates obtained from these plants, respectively; 10 isolates from beans, 15 from potatoes and 15 from sugar beets. Nine of the bean isolates and 14 of the potato and sugar beet isolates were determined as multinucleic. One isolate each isolated from the bean, sugar beet and potato was determined as binucleic. As a result, a total of 37 multinucleate (MN) and 3 binucleate (BN) isolates were obtained from all plants. As a result of pathogenicity tests, 3 R. solani isolates with the highest disease severity for each plant were amplified using ITS1F and ITS4B primers and anastomosis groups were determined. Accordingly, the anastomosis groups of Fa 3.2 (97%), Fa 2.2 (89%) and Fa 1 (86%) in beans were characterized as AG 4HGI. The isolates with the highest disease severity in potatoes (Pa 10, Pa 12.1 and Pa 15.2) were determined as AG 3 group. Disease severity was determined as 50% of Pa 15.2, 44% of Pa 10 and 42% of Pa 12.1. The disease severity of 9 of the isolates obtained from sugar beet was determined as 100%. The anastomosis group of 3 randomly selected isolates from these isolates was characterized as AG 2-2.

Determination of Disease Severity and Anastomosis Groups of Rhizoctonia solani Isolates from Chickpea Plant in Konya Province

This study was carried out to determine the anastomosis groups and disease severity of Rhizoctonia solani, which causes root and root collar rot in chickpea production areas in Konya. A total of eleven isolates of Rhizoctonia spp. were obtained in the isolations made from the root and root collar of the plants in the surveys. Rhizoctonia isolates examined microscopically were grouped as multinucleate, binucleate according to the number of nuclei. Ten isolates were multinucleate and identified R. solani, while one isolate was binucleate Rhizoctonia sp. In pathogenicity tests, five multinucleate isolates were found to be pathogenic. Disease severity was determined as 100% for 1.2 A and 1.2 B coded isolates, 86% for 1.1 A coded isolate, 75% for 1.1 B coded isolate, and 44% for 1.2 C coded isolate. It was observed that the other multinucleate five isolates caused only a shortening in plant heights. No disease symptoms were observed in the plants inoculated by the binucleate isolate. All isolates were characterized using the internal transcribed spacer (ITS) region of ribosomal DNA, and two different anastomosis groups were defined accordingly. It was determined that 5 virulence multinucleate R. solani isolates belonged to AG-4 HGII and 5 other multinucleate isolates to AG BI anastomosis group.

First report of Rhizoctonia solani AG-4 HG-I causing leaf blight disease on Cattleya × hybrid in Taiwan

First report of Rhizoctonia solani AG-4 HG-I causing leaf blight disease on Cattleya × hybrid in Taiwan

First Report of Target Spot Caused by Rhizoctonia solani AG-6 in Tobacco in China.

Tobacco is an annual and solanaceous crop, which is widely produced in China. In July 2020, tobacco target spot was observed on 50% of tobacco plants in a 5-ha commercial field of Bijie (27.32° N, 105.29° E), Guizhou province, China. Typical symptoms firstly appeared on the old leaves as round watery spots. Then the spots became a diameter of 2 to 20 cm, with concentric ring lines and dead spots. Fifteen small pieces (5 × 5 mm) of leaf tissue were cut from the edge of the lesions, surface sterilized and placed on potato dextrose agar (PDA) medium amended with kanamycin (0.1 mg/ml). Isolate J136, one of five isolates with similar morphology, was selected for pathogen identification. The culture of the isolate on PDA was brown and exhibited radial mycelial growth after incubation at 28 oC in darkness for 5 days. Hyphae of the fungus were white at the beginning, turned light brown to brown at the later stages, and finally became thick and separated. Sclerotia were brown and produced on PDA after 25 days of incubation in the dark. These characteristics were similar to the colony characteristics of R. solani. The genomic DNA of Isolate J136 was extracted using the CTAB method. PCR analyses were conducted using the following primers specifically designed for the detection of individual AGs or subgroups of R. solani: AG-1 IA, IB and IC (Kuninaga 2003), AG-2-1, AG-2-2, IIIB, IV and LP (Carling et al. 2002), AG-3 PT (Misawa 2015), AG-4 HG-I and HG-II (Kuninaga 2003), and AGs-5-6 (Arakawa and Inagaki 2014). Among the 12 specific primer pairs, only AG-6-specific primers amplified a fragment of ca. 230 bp product, indicating that the tested strain belonged to R. solani AG-6. The sequence was deposited in GenBank with accession no. MZ379468. Using BLASTN search, the sequence of the gene was aligned with the voucher specimen, R. solani AG-6. A phylogenetic tree was constructed based on these sequences. After wards, Isolate J136 was tested for hyphal anastomosis reaction using the R. solani AG-6 standard strain according to the method described by Ogoshi (1987). The hyphal diameter at the point of anastomosis was reduced, with obvious anastomosis point, and the death of adjacent cells, indicating their anastomosis reactions (Anderson 1982). Thus, based on the morphological and genetic analyses, the fungus was identified as R. solani AG-6. To verify its pathogenicity, six plants (cv. Yunyan87) at the 5-to-6 leaf stage were inoculated with mycelial PDA plugs (5 mm in diameter). Leaves inoculated with PDA-only plugs served as the controls. Treated tobacco plants were maintained at a temperature range of 15 to 25 oC in a greenhouse with 85% relative humidity. After 5 days inoculation, typical symptoms were observed on the inoculated leaves, whereas no symptoms were observed on the control leaves. Koch's postulates were fulfilled by re-isolation of the pathogen from the diseased leaves. R. solani AG-2-2 is the only previously reported group of R. solani, which causes tobacco target spot in the field (Gonzalez et al. 2011). Therefore, to our knowledge, this is the first report of R. solani AG-6 causing target spot of tobacco in the field in China. Since considerable losses caused by the disease have frequently happened in this region, addition of this new group pathogen in the disease pool can be more problematic. Proper disease control strategies are in need to be developed to prevent further losses.

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.

Thiamine and Piriformospora indica induce bean resistance against Rhizoctonia solani: The role of polyamines in association with iron and reactive oxygen species

In the present study, the protective effects of thiamine and Piriformospora indica against the pathogenic fungus Rhizoctonia solani AG-4 HGII, which causes root rot, have been investigated in bean plants. Our results revealed that thiamine and P. indica significantly reduced the disease progress in the leaf discs. Disease suppression via application of thiamine and the endophytic fungus, P. indica, was linked to biochemical and metabolic changes in bean plants. Accumalation of hydrogen peroxide (H2O2), superoxide anion (O2–), and iron ions decreased in the leaf discs pretreated with thiamine and/or P. indica, while the formation of polyamines (PAs) such as putrescine (Put), spermidine (Spd), and spermine (Spm) dramatically increased. We observed that occurrence of cell death, the levels of ferric (Fe3+) and ferrous (Fe2+) ions were positively correlated with H2O2 accumulation. Simultaneous increase in the generation of H2O2, Fe3+ and Fe2+ may play a role in induction of cell death. A positive correlation was also observed between PAs and H2O2 levels. Decreased accumulation of H2O2 in different treatments may be a result of an increase in the PAs contents. These findings suggest that formation of PAs may decrease progress of the disease via decreasing H2O2 accumulation in the plant cells. This is the first report on the crucial role of PA contents and their interplay with iron ions and reactive oxygen species in thiamine and P. indica mediated induction of bean defense responses against a necrotrophic fungal pathogen.

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.

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

The role of biological and chemical inducers in activating bean defense responses against Rhizoctonia solani

Rhizoctonia root rot is an important bean disease that causes significant yield losses. Increasing the levels of plant resistance using biological and chemical inducers is a sustainable strategy for plant disease management. In this study, the efficiency of thiamine, riboflavin, pyridoxine, folic acid, menadione, quercetin, naringenin, humic acid, fulvic acid, boric acid, zinc sulfate, manganese sulfate, Piriformospora indica and binucleate Rhizoctonia were evaluated to improve bean resistance to root rot pathogen Rhizoctonia solani AG-4 HGII. Under laboratory conditions, effect of various biological agents and chemical compounds on mycelial growth of the pathogen was evaluated. Results showed that applying boric acid, zinc sulfate and manganese sulfate at 1000 and 2000 mg L−1 concentrations and menadione at 2000, 5000, 10000 and 20000 μM completely inhibited the pathogen growth and 10000 and 20000 μM concentrations of thiamine reduced linear growth of R. solani. Under greenhouse conditions, application of the inducers significantly reduced the disease progress on treated plants compared to the non-treated plants. The highest level of disease reduction was observed using zinc sulfate and thiamine applied at 3 days before the pathogen inoculation, and also by using P. indica 14 days before inoculation with the pathogen. Thiamine, zinc sulfate and P. indica significantly reduced the disease index and improved plant growth parameters compared to the controls. Induction of resistance against R. solani by individual and combined treatments using thiamine, zinc sulfate and P. indica was associated with increased activity of various antioxidants and increased levels of membrane stability index (MSI), relative water content (RWC), lignin accumulation and total soluble phenolics in bean seedlings. Our findings revealed a practical possibility of inducing resistance and protection of bean plants against Rhizoctonia root rot with the help of biological and chemical resistance inducers.

First Report of Rhizoctonia solani AG4 HG-II Infecting Barley Stems in Idaho

First Report of <i>Rhizoctonia solani</i> AG4 HG-II Infecting Barley Stems in Idaho

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.

First report of Rhizoctonia solani AG-4 HG-I causing crown rot on Abelmoschus manihot in Italy

First report of Rhizoctonia solani AG-4 HG-I causing crown rot on Abelmoschus manihot in Italy